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Complex regulations of swarming and surfing motilities in pseudomonas aeruginosa Yeung, Amy Tsz Yan 2012

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COMPLEX REGULATIONS OF SWARMING AND SURFING MOTILITIES IN PSEUDOMONAS AERUGINOSA  by AMY TSZ YAN YEUNG  B.Sc., The University of British Columbia, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (MICROBIOLOGY AND IMMUNOLOGY)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  September 2012  © Amy Tsz Yan Yeung, 2012  Abstract To investigate how the complex adaptation process of swarming is regulated, a P. aeruginosa PA14 transposon mutant library was screened for mutants defective in swarming. As a result, 233 mutants exhibiting alterations in swarming phenotypes were identified and 35 of these genes encoded for regulators. Only a few of these regulatory mutants showed significant defects in the production of type IV pili, flagella, or rhamnolipid, each of which is known to be involved in swarming, suggesting that the majority of these regulators control other factors important in swarming. One regulatory mutant with a mutation in the cbrA gene was chosen to be investigated in detail. In addition to swarming motility and carbon source utilization, the sensor kinase CbrA was shown to play regulatory roles in other virulence and virulence-related processes of Pseudomonas, including biofilm formation, cytotoxicity, and antibiotic resistance. Microarray analysis revealed hundreds of dysregulated genes in the cbrA mutant that might contribute to the virulence and virulence-related phenotypes observed in the mutant. Phenotypic and genetic analyses of a cbrB mutant suggested that CbrA modulated swarming, biofilm formation, and cytotoxicity via the CbrB response regulator and that the CrcZ small RNA and the Crc protein are likely downstream of this two-component regulator. Little was known about the mode of motility P. aeruginosa uses to colonize the lungs of patients with cystic fibrosis (CF) since the viscous lung environment in vivo is influenced by mucin in the mucous. To investigate this, the nutritional composition of the CF sputum was mimicked using plates containing synthetic CF medium (SCFM) with mucin. Addition of small amounts (0.05%) of mucin to SCFM-swimming agar led P. aeruginosa to undergo accelerated motility on the surface of the agar. The surface motility colonies in the presence of mucin were circular with a green center surrounded by a thicker white edge. In contrast to swarming, bacterial cells at the edge of the mucin-promoted motility zone appeared piled up and lacked flagella. Using genetic and microscopic methods, it was demonstrated that mucin might be promoting a modified form of swarming or likely a new form of surface motility, termed “surfing”.  ii  Preface The majority of the research presented in this thesis was drawn from either published literature or submitted manuscripts. Below is a description of the contributions made by fellow scientists and collaborators. Chapter 2: A version of Chapter 2 has been published and copyright permission was granted. Yeung, A.T.Y., Torfs, E.C.W., Jamshidi, F., Bains, M., Wiegand, I., Hancock, R.E.W., and Overhage, J. (2009) Swarming of Pseudomonas aeruginosa is Controlled by a Broad Spectrum of Transcriptional Regulators including MetR. J. Bacteriol. 191: 5592-5602. Ellen Torfs and I are joint first authors in the paper. The initial high throughput swarming screen was performed by Ellen Torfs and Farzad Jamshidi. Although not described in this Chapter, detailed studies of a metR mutant were performed by Ellen and a metR microarray experiment was performed by Manjeet Bains. The remaining experiments described in this chapter were performed by me including performing swarming experiments to verify the swarming motility of 393 Pseudomonas aeruginosa transposon mutants. I also characterized 35 regulatory mutants in greater detail by screening the ability of these mutants to swim, twitch and form biofilm. Moreover, I wrote the initial version of the chapter. Bob Hancock and Irith Wiegand were involved in the original conception of the swarming screen. Bob and Joerg Overhage were involved in discussing the research plan and results and editing the manuscript. Chapter 3: A version of Chapter 3 has been published and copyright permission was granted. Yeung, A.T.Y., Bains, M., and Hancock, R.E.W. (2011) The sensor kinase CbrA is a global regulator that modulates metabolism, virulence and antibiotic resistance in Pseudomonas aeruginosa. J Bacteriol.193: 918-31. I performed almost all experiments described in the chapter with the exception of the DNA microarray experiment which was performed by Manjeet Bains. I also wrote 100% of the chapter. Bob Hancock was involved in discussing the research plan and experimental results and editing the manuscript. Chapter 4: Data presented in this chapter is part of a manuscript “Pseudomonas aeruginosa CbrA is required for full virulence in a murine acute lung infection model” currently in review. Laure iii  Janot and I are joint first authors of the manuscript. I performed all of the in vitro experiments described in the chapter involving human cells and am responsible for writing the initial draft of the paper. Laure Janot and Ashley Hilchie performed the mouse infection experiments and Laure Janot performed the in vitro experiments involving murine cells. Olga Pena collected blood from human donors, assisted me with the isolation of PBMCs, neutrophils and taught me how to differentiate monocytes into macrophages. She also helped me with the design and performance of LDH assays, phagocytosis assays, and macrophage/neutrophil killing assays. Laurence Madera helped with the blood collection from human donors and isolation of PBMCs and neutrophils. Irina Kukavica-Ibrulj performed the rat chronic lung infection experiments in the laboratory of Roger Levesque with his guidance. Bob Hancock was involved in designing and discussing the research plan and providing input regarding protocols and experimental results. Venous blood was collected from healthy adult donors in accordance with the ethical approval guidelines of the UBC Research Ethics Board. All mice experiments were conducted in accordance with the Animal Care Ethics Approval and Guidelines of the University of British Columbia (Certificate numbers of the Ethics Certificates obtained for the peritoneal infection model was A08-723 and for the acute lung infection model was A10-0261). Rats were used according to the ethics committee for animal treatment of Laval University (Certificate number of the Ethics Certificate obtained for the chronic lung infection model was 2011194-1). Chapter 5: A version of Chapter 5 has been published and copyright permission was granted. Yeung, A.T.Y., Parayno, A., and Hancock, R.E.W. (2012) Mucin promotes rapid surface motility in Pseudomonas aeruginosa. mBio 3(3): e00073-12. Alicia Parayno, a UBC undergraduate student, assisted me in testing the surface motility of P. aeruginosa under different carbon and nitrogen sources. She also assisted me with the screening of a total of 127 two-component sensor kinase and response regulator transposon mutants for their surface motilities on mucin plates. In addition, she helped me screen motility-defective regulatory mutants for biofilm production and growth. Lindsay Heller helped me in the studies involving examination of the surface motility zones using a light microscope. I performed all of the other experimental work described in the chapter. Moreover, I wrote 100% of the chapter. R. Hancock was involved in discussing the research plan, suggesting new approaches, discussing experimental results, and editing the manuscripts.  iv  Table of Contents Abstract .................................................................................................................................... ii Preface ..................................................................................................................................... iii Table of Contents ...................................................................................................................... v List of Tables ............................................................................................................................ix List of Figures............................................................................................................................ x List of Abbreviations .............................................................................................................. xii Acknowledgements ................................................................................................................. xiv Chapter 1: Introduction ........................................................................................................... 1 1.1 Pseudomonas aeruginosa and its pathogenesis ............................................................. 1 1.1.1 Prevalence of P. aeruginosa infections ........................................................................ 1 1.1.2 Acute infections ........................................................................................................... 2 1.1.3 Chronic infections........................................................................................................ 2 1.1.4 P. aeruginosa cell-associated virulence factors ............................................................ 3 1.1.4.1 Flagella ................................................................................................................... 4 1.1.4.2 Type IV pili ............................................................................................................ 6 1.1.4.3 Lipopolysaccharide ................................................................................................. 7 1.1.4.4 Alginate .................................................................................................................. 8 1.1.5 P. aeruginosa secreted virulence factors and associated secretion systems ................... 8 1.1.5.1 Pyocyanin ............................................................................................................... 8 1.1.5.2 Pyoverdine .............................................................................................................. 9 1.1.5.3 Rhamnolipid ........................................................................................................... 9 1.1.5.4 Proteases ............................................................................................................... 11 1.1.5.5 Type 2 secretion system ........................................................................................ 12 1.1.5.6 Type 3 secretion system ........................................................................................ 12 1.1.6 P. aeruginosa quorum sensing ................................................................................... 13 1.1.7 P. aeruginosa resistance to antibiotics ....................................................................... 15 1.1.7.1 Intrinsic resistance ................................................................................................ 16 1.1.7.2 Acquired resistance ............................................................................................... 17 1.1.7.3 Adaptive resistance ............................................................................................... 17 1.1.8 P. aeruginosa biofilm formation ................................................................................ 18 1.1.9 P. aeruginosa metabolic versatility ............................................................................ 20 1.1.10 P. aeruginosa two-component regulatory systems ..................................................... 21 1.2 P. aeruginosa motilities and their roles in pathgenesis ............................................... 23 1.2.1 Swimming ................................................................................................................. 23 1.2.2 Twitching .................................................................................................................. 24 1.2.3 Sliding ....................................................................................................................... 25 1.3 Swarming motility in P. aeruginosa ............................................................................ 25 1.3.1 Features required for swarming .................................................................................. 25 1.3.2 Role of rhamnolipids and its precursor 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs) ................................................................................................................................. 27 1.3.3 Role of swarming motility in the pathogenesis of P. aeruginosa ................................ 27 1.4  Nutritional environment of the CF sputum ................................................................ 28 v  1.4.1 Impact on P. aeruginosa pathogenesis ....................................................................... 28 1.4.2 Role of mucin in the CF sputum................................................................................. 29 1.5 Host strategies against P. aeruginosa infections ......................................................... 29 1.5.1 Mechanical defence mechanisms ............................................................................... 30 1.5.2 Innate immune response ............................................................................................ 30 1.6 Hypotheses and objectives ........................................................................................... 31 1.6.1 Hypotheses ................................................................................................................ 31 1.6.2 Specific objectives ..................................................................................................... 31 Chapter 2: Swarming motility, a complex adaptation controlled by a broad spectrum of transcriptional regulators ....................................................................................................... 33 2.1  Introduction ................................................................................................................. 33  2.2 Materials and methods ................................................................................................ 33 2.2.1 Bacterial strains and growth conditions ...................................................................... 33 2.2.2 Screening of P. aeruginosa strain PA14 transposon mutant library for swarming motility ................................................................................................................................. 33 2.2.3 Growth curves ........................................................................................................... 34 2.2.4 Swimming and twitching motility .............................................................................. 34 2.2.5 Biofilm formation ...................................................................................................... 35 2.2.6 Rhamnolipid production ............................................................................................ 35 2.2.7 Phage PO4 sensitivity test .......................................................................................... 35 2.3 Results .......................................................................................................................... 36 2.3.1 Identification of PA14 transposon mutants with altered swarming phenotypes ........... 36 2.3.2 Characterization of swarming-associated regulatory genes ......................................... 39 2.4  Discussion ..................................................................................................................... 41  Chapter 3: Role of P. aeruginosa CbrA sensor kinase in metabolism, swarming, biofilm formation, antibiotic resistance and virulence ....................................................................... 45 3.1  Introduction ................................................................................................................. 45  3.2 Materials and methods ................................................................................................ 46 3.2.1 Tissue culture, bacterial strains and growth conditions ............................................... 46 3.2.2 General DNA manipulations ...................................................................................... 46 3.2.3 Recombinant DNA manipulations.............................................................................. 47 3.2.4 Motility experiments .................................................................................................. 48 3.2.5 Biofilm and rapid attachment assays .......................................................................... 48 3.2.6 Congo red binding assays .......................................................................................... 48 3.2.7 Minimum inhibitory concentration determination and polymyxin B killing experiments ........................................................................................................................... 49 3.2.8 Cytotoxicity toward non-polarized HBE cells ............................................................ 49 3.2.9 Growth curves ........................................................................................................... 49 3.2.10 RT-qPCR ................................................................................................................... 50 3.2.11 DNA microarray experiment ...................................................................................... 50 3.2.12 Pyoverdine assay ....................................................................................................... 51 3.3 Results .......................................................................................................................... 52 3.3.1 Construction of a PA14 cbrA deletion mutant ............................................................ 52 3.3.2 Defect in swarming motility in the cbrA mutant ......................................................... 53 vi  3.3.3 Enhanced biofilm formation....................................................................................... 54 3.3.4 Enhanced cytotoxicity toward HBE cells ................................................................... 56 3.3.5 Increased resistance to polymyxins, aminoglycosides, and fluoroquinolones .............. 57 3.3.6 Microarray analysis of the cbrA mutant...................................................................... 60 3.3.7 Transcriptional analysis of the cbrA mutant under various growth conditions ............ 64 3.3.8 CbrA mediated regulation of swarming, biofilm formation, and cytotoxicity in conjunction with CbrB .......................................................................................................... 65 3.3.9 CbrAB system regulation of swarming, biofilm formation, and cytotoxicity via the downstream regulatory system CrcZ/Crc ............................................................................... 66 3.4  Discussion ..................................................................................................................... 70  Chapter 4: Requirement for P. aeruginosa CbrA for full virulence in a murine acute lung infection model ........................................................................................................................ 75 4.1  Introduction ................................................................................................................. 75  4.2 Materials and methods ................................................................................................ 75 4.2.1 Tissue culture, bacterial strains and growth conditions ............................................... 75 4.2.2 Mice models of peritoneal and acute lung infection .................................................... 76 4.2.3 Rat model of chronic lung infection ........................................................................... 77 4.2.4 Cytotoxicity assays .................................................................................................... 78 4.2.5 Growth curves ........................................................................................................... 78 4.2.6 In vitro phagocytosis assays (gentamicin protection assay) ........................................ 78 4.2.7 In vitro macrophage and neutrophil killing assays ...................................................... 79 4.3 Results .......................................................................................................................... 80 4.3.1 Lack of effect of varying carbon sources on cytotoxicity of the cbrA mutant toward HBE cells .............................................................................................................................. 80 4.3.2 Similar cytotoxicity towards PBMCs of PA14 WT and the cbrA mutant .................... 81 4.3.3 Reduced virulence of the cbrA mutant in mouse models of acute lung infection ......... 82 4.3.4 Reduced virulence of the cbrA mutant in a mouse model of peritoneal infection ........ 83 4.3.5 Lack of effect of the cbrA mutant on in vivo competitive growth................................ 86 4.3.6 Enhanced uptake of the cbrA mutant by macrophages ................................................ 87 4.3.7 Enhanced clearance of the cbrA mutant by macrophages and neutrophils ................... 88 4.4  Discussion ..................................................................................................................... 91  Chapter 5: Rapid surface motility promoted by mucin in P. aeruginosa ............................. 94 5.1  Introduction ................................................................................................................. 94  5.2 Materials and methods ................................................................................................ 94 5.2.1 Bacterial strains and growth conditions ...................................................................... 94 5.2.2 Mucin-promoted motility assays ................................................................................ 95 5.2.3 Growth curves ........................................................................................................... 95 5.2.4 Electron microscopy .................................................................................................. 96 5.2.5 Light microscopy ....................................................................................................... 96 5.2.6 RT-qPCR ................................................................................................................... 96 5.3 Results .......................................................................................................................... 96 5.3.1 Mucin promoted the same surface motility pattern for strains PA14 and PAO1 .......... 96 5.3.2 Mucin promoted rapid surface motility ...................................................................... 98 5.3.3 Increasing the concentration of mucin did not significantly enhance growth of P. aeruginosa ............................................................................................................................ 98 vii  5.3.4 Mucin-promoted surface motility was dependent on flagella expression but did not require type IV pili ................................................................................................................ 99 5.3.5 Mucin-promoted surface motility was dependent on cell-to-cell signalling............... 102 5.3.6 P. aeruginosa at the edge of mucin-promoted surface motility zone lacked flagella . 103 5.3.7 Promotion of mucin-mediated motility by amino acids and inhibition by ammonium .......................................................................................................................... 104 5.3.8 P. aeruginosa in the mucin-mediated motility zone upregulated the expression of certain genes involved in virulence and resistance ............................................................... 107 5.3.9 Mucin promotion of surface motility may involve lubricant-like action.................... 109 5.3.10 Regulation of mucin-mediated surface motility ........................................................ 110 5.3.11 Bacteria at the edge of the mucin-mediated motility zone appeared to stack ............. 112 5.4  Discussion ................................................................................................................... 113  Chapter 6: Concluding chapter ........................................................................................... 117 6.1  Introduction ............................................................................................................... 117  6.2  Complex regulation of swarming motility in P. aeruginosa ..................................... 118  6.3 The CbrAB TCS is a global regulator of swarming and various virulence-related processes in P. aeruginosa ..................................................................................................... 120 6.4  Requirement of acylhomoserine lactone acylase activity for swarming? ................ 121  6.5  The P. aeruginosa CbrA/CbrB TCS exhibits similarities to the GacS/GacA TCS .. 122  6.6  Interaction of CbrA with other sensor kinases? ....................................................... 122  6.7  Is mucin promoting P. aeruginosa to swarm or to surf? .......................................... 124  Bibliography .......................................................................................................................... 127 Appendix A ............................................................................................................................ 145 Appendix B ............................................................................................................................ 152 Appendix C ............................................................................................................................ 155  viii  List of Tables Table 2-1 Characteristics of transcriptional regulators involved in swarming. ........................... 37 Table 3-1 P. aeruginosa strains and plasmids used in this study. .............................................. 51 Table 3-2 Minimal inhibitory concentrations (µg/ml) of antibiotics toward P. aeruginosa grown in swarming medium. ............................................................................................................... 58 Table 3-3 Selected genes significantly dysregulated in the cbrA mutant as determined using microarrays. ............................................................................................................................. 61 Table 3-4 Dysregulated genes in the cbrA, cbrB, crcZ and crc mutants during HBE cell infections and growth on Congo red plates as determined by RT-qPCR. ................................... 65 Table 4-1 Average intracellular bacterial counts for PA14 WT and cbrA mutant recovered from macrophages or neutrophils at various time points. ................................................................... 91 Table 5-1 Average diameters of motility zones, and changes in diameter over time, of P. aeruginosa strain PA14 motilities in 0.3% (wt/vol) agar (swimming), 0.5% (wt/vol) agar (swarming) and 0.3% (wt/vol) agar with varying concentrations of mucin (surfing). ................ 99 Table 5-2 Expression of selected genes in P. aeruginosa strain PA14 taken from the surface motility zone with 0.3% agar and 0.4% mucin compared to bacteria swimming in 0.3% agar without mucin, as determined using RT-qPCR. .......................................................................108 Table 5-3 Characteristics of TCS sensor kinases or response regulators involved in surface motility on MSCFM-mucin plates.. .........................................................................................111 Table A-1 List of P. aeruginosa PA14 transposon insertion mutants displaying deficiencies in swarming motility. ..................................................................................................................145 Table C-1 List of P. aeruginosa genes with expression up- or down-regulated in the cbrA mutant compared to the PA14 WT. .....................................................................................................155  ix  List of Figures Figure 1.1 Cell-associated virulence factors in P. aeruginosa. .................................................... 4 Figure 1.2 Structure of P. aeruginosa LPS. ................................................................................ 7 Figure 1.3 Structures of HAA and rhamnolipids in P. aeruginosa. ........................................... 10 Figure 1.4 Stages of biofilm development in P. aeruginosa. ..................................................... 19 Figure 1.5 Schematic diagram of TCS signalling pathways in P. aeruginosa. ........................... 22 Figure 1.6 Representation of the various types of motilities in P. aeruginosa. .......................... 24 Figure 2.1 Selected P. aeruginosa PA14 transposon mutants displaying altered swarming and swimming motilities compared to PA14 WT. ........................................................................... 36 Figure 2.2 Biofilm formation by mutants with altered swarming motility. ................................ 42 Figure 3.1 Swarming motility of the PA14 cbrA and cbrB mutants. .......................................... 53 Figure 3.2 Biofilm formation of the PA14 cbrA and cbrB mutants............................................ 54 Figure 3.3 Congo red binding. .................................................................................................. 55 Figure 3.4 Influence of carbon source on biofilm formation of the PA14 cbrA deletion mutant. 56 Figure 3.5 In vitro cytotoxicity towards HBE cells. .................................................................. 57 Figure 3.6 Polymyxin B resistance in the PA14 cbrA deletion mutant. ..................................... 58 Figure 3.7 Growth curves of P. aeruginosa PA14 WT and cbrA deletion mutant. ..................... 60 Figure 3.8 Pyoverdine production of the PA14 cbrA deletion mutant........................................ 64 Figure 3.9 Swarming motility, biofilm formation, Congo red binding and in vitro cytotoxicity of the PA14 crc mutant. ............................................................................................................. 68 Figure 3.10 Swarming motility, biofilm formation and in vitro cytotoxicity of the PA14 crcZ mutant. ..................................................................................................................................... 69 Figure 3.11 Proposed model for the involvement of the CbrA/CbrB/CrcZ regulatory cascade in the regulation of swarming, biofilm formation, cytotoxicity and antibiotic resistance in P. aeruginosa. .............................................................................................................................. 71 Figure 4.1 In vitro cytotoxicity toward HBE cells. .................................................................... 81 Figure 4.2 In vitro cytotoxicity toward human PBMCs. ............................................................ 82 Figure 4.3 Bacterial load in BAL of mice infected with P. aeruginosa. .................................... 83 Figure 4.4 Bacterial load in the peritoneal lavage and blood of mice infected with P. aeruginosa. ................................................................................................................................................ 85 Figure 4.5 In vivo CI analysis of cbrA mutant in a rat model of chronic lung infection in competition with the PA14 WT strain....................................................................................... 86 x  Figure 4.6 Phagocytosis of P. aeruginosa by human macrophages. .......................................... 88 Figure 4.7 Killing of P. aeruginosa by phagocytes. .................................................................. 90 Figure 5.1 Swimming (A), Swarming (B, C) and mucin-promoted (D) motilities of P. aeruginosa. .............................................................................................................................. 97 Figure 5.2 Progression of P. aeruginosa PA14 surface motility zones over time. ...................... 98 Figure 5.3 Percentage fold changes of surface coverage of PA14 flagella (A) type IV pili (B) and quorum sensing (C) mutants compared to PA14 WT on MSCFM with 0.4% mucin and 0.3% agar. ........................................................................................................................................101 Figure 5.4 Example images of mucin-promoted surface motilities of PA14 WT and a fliC flagella mutant (A), chpB type IV pili mutant (B), fliCpilA flagella and type IV pili double mutant (C), and rhlI quorum sensing mutant with and without addition of C4-HSL (D). .........102 Figure 5.5 Electron microscopy images of P. aeruginosa strain PA14 WT from motility colonies on 0.5% agar or 0.4% mucin. ..................................................................................................104 Figure 5.6 Surface motility of PA14 WT on mucin plates supplemented with various carbon and nitrogen sources. .....................................................................................................................106 Figure 5.7 Growth and bacterial cell counts of PA14 WT using various amino acids as sole nitrogen sources. .....................................................................................................................107 Figure 5.8 Surface propagation of P. aeruginosa strain PA14 WT on wetting or viscosity enhancing agents. ....................................................................................................................109 Figure 5.9 Light microscopy images of PA14 WT surface motility zones on MSCFM-mucin plates.......................................................................................................................................113 Figure 6.1 Models for the expansion of the surfing motility zone.............................................126 Figure B.1 Classification of swarming-associated genes according to their predicted functions. ...............................................................................................................................................152 Figure B.2 Classification of genes from PA14 genome according to their predicted functions. 153 Figure B.3 Comparison of the functional composition of the swarming-associated genes to the composition of the PA14 genome. ...........................................................................................154  xi  List of Abbreviations aGM1 AHL AQ ASL BAL BHI BMDM CAA CDC cDNA CFU CI CMC COX CR Crc CF CFTR CFU DMEM DNA dNTP DTT EPS eDNA FBS FCS HAA HBE HBSS HHQ HIV Hpt ICU IL IP LB LDH LPS M-CSF MDR MDM MEM MHA MIC MOI  Asialo-gangloside M1 Acyl-homoserine lactone 2-alkyl-4-quinolone Airway surface liquid Bronchoalveolar lavage Brain heart infusion Bone marrow-derived macrophage Casamino acid Center for Disease Control Complementary deoxyribonucleic acid Colony forming unit Competitive index Carboxymethyl cellulose Cyclooxygenase Congo red Catabolite repression control Cystic fibrosis Cystic fibrosis transmembrane conductance regulator Colony forming unit Dulbecco's Modified Eagle's medium Deoxyribonucleic acid Deoxynucleoside triphosphates Dithiothreitol Extracellular polymeric substance Extracellular deoxyribonucleic acid Fetal bovine serum Fetal calf serum 3-(3-hydroxyalkanoyloxy) alkanoic acid Human bronchial epithelial Hank’s buffered salt solution 2-heptyl-4-quinolone Human immunodeficiency virus Histidine phosphotransferase Intensive care unit Interleukin Intraperitoneal Luria Bertani Lactate dehydrogenase Lipopolysaccharide Macrophage colony-stimulating factor Multidrug resistant Monocyte-derived macrophage Minimum essential medium Mueller Hinton agar Minimum inhibitory concentration Multiplicity of infection xii  mRNA MSCFM NET NFκB NIH NNIS PAMP PMBC PBS PCL PCR PQS PRR QS RNA RT-qPCR RPM RPMI SDS sRNA SCFM SV40 TEM T1SS T2SS T3SS TCA TCS TEM TLR TNFα TSB UBC US UTI VAP WT  Messenger ribonucleic acid Modified synthetic cystic fibrosis sputum medium Neutrophil extracellular trap Nuclear factor kappa B National Institutes of Health National Nosocomial Infections Surveillance Pathogen-associated molecular pattern Peripheral blood mononuclear cell Phosphate-buffered saline Periciliary liquid Polymerase chain reaction Pseudomonas aeruginosa quinolone signal Pathogen recognition receptor Quorum sensing Ribonucleic acid Quantitative real time polymerase chain reaction Revolutions per minute Roswell Park Memorial Institute medium Sodium dodecyl sulphate Small ribonucleic acid Synthetic cystic fibrosis sputum medium Simian virus 40 Transmission electron microscopy Type 1 secretion system Type 2 secretion system Type 3 secretion system Tricarboxylic acid Two-component system Transmission electron microscopy Toll-like receptor Tumor necrosis factor-alpha Tryptic soy broth University of British Columbia United States Urinary tract infection Ventilator-associated pneumonia Wild type  xiii  Acknowledgements Funding of this work has been provided through the Natural Sciences and Engineering Research Council (NSERC) Canada Postgraduate Scholarship and the Cystic Fibrosis Canada Studentship. This research was also funded by the Canadian Institute of Health Research (CIHR) Grants to Dr. Bob Hancock. I truly thank Dr. Bob Hancock for taking me on as a graduate student and providing me with tremendous amounts of encouragements, guidance and supervision over the years. I will never forget Bob’s enthusiasm for research and he remains my best role model for a scientist, mentor, and teacher. I am also extremely grateful to my committee members Drs. Michael Murphy, Rachel Fernandez and Erin Gaynor for their helpful career advice and support for these past 4 years. I would like to thank all the past and present members of the Hancock lab for lending me a hand when I needed one and making the lab such a wonderful place to work in. In particular I would like to thank Olga for her great emotional support during stressful times, Shaan for her help with formatting my thesis, Susan for handling the financial side of things and her advice on scholarship applications, Bernadette for her tremendous support during my job search, and Manjeet Bains for her assistance with the microarray slides. Finally, I would like to express my deepest gratitude to my mom, dad, and my younger brother for providing me with their endless patience, love, and care throughout the years. A special thanks to my fiancé, Aloysius, who has been a great supporter and mentor throughout my studies. Thank you for your unconditional love and standing by me during good and bad times. I especially thank you for having faith in me and my intellect even at times when I didn’t have faith in myself.  xiv  Chapter 1: Introduction 1.1  Pseudomonas aeruginosa and its pathogenesis P. aeruginosa is a Gram-negative, rod shaped bacterium that can survive in almost any  environment but is found living primarily in soil, water, and vegetation. This opportunistic human pathogen is responsible for a number of serious acute and chronic infections in patients with impaired immunity and mucosal defences (36, 241). The capacity of P. aeruginosa to cause these often detrimental infections is due to its arsenal of cell-associated (e.g. flagella, pili, alginate, LPS) and extracellular virulence factors (e.g. proteases, hemolysins, exotoxin A, exoenzyme S, pyocyanin) (155). P. aeruginosa is also equipped with highly complex cell-to-cell signalling systems to control expression of these virulence factors. In addition, the ability of P. aeruginosa to exhibit metabolic flexibility, resistance to antibiotics and to form biofilms enables the bacteria to persist and cause chronic infections. 1.1.1 Prevalence of P. aeruginosa infections P. aeruginosa is predominately associated with hospital-acquired infections, including pneumonia, urinary tract infections (UTI), bloodstream infections, surgical site infections and skin infections in the setting of burn injuries (60). P. aeruginosa can be isolated from nearly any conceivable source within hospitals (e.g. respiratory therapy equipments, antiseptics, soap, sinks, mops, medicines, and physiotherapy and hydrotherapy pools) (20, 199). According to the surveillance data collected by the CDC NNIS System from 1986 to 1998, P. aeruginosa is the 5th most frequently isolated nosocomial pathogen, accounting for 9% of all hospital-acquired infections in the US (1, 64). It is the 2nd leading cause of hospital-acquired pneumonia, healthcare-associated pneumonia and ventilator-associated pneumonia (VAP) (78, 129). It is also the 3rd most common cause of UTI (7-11% of cases), 4th most frequently isolated pathogen in surgical site infections (8% of cases) and 7th leading contributor to bloodstream infections (2-6% of cases). P. aeruginosa is the most frequent infectious isolate in burn units accounting for large percentage of wound infections, bacteraemia and VAP in these units (136, 232, 277). This bacterium is also the leading cause of pneumonia among pediatric patients in the ICU (211). P. aeruginosa is especially problematic for seriously ill patients in ICU as data from NNIS System showed this pathogen was responsible for 13.2-22.6% of all ICU infections (67, 75, 119, 154). It is also a prevalent pathogen causing infections in patients with primary and acquired immunodeficiencies. It is the most common cause of septicaemia and bacteraemia in patients 1  with acute leukaemia (144). In a review of 233 autopsies of HIV-1 patients, P. aeruginosa was identified as the most common cause of bacterial bronchopneumonia, accounting for 16 of 98 cases (5). Chronic sinopulmonary colonization and recurrent infections from P. aeruginosa are commonly observed in Cystic Fibrosis (CF) patients (60). In 2004 the US CF Foundation Patient Registry reported 57.3% of all reported respiratory cultures contained P. aeruginosa. Up to 97.5% of CF patients were found to be infected with P. aeruginosa by the age of 3 years (30). CF patients colonized with P. aeruginosa are associated with higher rates of mortality, increased frequency of hospitalization, decreased lung function and lower weight (63). In addition to hospitals, P. aeruginosa can be found in community reservoirs including swimming pools, whirlpools, hot tubes, contact lens solutions, home humidifiers, soil and rhizosphere and vegetables (97). This bacterium is responsible for variety of community-acquired infections, including ulcerative keratitis (associated with contact lens use), otitis externa (in immunocompromised hosts such as those with diabetes mellitus), and skin and soft tissue infections (including diabetic foot infections) (90). Despite abundant opportunities for spread, P. aeruginosa is rarely a member of the normal microbial flora in humans and seldom causes infections in healthy individuals (175). 1.1.2 Acute infections Acute P. aeruginosa infections are typified by bacterial penetration of the host epithelium and systemic spread, such as in severe burn wound infections. These types of infections are invasive, cytotoxic and frequently result in systemic infection, sepsis, and mortality. P. aeruginosa requires expression of many virulence factors, including various toxins, proteases via Type 2 secretion system (T2SS) and Type 3 secretion system (T3SS) (140). P. aeruginosa isolates responsible for acute infections predominantly express full length LPS side chains, are motile and do not produce significant amounts of alginate (73, 82). 1.1.3 Chronic infections CF patients are particularly susceptible to chronic infection by P. aeruginosa. CF is a genetic disorder resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a transmembrane chloride channel in secretory epithelia (99). The mutation results in abnormal transport of chloride and sodium across the epithelium leading to accumulation of thick mucous (or sputum) in the airways of CF patients. The thick mucous becomes an ideal growth medium for multiple opportunistic pathways, with P. 2  aeruginosa being the predominant pathogen (178). To establish P. aeruginosa chronic lung infection in CF patients, the first step involves the inhalation of P. aeruginosa from the environment. The bacteria initially attach to the upper respiratory tract and then gain access to the lung parenchyma due to failure of normal mucociliary clearance mechanisms (178, 269). Finally, to establish a chronic infection, P. aeruginosa must undergo a number of genetic changes to assist the ability of the pathogen to survive in the host and to evade detection and clearance by the immune system. In contrast to acute infections, P. aeruginosa isolates obtained from CF patients during chronic infection often exhibit loss of motility (162) and become rough thorough loss of their O-antigens (96). During chronic infections, P. aeruginosa often exhibits a mucoid phenotype due to overproduction of alginate and grows as sessile biofilm aggregates (237). It was speculated that P. aeruginosa loses its immunogenic features such as pili and flagella to avoid detection and clearance by the host immune system. P. aeruginosa isolates from chronic infections often exhibit lack of expression of the T3SS and its associated effectors suggesting these virulence factors are no longer required for the pathogen to persist in the host (113). In addition to the lack of virulence gene expression, P. aeruginosa isolates often exhibit decreased production of pyoverdine, O-antigen polysaccharide and pyocins (48). These isolates are also more likely to exhibit hypermutability, increased phage resistance, increased resistance to antibiotics and convert to small colony variants (85, 185). 1.1.4 P. aeruginosa cell-associated virulence factors To initiate infections after the primary defence barriers of the host are breached, P. aeruginosa must colonize the altered epithelium. This colonization step is mostly dependent on cell-associated virulence factors, including flagella, pili, alginate and LPS (Fig. 1.1). These virulence factors facilitate P. aeruginosa adherence to the epithelium. In addition, they play important roles in mediating P. aeruginosa motility, invasion, biofilm formation, and host inflammatory response.  3  Figure 1.1 Cell-associated virulence factors in P. aeruginosa. Image adapted, with permission, from (123). 1.1.4.1 Flagella P. aeruginosa are typically equipped with a single polar flagellum, enabling the bacteria to swim in aqueous environments (186). The expression of flagella in P. aeruginosa is tightly regulated, involving 41 genes encoding structural, assembly, and regulatory components organized into a four-tiered hierarchy (45). Each flagellum is comprised of three sections- the basal body, the hook and the filament. The basal body is embedded within the cell surface and is comprised of 3 rings- the cytoplasmic membrane supramembrane ring, the peptidoglycan ring and the outer membrane lipopolysaccharide ring, encircling a rod that transverses the periplasm(45). The hook is the joint between the basal body and the filament. The filament is made up of polymerized flagellin subunits and is capped by the flagellar cap, FliD, which acts as a mucin adhesion (45).  4  Interestingly, comparison of the ultrastructure of the basal bodies of the flagellum and the T3SS machinery revealed significant similarities (19). In addition, inverse regulation of flagella expression and T3SS expression were demonstrated. Soscia et al. showed that in a nonflagellated fliC mutant, the T3SS regulon was upregulated resulting in an increase in T3SS effector secretion and in vitro cytotoxicity (235). The group also showed that global activation of the T3SS had an inverse effect on flagellar-gene expression as flagellar gene expression and motility were decreased in an ExsA-overproducing strain (ExsA is a positive regulator of the T3SS) (235). Moreover, upregulation of the T3SS was not observed in ∆motAB and ∆motY mutants (mutants expressing non-functional flagella) suggesting that it is the absence of the flagella structure that result in upregulation of the T3SS (235). The flagella play a critical role in the pathogenesis of P. aeruginosa. An isogenic P. aeruginosa mutant that lacked flagella exhibited significant reduction in virulence compared to the wild type (WT) strain in a mouse burn model of infection and neonatal mouse model of pneumonia (59, 68). While the non-motile P. aeruginosa mutants proliferated in the wound, the characteristic bacteremia and systemic invasion typically observed with the WT strain were markedly absent. Moreover, a study demonstrated passive transfer of anti-flagellar serum increased protein and opsonophagocytosis against P. aeruginosa infections (59). A primary role of flagella is to mediate adhesion to epithelial cells. Studies showed that P. aeruginosa mediate epithelial cell contact through binding to a common epithelial surface glycolipid asialogangloside M1 (aGM1) (68). aGM1 is maximally expressed in the host during epithelial cell repair processes, which may explain why P. aeruginosa primarily adhere to injured respiratory epithelium. Flagellar genes (e.g. fliD) have also been demonstrated to contribute to binding to respiratory mucin, a major glycoprotein found in sputum (11). In addition to swimming, flagella are also required for P. aeruginosa to undergo swarming (128) and to form biofilms on biotic and abiotic surfaces (186). Flagella have been suggested to play a role in biofilm formation by facilitating initial surface interaction. The role of flagella in host inflammatory responses has also been documented (3, 54). Toll-like receptor 5 (TLR5), a sensor for monomeric flagellin, is one of the many TLRs employed by the host as key components of the immune system to detect microbial infection and trigger antimicrobial host defence responses (3). Flagellin released from P. aeruginosa trigger the airway epithelial TLR5 signalling via the nuclear factor kappa B (NFκB) to result in production and release of proinflammatory cytokines to recruit neutrophils to the infected region (3). Optimally, neutrophils play roles in helping to clear microorganisms from mucosal surfaces, however, in CF patients, chronic P. aeruginosa infection lead to a 5  hyperinflammatory response that is detrimental in part because a large number of neutrophils accumulate damaging the CF lung tissue. 1.1.4.2 Type IV pili P. aeruginosa type IV pili are flexible and strong surface filaments made up of thousands of copies of a single pilin subunit, PilA (165). They can occur at one or both poles of the bacterium with multiple pili usually present on the bacterial surface. They are typically 6 nm in diameter and up to 4 µm in length. The assembly and function of the type IV pili is a complex process involving over 40 different proteins (165). In brief, the assembly of type IV pili is facilitated by a complex molecular machinery, the core of which consists of a traffic adenosine triphosphatase (ATPase), a polytopic inner membrane protein, and an outer membrane channel. The pilin subunits, PilA, are made in the cytosol and transported across the inner membrane via the Sec machinery. The pilin monomer becomes embedded in the inner membrane where the pre-pilin peptidase, PilD, cleaves the pilin signal sequence (165). The pilus filament is then assembled from a molecular platform composed minimally of an assembly ATPase, PilB, and an inner membrane protein. While polymerization requires energy provided by the PilB ATPase, during pilus retraction, the retraction ATPase, PilT, is required for rapid depolymerization. Subsequently the pilus is extruded through the outer membrane via a pore composed of multimeric PilQ (165). Interestingly, a number of genes involved in pilus assembly exhibit homology to those genes involved in T2SS of P. aeruginosa (62). The type IV pili play an important role in P. aeruginosa pathogenesis by mediating motility, adherence, and biofilm formation. Mutants defective in type IV pilus biogenesis were able to attach but were unable to form characteristic biofilm microcolonies (126). Type IV pili is required for P. aeruginosa to undergo twitching motility (165) and in some cases, swarming motility (128). Earlier studies showed that piliated P. aeruginosa exhibited increased adherence to murine respiratory epithelial cells in vitro compared to non-piliated P. aeruginosa mutants (57). It was also demonstrated that piliated strains of P. aeruginosa caused more severe and diffuse pneumonia than non-piliated mutants in an infant mouse model of lung infection (247). In vitro studies showed a pilA mutant exhibited reduced cytotoxicity toward epithelial cells (42). Moreover, preliminary studies showed that vaccination with purified pili conferred protection against infections by P. aeruginosa of the same pilus type (224). Type IV pili can function in bacterial adhesion via direct interactions of the assembled pilin subunit (exposed at the tip of the pilus) with host receptors (aGM1) (87, 91). It is proposed that the type IV pili facilitated the 6  ability of P. aeruginosa to establish an initial long-range contact between the bacterium and the epithelial cell surface. Retraction of its pili pulls the bacterium close to the host cell surface to enable binding of other surface-located adhesins, including polysaccharides, alginate, exoenzyme S and LPS, to result in irreversible attachment (91, 156). In addition to adherence, pili serve as targets recognized by phagocytes (118, 243). Studies have shown that the presence of pili on the surface of P. aeruginosa was required for non-opsonic phagocytosis by human neutrophils and monocyte-derived macrophages (118). Stimulated macrophages were shown to phagocytose P. aeruginosa under conditions known to promote maximal piliation of the bacteria. In contrast, the bacteria were not taken up by macrophages under conditions that caused shearing of the surface pili. Furthermore, a pilA mutant was not phagocytosed (243). 1.1.4.3 Lipopolysaccharide P. aeruginosa LPS are found at the outer face of the bacterial outer membrane. It is comprised of a hydrophobic domain, Lipid A, inserted into the phospholipid bilayer, to a hydrophilic tail composed of the core polysaccharide and the O-specific polysaccharide (Fig. 1.2) (123). The LPS plays an important role in virulence of P. aeruginosa (248). The Lipid A component can activate multiple proinflammatory pathways in the host (267). Often, P. aeruginosa isolates obtained from CF patients become rough thorough loss of their O-antigens (96). Moreover, P. aeruginosa in CF patients often adapt by selecting mutants with specific Lipid A modifications that allow resistance to host antimicrobial peptides and increase TLR4 activation (92). LPS also mediates serotype specificity and resistance to complement.  Figure 1.2 Structure of P. aeruginosa LPS.  7  1.1.4.4 Alginate P. aeruginosa can synthesize alginate, which is an exopolysaccharide made up of repeating polymers of mannuronic and guluronic acid (166). Alginate primarily plays a role in bacterial adhesion (56) and biofilm formation (107). A common occurrence in chronic infections such as in the CF airway is the conversion of P. aeruginosa from non-mucoid to mucoid (alginate overproducing) phenotype (166). The mucoid phenotype typically arose from mutation in the mucA gene. MucA represses the sigma factor, AlgU (21). In the absence of MucA, it leads to derepression of AlgU, which in turn activates genes for alginate biosynthesis. It was demonstrated that conversion to a mucoid phenotype facilitated the ability of P. aeruginosa to persist during chronic infections as the overexpression of alginate can protect the bacteria from phagocytosis and antibiotics and attenuate the host response (123). Alginate has been widely considered to participate in the architecture of P. aeruginosa biofilm, but recent studies have shown that alginate is not essential for biofilm formation (238). 1.1.5 P. aeruginosa secreted virulence factors and associated secretion systems Upon successful colonization, P. aeruginosa secrete a variety of virulence factors to cause extensive tissue damage, bloodstream invasion and dissemination in the host. P. aeruginosa employ a variety of secretion systems to secrete virulence factors into the extracellular milieu and/or deliver toxins directly into host cells. 1.1.5.1 Pyocyanin Pyocyanin is a blue redox-active secondary metabolite produced by P. aeruginosa (271). It is zwitterionic and can easily penetrate biological membranes. In animal models of acute and chronic lung infections, pyocyanin was shown to be essential to P. aeruginosa virulence (138). Pyocyanin has been recovered in large quantities from the sputum of CF patients infected with P. aeruginosa as well as from ear-secretions of P. aeruginosa-mediated chronic otitis media (137). It causes a wide spectrum of cellular damage, including inhibition of host cellular respiration, ciliary function, epidermal cell growth and prostacyclin release, and the disruption of calcium homeostasis, all of which contribute to the ability of P. aeruginosa to persist in the CF lung (234). Pyocyanin can also alter the host immune system by modulating the levels of interleukin 8 (IL8), depressing the host response and inducing apoptosis in neutrophils (254). Moreover, the oxidoreductive property of pyocyanin enables it to cause oxidative-stress related damage by oxidizing glutathione and inactivating catalases in respiratory epithelial cells (138). 8  1.1.5.2 Pyoverdine Pyoverdines are a group of diffusible green-fluorescent compounds synthesized by fluorescent Pseudomonads (258). It is a siderophore that represents a major iron uptake system used by P. aeruginosa to chelate iron from its environment. The concentration of free iron in mammalian hosts is too low to support the growth of P. aeruginosa, therefore, the bacteria must express iron acquisition systems to acquire iron. P. aeruginosa also expresses other siderophores including pyochelin and quinolobactin (258). Pyoverdine plays an important role in the virulence of P. aeruginosa since pyoverdine mutants have been demonstrated to exhibit a reduced ability to cause infections (170). Moreover, a role for pyochelin in the virulence of P. aeruginosa has been demonstrated in various mouse infection models (44, 231). Also sputum samples from CF patients have been found to contain significant quantities of pyoverdine (89). Moreover, pyoverdine was found to not only regulate its own production but also regulate the production of several other P. aeruginosa virulence factors, such as exotoxin A (123). The role of pyoverdine in iron acquisition is also important in biofilm maturation. Banin et al. (12) showed that in an iron-poor media, functional pyoverdine was necessary for biofilm maturation into large mushroom-like structures in vitro. However, in the absence of pyoverdine, iron delivery to the innermost biofilm layers was impaired resulting in thin layers of bacterial aggregates that did not develop further. 1.1.5.3 Rhamnolipid P. aeruginosa are able to synthesize rhamnose-containing glycolipid biosurfactants known as rhamnolipids. Rhamnolipid biosynthesis begins with RhlG-mediated synthesis of the lipid component of rhamnolipids from the general pool of fatty acids (163). The acyltransferase activity of RhlA subsequently converts these to the rhamnolipid precursor, 3-(3hydroxyalkanoyloxy) alkanoic acids (HAAs). Finally the rhamnosyl transferase activities of RhlB and RhlC catalyze the addition of dTDP L-rhamnose to the HAAs to give mono- and dirhamnolipids, respectively (163).  9  Figure 1.3 Structures of HAA and rhamnolipids in P. aeruginosa. The enzymatic addition of one or two rhamnose groups to HAAs (A) give the products mono-rhamnolipid (B) or dirhamnolipid (C), respectively. Rhamnolipids produced by P. aeruginosa have a variety of functions, including assimilation of insoluble substrates, especially hydrocarbons, changing the hydrophobicity of the cell surface, antimicrobial activity, solubilization of Pseudomonas aeruginosa quinolone signal (PQS), and reducing the surface tension to promote swarming motility (128, 230). Rhamnolipids have been associated with lung deterioration in patients with VAP and contribute to the establishment and maintenance of P. aeruginosa infections in CF patients (17). Rhamnolipids have hemolytic activity to solubilize phospholipids of lung surfactants to make them more accessible to cleavage by phospholipase C (255). Loss of lung surfactant is associated with atelectasis (alveolar collapse) in patients with acute and chronic P. aeruginosa lung infections. Rhamnolipids also inhibit mucociliary transport and ciliary function of human respiratory epithelium to further prevent clearing of mucous (208). Rhamnolipids can also cause necrosis and elimination of neutrophils and macrophages to resist phagocytosis (114). Rhamnolipids have been demonstrated to play multiple roles in the establishment and maintenance of P. aeruginosa biofilms, including microcolony formation, maintenance of open channels, mushroom cap formation and detachment of cells from the biofilm. Rhamnolipids are not required for initial microcolony or channel formation but play an important role in keeping the biofilm channels open (46). These channels are found in mature biofilms and they allow bacterial cells to access nutrients and oxygen, and remove waste products. Rhamnolipids are also involved in dispersal of bacterial cells from the biofilm as evidenced by a hyperdetachment  10  mutant that was found to overexpress rhamnolipids and inactivation of the rhlAB operon in this mutant abolished accelerated detachment (116). 1.1.5.4 Proteases Extracellular proteases (alkaline protease, protease IV, elastase) have been shown to degrade both structural and soluble host proteins contributing to the pathogenesis of P. aeruginosa. Alkaline protease is a fibrin degrading protease secreted via the Type 1 secretion system (T1SS). Its pathogenic role is well known in corneal infections but it has also been shown to participate in the pathogenesis of acute lung injury (88). It has been shown that in an animal model of P. aeruginosa acute lung injury, there is an early massive intra-alveolar formation of fibrin and that inhibition of this initial fibrin formation by the action of alkaline protease is detrimental to the host (124). Protease IV, a serine protease, is a significant virulence factor in the pathogenesis of eye and lung infections (43). P. aeruginosa mutants deficient in protease IV production exhibit reduced virulence compared to WT strains (65). Mature protease IV degrades host immunological proteins such as C3, C1q, IgG, fibrinogen, plasmin, and plasminogen. It can also degrade host iron binding proteins including lactoferrin and transferrin to facilitate acquisition of iron by P. aeruginosa (31). Elastase, encoded by the lasB gene, is a metalloproteinase secreted via the Type 2 secretion pathway (123). The concerted activities of elastase and LasA protease are responsible for the elastolytic activity destroying elastin-containing lung tissue and causing pulmonary hemorrhages during invasive P. aeruginosa infections (31). In addition to elastin, elastase can also degrade fibrin and collagen. During P. aeruginosa respiratory infections, elastase contributes to the rupturing of the respiratory epithelium through tight-junction destruction. Elastase not only destroys tissue components but also interferes with host defence mechanisms such as inactivating IgG, IgA, airway lysozyme, complement components and factors involved in protecting the respiratory tract such as α-1-proteinase inhibitor and bronchial mucous proteinase inhibitor (255). The enzyme also decreases the host immune response through cleavage of respiratory tract surfactant proteins A and D and proteinase-activated receptor 2 into their inactive forms. Elastase has been found in the sputum of CF patients during pulmonary exacerbation and P. aeruginosa mutants defective in elastase production are less virulent in animal models (246, 248). 11  1.1.5.5 Type 2 secretion system In addition to elastase, other virulence factors, including lipase, alkaline phosphatase, exotoxin A, and phospholipase C, are also secreted via the T2SS into the extracellular space. These virulence factors are secreted via the T2SS in a 2-step process (18). First step involves undergoing a signal-sequence-dependent translocation across the inner membrane mediated by the Sec machinery. The subsequent transport of the products across the outer membrane is mediated by a machinery composed of more than 12 proteins. Generally, the virulence factors secreted via the T2SS participate in invasion by destroying the protective glycocalyx of the respiratory epithelium and exposing epithelial ligands to P. aeruginosa (123). Specifically, phospholipase C has hemolytic activity and targets eukaryotic membrane phospholipids. It plays a major role in the pathogenesis of P. aeruginosa during acute lung injury and in inflammation (130, 268). It can suppress host neutrophil oxidative burst response, as well as inactivate host surfactant proteins (249). Exotoxin A is an ADP-ribosyl transferase encoded by the exoA gene. It plays a major role in the pathogenesis of P. aeruginosa since studies showed that an exoA mutant was 20 times less virulent than the WT strain in mice (173). Exotoxin A destroys host cells by inhibiting elongation factor 2 (EF2) to prevent host protein synthesis (196). It can also depress the host response during P. aeruginosa infections (220). 1.1.5.6 Type 3 secretion system In contrast to the T2SS, the T3SS delivers virulence factors directly into the host cytoplasm. T3SS gene expression is generally activated in response to low-calcium growth conditions or contact with host cells (18). The T3SS is a complex apparatus that allows the translocation of effector proteins from the bacteria, across the bacterial membranes and into the eukaryotic cytoplasm through a needle-like appendage forming a pore in the eukaryotic membrane. Four T3SS-secreted effectors have been identified in P. aeruginosa: ExoT, ExoY, ExoS and ExoU (123). These effector proteins are variably expressed in different strains of P. aeruginosa (e.g. P. aeruginosa strain PA14 does not express ExoS). Generally, the T3SS and its effectors are associated with acute and invasive infections (60). The delivered toxins can alter host immune responses, and induce cell injury and cell death to promote bacterial invasion and dissemination (255). The expression of the T3SS in P. aeruginosa isolates has been associated with increased mortality in patients with pneumonia, sepsis, and respiratory failure (215).  12  ExoU is a major cytotoxin that is 100 times more cytotoxic than ExoS (123). ExoU secretion alone confers cytotoxicity in animal models of lung injury leading to sepsis. It is responsible for decompartmentalization of the inflammatory response in a model of acute lung injury leading to sepsis (135). In P. aeruginosa-induced VAP, ExoU was secreted by isolates from more severely ill patients, and patients infected with a strain of P. aeruginosa expressing ExoU  are  associated  with  increased  risk  of  mortality  (101).  ExoU  has  phospholipase/lysophospholipase activity disrupting the host cell membrane after translocation into the cell (255). ExoS, a major cytotoxin, is bifunctional with two active domains, C-terminal ADPribosyltransferase domain and an N-terminal Rho GTPase-activating protein (GAP) domain (123). The pathogenic role of ExoS is mainly attributable to it ADP-ribosyltransferase activity resulting in disruption of normal cytoskeletal organization (131). Its C-terminal domain binds to TLR2 while its N-terminal can bind to TLR4 to modulate host immune and inflammatory response (66). ExoT, a minor cytotoxin, also has dual ADP-ribosyltransferase and GAP activities (123). This effector has similar effects on the eukaryotic cytoskeleton as ExoS. Moreover, ExoT can inhibit the repair of the wounded epithelium and has anti-internalization activities (74, 76). ExoY is an adenylate cyclase. Its role in pathogenesis is associated with increasing cytosolic cAMP leading to increased pulmonary microvascular intercellular gap formation and increased lung permeability (219). ExoY is considered a minor cytotoxin as most in vitro and in vivo models of cytotoxicity only showed a minor effect of ExoY (123). Interestingly, the T3SS apparatus also confers cytotoxicity since mutants expressing the T3SS but not the effectors are still cytotoxic (143). Furthermore, studies have shown the efficacy of anti-PcrV immunotherapy against P. aeruginosa infections in animal models (84). PcrV is a structural protein of the translocation structure of the T3SS and plays a role in the assembly of the translocation pore in the eukaryotic cell membrane. 1.1.6 P. aeruginosa quorum sensing Quorum sensing (QS) is a complex cell-to-cell signalling mechanism employed by P. aeruginosa to sense their own cell density and to communicate with each other, resulting in coordinated production of hundreds of genes, among them many virulence factors (255). QS relies on small, freely diffusible molecules known as acyl homoserine lactones (AHLs). When a certain bacterial density is reached, these molecules reach a threshold concentration and bind to 13  their corresponding transcriptional regulators in the cell to result in coordinated gene expression in an entire bacterial population (262). In P. aeruginosa, QS involves 3 intertwined QS systems and an orphan autoinducer receptor (10). The las system is the first QS system identified in P. aeruginosa shown to regulate expression of elastase. The las system is comprised of lasI, the autoinducer synthase gene responsible for the synthesis of 3-oxo-C12-HSL (N-[3-oxododecanoyl]-L-homoserine lactone), and lasR, gene that encodes the transcriptional activator protein (221). The rhl QS system was initially named due to its ability to regulate the biosynthesis of rhamnolipids. The rhl system is comprised of rhlI, gene responsible for synthesis of the signal C4-HSL (N-butyryl-homoserine lactone), and rhlR, gene encoding the transcriptional activator. Transcriptiome analyzes of genes regulated by the las and rhl systems revealed las-specific genes, rhl-specific genes, as well as genes regulated by both systems. Moreover, the las and rhl QS systems are arranged in a hierarchical fashion where the las system activates the rhl system (221). Mutants deficient in las and/or rhl QS systems have been shown to be significantly less virulent in mouse models of acute pneumonia and burn wound infections (229). The las and rhl systems regulate the timing and production of multiple virulence factors, including elastase, alkaline protease, exotoxin A, rhamnolipids, pyocyanin, lectins and superoxide dismutase (221). Moreover, QS plays an important role in swarming, biofilm formation, and modulating host immune response (47, 128, 229). A mutant defective in 3-oxo-C12-HSL production was shown to form abnormal biofilms that were more sensitive to low concentrations of the detergent SDS (47). Exogenous addition of 3-oxo-C12-HSL to the culture media restored production of differentiated, SDS-resistant biofilms by the mutant. QS has been shown to control bacterial gene expression in biofilms, including genes involved in iron scavenging or metabolism (16). QS also regulates the expression of the pel operon, that encodes a matrix polysaccharide of biofilms, and contributes to the release of extracellular DNA (eDNA) into the biofilm (217). Interestingly, the signal, 3-oxo-C12-HSL, alone has been suggested to contribute to the virulence of P. aeruginosa due to its ability to modulate the host immune response to infection (229). For instance, 3-oxo-C12-HSL has been shown to induce secretion of IL8 from human bronchial epithelial (HBE) cells and induce COX-2 production, inhibit lymphocyte proliferation, downregulate production of tumor necrosis factor alpha (TNFα) and IL12, induce apoptosis in macrophages and neutrophils, and activate T cells to produce interferon gamma (IFNγ) (167, 229). 14  A third P. aeruginosa signalling molecule is the Pseudomonas aeruginosa quinolone signal (PQS, 2-hepta-3-hydroxyl-4-quinolone). This signalling molecule belongs to the 2-alkyl4-quinolone (AQ) family and is synthesized via the pqsABCD genes in the pqsABCDE operon responsible for synthesis of the PQS precursor, HHQ (2-heptyl-4-quinolone) (197). Both HHQ and PQS are involved in cell-to-cell signalling. PqsR, a transcriptional factor activated by HHQ and PQS, activates many virulence factors, including those controlled by the las and rhl systems (260). Moreover, the expression of PqsR is positively regulated by the las system. The pqs system has also been shown to affect biofilm formation (102). Significant concentrations of PQS molecules have been detected in the sputum, bronchoalveolar fluid, and mucopurulent fluid from CF patients. The pqs system has also been shown to be required for full virulence in multiple hosts (205). In a mouse infection model, a pqsA mutant was unable to disseminate in the lung tissue as well as the WT strain (120). PQS and HHQ also affect NFκB to downregulate the host immune response. The P. aeruginosa genome also encodes for an orphan receptor, QscR (QS control repressor), for which there is no cognate AHL synthetic enzyme (39). A qscR mutant is hypervirulent. Studies showed QscR transiently represses a few genes controlled by the las and rhl systems, including genes involved in hydrogen cyanide biosynthesis and phenazine biosynthesis (39). Transcriptome analysis revealed that the QscR regulon is comprised of more than 400 genes, most of which are not affected by the las and rhl systems (146). Studies have also shown that QscR can use the signal 3-oxo-C12-HSL to control its own regulon. The QS systems are embedded in a network of global regulation with a high potential for integrating and responding to a wide range of environmental signals to influence target gene expression beyond population density. Studies with reporter gene fusions showed that several QS controlled genes exhibited a delayed response to exogenous added QS signals until the stationary phase of growth was reached while only a small number of genes were induced early in growth, suggesting that the activation of most QS controlled genes is not solely triggered by the accumulation of QS signals but requires additional factors, such as the stationary phase sigma factor, RpoS (221). 1.1.7 P. aeruginosa resistance to antibiotics P. aeruginosa are particularly difficult to control with antibiotics or disinfectants. In addition to its intrinsic resistance to multiple classes of antibiotics, their ability to acquire resistance and exhibit adaptive resistance during a therapeutic course, greatly limits the 15  therapeutic options available for treatment of these life-threatening infections (242). Infections caused by drug-resistant P. aeruginosa are associated with significant increase in morbidity, mortality, need for surgical intervention, length of hospital stay and chronic care, and overall cost of treating the infection (153). Data published by the NNIS reported P. aeruginosa isolates from ICU patients in 2003 exhibited resistance rates to imipenem, fluoroquinolones and thirdgeneration cephalosporins of 21.1%, 29.5% and 31.9%, respectively (153). Moreover, the emergence of multidrug-resistant (MDR) P. aeruginosa, resistant to three or more drug classes, poses a serious therapeutic challenge for treatment of P. aeruginosa infections. MDR P. aeruginosa has become relatively common in ICUs where between 1997 and 2002, it was reported that 10.4% of ICU bloodstream P. aeruginosa isolates were MDR (153). 1.1.7.1 Intrinsic resistance P. aeruginosa is intrinsically resistant to multiple antibiotics, including many β-lactams, macrolides,  tetracyclines,  co-trimoxazole  (trimethoprim/sulfamethoxazole)  and  most  fluoroquinolones (60). The intrinsic resistance of P. aeruginosa is attributed to the low permeability of its outer membrane, efflux pumps, and β-lactamase expression. The permeability of the outer membrane of P. aeruginosa is 1/100th of the permeability of Escherichia coli outer membrane (95). The low permeability in P. aeruginosa limits the rate of antibiotic penetration into bacterial cells. However, in order to survive, P. aeruginosa must allow passage of nutrients into the cell and this is accomplished through the channels of water-filled porins (245). In addition to mediating the transport of sugars, amino acids, phosphates, divalent cations and siderophores, certain hydrophilic antibiotics, such as β-lactams, tetracyclines and some fluoroquinolones have been shown to traverse the outer membrane through these porin channels (245). Carbapenems can enter into the bacterial cells via the OprD porin, which is a substratespecific porin used by P. aeruginosa to transport basic amino acids, small peptides (191). Low outer membrane permeability works in synergy with secondary mechanisms that take advantage of the slow uptake of antibiotics into the cell. For example, antibiotics may be actively extruded from P. aeruginosa via multidrug efflux pumps (60). These systems are tripartite structures that span both the inner and outer membranes, as well as the periplasmic space between the membranes. Four multidrug efflux pumps have been well characterized: MexA-MexB-OprM, MexC-MexD-OprJ, MexE-MexD-OprN and MexX-MexY-OprM, with the first and last of these systems being expressed constitutively and having an impact on intrinsic resistance. Of the four efflux pumps, the MexAB-OprM has the broadest substrate profile for the 16  β-lactam class, with an ability to export β-lactams, such as the carboxypenicillins, aztreonam, extended-spectrum cephalosporins, penems, and the carbapenems meropenem and panipenem (4). P. aeruginosa genome also encodes an inducible chromosomal Class C β-lactamase AmpC, enzyme that degrades β-lactams by hydrolysis (60) which is also influential in intrinsic resistance to substrate β-lactams. The expression of AmpC is induced in the presence of βlactams. 1.1.7.2 Acquired resistance In addition to intrinsic resistance, P. aeruginosa can also develop resistance to antibiotics through the acquisition of resistance genes on mobile gene elements (e.g. plasmids) or through mutational processes that alter the expression and/or function of chromosomally encoded mechanisms (106). For example, P. aeruginosa can acquire mutations leading to derepression of chromosomal AmpC to confer enhanced resistance to β-lactams (153). Also P. aeruginosa can acquire plasmids encoding new β-lactamases conferring resistance to antibiotics. Horizontal transfer of integrin-encoded extended-spectrum β-lactamases gives rise to P. aeruginosa are resistant to β-lactamase inhibitors such as clavulanic acid (263). Acquired plasmid-encoded metallo-β-lactamases, that possess carbapenemase activity, and mutations giving rise to decrease or absence in expression of OprD have been identified in carbapenem-resistant P. aeruginosa clinical isolates (153, 216). Mutations in DNA gyrase (e.g. gyrA, pyrB) and topoisomerase IV (e.g. parC, parE) are commonly found in isolates that exhibit fluoroquinolone resistance (61). Mutations resulting in derepression and overexpression of multidrug efflux pump (e.g. mutations in mexZ resulting in overexpressing of MexXY-OprM) are also found in aminoglycoside resistant P. aeruginosa isolates (4). MDR strains typically exhibit several resistance mechanisms simultaneously (60). 1.1.7.3 Adaptive resistance Adaptive resistance plays a significant role in antibiotic resistance in P. aeruginosa. This type of resistance is distinct from intrinsic and acquired resistance, both of which are relatively independent of the presence of an antibiotic or the bacterium's surrounding environmental conditions (70). Adaptive resistance is inducible and dependent on the presence of sub-inhibitory concentrations of an antibiotic or particular environmental stimuli or growth states. In addition to antibiotics, a number of triggering factors have been identified, including pH, anaerobiosis, and cation levels, as well as social activities such as biofilm formation and swarming motility (49). 17  These environmental cues and/or sub-inhibitory concentrations of antibiotics lead to changes in the expression of many genes (e.g. upregulation of genes encoding efflux pumps) in P. aeruginosa to allow the bacterium to withstand subsequent exposure to otherwise lethal concentrations of the inducing or related antibiotics (49). Moreover, adaptive resistance generally reverts upon removal of the triggering factor. A well-studied example is the adaptive resistance of P. aeruginosa to polymyxins and cationic antimicrobial peptides in low extracellular concentrations of divalent cations (e.g. Mg2+, Ca2+) (26). The low external Mg2+ levels activate 2 two-component regulatory systems (TCSs), PhoPQ and PmrAB, leading to induction of the arnBCADTEF operon. The arn operon mediates the addition of aminoarabinose to Lipid A, thereby reducing the negative charge on the LPS. As a result, this reduces interactions of polycationic antimicrobials with the bacterial outer membrane. Adaptive resistance can also be induced by peptides themselves. 1.1.8 P. aeruginosa biofilm formation Biofilms are of clinical relevance as the high prevalence of biofilm-associated infections is more appreciated. The Centre for Disease Control in Atlanta reported that biofilms are responsible for more than 60% of all microbial infections (148). Some common biofilmmediated infections occur on medical devices such as intravascular catheters, urinary catheters, orthopedic devices, and dialysis machines (152). There is also evidence that chronic infections, such as recurrent ear infections (otitis media) and lung infections in CF patients, are caused by bacterial biofilms (261). Biofilms consist of large numbers of bacteria organized in a structured community composed of microcolonies embedded in a self-produced matrix. The matrix encapsulating the bacteria is also known as the extracellular polymeric substance (EPS) (71). EPS consists of polysaccharides, eDNA and other macromolecular components such as proteins, lipids and biosurfactants. The Pel and Psl polysaccharides have been shown to be important components of the matrix (156). The Pel polysaccharide is a glucose-rich polymer that primarily plays a role after surface attachment. Psl polysaccharide is a mannose-rich polymer that is important for attachment to surfaces as well as maintaining biofilm structure post-attachment (156). The matrix protects the bacterial biofilm from shear forces in fluid environments. Bacteria in biofilms exhibit increased resistance to antimicrobials and increased tolerance to the antimicrobial properties of the host immune defences. P. aeruginosa biofilms can be formed on biotic or abiotic surfaces. However, attachment to a surface has been suggested to not represent an 18  absolute requirement, particularly for biofilms formed within CF airways (99). Assuming this is correct, P. aeruginosa biofilms in the CF airways were first shown by Niels Hoiby and colleagues 40 years ago as completely unattached communities of microorganisms surrounded by exopolysaccharide matrix enmeshed within the thick mucous of CF sputum. Typically, biofilm development on solid substrates (e.g. on a glass substratum) occurs as a 5-stage process (Fig. 1.4) (240). The first stage involves the initial attachment of free (planktonic) bacterial cells to a surface. Then the production of EPS results in more firmly adhered attachment. Stage 3 involves early development of the biofilm architecture and stage 4 is the maturation of the biofilm characterized by a complex architecture of bacterial microcolonies separated by water channels. Finally, the last stage involves the dispersal of single cells or small microcolonies from the biofilm. Although biofilms are thought to maintain a ‘steady state’ (balance of biofilm growth and biofilm dispersal), several factors are known to promote biofilm dispersal, including phage induction, carbon starvation and nitric oxide signalling (116).  Figure 1.4 Stages of biofilm development in P. aeruginosa. Image reproduced, with permission from (72). P. aeruginosa biofilm cells have been shown to exhibit tens to thousands of fold increase in resistance to antibiotics compared to their planktonic counterparts. A study showed that a P. aeruginosa isolate from a patient with UTI exhibited a 1,000-fold increase in tobramycin resistance in the biofilm state (181). Moreover, they showed that the tobramycin-sensitive phenotype was recovered after growing the biofilm cells in liquid media. P. aeruginosa employ several mechanisms of antibiotic resistance in biofilms, including restricted penetration, 19  expression of antimicrobial destroying enzymes, limited or anaerobic growth at the base of biofilms, specific QS-regulated resistance mechanisms, the presence of antibiotic-tolerant persister cells, and the general stress response (69). The extracellular matrix confers not only stability to the biofilm structure, but also protection from external agents. The extracellular matrix is also suggested to concentrate extracellular enzymes, such as β-lactamase, produced by P. aeruginosa, thereby resulting in inactivation of β-lactams before reaching the cells (160). The eDNA in the biofilm is mostly derived from random pieces of released and fragmented chromosomal DNA and can function as a cell-to-cell interconnecting component in the biofilm (156). Furthermore, eDNA can chelate cations to destabilize the LPS leading to upregulation of PhoPQ and PmrAB expression. This results in resistance toward cationic peptides and aminoglycosides. Biofilm cells have also been demonstrated to exhibit an increased mutation frequency, thereby increasing the rate of development of acquired resistance to antimicrobials than their planktonic counterparts (69). The gradients of nutrients and oxygen also result in cells being in distinct growth states in the biofilm. This means cells at the biofilm surface are metabolically active due to greater access to nutrients and oxygen, while cells at the biofilm center grow more slowly (77). Therefore, those antibiotics (including most β-lactams) that target metabolic processes in bacteria would be effective only against the actively growing cells. 1.1.9 P. aeruginosa metabolic versatility The metabolic versatility and flexibility of P. aeruginosa enables the bacteria to survive on minimal nutritional requirements and a wide variety of carbon and nitrogen sources and contributes to the ability of this microorganism to persist in both the community and hospital settings. P. aeruginosa are able to use a wide range of organic compounds, including sugars, amino acids (e.g. arginine, histidine, proline), sugar alcohols, polyamines, small aromatic compounds and organic acids as carbon and/or nitrogen sources. The majority of these compounds are initially processed via specific metabolic pathways into intermediates that are funnelled into the tricarboxylic acid (TCA) cycle. Approximately 10% of the genes encoded by the P. aeruginosa genome are involved in metabolism. These genes encoding enzymes that include those involved in central metabolic pathways, such as the TCA cycle, the EntnerDoudoroff pathway, the pentose phosphate pathway, and gluconeogenesis. A number of these genes are reported to be involved in the initial catabolic steps of these compounds. These include genes encoding aldolases, amidases, hydratases, and transferases. P. aeruginosa genome also encodes about 200 cytoplasmic membrane transport systems involved in the import of nutrients. 20  Expression of the components of catabolic pathways involved in the utilization of these compounds is subject to catabolite repression (41). In contrast to E. coli and Bacillus subtilis, succinate and other TCA cycle carboxylates are preferred to glucose as carbon sources for Pseudomonas spp. The presence of intermediates of the TCA cycle generally causes catabolite repression of degradative pathways for sugars, amino acids, and other carbon sources (233). Moreover, studies have shown that Pseudomonads utilize a different mechanism of catabolite regulation, which involves the Crc protein that binds to mRNA and acts as a translational repressor (108, 159, 272). Wolff et al. showed that a crc mutant was pleiotropically impaired in catabolite repression of several catabolic pathways by carboxylic acids (272). 1.1.10 P. aeruginosa two-component regulatory systems The ability of P. aeruginosa to exhibit considerable nutritional and metabolic versatility is due to its ability to sense and respond quickly to many different stimuli (213). This versatility is mediated in part by its large repertoire of two-component regulatory systems (TCSs): 64 sensor kinases and 72 response regulators (79) that mediate simple signal transduction processes. A number of these TCSs are involved in nutritional adaption, including the NtrB/NtrC (nitrogen assimilation), CbrA/CbrB (carbon and nitrogen metabolism) and PhoR/PhoB (phosphate assimilation). In addition to metabolism, TCSs play important roles in regulating diverse virulence, virulence-associated and resistance processes in P. aeruginosa, contributing to its ability to cause infections in multiple hosts (79). A TCS is generally comprised of an inner membrane-spanning sensor histidine kinase and a cytoplasmic response regulator (239). Functionally coupled sensor kinase and response regulator genes are often encoded adjacent to one another on the genome, forming an operon (213). Some of the environmental signals sensed by the sensor kinase components include changes in oxygen availability, nutrient limitation, osmolarity, antimicrobial peptides, and signalling molecules such as homoserine lactones, but often the specific signal is not known. Despite the variability of the input domain enabling detection of different environmental signals, the phosphorylation cascade resulting from the detection of the signal is a conserved process and the structures and sequences of the histidine kinases exhibit high similarity (213). Moreover, the receiver domain of the response regulators is highly conserved. There are three types of TCSs, including the classical, unorthodox and hybrid systems (Fig. 1.5) (239). In the classical system (most common), the signal transduction involves a cognate sensor-regulator pair. In the presence of an environmental signal, the sensor kinase is autophosphorylated at its conserved histidine 21  residue. The sensor kinase then phosphorylates the conserved aspartate residue of the DNAbinding response regulator to activate it (239). Upon activation, the regulator can activate or repress its target genes by binding to specific regulatory DNA sequences. Subsequently, dephosphorylation of the response regulator by the sensor kinase returns the system to its preactivation state. In the unorthodox and hybrid systems, the phosphotransfer between the sensor kinase to the response regulator requires an intermediate receiver domain that is part of the sensor kinase, enabling intermolecular phosphotransfer, and a histidine phosphotransfer (Hpt) domain (239). The Hpt domain may either be bound to the additional receiver domain of the histidine kinase (unorthodox system) or present as a low molecular weight free Hpt protein (hybrid system). In the hybrid system, the multistep phosphorelay enables enhanced cross talk between the different hybrid kinases and response regulators via the separate Hpt domain (239).  Figure 1.5 Schematic diagram of TCS signalling pathways in P. aeruginosa. A) Classical system, B) Unorthodox system, C) Hybrid system. H is the invariant histidine reside that is phosphorylated (P) and D is the conserved aspartate residue. Among the many P. aeruginosa TCSs, only a few have been characterized in great detail and for most of these systems, the sensed stimulus remains unknown. A well-characterized TCS associated with the pathogenesis of P. aeruginosa is the GacAS TCS (205). Although the signal remains unknown, the GacAS system is part of an intricate network and is the master regulatory system in the control of P. aeruginosa virulence in multiple hosts (256) including in plant (Arabidopsis thaliana), nematode (Caenorhabditis elegans), insect (Drosphila melanogaster, 22  Galleria mellonella, Bombyx mori), and mouse models of acute infection (79, 204). GacA also contributes to chronic infections in a CF murine model (40). The GacAS TCS plays a role in regulating the expression of multiple extracellular virulence factors as well as chronic persistence genes, including those required for biofilm formation and AHL inducers (195, 209). This TCS is also involved in antibiotic resistance and swarming motility by regulating rhamnolipid production (28). The GacA/GacS signalling pathway involves GacS (sensor kinase) activating GacA (response regulator) via phosphorylation (79) but in this case the response regulator work largely to trigger post-transcriptional regulatory mechanisms. This activated GacA positively regulates the transcription of two small regulatory RNAs, RsmZ and RsmY, which in turn jointly antagonize RsmA, a small RNA-binding protein. Sequestration of RsmA by RsmY/RsmZ alleviates the repressing activity of RsmA on the expression of its target mRNAs. Free RsmA normally promotes the production of virulence factors associated with acute infections, such as the T3SS and its associated effectors, while repressing genes involved in chronic persistence, including biofilm formation and production of AHL autoinducers (256). Thus, the GacA/GacS TCS controls the reciprocal expression of acute and chronic virulence determinants in P. aeruginosa. 1.2  P. aeruginosa motilities and their roles in pathgenesis Motility in P. aeruginosa requires a considerable expenditure of cellular energy to  synthesize and assemble various flagellar and pili components as well as to fuel the flagellar and pili motors. However, motility is strongly associated with the pathogenesis of P. aeruginosa. Its roles include increasing the efficiency of nutrient acquisition, evasion of toxic substances, facilitation of infection of preferred hosts and access to optimal colonization sites within them, as well as dispersal within the environment. P. aeruginosa is unique in that prior to the work here it was known to be capable of 3 major types of motilities: swimming, twitching, and swarming (Fig. 1.6). Moreover, P. aeruginosa was recently observed to undergo a passive form of surface spreading called sliding motility (176). 1.2.1 Swimming P. aeruginosa swim via rotation of its single polar flagellum in an aqueous environment and in low agar (< 0.4%) medium (98). The contribution of flagellar motility in virulence was tested in a burned mouse model of infection. Non-motile mutants or mutants with paralyzed flagella were less virulent than the WT strain. In support, another study by Rustin et al. 23  demonstrated that it is flagellum-mediated swimming motility, rather than flagellar expression itself, that results in bacterial resistance to phagocytosis by murine and human phagocytes (8). In their study, they used a ∆motAB∆motCD strain deficient for four stator genes which comprise the stationary components of the flagellar motor (8). The motor provides energy for rotation of the flagellum. In this strain, flagella are assembled and are found intact on the surface of the bacteria but these flagellar structures cannot rotate (are non-functional) therefore these bacteria are defective in swimming and swarming motilities (250).  Figure 1.6 Representation of the various types of motilities in P. aeruginosa. Schematic and actual images of motility colonies on agar plates are shown. (A, B) Swarming on a semisolid surface; (C, D) Swimming in an aqueous environment; (E, F) Twitching on a solid surface. 1.2.2 Twitching The role of type IV pili in virulence involves not only its role in adherence and biofilm formation, but also includes type IV pilus-mediated twitching motility (6, 279). Twitching motility is a highly organized process that can occur on smooth solid surfaces or at the interstitial surface between agar (≥1% wt/vol) and plastic or glass (223), and in P. aeruginosa is mediated by the extension and retraction of its polar type IV pili (223). Studies by Zolfaghar and colleagues (279) demonstrated a role for twitching motility in P. aeruginosa corneal disease in vivo. After initial bacterial adherence to corneal epithelial cells, the development of P. 24  aeruginosa keratitis requires bacterial entry into the corneal stoma underlying the multilayered epithelium. This group tested the virulence of pilA, pilT and pilU mutants in murine models of keratitis. The gene, pilA, encodes for type IV pilin, and the genes, pilT and pilU, encode proteins that provide energy for type IV pili retraction. While the pilA mutant was non-piliated and the pilT and pilU mutants were hyperpiliated, all 3 mutants exhibited twitching deficiencies (86). In the study, it was demonstrated that corneal colonization by the twitching-deficient and hyperpiliated mutants was reduced at 48 hours, but not after 4 hours of infection (279). These results implied a role for twitching motility in tissue penetration or bacterial dissemination. Indeed, a subsequent study showed that twitching motility was required for P. aeruginosa to traverse the multilayered corneal epithelium and contributed to epithelial cell exit after internalization of bacteria inside invaded cells (6). 1.2.3 Sliding In addition to these 3 types of well-established motilities, sliding motility in P. aeruginosa was first described by Murray et al. whereby they showed that a PAO1 fliCpilA double mutant, which did not express flagella nor type IV pili, exhibited sliding motility on semi-solid surfaces under in vitro conditions that would otherwise promote swarming motility (176). In contrast to other forms of motility, sliding appears to be a form of passive translocation that does not require an active motor. Instead this form of motility relies on surfactants to reduce surface tension, enabling the colony to spread away from the origin, driven by the expansion of the growing culture. Murray et al. demonstrated that sliding motility was inhibited when type IV pili expression was restored in the double mutant. Moreover, they showed that sliding motility requires the production of rhamnolipids and that the GacA/GacS/RetS system plays an important role in this form of spreading motility (176). 1.3  Swarming motility in P. aeruginosa Swarming is a social phenomenon that involves rapid and coordinated movement of cells  across a hydrated semi-solid surface (0.5-0.7% agar), often typified by dendritic (strain PA14) or solar flare (strain PAO1) colonial appearances. 1.3.1 Features required for swarming Flagella are required for swarming in P. aeruginosa as mutants defective in flagella biosynthesis or function exhibit impaired swarming motility (128). P. aeruginosa swarmer cells are elongated and express 2 polar flagella (128). Studies showed that during swarming, P. 25  aeruginosa retains its polar flagella and synthesizes an alternative motor that is specially required to propel movement on semi-solid surfaces (58). Although evidence for the exact role of flagella in swarming motility is lacking, it is postulated that the flagella are used either to sense the viscosity of the surface and/or to provide more efficient propagation on semi-solid surfaces (98). Interestingly, the requirement of type IV pili in swarming appears to be variable among different P. aeruginosa strains and may even vary for the same strain coming from different laboratories. For example, while our laboratory (190, 276) and some laboratories (128, 210) have shown that both PAO1 and PA14 strains require type IV pili for swarming, there are other laboratories (207, 250) that showed type IV pili was dispensable for swarming for both strains of P. aeruginosa. Swarming motility is dependent on cell-to-cell signalling, particularly the las and rhl QS systems, at least in part for the production of the biosurfactants, such as rhamnolipids and its precursors, HAAs (128). HAAs and rhamnolipids synthesized by P. aeruginosa serve to reduce the surface tension between the bacterial cells and its environment to promote swarming. In addition to cell-to-cell signalling and surface moistness (or appropriate viscosity), swarming is highly dependent on the nutrient growth medium (128). Swarming in P. aeruginosa is typically not observed on rich media but is promoted under conditions of nitrogen limitation and in response to certain amino acids, namely histidine, proline, glutamate and aspartate, when provided as the sole source of nitrogen (128). Studies have shown that limitation of nitrogen promotes rhamnolipid production (50). Moreover, swarming was shown to be inhibited when a high concentration of ammonium was added to the swarm medium (128). Swarming is also dependent on the carbon source. Kohler et al. showed that while glucose permitted optimal swarming, glycerol and succinate were less efficient at promoting swarming. The less than favourable nutritional conditions that promote swarming motility indicate P. aeruginosa may use this form of motility as a means to find a new niche with more favourable nutrients. A microarray of swarmer cells identified more than 70 differentially regulated genes relating to various types of bacterial metabolism, including cofactor biosynthesis, energy, and carbon source, fatty acid, and amino acid metabolism (189). Various genes associated with glucose metabolism and uptake, including those encoding for the carbohydrate-selective sugar porin, OprB, and other carbohydrate transporters were downregulated (189). Genes involved in fatty acid metabolism and nitrite reduction were upregulated. Many genes involved in energy metabolism including those encoding cytochromes involved in the production of ATP via the respiratory electron transport chain were upregulated in swarmer cells (189, 251). Genes 26  encoding ATP synthases were also upregulated possibly implying that swarmer cells require more energy than non-swarming cells. 1.3.2 Role of rhamnolipids and its precursor 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs) Recent studies demonstrated that HAAs and rhamnolipids have distinct roles in swarming modulation (32, 252). Caiazza et al. showed that a rhlA mutant, unable to synthesize HAAs, was completely unable to swarm. Interestingly, a rhlB mutant, capable of making HAAs but not rhamnolipids, was still able to swarm. However, instead of long isolated tendrils radiating from a small core of cells, as observed with WT strains, a rhlB mutant produced a dense core of cells on the swarm agar (32). A rhlC mutant, that can still make monorhamnolipids, was able to swarm but the swarm colony produced fewer defined tendrils than the WT. Along with other supporting data, these results suggest that rhamnolipids are required to maintain tendril organization and prevent colonization of the area between tendrils, while HAAs are the primary surface-wetting agent for swarming (32). It was suggested that rhamnolipids diffuse from neighbouring tendrils during swarming, and that these molecules accumulate between tendrils, thereby maintaining spacing between neighbouring tendrils. Due to the low concentration of rhamnolipids directly in front of the advancing tendrils, the tendrils continue to travel outward until they approach another swarm tendril, at which point the level of rhamnolipids accumulate at both tendril tips, resulting in directional changes (32). 1.3.3 Role of swarming motility in the pathogenesis of P. aeruginosa Although the exact role for swarming motility during in vivo infection or colonization still remains to be established, a study of P. aeruginosa transposon insertion mutants attenuated for virulence in a rat chronic pulmonary infection model identified several genes required for swarming motility, including those encoding for type IV pili and rhamnosyl transferases. (201). Swarming might also protect pathogens from macrophages as it was demonstrated that swarmer cells exhibited elevated resistance to engulfment (9). Mucosal surfaces of the body represent regions of high viscosity, which is analogous to the conditions that promote swarming in vitro (189). Thus, swarming motility is thought to be clinically relevant since P. aeruginosa could use it to move across the mucosal surfaces of the body, such as the epithelial surfaces of the lung. Moreover, several cues required for swarming in vitro, namely rhamnolipids and amino acids as weak nitrogen sources, are present in the sputum of CF patients (227). A microarray study of 27  swarmer cells by Overhage et al. revealed upregulation of a large number of virulence-associated genes, including those encoding the T3SS, alkaline protease, protease IV, elastase, pyoverdine and pyochelin (189). The microarray also showed upregulation of lasR and rhlR, providing further support for the involvement of QS in swarming motility. Another group, Murray et al. found that clinical isolates of P. aeruginosa displaying large swarm zones secrete increased levels of proteases compared to isolates that displayed reduced or absent swarming (177). They showed that isolates with the largest swarming zones were significantly more likely to be T3SSpositive, and were more likely to secrete T3SS effectors. P. aeruginosa also exhibit adaptive resistance toward antimicrobials during swarming (189). Swarmer cells were shown to be more resistant to the action of antibiotics (e.g. polymyxin B, gentamicin, tobramycin, ciprofloxacin) than planktonic cells. However, resistance to these antibiotics reverted back to WT level after transfer of swarmer cells to liquid medium (189). Although the molecular mechanisms underlying this resistance are still largely unknown in P. aeruginosa, some clues can be obtained from the microarray study. The altered expression of the ATP-dependent Lon protease might contribute to ciprofloxacin resistance (189). The lon gene encodes for an ATP-dependent protease involved in the degradation and refolding of abnormal proteins (189). Studies have shown that in addition to virulence-related phenotypes, a lon mutant was supersusceptible to fluoroquinolones (25). The microarray also revealed increased expression of the efflux pump systems, such as the multidrug efflux pump, MexGHI-OpmD, and decreased expression of outer membrane components, including certain porins (189). 1.4  Nutritional environment of the CF sputum The CF lung sputum (mucous) is a thick, viscoelastic gel comprised of glycoproteins,  especially mucins, proteins, amino acids, DNA and lipids (122). It is the secretory product of globlet cells and submucosal glands (269). The CF sputum serves as a nutritionally rich environment with carbon and energy sources to support high-density growth of P. aeruginosa. 1.4.1 Impact on P. aeruginosa pathogenesis In addition to the fitness of P. aeruginosa, the nutritional environment of the CF sputum has an impact on the pathogenesis of the bacteria (192). Individual carbon and nitrogen sources have been shown to modulate biofilm formation, surface motility and QS (128, 193, 225). For example, aromatic acids in the CF sputum promote the synthesis of PQS P. aeruginosa, thereby inducing the production of several PQS-controlled virulence factors (193). Moreover, aromatic 28  amino acids, present in CF sputum, would act as important inducers for the production of staphylolytic compounds by P. aeruginosa (193). It is speculated that during the initial stages of P. aeruginosa colonization of the CF lung, the production of antimicrobial factors, along with other factors, facilitate the ability of P. aeruginosa to outcompete and displace other bacterial inhabitants of the CF lung, such as Staphylococcus aureus, to become the predominant bacteria in the CF lung (192). Transcriptional analysis revealed that genes involved in branched chain and aromatic amino acid catabolism were upregulated during growth in CF sputum medium, while genes involved in the biosynthesis of these amino acids were downregulated (193). Genes involved in the transport and metabolism of glucose were also downregulated in CF sputum. In agreement with the high levels (15 to 20 mM) of total amino acids observed in the CF sputum, this seems to suggest that amino acids are the likely sources of carbon for P. aeruginosa in the CF sputum (193). Biofilm production by P. aeruginosa is thus promoted by the hypersecretion of components of the viscous mucous layer in the CF airway, and the thickened mucous provides a low oxygen environment, as well as the presence of DNA resulting from the necrosis of recruited neutrophils (172). 1.4.2 Role of mucin in the CF sputum The inherent viscosity of the CF sputum is substantially due to its high mucin content. Mucins are the major macromolecular component of mucous. They are large, heavily glycosylated proteins with a defining feature of tandemly repeating sequences of amino acids rich in serine and threonine (259). Mucins are composed of two major families, secreted and membrane-associated mucins. The membrane-associated mucins function as cell surface receptors for pathogens and activate intracellular signalling pathways (262). The secreted mucins form large oligomeric gels that impart viscoelastic properties to mucous. In the CF airway, the presence of pathogens leads to upregulation of the expression of mucin in the host (214). 1.5  Host strategies against P. aeruginosa infections The high adaptability and opportunistic nature of P. aeruginosa allows the bacteria to  cause serious infections in immunocompromised patients. Despite these capabilities, P. aeruginosa rarely infects healthy human beings (175). This is likely due to the highly evolved host defence mechanisms that efficiently remove inhaled or aspirated Pseudomonas. These host defence strategies include mechanical barrier functions and the innate immune response.  29  Understanding the strategies that healthy hosts employ to defend against P. aeruginosa infections will help us to understand the failures of the host defence in susceptible patients. 1.5.1 Mechanical defence mechanisms The mucociliary clearance mechanism serves as a critical mechanism of prevention of colonization of the respiratory tract by bacteria such as P. aeruginosa (269). This clearance mechanism prevents P. aeruginosa from attaching to host cells or remain long enough to express and secrete a sufficient concentration of toxic products to harm host cells. The mucociliary clearance mechanism employed in the human airway comprise a viscous mucous layer called the Airway Surface Liquid (ASL), composed of water, ions and mucin, which ‘floats’ on a lower viscosity Periciliary Liquid layer (PCL) that spans the height of the cilia (127). The ASL layer entraps inhaled pathogens and subsequently the beating (concerted wave-like motion oriented in the direction of the upper airways) of the cilia of the airway epithelial elevates the floating mucous layer out of the lung and into the central airways where secretions are either elevated to the throat and swallowed, or expectorated. In contrast to most non-CF individuals, there is the accumulation of large volumes of sputum within the lungs of CF patients, impairing mucociliary clearance and resulting in airway obstruction and failure of the host to clear the bacterial infection (269). 1.5.2 Innate immune response Innate immunity is the first line of defence once the mechanical barriers of the epithelia have been breached. The innate immune defence mechanisms are complex, involving components such as phagocytic and other innate immune cells, activated complement, activation through pathogen recognition receptors (PRRs), and chemokines and cytokines. PRRs are found on innate immune cells to detect pathogens via recognition of conserved microbial signatures, such as LPS, flagellin, pili (94). For example, transmembrane Toll-like receptors (TLRs) are found on inflammatory cells (e.g. TLR4 that recognizes LPS) and epithelial cells (e.g. TLR5 that recognizes flagella) (14). Upon binding their bacterial ligands, they amplify innate immune protective responses to P. aeruginosa infections. During P. aeruginosa lung infections, neutrophils are massively recruited into the airways and can potentially play a primary role in bacterial clearance (139). Neutrophils can mediate killing of pathogens via phagocytosis, release of soluble antimicrobials, and generation of neutrophil extracellular traps (NETs). Neutrophils generate a number of important 30  microbicidal molecules, such as reactive oxygen and nitrogen species, serine proteases, elastases, lactoferrins, lysozyme and antimicrobial peptides (269). Neutrophils also express and release cytokines, which in turn amplify inflammatory reactions by several other cell types. Thus while neutrophils can confer protection, inappropriate or prolonged activation can be very destructive to host tissues. Alveolar macrophages are resident leukocytes of the lung. They have PRRs to recognize pathogens (139). Upon recognition, pathogens can be internalized and killed. Macrophages also express monocyte and neutrophil-recruiting chemokines enabling them to recruit other phagocytic defences. In addition to acting as a fairly impenetrable cell barrier, epithelial cells produce antibacterial compounds in the ASL layer (269), although the importance of these is not well understood. Moreover, epithelial cells contain surface-bound PRRs and can produce certain proinflammatory cytokines. Epithelial cells can also induce apoptosis and/or be sloughed off in patches to remove cells that have been invaded by pathogens (269). Cytokines and chemokines secreted by various cell types play an important role in host defence against P. aeruginosa infections. TNFα is a major proinflammatory cytokine produced by macrophages and epithelial cells in the lung in response to P. aeruginosa. IL8 is a proinflammatory chemokine produced by epithelial cells and alveolar macrophages that plays an important role in neutrophil recruitment (83, 253). 1.6  Hypotheses and objectives  1.6.1 Hypotheses 1. Swarming in P. aeruginosa is a complex motility mechanism influenced by a large number of cooperating genes 2. CbrA is involved in substantially more than just catabolite regulation in P. aeruginosa but also plays a critical role in the complex regulatory network that controls swarming motility of this bacterium 3. Within the host, the presence of mucin promotes a new form of surface motility termed surfing in P. aeruginosa 1.6.2 Specific objectives 1. To identify all of the genes in P. aeruginosa strain PA14 involved in swarming motility 2. To screen the swarming-deficient regulatory mutants for alterations in known factors required for swarming, such as flagella, type IV pili and rhamnolipid production  31  3. To investigate the involvement of CbrA in various virulence and virulence-related properties of P. aeruginosa 4. To identify the CbrA regulon under swarming conditions 5. To investigate the roles of downstream regulatory components, CbrB, CrcZ and Crc in the CbrA-mediated regulation of swarming, biofilm formation, and cytotoxicity 6. To investigate the in vivo virulence of the cbrA mutant 7. To investigate a novel form of surface motility on mucin-containing MSCFM agar plates  32  Chapter 2: Swarming motility, a complex adaptation controlled by a broad spectrum of transcriptional regulators 2.1  Introduction A recent screen of the 1,200 genes for which mutants are available in the P. aeruginosa  PAO1 mini-Tn5-luxCDABE transposon mutant library (147) identified 36 mutants with altered swarming phenotypes (190). Moreover, a comparison of the gene expression profiles of swarmer cells and bacteria growing in the same medium under planktonic conditions showed that there was a > 2-fold change in 7.5% of all P. aeruginosa genes. The 417 genes that were differentially regulated in swarmer cells included no less than 18 predicted or known transcriptional regulator genes. Furthermore, differentially regulated genes were found to function in processes as diverse as transport, secretion, metabolism, and motility, suggesting that the regulation of swarming behaviour is complex (190). To study the multifaceted regulation of swarming, I screened a P. aeruginosa strain PA14 transposon insertion mutant library (150) that covers about 75% of the predicted 5,962 PA14 genes to look for mutants with altered swarming phenotypes. As a result, 35 transcriptional regulators were found to be involved in swarming, including a variety of twocomponent sensors and response regulators. Interestingly, in contrast to the results of a small study described previously (190), an inverse relationship between the ability of mutants to swarm and their ability to form biofilms was observed in this study. 2.2 2.2.1  Materials and methods Bacterial strains and growth conditions The bacteria used included P. aeruginosa PA14 (203), as well as the entire P. aeruginosa  transposon mutant library (5,800 mutants) from Harvard University (150). When required for transposon maintenance, gentamicin was added to a final concentration of 15 µg/ml. 2.2.2 Screening of P. aeruginosa strain PA14 transposon mutant library for swarming motility To investigate the genes involved in swarming motility in P. aeruginosa, the Harvard PA14 transposon insertion mutant library was screened for swarming defects as described elsewhere (190). Briefly, mutants were grown overnight in Luria-Bertani (LB) broth, and 1 µl of each culture was transferred using a custom 96-well pin device onto the surface of brain heart infusion (BHI) agar plates containing 0.5% (wt/vol) agar (Difco). Colonies were scored for 33  differences in swarming motility compared to the WT after 48 h of incubation at room temperature. Two independent experiments were performed with 2 replicates of each mutant. The preliminary screen was largely performed by Ellen Torfs and Farzad Jamshidi. The mutants displaying swarming deficiencies identified in the preliminary screen were independently verified by performing individual swarming experiments on BM2-swarming agar plates (62 mM potassium phosphate buffer [pH 7], 2 mM MgSO4, 10 µM FeSO4, 0.4% [wt/vol] glucose, 0.1% [wt/vol] Casamino acid (CAA), 0.5% [wt/vol] agar) (189). One-microliter aliquots of mid-log-phase cultures grown in BM2 minimal medium [62 mM potassium phosphate buffer (pH 7), 7 mM (NH4) 2SO4, 2 mM MgSO4, 10 µM FeSO4, 0.4% (wt/vol) glucose] were inoculated onto the plates. Each experiment was carried out once with 3 replicates for each mutant. The verification experiments performed for transcriptional regulator or two-component sensor and response regulator mutants, that were identified in the preliminary screen as being swarming deficient, were performed 3 times, employing 3 to 5 replicates each time. All of the resulting dendritic colonies were analyzed by measuring the surface coverage on agar plates after 20 h of incubation at 37°C. 2.2.3 Growth curves P. aeruginosa PA14 mutants and the WT strain were grown overnight in BM2-swarming medium (62 mM potassium phosphate buffer, [pH 7], 2 mM MgSO4, 10 µM FeSO4, 0.4% [wt/vol] glucose, 0.1% [wt/vol] CAA). If necessary, cultures were diluted to obtain equal optical densities. Five-microlitre portions of these cultures were added to 195 µl of fresh swarming medium in 96-well microtiter plates. The growth of these cultures at 37°C under shaking conditions was monitored with a TECAN Spectrofluor Plus by determining the absorbance at 620 nm every 20 min for 24 h. Two independent experiments were performed with 3 replicates for each mutant. 2.2.4 Swimming and twitching motility As P. aeruginosa PA14 exhibits very poor swimming motility on BM2-glucose swimming plates, which are used for PAO1 (189), LB medium plates with 0.3% (wt/vol) agar were used instead. One-microliter aliquots of mid-log-phase cultures grown in LB broth were inoculated onto the plates. The diameters of the swimming zones within the agar were measured after incubation for 20 h at 37°C. Twitching motility was determined by measuring the diameters of the twitching zones in the interface between the agar and the plastic surface of the plate after 34  24 h and 48 h of incubation of PA14 mutants on LB medium plates with 1% (wt/vol) agar as described previously (49). For both swimming and twitching assays, 3 independent experiments were performed with 3 replicates for each mutant. 2.2.5 Biofilm formation Biofilm formation was analyzed using an abiotic solid surface assay as described elsewhere (71). Dilutions (1/100) of overnight cultures were incubated in BM2-adjusted medium [62 mM potassium phosphate buffer (pH 7), 7 mM (NH4) 2SO4, 2 mM MgSO4, 10 µM FeSO4, 0.4% (wt/vol) glucose, 0.5% (wt/vol) CAA] in polystyrene microtiter plates (Falcon, US) for 20 h at 37°C. Biofilms were stained with crystal violet, and the absorbance at 595 nm was measured using a microtiter plate reader (Bio-Tek Instruments Inc., US). 2.2.6 Rhamnolipid production Rhamnolipid biosynthesis was analyzed by the agar plate method as described previously (50-52). Briefly, P. aeruginosa PA14 mutants were grown overnight in LB medium and spot inoculated onto agar plates containing iron-limited salt medium (0.7 g/l KH2PO4, 0.9 g/l NaHPO4, 2.0 g/l NaNO3, 0.4 g/l MgSO4·H2O, 0.001 g/l CaCl2·2H2O, 0.001 g/l FeSO4·7H2O) supplemented  with  1%  (wt/vol)  glucose,  0.5%(wt/vol)  CAA,  0.02%  (wt/vol)  cetyltrimethylammonium bromide (CTAB), 0.0005% (wt/vol) methylene blue, and 1.6% (wt/vol) agar. The plates were incubated for 37°C for 24 h and then at room temperature for an additional 24 h. Rhamnolipid production was determined by measuring the diameter of the dark blue halo that formed around a colony. Three independent experiments were performed for each mutant. 2.2.7 Phage PO4 sensitivity test Phage sensitivity assays were performed as previously described (29). Briefly, bacterial lawns were produced by adding 100 µl of an overnight P. aeruginosa LB broth culture to 3 ml of LB soft agar. The inoculated top agar was overlaid on an LB agar plate and allowed to solidify. Five l of a lysate of phage PO4 (which binds to pilus as its cellular receptor) were spotted in duplicate onto lawns of P. aeruginosa WT and mutant strains. Zones of lysis (or clearing) on the plates were detected after incubation of the plates overnight at 37°C. Three independent experiments were performed for each mutant.  35  2.3  Results  2.3.1 Identification of PA14 transposon mutants with altered swarming phenotypes During a preliminary screen of the PA14 transposon insertion mutant library, 375 of approximately 5,600 mutants were identified as swarming deficient (data not shown). The phenotypes of these mutants were examined in more detail by performing 3 to 5 individual swarming experiments with each of the mutants on BM2 swarming agar plates (e.g. Fig. 2.1).  Figure 2.1 Selected P. aeruginosa PA14 transposon mutants displaying altered swarming and swimming motilities compared to PA14 WT. (A to C) Mutants displaying altered swarming motility on plates containing BM2 swarming-medium with 0.5% (wt/vol) agar. (A) cbrA mutant displaying no swarming phenotype. (B) pilH mutant displaying a significantly reduced swarming phenotype. (C) pqsH mutants displaying a hyperswarming phenotype. (D) hutC mutants displaying a strong swimming motility defect on a plate containing LB medium with 0.3% (wt/vol) agar. As a result, 233 transposon insertion mutants from the PA14 mutant library were confirmed to have alterations in swarming motility (Appendix Table A-1). As expected from previous observations (128, 190), I found swarming-deficient mutants with transposon insertions in flagellum and type IV pilus biosynthesis and QS genes (e.g., rhlR, rhlI, pqsD, and pqsH). Mutants with transposon insertions in genes belonging to a wide variety of functional classes other than the motility and QS classes were also identified, and large numbers of these genes had functions in metabolism, transport of small molecules, adaptation, and protection. Moreover, 35 transcriptional regulators, including two-component sensors and response regulators, were 36  identified as regulators involved in swarming motility (Table 2-1). To further probe the basis for the complex adaptive behaviour in response to a viscous environment, I focused on the regulation of swarming motility. Table 2-1 Characteristics of transcriptional regulators involved in swarming. Polar Fold change relative to PA14 WT effect Mutant Product (Mean ± SD) possiblea Swarming Swimming Twitching PA0037 (trpI)* transcriptional regulator N 0.03 ± 0.01# 0.60 ± 0.02# 0.98 ± 0.07 PA0409 (pilH)* type IV pilus response Y 0.17 ± 0.12# 1.10 ± 0.03 0.03 ± 0.03# regulator PA0475* putative transcriptional N 0.05 ± 0.02# 0.59 ± 0.01# 0.95 ± 0.05 regulator PA0479* putative transcriptional N 0.04 ± 0.01# 0.70 ± 0.02# 0.94 ± 0.05 regulator PA0831 (oruR)* transcriptional regulator N 0.05 ± 0.03# 0.53 ± 0.05# 0.89 ± 0.07 OruR PA0905 (rsmA) regulator of secondary N 0.12 ± 0.12# 0.87 ± 0.01 0.78 ± 0.06 metabolites PA0928 (gacS) sensor/response regulator Y 0.03 ± 0.01# 0.68 ± 0.01# 0.90 ± 0.10 hybrid PA1180 (phoQ) two-component sensor N 0.21 ± 0.22# 0.87 ± 0.01 0.93 ± 0.10 PA1196* putative transcriptional N 0.04 ± 0.03# 0.50 ± 0.06# 0.87 ± 0.05 regulator PA1347* putative transcriptional N 0.05 ± 0.03# 0.89 ± 0.00 0.85 ± 0.03 regulator PA1422 (gbuR)* transcriptional regulator N 0.03 ± 0.01# 0.77 ± 0.03 0.85 ± 0.04 PA1431 (rsaL) regulatory protein N 0.40 ± 0.06# 0.98 ± 0.02 0.95 ± 0.11 PA1458* putative two-component N 0.12 ± 0.03# 0.09 ± 0.01# 0.98 ± 0.08 sensor PA1544 (anr)* transcriptional regulator N 0.66 ± 0.16# 0.92 ± 0.01 0.74 ± 0.01# PA1976* putative sensor histidine N 0.37 ± 0.16# 1.00 ± 0.01 1.03 ± 0.14 kinase PA2072* putative sensory box N 0.11 ± 0.03# 0.50 ± 0.05# 0.95 ± 0.06 protein PA2332* putative AraC family N 0.50 ± 0.07# 0.99 ± 0.03 1.01 ± 0.04 regulator, PA2571* putative histidine kinase N 0.09 ± 0.08# 0.73 ± 0.07 0.10 ± 0.01# PA2586 (gacA) response regulator GacA Y 2.28 ± 0.77# 1.05 ± 0.03 0.94 ± 0.11 PA2622 (cspD)* cold-shock protein CspD N 1.72 ± 0.78# 1.07 ± 0.03 0.89 ± 0.05 PA2957* putative transcriptional N 0.08 ± 0.05# 0.56 ± 0.02# 0.94 ± 0.09 regulator PA3391 (nosR)* regulatory protein for N 0.09 ± 0.08# 0.71 ± 0.30 0.60 ± 0.06# NO2 reductase  37  Mutant  Product  Polar Fold change relative to PA14 WT effect (Mean ± SD) a possible Swarming Swimming Twitching N 0.08 ± 0.02# 1.05 ± 0.02 0.83 ± 0.06 N 0.18 ± 0.08# 0.87 ± 0.02 0.75 ± 0.07 N 0.05 ± 0.03# 0.54 ± 0.02# 0.91 ± 0.11  PA3477 (rhlR) transcriptional regulator PA3587 (metR)* transcriptional regulator PA3895* putative transcriptional regulator PA4315 (mvaT) transcriptional regulator N 0.55 ± 0.17# 0.99 ± 0.02 0.87 ± 0.09 MvaT PA4398* putative two-component Y 0.38 ± 0.12# 0.96 ± 0.02 1.08 ± 0.08 response regulator PA4725 (cbrA)* two-component sensor N 0.03 ± 0.01# 0.97 ± 0.08 0.98 ± 0.05 CbrA PA4778* putative transcriptional N 0.04 ± 0.02# 0.54 ± 0.01# 0.85 ± 0.05 regulator PA4853 (fis)* DNA-binding protein Fis N 0.07 ± 0.04# 0.75 ± 0.06 0.99 ± 0.09 PA5105 (hutC)* histidine utilization genes N 0.09 ± 0.07# 0.50 ± 0.04# 0.97 ± 0.06 repressor PA5124 (ntrB)* two-component sensor Y 0.03 ± 0.01# 0.90 ± 0.04 0.30 ± 0.17# NtrB PA5125 (ntrC)* two-component response N 0.12 ± 0.07# 0.98 ± 0.01 1.00 ± 0.05 regulator NtrC PA5261 (algR) alginate biosynthesis N 0.45 ± 0.18# 0.99 ± 0.01 0.34 ±0.03# regulator PA5536* putative C4-type zinc N 0.49 ± 0.15# 1.01 ± 0.01 1.00 ± 0.03 finger protein, DksA/TraR family * Regulatory mutant identified as swarming deficient in this study. # The phenotype was statistically significantly altered (P < 0.05 using Student’s t test) with a ˃ 25% compared to P. aeruginosa PA14 WT. a Possibility that a transposon insertion affects expression of downstream genes. Y, yes; N, no. Closer study of the mutants with transposon insertions in transcriptional regulator genes confirmed that 33 mutants exhibited reduced swarming and 2 mutants (gacA and cspD mutants) exhibited hyperswarming (Table 2-1). To our knowledge, only the swarming phenotypes of mutants with mutations in gacS (PA0928) (28), rhlR (PA3477) (128), rsmA (PA0905) (109), phoQ (PA1180) (28), rsaL (PA1431) (262), algR (PA5261) (190), mvaT (PA4315) (53), and gacA (PA2586) (117) had been described previously. Therefore, 27 swarming-deficient transcriptional regulators, 10 of which were two-component sensors or response regulators, were newly identified in this study (Table 2-1). In summary, I confirmed that 27 mutants (22 new mutants) showed either no swarming or minimal swarming, 6 mutants (4 new mutants) showed reduced (34 to 62% reduced) swarming, and 2 mutants (1 new mutant) had a hyperswarming phenotype. 38  To test whether swarming deficiency may have resulted from a growth defect, all mutants were tested to determine their growth behaviour in liquid swarming medium. Most of the mutants grew like P. aeruginosa WT strain PA14 (data not shown). Mutants with transposon insertions in the cbrA (PA4725), gacS (PA0928), fis (PA4853), and PA4778 genes showed significant reductions in the growth rate in this medium (data not shown); however, it seems unlikely that these defects, which reduced the growth rate up to twofold, could entirely explain the 93 to 97% decrease in swarming motility observed for these mutants. The possibility that polar effects on downstream genes, rather than the genes in which the insertions occurred, might be responsible for the observed swarming phenotype of the transposon mutants was examined (Appendix Table A-1, summarized in Table 2-1). In particular, the swarming abilities were examined for available mutants with transposon insertions in genes that were part of a predicted or known operon and downstream of the mutations of the 35 regulatory mutants, based on the operon prediction tool at www.pseudomonas.com (270). As a result, the swarming phenotypes of 5 of the 35 regulatory mutants (pilH, gacA, gacS, ntrB, and PA4398 mutants) might have been affected by the altered expression of downstream genes, but the remainder of the regulatory mutants were either mutants with orphan genes or mutants for which transposon insertions in downstream genes did not result in swarming defects (Table 2-1). 2.3.2 Characterization of swarming-associated regulatory genes Swarming-deficient mutants with transposon insertions in transcriptional regulator genes were characterized further to investigate how these genes influence swarming behaviour. One way in which swarming may be regulated is by modulating flagellum biosynthesis or function, as flagella have been shown to be essential for swarming motility in P. aeruginosa. As swimming motility depends entirely on a functional flagellum, I tested swarming-defective mutants to determine their abilities to swim in 0.3% agar. Only 1 mutant with a mutation in the putative two-component sensor gene PA1458 had a major defect in swimming motility. Another 10 mutants (including the PA0037/trpI, PA0475, PA0831/oruR, PA1196, PA2072, PA3895, PA4778, and PA5105/hutC mutants) had significant (P ≤ 0.05, Student’s t test) but modest (40 to 50%) swimming defects (Table 2-1) that are unlikely per se to explain the much more extreme swarming defect. Also, it was observed that most mutants that were completely unable to swarm also showed at least mild swimming defects (range, 10 to 50% inhibition). Thus, it seems that the swarming defects were generally much more extreme than the swimming defects and that the loss of flagellar function could not fully 39  explain the substantial swarming alterations observed (> 92% for 19 of the regulatory mutants). Mutants that showed more moderate deficiencies in swarming (40 to 60%) exhibited minor (anr and PA4398 mutants) or no (rsaL, mvaT, PA1976, algR, PA5536, and PA2332 mutants) swimming defects. Particularly in the case of the cbrA, ntrB, ntrC, rhlR, and PA1347 mutants, absolutely no association was observed between swarming and swimming, since the swarming of these mutants was strongly impaired but their swimming motility was (almost) normal. In addition to flagella, type IV pili are known to be important for swarming motility, although the dependence on type IV pili has been reported to be different in different strains. For PAO1, knockout mutants with mutations in pilus synthesis genes were found to be swarming deficient (128, 190). In contrast, in one study, specific type IV pilus synthesis genes were shown to be dispensable for this type of migration in P. aeruginosa PA14 (250). In our screen of the PA14 mutant library for swarming-deficient mutants, I identified 10 genes that were known to be involved in type IV pilus biogenesis and twitching motility (a type of motility mediated by type IV pili). This indicates that type IV pili are involved in the swarming motility of P. aeruginosa strain PA14 under our experimental conditions. The twitching motility of swarming-deficient regulators was largely unaffected, except for strong effects (P ≤ 0.05, Student’s t test) for two mutants with transposon insertions in pilH and PA2571 and intermediate effects for algR, ntrB, and nosR mutants compared to the WT (Table 2-1). To further investigate whether the deficiencies in twitching observed in the pilH, PA2571, algR, ntrB, and nosR mutants were related to the inability of these mutants to generate functional type IV pili, the sensitivity to pilus-dependent bacteriophage PO4 was assessed for all 35 regulatory mutants (23). As expected, the pilH, PA2571, algR, ntrB, and nosR mutants showed resistance to phage PO4 infection, while the rest of the regulatory mutants showed zones of lysis where phage PO4 was spotted. Another factor known to be involved in the swarming of P. aeruginosa is rhamnolipid production. Rhamnolipids are synthesized and excreted into the external medium by P. aeruginosa to aid in overcoming the surface tension between bacterial cells and their environment and thus facilitate movement of the bacteria across semi-viscous surfaces. Previous studies showed that mutants with defects in genes involved in the rhamnolipid biosynthesis pathway displayed major defects in swarming motility (e.g., rhlA) or showed an altered swarming pattern (e.g., rhlB) (37). To test whether the swarming-deficient phenotype of the regulatory mutants was related to a lack of rhamnolipid biosynthesis, agar plate assays were performed. On the agar plates inoculated with the rhlR, rsaL, and anr regulatory mutants, a lack 40  of visible haloes around the colonies was observed, indicating that the mutants were not able to excrete rhamnolipid. The PA1347 and PA4398 mutants consistently had larger and smaller halos, respectively, around their colonies, while for the remaining 30 regulatory mutants the formation of a blue halo around a colony was the same as that for the WT strain (data not shown). A characteristic of P. aeruginosa that has been associated with swarming motility is its ability to form biofilms. Intercellular signalling through QS systems is thought to play a role in both types of surface-associated behavior, although the contribution of QS to biofilms seems to depend on environmental conditions (125). Simple biofilm formation was measured in 96-well microtiter plates after 20 hours of incubation as described above. All of the mutants except the PA1458, PA1976, PA5536, PA2332, fis, ntrB, ntrC, pilH, cspD, and anr mutants showed significant (P ≤ 0.05, Student’s t test) changes in biofilm formation (Fig. 2.2). Mutants displaying severe swarming defects generally showed biofilm overproduction. In contrast, mutants with more moderate deficiencies in swarming or hyperswarmers showed normal or decreased biofilm formation, and only the phoQ, rsaL, gacA, and nosR mutants had major deficiencies in biofilm formation. Thus, while swarming motility and biofilm formation seem to be related, the data seem to imply that there is an inverse relationship between these processes. 2.4  Discussion In this study, 233 mutants from the P. aeruginosa strain PA14 transposon insertion  mutant library were verified to have alterations in swarming motility. The large number of genes (4% of the PA14 genome) found to be involved in this form of motility and the variety of functions performed by these genes support the previous finding that swarming is a complex adaptive behaviour.  41  Figure 2.2 Biofilm formation by mutants with altered swarming motility. Mutants were sorted on the basis of increasing swarming motility. Bacteria were incubated in BM2-adjusted medium in polystyrene microtitre plates for 20 h at 37 oC, and biofilms were stained with crystal violet. The data are representative of 3 independent experiments with 8 replicates for each mutant and are expressed as the fold change in the optical density at 595 nm (OD 595-nm) of the mutant compared to the WT. Asterisks indicate mutants that showed a statistically significant change in biofilm production (P ≤ 0.05 in Student’s t test). A classification of all 233 swarming-associated genes according to their predicted functions (www. pseudomonas.com) is shown in Appendix Fig. B.1. When these genes were compared with the functional composition of the entire PA14 genome (Appendix Fig. B.2), they were found to be in almost all of the functional categories for PA14 except the “non-coding RNA gene” and “related to phage, transposon, or plasmid” categories. Interestingly, a significant proportion of the swarming-associated genes (12%) were regulator genes (e.g. transcriptional regulator or two-component system genes). As well, a comparison of the functional composition of the swarming-associated genes with that of the PA14 genome revealed that 6% of all known or putative regulator genes in the PA14 genome are involved in swarming (Appendix Fig. B.3). The relatively large number of regulators involved in this form of motility supports the hypothesis that swarming may be highly regulated. As well, this study revealed that the regulatory system for swarming overlapped with other complex adaptations, such as biofilm and QS. A greater effort to study the functions of genes in the PA14 genome is important as nearly 30% of the swarming-associated genes identified are currently labelled hypothetical genes. In this study, I confirmed that 35 P. aeruginosa PA14 mutants showed altered swarming motility and had transposon insertions in genes encoding transcriptional regulators. Twentyseven of these mutants were newly identified in this study. There are hints that some of these 42  regulators might be involved in part in controlling flagellum biosynthesis or function, but only a single mutant showed a strong swimming motility defect. Thus, the great disparity in the extents of the defects of all mutants except the PA1458 mutant led to the conclusion that other factors must be important in determining swarming deficiency in most mutants. Similarly, only 5 mutants (pilH, PA2571, algR, nosR, and ntrB mutants) had substantial or moderate deficiencies in twitching motility and displayed resistance to phage PO4, indicating that most regulators are not obligately involved in type IV pilus functioning. With the exception of the rhlR, rsaL, anr, PA4398, and PA1347 mutants, all the regulatory mutants showed the same formation of a dark blue halo around a colony as the WT strain, indicating that there was efficient production of the biosurfactant. Evidently, mutations in the rhlI and rhlR genes, lead to the inability of the mutants to swarm (Appendix Table A-1). The rsaL gene is located between the lasR and lasI genes, and its expression is directly activated by LasRI. In turn, the RsaL protein can specifically represses the transcription of lasI (206). I was unable to identify a mechanism explaining how this regulator decreased rhamnolipid synthesis, as observed in this study. As recent studies suggested HAAs (rhamnolipid precursors) are the minimal surfactants required for swarming in P. aeruginosa (32), it would be interesting to study whether rsaL, anr, PA1347, and PA4398 are involved in the biosynthesis of HAAs. While it was reported that mutations in O-antigen or LPS synthesis eliminate the swarming motility of Salmonella enterica serovar Typhimurium (37), in our study, transposon insertions in O-antigen and LPS synthesis genes in P. aeruginosa did not seem to affect the ability of strains to swarm (Appendix Table A-1). It is possible that the production of secreted biosurfactants, rhamnolipids and HAAs, make P. aeruginosa less dependent on LPS and Oantigen to improve the surface “wettability” that is required for swarming colony expansion. In contrast, LPS and O-antigen play an important role in the swarming motility of Salmonella strains, as these bacteria do not excrete biosurfactants and most likely utilize their outer surface as a biosurfactant (37). For some of the swarming-deficient mutants, previous studies provided greater insights into how the corresponding regulators might affect swarming. The gbuR mutant was unable to swarm in our assay. Expression of the gbuR gene is normally induced by the alternative sigma factor RpoS (222). Interestingly, RpoS is involved in the control of expression of a large number of QS-regulated genes. More specifically, many of the genes that are controlled by RpoS are also under the control of rhlR and lasR (222). 43  A strong swarming deficiency was also observed for the PA1458 mutant. The PA1458 gene is predicted to be involved in chemotaxis. PA1458 in P. aeruginosa is homologous to cheA in Pseudomonas putida and E. coli. Upon chemoattractant recognition at the cell surface by a methyl-accepting chemotaxis protein, CheA is autophosphorylated. CheY is then phosphorylated by CheA and interacts with switch proteins in the flagellar motor. Disruption of the cheA gene in P. putida resulted in a non-swarming, non-chemotactic mutant that was unable to change the direction of flagellar rotation but was still able to swim (55). I found an even stronger effect on flagellar function in the P. aeruginosa PA1458 mutant, as this mutant displayed no swimming and almost no swarming motility. Another indication that PA1458 is involved in flagellar functioning is the observation that PA1458 expression depends on FleQ, a key regulator of flagella biosynthesis (45). An interesting trend was found for the relationship between swarming and biofilm formation. Both of these surface-associated behaviours depend on QS, flagella, and type IV pili (125, 186). For some P. aeruginosa mutants, defects in swarming coincide with deficiencies in biofilm formation (2, 262). The results obtained here, however, support the idea that there is generally an inverse relationship between the regulation of swarming and biofilm formation, which has also been proposed in other studies (33, 169). Most of the non-swarming mutants with transposon insertions in transcriptional regulator genes showed strong increases in biofilm formation. In contrast, the hyperswarming gacA mutant is deficient in biofilm formation (195). Importantly, swarming has been proposed to influence the early stages of biofilm formation, with actively swarming cells forming flat biofilms and swarming-deficient cells forming structured biofilms (225). As I observed an increase in the total biofilm mass in our solid-surface biofilm assays for most mutants with strong swarming defects, this may be explained by lessmotile cells forming structured biofilms (46, 145). By performing preliminary screening of the P. aeruginosa PA14 transposon insertion mutant library, I found that approximately 4% of the genes for which mutants are present in this library are involved in swarming behaviour. The complexity of this form of motility is reflected by the high number of regulators that were found to be involved in swarming behaviour. Seventeen regulators and 10 two-component sensors and response regulators were newly identified in this study. Furthermore, regulatory components that control swarming motility also played important roles in other virulence-related processes in P. aeruginosa, such as biofilm formation. Therefore, to understand how swarming behaviour is regulated, it will be important to study the hierarchy of the transcriptional regulators involved. 44  Chapter 3: Role of P. aeruginosa CbrA sensor kinase in metabolism, swarming, biofilm formation, antibiotic resistance and virulence 3.1  Introduction A screen of the P. aeruginosa strain PA14 transposon mutant library revealed 233  mutants involved in swarming motility, with 35 of these genes encoding transcriptional regulators, two-component sensor kinases or response regulators. Moreover, 22 of these swarming-associated regulators that displayed major swarming deficiency exhibited normal swimming, twitching motilities and rhamnolipid production. To begin to understand how these signalling components promote swarming motility, one interesting mutant with a mutation in the cbrA gene was chosen to be investigated in great detail. Metabolically versatile pseudomonads effectively utilize a broad range of organic compounds as carbon and/or nitrogen sources. The CbrAB TCS in P. aeruginosa was first identified to be involved in controlling the expression of a number of catabolic pathways involved in carbon and nitrogen utilization (149, 183). Mutations in the sensor kinase CbrA or the response regulator CbrB rendered the bacterium incapable of growing on a variety of organic compounds as the sole carbon source (183). Recently, Sonnleitner et al. discovered that the P. aeruginosa genome encodes a small RNA (sRNA), CrcZ, which binds to and sequesters the Crc protein with high affinity and thus relieves catabolite repression of a variety of degradative genes, such as amiE (233). The same group also found that the expression of CrcZ is controlled by the CbrAB system. In addition to its role in metabolism, the CbrAB system has been demonstrated to be involved in the metabolic regulation of the T3SS and its effectors, exoenzymes S and T (212). A TCS, LipQR, was discovered in Pseudomonas alcaligenes and demonstrated to be involved in the regulation of lipase expression (132). The LipQR system exhibits significant homology to the CbrAB system in P. aeruginosa (132). The PA14 screen in Chapter 2 revealed that a cbrA mutant is swarming deficient and exhibits hyperbiofilm formation (276). Also analysis of the transcriptome profile of P. aeruginosa exposed to sublethal concentrations of tobramycin revealed the downregulation of the cbrA gene (121). Furthermore, these results led us to propose that CbrA may be involved in substantially more than just catabolite regulation in P. aeruginosa. In this study, I demonstrated that CbrA is involved in the regulation of not only carbon and nitrogen metabolism, but also various virulence and virulence-related processes in P. aeruginosa. I constructed a cbrA deletion mutant and showed that this mutant displayed 45  swarming deficiency while exhibiting increased biofilm formation and in vitro cytotoxicity toward HBE cells. The cbrA mutant also demonstrated increased resistance to a variety of clinically important antibiotics. Microarray analysis of the cbrA mutant provided insight into the basis for these observed phenotypes. Based on detailed phenotypic and genetic studies of the cbrB mutant, I proposed that CbrA most likely modulates swarming, biofilm formation, and cytotoxicity via the response regulator CbrB, while CbrA may cross talk with another regulatory system to modulate antibiotic resistance. 3.2  Materials and methods  3.2.1 Tissue culture, bacterial strains and growth conditions Bacterial strains and plasmids used in this study are described in Table 3-1. Cultures were routinely grown in LB broth, tryptone broth (10 g/l Bacto tryptone), BM2 minimal medium, or BM2-swarming medium. E. coli S17-1 λpir was used as the donor strain in bacterial conjugations (226). P. aeruginosa competent cells were prepared as previously described (38). For plasmid or transposon selection or maintenance, antibiotics were added to growth media at the indicated concentrations: E. coli, 10 µg/ml gentamicin and 100 µg/ml ampicillin; P. aeruginosa, 30 µg/ml gentamicin, 100 µg/ml tetracycline, and 500 µg/ml carbenicillin. The SV40-transformed, immortalized HBE cell line, 16HBE14o-, was a gift from D. Gruenert (University of California, San Francisco, CA). 16HBE14o- cells were cultured in Minimum Essential Medium (MEM) with Earle’s salts (Life Technologies Invitrogen), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies Invitrogen) and 2 mM L-glutamine (Life Technologies Invitrogen). 16HBE14o- cells were routinely cultured to 85 to 90% confluence in 100% humidity and 5% CO2 at 37°C and were used between passages 9 and 15. 3.2.2 General DNA manipulations Routine genetic manipulations were carried out using standard procedures (218). Primers were synthesized by AlphaDNA Inc. (Montreal, QC, Canada), and their sequences are available from us on request. Plasmid DNA was isolated using QIAprep spin miniprep kits (Qiagen Inc., Mississauga, ON, Canada), and agarose gel fragments were purified using a QIAquick gel extraction kit (Qiagen). Restriction endonucleases were from New England BioLabs (Mississauga, ON, Canada). DNA sequencing was carried out by the UBC NAPS unit.  46  3.2.3 Recombinant DNA manipulations For construction of a cbrA deletion mutant, an in-frame deletion of cbrA was obtained via splicing by an overlap extension PCR strategy (111). Briefly, primers were designed to amplify the gentamicin cassette of pPS858. Primers were also designed to amplify approximately 1-kb fragments located upstream and downstream of cbrA from PA14 genomic DNA with additional short sequences of overlap with the gentamicin cassette. Next, the 3 DNA fragments were fused together and the final product was boosted by a third PCR. The resulting fragment was cloned into pEX18Ap carrying a sacB sucrose sensitivity gene (110). This plasmid was transformed into E. coli S17-1 λpir and conjugated into P. aeruginosa PA14 to generate an in-frame deletion of the cbrA gene in the PA14 strain by allelic exchange. Selection for double recombinants was performed on plates containing gentamicin and 5% (wt/vol) sucrose. The deletion was confirmed by PCR and sequencing. The suicide vector pME9673, obtained from Dieter Haas’s laboratory, contained the deleted crcZ promoter and the 5’ region of crcZ (233). To construct a crcZ deletion mutant, a crcZ deletion was introduced from plasmid pME9673 into the chromosomal crcZ locus of P. aeruginosa strain PA14 by gene replacement as described previously (233). Briefly, the plasmid pME9673 was mobilized from E. coli DH5α into P. aeruginosa PA14 with the help of E. coli HB101/pRK2013. PA14 transconjugates carrying a chromosomally integrated copy of pME9673 were selected on tetracycline. Excision of the vector by a second crossover (e.g tetracyclinesensitive derivatives) was subsequently obtained by enrichment with carbenicillin. The chromosomal crcZ deletion was confirmed by PCR and sequencing. The PA14 cbrA deletion mutant and cbrB and crc transposon mutants were complemented by amplifying cbrAB, cbrB, or crc, including an upstream region of 400 base pairs, from PA14 genomic DNA by PCR and cloning each fragment into the broad-host-range vector pUCP18 or pUCP19 (264). The resulting hybrid plasmids, pUCP18::cbrAB+, pUCP19::cbrB+, and pUCP18::crc+, were transferred into the cbrA, cbrB, and crc mutants, respectively, by electroporation (38). The crcZ deletion mutant was complemented by chromosomal insertion of a mini-Tn7 carrying the functional crcZ+ gene into the Tn7 attachment site of the mutant. The suicide plasmid, pME9818, which contained the cloned crcZ+ gene including the promoter region, was kindly provided by the laboratory of Dieter Haas (L. Abdou and D. Haas, unpublished data). Briefly, pME9818, carried by host E. coli S17-1, was transferred into the PA14 crcZ mutant by conjugation. Transposition of the mini-Tn7 carrying the crcZ+ gene into the chromosome of the 47  crcZ mutant was facilitated by the E. coli SM10 λpir helper carrying pUXBF-13. The P. aeruginosa strain carrying a crcZ+ insertion was selected with gentamicin and chloramphenicol and confirmed by PCR. 3.2.4 Motility experiments Swimming, twitching, and swarming of P. aeruginosa PA14 WT and mutants were examined as previously described in Materials and Methods in Chapter 2. To test the effects of the carbon source on the swarming motility of the cbrA and cbrB mutants, glucose was replaced with 0.4% (wt/vol) glycerol, 0.4% (wt/vol) mannitol, or 20 mM succinate. For iron complementation studies, Fe(II) sulfate was added to the swarming medium to a final concentration of 100 µM. For each form of motility, 3 independent experiments were performed with 3 replicates for each mutant. 3.2.5 Biofilm and rapid attachment assays Biofilm formation was analyzed as previously described in Materials and Methods in Chapter 2. To test the effects of the carbon source on biofilm formation of the cbrA and cbrB mutants, glucose was replaced with 0.4% (wt/vol) glycerol, 0.4% (wt/vol) mannitol, or 20 mM succinate. For iron complementation studies, Fe(II) sulfate was added to the biofilm medium to a final concentration of 100 µM. Rapid attachment was assayed as described previously with modifications (187). Overnight cultures were first diluted 1/100 into fresh BM2-biofilm medium and grown to an optical density at 600 nm of 0.5, and 100 µl was added to each well of a 96-well polystyrene microtiter plate. Cells were allowed to attach for 30 min at room temperature prior to staining with crystal violet as described above. 3.2.6 Congo red binding assays Congo red (CR) binding assays were performed as previously described (71). Briefly, tryptone-grown overnight cultures were diluted to OD600-nm of 0.025 and 1, 5, and 10 µl were spotted onto CR plates (10 g/l tryptone broth with 10 g/l agar, 40 µg/ml Congo red, and 20 µg/ml Coomassie brilliant blue). The plates were incubated for 24 h at 37°C, followed by 48 h at room temperature to assess colony morphology.  48  3.2.7 Minimum inhibitory concentration determination and polymyxin B killing experiments Minimum inhibitory concentrations (MICs) were measured using standard broth microdilution procedures (158) in BM2-swarming medium. Growth was scored following 24 h of incubation at 37°C. For measuring MICs against polymyxin B, a modified assay was used to prevent artificially high MICs due to aggregation of the antibiotic and binding to polystyrene (266). To perform killing experiments, cells of P. aeruginosa were grown to an OD600-nm of 0.5 in BM2-swarming liquid medium or on BM2-swarming agar plates for 18 h (189). These cultures were diluted into 1 X BM2-salts containing 1 µg/ml polymyxin B sulfate (Sigma). Samples were shaken at 37°C, and aliquots were taken at specified times, plated for survivors on LB agar, and grown overnight at 37°C. All experiments were repeated at least 3 times. 3.2.8 Cytotoxicity toward non-polarized HBE cells For the interaction assay, 16HBE14o- cells were seeded in 96-well plates (Corning Life Science, Corning, NY) at a density of 2 X 10 4 cells/well and grown at 37°C with 5% CO2 until 100% confluent (~48 h). Bacteria were grown in LB broth until mid-logarithmic phase, washed with PBS, and resuspended and diluted in MEM containing 1% FBS and 2 mM L-glutamine. The interaction assay was performed at a MOI of 2 bacteria/cell in MEM containing 1% FBS and 2 mM L-glutamine, and the assay mixture was incubated at 37°C with 5% CO 2. At post-infection time points, medium was removed from the wells, placed in microtiter plates, and spun for 10 min at 3,000 rpm to pellet the bacteria and host cell debris. The level of lactate dehydrogenase (LDH) in the supernatant was then assayed in triplicate using a colorimetric cytotoxicity detection kit (Roche, Mannheim, Germany). As a positive control for maximum LDH release, cells were treated with 1% Triton X-100 (Sigma, Oakville, Canada), resulting in complete cell lysis, while untreated cells were used to assess background LDH release. 3.2.9 Growth curves Growth curves of P. aeruginosa mutants and WT grown in LB medium, BM2-swarming medium, or BM2-biofilm medium were performed as discussed in Materials and Methods in Chapter 2. Two independent experiments were performed with 3 replicates for each mutant.  49  3.2.10 RT-qPCR Total RNAs from the cbrA, cbrB, crcZ, and crc mutants were harvested under various conditions as follows: (i) for the swarming condition, cells were obtained from the leading edge of the dendritic swarm colonies of the PA14 WT and the entire non-swarming colonies of the PA14 cbrA transposon mutant (ID33836), the cbrA deletion mutant, the cbrB transposon mutant, and the crcZ deletion mutant; (ii) for the polysaccharide synthesis-inducing condition, cells for RNA isolation were obtained by spotting 10 µl of diluted cultures grown in tryptone broth onto CR plates without Congo red or Coomassie brilliant blue; (iii) for the HBE cell infection condition, 16HBE14o- cells were seeded in tissue culture-treated Petri dishes (Corning Life Science, Corning, NY) at a density of 2 X 10 4 cells/well and grown at 37°C with 5% CO2 until 100% confluent (~48 h). Bacteria were grown in LB medium until mid-log phase, washed with PBS, and resuspended and diluted in MEM containing 1% FBS and 2 mM L-glutamine. The interaction assay was performed at an MOI of 100 bacteria/cell in MEM containing 1% FBS and 2 mM L-glutamine, and the assay mixture was incubated at 37°C with 5% CO 2. At 4 h postinfection, medium was removed from the dishes, placed in sterile Falcon tubes, and spun for 10 min at 3,000 rpm to pellet the bacteria. Subsequently, RNAs were isolated using RNeasy minicolumns (Qiagen) treated with DNase I (Invitrogen) to remove contaminating genomic DNA. Three micrograms of total RNA was combined with 0.5 µM dNTPs, 500 U SuperaseIN/ml (Ambion), and 10 µM DTT in 1 X reaction buffer and reverse transcribed with Superscript II reverse transcriptase (Invitrogen). The resultant cDNA was used as a template for qPCR. Analysis was carried out in the ABI Prism 7000 sequence detection system (Applied Biosystems) using the 2-step RT-qPCR kit with SYBR green detection (Invitrogen). Fold change was determined using the comparative threshold cycle (Ct) method by comparison to the PA1544 housekeeping gene. 3.2.11 DNA microarray experiment Microarray experiments were performed, with the assistance of Manjeet Bains, on 3 independent cultures. The cbrA deletion mutant and WT PA14 were grown on a BM2-swarming plate containing 0.5% (wt/vol) agar for 18 h at 37°C. RNA was harvested from the leading edge of the dendritic swarm colonies of the PA14 WT and of the entire non-swarming colonies of the cbrA mutant. As described previously (189), cells were resuspended in BM2-swarming medium supplemented with RNAprotect reagent (Qiagen, Germany). Harvesting of cells, RNA isolation, cDNA synthesis, hybridization to P. aeruginosa PAO1 DNA microarray slides (aminosilane 50  coated) from the Institute for Genomic Research (TIGR) Pathogenic Functional Genomics Resource Center, analysis of microarray slides using ArrayPipe version 1.7, and RT-qPCR were performed as described previously. 3.2.12 Pyoverdine assay Bacterial strains were grown in CAA medium (5 g/l low-iron Bacto CAA [Difco], 1.54 g/l K2HPO2·3H2O, 0.25 g/l MgSO4 ·7H2O) at 37°C for 48 h. The supernatants were diluted 1/75 in 10 mM Tris-HCl, pH 7.5, and excited at 400 nm with a spectrofluorimeter (80). Table 3-1 P. aeruginosa strains and plasmids used in this study. Strain or plasmid Description P. aeruginosa PA14 WT Wild type P. aeruginosa PA14 cbrA mutant PA14 transposon insertion mutant, ID33836 PA14 transposon insertion mutant, ID44074 cbrB crc PA14 transposon insertion mutant, ID44185 cbrA chromosomal deletion mutant of PA14; cbrA GmR  Reference 150 150 150 150 This study  crcZ  crcZ chromosomal deletion mutant of PA14  This study  cbrA/B+  cbrA mutant with pUCP18::cbrAB+; CbR  This study  cbrB +  cbrB mutant with pUCP19::cbrB+; CbR  This study  crc mutant with pUCP18::crc ; Cb crcZ mutant with crcZ+::mini-Tn7 chromosomal integration; GmR  This study  +  crc  crcZ+ E.coli  TOP10  DH5α  S17-1 HB101 SM10/λpir  +  R  F- mcrA (mrr-hsdRMS-mcrBC) recA1 ara139 (ara-leu)7697 galU galK rpsL (StrR) endA1 nupG φ80lacZM15lacX74 F-φ80lacZM15 lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK-mK+) supE44 - thi-1 gyrA96 relA galU galK rpsL(StrR) endA1 nupG thi pro hsdR hsdM+ recA (RP4-2Tc::Mu Km::Tn7) pir thi-1 hsdS20(rB-, mB-) supE44 recA13 ara-14 leuB6 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20 thi-1 thr-1 leu-6 tonA21 lacY1 supE44 recA chromosomal RP4–2 [Tcr::Mu Kmr::Tn7]λpir  This study  Invitrogen  Invitrogen, 46  45, 233 233 233  51  Strain or plasmid Plasmids  Description  Reference  pUCP18, pUCP19  E. coli-Pseudomonas shuttle vector, ApR, CbR  264  pUCP18 with cbrAB fragment  This study  pUCP19 with cbrB fragment  This study  pUCP18::crc  pUCP18 with crc fragment  This study  pEX18Ap  Suicide plasmid carrying sacBR, ApR  110  pME3087  Suicide vector, ColE1 replicon, Mob; TcR  233  pME9673  pME3087 with a 160-bp deletion in crcZ  233  pUCP18::cbrAB pUCP19::cbrB +  pRK2013 pME3280a  +  +  R  Helper plasmid, ColE1 replicon, Tra; Km Chromosomal integration vector, mini-Tn7; GmR, ApR  233 233  pME9818  pME3280a with crcZ in mini-Tn7 Laetitia Abdou Helper plasmid containing Tn7 transposition pUXBF-13 functions, R6K replicon; Ap R 233 a Ap, ampicillin; Cb, carbenicillin; Gm, gentamicin; Km, kanamycin; Tc, tetracycline.  3.3  Results  3.3.1 Construction of a PA14 cbrA deletion mutant The previous study (Chapter 2) showed that the PA14 cbrA transposon mutant (ID33836), obtained from Harvard University, was swarming deficient (276). As cbrA is located directly upstream of its cognate response regulator gene cbrB, it is possible that the swarming defect observed in the cbrA transposon mutant was simply due to a polar effect. Therefore, I investigated whether or not the transposon insertion in the PA14 cbrA gene affected the expression of cbrB. RNA was harvested from the PA14 WT and the PA14 cbrA transposon mutant and then reverse transcribed into cDNA. Changes in the expression levels of cbrA and cbrB were determined via RT-qPCR. In the PA14 cbrA transposon mutant, cbrB expression was slightly downregulated (fold change of -1.9 ± 0.1 relative to the WT), suggesting that the transposon insertion in cbrA affected not only the expression of CbrA but also that of CbrB. Since the expression of CbrB was affected in the cbrA transposon mutant, it was necessary to generate a non-polar, in-frame deletion of cbrA in the PA14 WT in order to study the role of CbrA in P. aeruginosa. To generate a P. aeruginosa cbrA deletion mutant, I utilized the sacB-based method (110) that involved amplifying 3 overlapping DNA fragments, splicing these fragments together by overlap extension PCR, and cloning the resultant fragment into a suicide vector, pEX18Ap. The plasmid-borne deletion was then transferred to the P. aeruginosa 52  strain PA14 chromosome by homologous recombination and selected on medium containing gentamicin and 5% sucrose. The resultant cbrA deletion mutant was verified by sequencing and PCR. RT-qPCR was performed to ensure that the expression of CbrB was not affected in the cbrA deletion mutant (data not shown). 3.3.2 Defect in swarming motility in the cbrA mutant The abilities of the cbrA deletion mutant to swim, twitch, and swarm on 0.3%, 1%, and 0.5% agar, respectively, were examined. The cbrA deletion mutant exhibited minor defects in flagellum-mediated swimming and type IV pilus-mediated twitching motilities (data not shown). In contrast, this mutant’s ability to swarm was completely abolished. The swarming-defective phenotype of this mutant could be restored to the WT level by introducing the WT cbrAB genes into the mutant (Fig. 3.1A).  Figure 3.1 Swarming motility of the PA14 cbrA and cbrB mutants. Defective swarming motilities of the cbrA deletion mutant (cbrA) and the cbrB transposon mutant (cbrB) were restored to levels similar to that of the WT, P. aeruginosa PA14, by transforming these mutants with plasmids containing the WT cbrAB genes or the WT cbrB gene. I also investigated whether the inability of the mutant to swarm was influenced by differences in production of rhamnolipids. Using the rhamnolipid agar plate method (51, 52), no difference was observed between the diameters of the halos formed due to rhamnolipid produced from the cbrA mutant and the PA14 WT (data not shown). To investigate whether the inability of the cbrA mutant to swarm was due to its poor ability to utilize glucose as the carbon source, I replaced glucose, in the swarming medium, with succinate, a carbon source that had been demonstrated, and confirmed in our growth studies, to sustain the WT growth of the P. aeruginosa cbrA mutant. Furthermore, swarming of the cbrA mutant was tested on other carbon  53  sources, including glycerol and mannitol. Replacement of glucose with other carbon sources did not restore the ability of the cbrA mutant to swarm (data not shown). 3.3.3 Enhanced biofilm formation P. aeruginosa forms biofilms on a number of surfaces, including tissues of the human host. Consequently, biofilm infections are virtually impossible to eradicate due to the inherent resistance of biofilm to conventional antibiotic therapies (161). Therefore, I investigated here the ability of the cbrA deletion mutant to form simple biofilms using static microtitre biofilm methods. These experiments demonstrated that the cbrA mutant showed a significant (P < 0.01 by Student’s t test) but moderate (~40 to 60%) enhancement in biofilm formation as early as 8 h (Fig. 3.2A and B). This biofilm phenotype could be successfully complemented by introducing the WT cbrAB operon into the mutant.  Figure 3.2 Biofilm formation of the PA14 cbrA and cbrB mutants. Cells were incubated in 96-well microtiter plates containing BM2-biofilm media for (A) 8 h and (B) 20 h at 37 oC for the cbrA mutant and (C) 20 h at 37oC for the cbrB mutant. Surface-associated biofilm formation was analyzed by crystal violet staining of the adherent biofilm followed by ethanol solubilization of the crystal violet and quantification (A600) of stained well. Results shown are means with standard deviations for 3 biological experiments, each with 8 technical repeats. **, statistically significant difference (P < 0.01) between the mutants and WT as determined by Student’s t test. To determine whether the biofilm formation phenotype occurred during initial attachment stage or later during biofilm development, a rapid (30 min) attachment assay was performed. No difference in early attachment was observed between the PA14 WT and the cbrA mutant (data not shown). The excess biofilm-forming phenotype of the cbrA mutant led me to investigate further whether this mutant was altered for other biofilm-related functions. Chemical analyses of P. 54  aeruginosa biofilms have suggested that the matrix is comprised of EPS, DNA, RNA, proteins, and ions (244, 265). In P. aeruginosa, the psl and pel loci have been suggested to be involved in the production of the polysaccharide component of the matrix (71). While alginate is a component of the extracellular matrix, studies have suggested that alginate is not a significant component of the extracellular polysaccharide present in the matrix of biofilms formed by P. aeruginosa under commonly used laboratory growth conditions (107, 273). Also, while P. aeruginosa strain PAO1 has both pel and psl loci, only the pel locus has been identified in the PA14 strain (71). To investigate whether the increased-biofilm-forming phenotype of this mutant was due to increased production of the pel-encoded extracellular matrix, I performed CR assays, as Congo red has been shown to bind the pel encoded polysaccharide of P. aeruginosa PA14. The cbrA mutant showed substantially increased binding to Congo red compared to the PA14 WT (Fig. 3.3).  Figure 3.3 Congo red binding. Strains were spotted on Congo red and Coomassie brilliant blue plates and incubated for 24 h at 37oC, followed by 48 h at room temperature. Representative images of the colony morphology of P. aeruginosa PA14 WT, cbrA mutant (cbrA), cbrB mutant (cbrB), complemented cbrA/B+ strain, and complemented cbrB+ strain are shown. Furthermore, biofilm production of the cbrA mutant was assessed in different carbon sources, including succinate, mannitol, and glycerol. Interestingly, when glucose was replaced with succinate as the major carbon source, the cbrA mutant produced significantly less biofilm than in glucose at 20 h, while no such difference was observed for the WT (Fig. 3.4). When mannitol or glycerol was provided as the carbon source, the cbrA mutant still produced significantly more biofilm than did the WT (data not shown). 55  Figure 3.4 Influence of carbon source on biofilm formation of the PA14 cbrA deletion mutant. Cells were incubated at 37oC for 20 h in 96-well microtiter plates containing BM2biofilm media supplemented with 0.4% (wt/vol) glucose (open bars) or 20 mM succinate (filled bars). Surface-associated biofilm formation was analyzed by crystal violet staining of the adherent biofilm followed by ethanol solubilization of the crystal violet and quantification (A600) of stained well. Results shown are means with standard deviations for three biological experiments, each with eight technical repeats. **, statistically significant difference (P < 0.01) between the mutants and WT as determined by Student’s t test. 3.3.4  Enhanced cytotoxicity toward HBE cells P. aeruginosa possesses a large arsenal of virulence factors. One of the major virulence  mechanisms employed by P. aeruginosa to intoxicate eukaryotic cells is the type 3 secretion system (T3SS). The CbrAB system was previously demonstrated to be involved in the metabolic regulation of the T3SS and its effectors (212). Therefore, I examined the ability of the cbrA mutant to infect and destroy a monolayer of cultured 16HBE14o- epithelial cells. To measure the cytotoxic effects of the PA14 WT and cbrA mutant on the epithelial cells, the amount of LDH released from the 16HBE14o- cells was quantified using an enzyme assay. The cbrA mutant displayed 3.5- and 2.2-fold-greater cytotoxicity than did the WT at 4 h and 6 h post-infection, respectively (Fig. 3.5A). Introducing the WT cbrAB operon into the cbrA mutant restored cytotoxicity to WT levels at both time points. Furthermore, to investigate whether the cbrA mutant was able to infect the epithelial cells better than the WT, due to an improved ability to adhere to the epithelial cells, adhesion assays were performed. No significant differences in adherence to 16HBE14o- cells at 1 h were observed between the WT and the mutant (data not shown).  56  Figure 3.5 In vitro cytotoxicity towards HBE cells. The abilities of the PA14 WT, the mutants (cbrA mutant [cbrA] and cbrB mutant [cbrB]), and complemented strains (cbrA/B+ and cbrB+) to induce cell damage were determined by monitoring the release of LDH into the supernatant from HBE cells. Bacteria were co-cultured with the cells and LDH release was monitored at the time points indicated. Each result represents the mean of 3 independent biological repeats, each assayed in triplicate. 3.3.5  Increased resistance to polymyxins, aminoglycosides, and fluoroquinolones Recently, a microarray analysis indicated that among the many transcriptional changes,  the cbrA gene was 2-fold downregulated (P < 0.05) in response to sublethal concentrations of tobramycin (121). Therefore, I was interested in investigating the role of CbrA in modulating resistance to a variety of clinical antibiotics, including aminoglycosides, cationic peptides, fluoroquinolones, cephalosporins, and carbapenems (Table 3.2). Intrinsic resistance of the mutant to these antibiotics was assessed by MIC assay in cells growing in BM2- swarming medium containing high (2 mM) Mg2+ to suppress the possibility of induction of genes by limiting Mg2+. Compared to the PA14 WT, the cbrA mutant reproducibly exhibited a 2-foldincreased resistance to polymyxin B and colistin and 4-fold-increased resistance to ciprofloxacin and tobramycin. No differences were observed between the MIC values of the PA14 WT and the mutant to piperacillin, tetracycline, cefepime, ceftazidime, and imipenem. Although the 2-fold change in MIC observed for the cbrA mutant to polymyxin B is often considered to be within the acceptable range of error of these assays, I confirmed the increased resistance of the cbrA mutant to polymyxin B by performing kill curve assays with cells taken from liquid swarm medium and from swarm plates (Fig. 3.6). The antibiotic susceptibility phenotype could be complemented to WT levels by introducing the WT cbrAB genes into the mutant.  57  Table 3-2 Minimal inhibitory concentrations (µg/ml) of antibiotics toward P. aeruginosa grown in swarming medium. Results are shown as the mode of 4-6 independent experiments. MIC (µg ml-1) Antibiotic Polymyxin B Colistin Tobramycin Piperacillin Tetracycline Ciprofloxacin Cefepime Ceftazidime Imipenem  PA14 (WT)  cbrA mutant  cbrB mutant  1 1 2 4 64 0.2 2 4 2  2 2 8 4 64 0.8 2 4 2  1 1 2 4 64 0.2 1 4 2  Figure 3.6 Polymyxin B resistance in the PA14 cbrA deletion mutant. Sensitivity to polymyxin B at 1 µg/ml was analyzed using cells (A) from mid-log in swarm medium or (B) directly off of swarm plates and then plating diluted aliquots for survivors. For each condition, 1 representative experiment of 4 independent experiments that produced identical results is shown. Previous studies identified CbrAB as an important regulatory element for the expression of several catabolic pathways and utilization of a variety of organic compounds as the sole carbon source (183). Li and Lu showed that a cbrAB mutant displayed weak growth when 58  glucose was used as the sole carbon source, while growth on TCA cycle intermediates was sustained (149). Therefore, I investigated whether any of the phenotypes observed for the cbrA mutant could be related to growth impairment when glucose was provided as the major carbon source in the swarming, MIC, and biofilm media. The growth of the cbrA mutant and the PA14 WT was measured in the appropriate medium (BM2-swarm medium, LB medium, or BM2biofilm medium) at 37°C using a Tecan Spectrofluor Plus to measure the absorbance at 620 nm every 20 min for 15 to 20 hours under shaking conditions. The growth of the mutant and WT was also determined by measuring the absorbance at 600 nm every 20 min during infection of 16HBE14o- cells. As shown in Fig. 3.7A, the cbrA mutant exhibited a very minor growth defect in LB medium. A moderate defect in growth under swarming conditions was observed for the mutant (Fig. 3.7B). The moderate growth defect of the mutant in this medium (~50% change in growth rate), however, seemed insufficient to explain the complete abolition of swarming motility. The cbrA mutant also exhibited slight growth defects under biofilm-inducing (Fig. 3.7C) and HBE infection conditions, but these growth defects also seemed insufficient to explain the increased biofilm production and cytotoxicity of the mutant. Furthermore, I investigated whether changing the carbon source from glucose to succinate would restore the ability of the mutant to swarm and form biofilms at the WT level. The cbrA mutant indeed showed WT growth when glucose was replaced with succinate in the BM2-swarming and BM2- biofilm media, as suggested from previous studies (149), but exhibited the same swarming and biofilm defects as in glucose medium.  59  Figure 3.7 Growth curves of P. aeruginosa PA14 WT and cbrA deletion mutant. Cells were diluted and grown in (A) LB broth, (B) BM2-swarm medium, and (C) BM2-biofilm medium. Growth was measured at 37oC using a TECAN Spectrofluor Plus. 3.3.6  Microarray analysis of the cbrA mutant To investigate how CbrA contributed to the various phenotypes observed, microarray  studies were performed comparing the cbrA mutant to the PA14 WT. For the microarray, RNAs from the PA14 WT and the cbrA mutant were taken directly from BM2-swarm plates that had been incubated at 37°C for 20 hours. The microarray revealed 236 genes that were differentially regulated by more than 2-fold (P ≤ 0.06) with 145 transcriptionally upregulated and 91 transcriptionally downregulated genes (Appendix Table C-1). A selection of these genes is 60  presented in Table 3.3. Of note, PAO1 DNA microarray slides were used to analyze gene expression of PA14 cbrA mutant and PA14 WT, as there are no PA14-specific microarray slides available. The PA14 genome (6.5 Mbp) is slightly larger than that of PAO1 (6.3 Mbp), but the PA14 and PAO1 genomes are very similar, with greater than 92% of all genes in PA14 also present in PAO1. The additional genes in the PA14 genome that are absent in PAO1 have been suggested to contribute to its enhanced pathogenicity, as PA14 is more virulent than PAO1 (104). Analysis of the microarray data revealed dysregulation of genes involved in amino acid biosynthesis and metabolism, carbon compound catabolism, and central intermediary metabolism, consistent with the proposed role of CbrAB in the utilization of a variety of organic compounds as sole carbon source (149, 183). For example, hutU (urocanase), part of the hutUHIG operon involved in histidine catabolism, was moderately downregulated in the cbrA mutant (RT-qPCR revealed a fold change of -6.0 ± 0.6 relative to the WT). Table 3-3 Selected genes significantly dysregulated in the cbrA mutant as determined using microarrays. * indicates confirmation of gene regulation by RT-qPCR. Gene Fold Name P value Function IDa changeb Adaptation and protection PA1159 -2.36 0.0353 probable cold-shock protein 3-oxo-C12-homoserine lactone acylase and PA2385 pvdQ -3.01* 0.002 pyoverdine biosynthesis PA2386 pvdA -2.39* 0.0011 L-ornithine N5-oxygenase PA2397 pvdE -3.44 0.002 pyoverdine biosynthesis protein PA2399 pvdD -2.24 0.0061 pyoverdine synthetase D PA2920 2.14 0.0068 probable chemotaxis transducer PA3349 2.15 0.0013 probable chemotaxis protein probable ATP-binding component of ABC PA4223 pchH -4.16* 8.59E-05 transporter PA4225 pchF -2.53 0.0005 pyochelin synthetase PA4231 pchA -2.48 0.0009 salicylate biosynthesis isochorismate synthase PA4356 xenB 2.27 0.0039 xenobiotic reductase PA4468 sodM 3.55 8.86E-05 superoxide dismutase PA4876 osmE -2.00 0.0161 osmotically inducible lipoprotein  61  Gene Fold Name P value IDa changeb Chemotaxis; Cell wall / LPS / capsule PA1423 2.55 0.0007 PA2920 2.14 0.0068 PA3157 wbpC -2.3 0.0186 PA3349 2.15 0.0013 PA3545 algG 2.08 0.0068 Chaperones & heat shock proteins PA1596 htpG 2.21 0.0016 PA2830 htpX 3.27 0.0001 PA3126 ibpA 3.26 6.23E-05 PA4352 2.65 0.0003 PA4761 dnaK 3.57* 5.33E-05 PA4762 grpE 2.95 0.0004 PA5053 hslV 4.9 9.70E-05 PA5054 hslU  3.07  0.0054  Function probable chemotaxis transducer probable chemotaxis transducer probable acetyltransferase probable chemotaxis protein alginate-c5-mannuronan-epimerase heat shock protein 90 heat shock protein heat-shock protein putative universal stress protein molecular chaperone heat shock protein ATP-dependent protease peptidase subunit ATP-dependent protease ATP-binding subunit  Antibiotic resistance and susceptibility PA1178 oprH  9.36*  1.63E-06  PA1170 phoP  4.16*  2.74E-05  PhoP/Q and low Mg2+ inducible outer membrane protein H1 precursor  two-component response regulator putative function in adaptive polymyxin PA1797 10.37* 1.16E-06 resistance putative antibiotic biosynthesis PA2198 3.73 0.001 monooxygenase PA3552 arnB 6.16* 6.84E-06 hypothetical protein PA3553 arnC 3.61 5.17E-05 probable glycosyl transferase PA3554 arnA 2.44 0.0021 hypothetical protein PA3555 arnD 4.49 1.57E-05 hypothetical protein PA3556 arnT 3.13 0.0001 inner membrane L-Ara4N transferase PA3557 arnE 4.51 2.97E-05 hypothetical protein PA3558 arnF 2.45 0.0028 hypothetical protein a ID, identification. Information is according to the P. aeruginosa genome website (http://www.pseudomonas.com). b Fold regulation of genes differentially expressed in cbrA mutant relative to PA14 WT. A positive number indicates transcript upregulation and a negative number indicates transcript downregulation in the cbrA mutant. An asterisk indicates confirmation of gene regulation by RTqPCR. The cbrA microarray results were examined to identify genes that might influence antibiotic resistance of the mutant (since the microarray and MIC experiments utilized similar growth conditions). Moderate (3 to 10-fold) upregulation of the operons encompassing the two62  component regulators oprH-phoPQ and pmrAB and of the downstream arnBCADTEF (LPS modification) operon was observed in the microarray. To confirm the microarray results, RTqPCRs were performed and revealed 9.3 ± 1.8-fold upregulation of oprH, 3.7 ± 0.5-fold upregulation of phoP, 3.1 ± 0.3-fold upregulation of phoQ, 10.2 ± 1.9-fold upregulation of arnB, and 4.0 ± 1.4-fold upregulation of pmrB. Activation of these operons is known to trigger bacterial resistance to cationic peptides and polymyxins in response to low-Mg2+ conditions by controlling the addition of aminoarabinose to lipid A, thereby reducing the net negative charge of LPS and limiting its interaction with polycationic peptides such as polymyxin B (168); however, their link to CbrAB had not been revealed previously. The involvement of the PhoPQ system in aminoglycoside resistance has also been defined (157, 200). Although the details of PhoPQ involvement in aminoglycoside resistance remain to be fully elucidated, PhoPQ appears to mediate resistance to aminoglycosides via a mechanism different from that involved with the polycationic peptides. The cbrA microarray also identified the upregulation of several heat shock protein genes, including htpG, ibpA, dnaK, grpE, hslV, and hslU. The upregulation of dnaK was confirmed by RT-qPCR, revealing 3.6 ± 0.2-fold upregulation in the mutant relative to the WT. Recent studies have demonstrated that upregulation of heat shock genes prior to treatment with tobramycin led to increased resistance of P. aeruginosa to tobramycin (121). Although the cbrA microarray indicated upregulation of the mexX gene, known to be involved in involvement in aminoglycoside resistance (200), RT-qPCR failed to detect any significant changes in the transcriptional expression of this gene (fold change of 1.1 ± 0.3) in the cbrA mutant compared to the WT. Analysis of the microarray data also revealed moderate downregulation of several genes, PA0621, pvdD, PA3784, pchH, and pchF, which were possibly involved in the ciprofloxacin resistance phenotype observed in the cbrA mutant. A previous ciprofloxacin screen of the PA14 transposon mutant library by Breidenstein et al. showed that transposon mutants of PA0621, pvdD, PA3784, pchH, and pchF are more resistant than their WT parent strain toward ciprofloxacin (27). Flagella and type IV pili play important roles in biofilm and microcolony formation and are also required for swarming (128). Furthermore, studies have suggested that the flagellum secretion system plays a role in P. aeruginosa invasion of epithelial cells (70). However, the microarray did not reveal dysregulation in any flagellum- or type IV pilus-related genes. Furthermore, there was no dysregulation of the type 3 secretion apparatus or effector genes in the cbrA mutant. 63  This transcriptome analysis also highlighted several other interesting genes. These findings included moderate downregulation of the pyoverdine and pyochelin biosynthesis genes, including pvdA, pvdQ, and pchF. RT-qPCR confirmed downregulation of these genes in the mutant with values of -9.9 ± 1.3 for pvdA, -16.5 ± 2.4 for pvdQ, and -4.2 ± 0.6 for pchF. The downregulation of pvdQ in the cbrA microarray was of interest since a pvdQ mutant is swarming deficient (189) and has been suggested to play a role in biofilm formation and virulence of P. aeruginosa (180). Consistent with the qPCR data, I grew the cbrA mutant and the WT in a lowiron medium to induce pyoverdine production and observed, using a spectrofluorimeter, that the mutant secreted less pyoverdine into the supernatant than did the WT (Fig. 3.8). In a previous study, Jimenez et al. showed that addition of iron in swarm plates restored the swarming of a swarming-impaired pvdQ mutant to the WT level (180). Therefore, I also tested whether addition of iron had any effect on swarming motility or the biofilm formation of the cbrA mutant. However, addition of iron did not restore swarming or biofilm formation of the cbrA mutant to the WT level (data not shown).  Figure 3.8 Pyoverdine production of the PA14 cbrA deletion mutant. Cells grown in CAA medium for 48 h at 37oC were diluted, and the resultant sample was excited at 400 nm with a spectrofluorimeter. 3.3.7 Transcriptional analysis of the cbrA mutant under various growth conditions The microarray experiment of the cbrA mutant versus PA14 WT did not reveal transcriptional changes in the expression of genes known to be involved in virulence (e.g., the T3SS) or genes involved in the production of exopolysaccharides in P. aeruginosa. This was not completely unexpected, since the growth conditions used for the microarray experiment and the conditions used to assay exopolysaccharide production or in vitro cytotoxicity experiments were different and it is known that CbrAB influences the utilization of many different carbon and 64  nitrogen sources. Therefore, I isolated RNA from bacterial cells during in vitro infection of HBE cells and cells growing on CR plates. Consistent with the increased binding phenotype observed on the CR plates (Fig. 3.3), RT-qPCR revealed 2.5 to 5-fold upregulation of the exopolysaccharide pelD and pelF genes in the cbrA mutant compared to the PA14 WT (Table 34). Bacterial cells obtained during HBE cell infection revealed moderate upregulation of the type 3 secretion apparatus and effectors (exoT, exoY, exoU, pcrV, exsA, and popD) and the type 1 secretion apparatus, aprD (Table 3-4), consistent with the enhanced cytotoxicity of this mutant. Table 3-4 Dysregulated genes in the cbrA, cbrB, crcZ and crc mutants during HBE cell infections and growth on Congo red plates as determined by RT-qPCR. Values are represented as fold changes relative to the PA14 WT. Fold changes relative to PA14 WTa cbrA cbrB crcZ crc During HBE cell infection exoT 2.9± 0.3 3.4± 1.0 3.8± 0.9 -3.8± 1.2 exoY 2.7± 0.3 2.3± 0.6 2.1± 0.5 -4.3± 0.3 exoU 2.5± 0.4 2.7± 0.3 2.0± 0.4 -9.8± 1.0 pcrV 2.6± 0.3 2.2± 0.1 2.0± 0.3 -5.8± 0.9 exsA 2.2± 0.2 2.7± 0.5 1.7± 0.6 -2.7± 0.9 pcrD 1.4± 0.1 1.2± 0.5 1.2± 0.8 -1.4± 0.5 popD 2.9± 0.5 2.3± 0.5 2.4± 0.4 -6.6± 0.7 pscF 1.2± 0.1 1.4± 0.5 1.6± 0.9 -2.4± 0.3 pcrR 1.3± 0.3 1.8± 1.1 1.2± 0.9 -1.5± 0.5 lasB 1.2± 0.2 1.1± 0.6 1.5± 0.8 -1.2± 0.5 lipC 1.2± 0.1 -1.1± 0.3 1.1± 0.5 -1.3± 0.3 plcB 1.6± 0.5 1.3± 0.3 1.7± 0.6 -1.8± 0.6 PA4528 1.5± 0.3 1.3± 0.1 1.1± 0.3 -1.0± 0.5 aprD 2.5± 0.5 2.9± 0.4 2.4± 0.3 -6.2± 0.8 Congo red condition pelD 2.6± 0.5 5.1± 0.5 2.7± 0.5 -4.1± 1.0 pelF 2.5± 0.4 4.5± 0.4 2.8± 0.5 -2.6± 0.8 a Values are represented as averages of at least 3 biological samples.  3.3.8 CbrA mediated regulation of swarming, biofilm formation, and cytotoxicity in conjunction with CbrB The CbrB response regulator has been identified to play a role along with its cognate sensor kinase CbrA in the global metabolic regulation of carbon and nitrogen utilization in P. aeruginosa (149). Therefore, I examined whether CbrB also interacted with CbrA to modulate virulence and virulence-related processes in P. aeruginosa. Similarly to the cbrA mutant, the cbrB mutant displayed impairment in swarming and excessive biofilm formation (Fig. 3.1B and 65  3.2C). These phenotypes could also be complemented by introducing the WT cbrB allele into the cbrB mutant. CR assays revealed increased binding of Congo red to the cbrB mutant compared to the WT (Fig. 3.3). Interestingly, on the CR plates, the cbrB mutant also showed wrinkled morphology while the cbrA mutant remained smooth like the WT. Furthermore, the cbrB mutant exhibited 2-fold-enhanced in vitro cytotoxicity toward HBE cells (Fig. 3.5B). Similarly to the cbrA mutant, the cbrB mutant also exhibited moderate growth defects in swarming/MIC and biofilm media (data not shown). However, while the cbrA mutant showed increased resistance to a variety of antibiotics, the cbrB mutant displayed WT MIC values for all antibiotics tested (Table 3-2). Based on the microarray results of the cbrA mutant, I examined the expression level of a variety of genes in the cbrB mutant under swarming conditions by RT-qPCR. As expected, RT-qPCR revealed no dysregulation in the genes that were thought to explain the altered susceptibility of the cbrA mutant; these findings included a lack of dysregulation of the oprHphoPQ operon, the pmrAB operon, and the LPS modification operon (data not shown). Similarly to the cbrA mutant, upregulation of energy metabolism genes, such as nirS and norC, was observed in the cbrB mutant. The expression of pvdQ was also downregulated in the cbrB mutant, while other genes involved in pyoverdine biosynthesis, such as pvdA, were not dysregulated in the cbrB mutant (data not shown). However, similarly to the cbrA mutant, pyochelin biosynthesis genes, such as pchG, were downregulated in the cbrB mutant. In addition, RNA was isolated from the cbrB mutant from CR plates, and during HBE cell infection, RT-qPCRs were performed. The cbrB mutant revealed upregulation of the pel operon under the CR condition, but to a greater extent than that with the cbrA mutant (Table 3-4). This result was consistent with the wrinkled morphology observed only in the cbrB mutant and the increased biofilm formation of the cbrB mutant compared to the cbrA mutant. RT-qPCR analysis of the cbrB mutant during HBE cell infection also revealed upregulation of the T3SS during in vitro infection (Table 3-4). 3.3.9 CbrAB system regulation of swarming, biofilm formation, and cytotoxicity via the downstream regulatory system CrcZ/Crc Recently, Sonnleitner et al. found that the CbrAB/CrcZ/Crc system enables P. aeruginosa to utilize various carbon sources (233). In addition to its role in catabolite repression, O’Toole et al. showed that Crc is required for biofilm formation in P. aeruginosa (188). Furthermore, Linares et al. demonstrated that the Crc protein plays a role in the regulation of virulence in P. aeruginosa (151). They showed that a crc mutant is defective in type 3 secretion 66  and is less virulent in a Dictyostelium discoideum model (151). Thus, these results led me to investigate whether the CbrAB system regulated swarming, biofilm formation, and cytotoxicity through the CrcZ/Crc system. First, I examined the ability of a PA14 crc transposon mutant to swarm, form biofilm, and infect HBE cells. As shown in Fig. 3.9A, the crc mutant was able to swarm as well as the WT but consistently exhibited reduced branching of the swarming tendrils compared to the WT. The crc mutant was also defective in biofilm formation and showed a reduced ability to bind CR (Fig. 3.9B and D). The crc mutant also displayed a reduced ability to infect and destroy a monolayer of cultured 16HBE14o- epithelial cells (Fig. 3.9C). As well, the crc mutant showed WT growth in swarm and biofilm media (data not shown). These phenotypes could also be complemented by introducing the WT crc allele into the crc mutant. These results suggest that the Crc protein had different effects than did the cbrA and cbrB mutants on swarming motility (modest effects on branching), while positively regulating biofilm formation and virulence of P. aeruginosa. RT-qPCR experiments on the crc mutant demonstrated downregulation of the T3SS during HBE cell infection and downregulation of the pel operon expression from CR plates (Table 3-4).  67  Figure 3.9 Swarming motility, biofilm formation, Congo red binding and in vitro cytotoxicity of the PA14 crc mutant. (A) Swarming motility was assessed by spotting 1 µl of mid-log cell cultures onto BM2-swarming plates with 0.5% (wt/vol) agar and incubated at 37oC for 20 h. (B) Biofilm formation was assessed by diluting overnight cultures in BM2-biofilm media and incubating at 37oC for 20 h. (C) Cytotoxicity towards HBE cells was determined by infecting a monolayer of HBEs with mid-log bacterial cultures at an MOI of 2. (D) CR binding was assessed by spotting 1, 5 or 10 µl of diluted cultures onto CR plates and incubating for 24 h at 37 oC and for an addition 48 h at room temperature. I also tested the ability of a crcZ deletion mutant to swarm, form biofilm, and infect HBE cells. In contrast to the crc mutant, the crcZ mutant exhibited phenotypes very similar to those of the cbrA and cbrB mutants, including substantial swarming deficiency, excessive biofilm formation, and enhanced cytotoxicity toward HBE cells (Fig. 3.10). These phenotypes could be complemented by introducing the WT crcZ allele into the crcZ mutant. In addition, growth studies revealed that the crcZ mutant exhibited moderate growth defects in swarming media (data not shown). RT-qPCR analysis of the RNA isolated from the crcZ mutant cells grown on  68  CR plates revealed upregulation of the pel operon and upregulation of the T3SS during HBE cell infection (Table 3-4). In addition, I tested the susceptibility/resistance of the crcZ and crc mutants toward the antibiotics tested for the cbrA mutant. However, no differences between the mutants and WT were observed for any of the tested antibiotics (data not shown). These results indicate that the CbrAB system might be regulating swarming, biofilm formation, and cytotoxicity via CrcZ, with a partial inverse regulation by Crc.  Figure 3.10 Swarming motility, biofilm formation and in vitro cytotoxicity of the PA14 crcZ mutant. (A) Swarming motility of the PA14 WT and crcZ mutant was determined by inoculating 1 µl of mid-log cell cultures onto BM2-swarming plates with 0.5% (wt/vol) agar and incubated at 37oC for 20 h. (B) Biofilm formation of the PA14 WT, PA14 crcZ mutant and PA14 crcZ+ complemented were determined by diluting overnight cultures in BM2biofilm media and incubating at 37oC for 20 h. (C) Cytotoxicity towards HBE cells was determined by infecting a monolayer of HBEs with mid-log bacterial cultures at an MOI of 2 for 6 h at 37C + 5% CO2. **, statistically significant difference (P < 0.05) between the mutants and WT as determined by Student’s t test. (D) CR binding was assessed by spotting 1, 5, or 10 µl of diluted cultures onto CR plates and incubating the plates for 24 h at 37oC and an addition 48 h at room temperature.  69  3.4  Discussion In this study, I examined the role of the sensor kinase CbrA in motility, biofilm  formation, cytotoxicity, and antibiotic resistance. A cbrA deletion mutant was swarming defective but exhibited enhanced biofilm formation and in vitro cytotoxicity toward human bronchial epithelial cells. Furthermore, the cbrA mutant exhibited increased resistance toward several common clinical antibiotics, including polymyxin B, tobramycin, and ciprofloxacin. The sensor kinase CbrA and adjacently encoded response regulator CbrB have been proposed to work together in regulating the utilization of a variety of organic compounds as sole carbon sources. Phenotypic and genetic analyses performed here indicated that CbrA regulated swarming, biofilm formation, and cytotoxicity via its cognate response regulator CbrB. In addition to CbrB, I provided evidence for the involvement of the sRNA CrcZ in the regulation cascade of these virulence and virulence-related phenotypes. In contrast, the antibiotic resistance phenotype observed in the cbrA mutant was absent in the cbrB and crcZ mutants, and I speculate that CbrA may cross talk with other response regulators to regulate antibiotic resistance. A proposed model for the regulation of swarming, biofilm formation, cytotoxicity, and antibiotic resistance by the CbrA/ CbrB/CrcZ pathway is shown in Fig. 3.11. The situation is not as clear with Crc, which is known to be negatively regulated by crcZ RNA and to fully participate in regulation of carbon source utilization. If Crc were fully involved, I would expect there to be a reciprocal phenotype in the crc mutant. Instead, there was only a partial effect on swarming in our study, but a substantial decrease in biofilm formation and a complete absence of epithelial cell toxicity. Thus, downstream effects on crc may be part of the phenotype but do not appear to fully explain the phenotypes observed. Complementation of the cbrA deletion mutant with the WT cbrAB operon was necessary since complementation with the PA14 WT cbrA allele alone only moderately but incompletely restored the phenotypes. I speculate that the reason that the cbrA mutant was unable to be fully complemented with cbrA alone is that in the cbrA complemented strain, there were multiple copies of the cbrA gene due to the presence of the multicopy vector. Hence, when CbrA was expressed, the disproportionate amount of CbrA relative to the amount of CbrB in the complemented strain, compared with the WT (confirmed by RT-qPCR), might have changed the functional interaction of these components and even shifted the balance from phosphorylation to dephosphorylation (both functions of sensor kinases).  70  Figure 3.11 Proposed model for the involvement of the CbrA/CbrB/CrcZ regulatory cascade in the regulation of swarming, biofilm formation, cytotoxicity and antibiotic resistance in P. aeruginosa. Under conditions that activate the CbrAB TCS, the phosphorylated response regulator CbrB will activate the expression of CrcZ. In turn, CrcZ has a high affinity for the RNA binding protein Crc. The binding of CrcZ to Crc sequesters Crc, resulting in the inhibition of expression of mRNAs that encode for factors to promote biofilm formation and virulence. In addition to sequestration of Crc, CrcZ is involved in the direct or indirect activation of the expression of genes to promote swarming motility. Antibiotic resistance, however, involves only the CbrA sensor kinase. Several physical factors are currently known to be required for swarming, including flagella, type IV pili, and the production of the biosurfactant rhamnolipids. The ability of the cbrA mutant to swim and twitch at close to WT levels (276), and in microarrays to express relevant genes at the same level as that for the WT, suggested that the cbrA mutant was not impaired in flagellum or type IV pilus biosynthesis. Furthermore, the cbrA mutant produced a WT level of rhamnolipids. These results indicate that CbrA normally promotes the expression of additional factors required for swarming in P. aeruginosa. Microarray analysis of the cbrA mutant, compared to the PA14 WT under swarming conditions, revealed the downregulation of a number of genes that have been identified to be upregulated in swarmer cells (189), suggesting that these genes may be regulated through CbrA 71  and involved in the swarming growth state. Among others, these genes included a probable ATPbinding component of the ABC transporter, PA4223; genes involved in pyochelin biosynthesis, pchG and pchF; genes involved in pyoverdine biosynthesis, pvdQ, pvdE, and pvdD; and a gene encoding an ammonium transporter, amtB. Of these genes, only the PA4223 and pvdQ mutants displayed swarming deficiencies, consistent with previous studies (189, 276). PvdQ is of particular interest, as this protein plays a dual role in pyoverdine biosynthesis, as well as QS, with an acylhomoserine lactone acylase activity. Recently, the role of PvdQ in swarming was further studied by Jimenez et al. (180). This group showed that under iron limitation, the pvdQ mutant was defective in swarming, and they indicated that swarming could be restored by exogenous addition of iron, suggesting that the role of PvdQ in swarming was closely linked to the pyoverdine/iron pathway (180). In our current study and the study by Overhage et al. (189), it was found that, with the exception of pvdQ, the genes required for pyoverdine biosynthesis, such as pvdE and pvdD, exhibited WT swarming behavior. Moreover, addition of iron to the swarming medium did not change the swarming phenotype of the cbrA mutant. Therefore, I suggest that the role of PvdQ as a quorum signal quencher might be more influential in swarming. As suggested previously, PvdQ may play a role in maintaining the relative concentrations of the two homoserine lactone QS signals, required for swarming differentiation. Alternatively, the degradation product induced by PvdQ may act a signal during swarmer cell differentiation (189). I am currently investigating these possibilities. The cbrA deletion mutant produced significantly more biofilm than did the WT strain in an abiotic biofilm assay (Fig. 3.2A and B). CR assays revealed that the hyperbiofilm phenotype of the cbrA mutant might be due to increased production of the pel-encoded exopolysaccharide (Fig. 3.3). CbrA did not appear to play a role during initial attachment stage, as a rapid attachment assay revealed no difference in the abilities of the mutant and the WT to attach to the wells of the polystyrene plates. This result was expected, since the cbrA mutant had a functional flagellum and pilus. Overall, these results suggested that CbrA normally negatively regulates biofilm formation. In contrast, a recent screen of the PA14 transposon mutant library by Musken et al. revealed that a cbrA mutant exhibited a reduced-biofilm phenotype (179). The reason for the difference in biofilm phenotype may be due to the different media used to grow the biofilms. While minimal medium was used here to cultivate formation of biofilm at the air-liquid interface, Musken’s group used a rich medium (LB broth) to promote biofilm formation at the bottom of the microtiter plate. The intricate relationship between swarming motility and biofilm formation in P. aeruginosa is complex. Although biofilm formation is a surface-associated 72  sessile behaviour and swarming is a surface-associated motile behaviour, both processes are suggested to involve similar components at certain stages and under specific conditions (257). For example, both swarming motility and the initiation of biofilm formation have been shown to require flagella (187). Moreover, there is evidence that swarming motility can contribute to the early stages of P. aeruginosa biofilm formation (225). There are a number of studies that suggest that these surface-associated behaviours are inversely regulated and mediated through the signalling molecule cyclic-di-GMP (c-di-GMP) (133, 169, 257). It has been demonstrated that the intracellular levels of this signalling molecule influence a number of bacterial behaviours, with the common theme being that the accumulation of c-di-GMP promotes sessile behaviours, such as biofilm formation, while the degradation of c-di-GMP favours motile behaviours, such as swarming. Recent studies have shown that BifA, a c-di-GMP phosphodiesterase, participates with SadC, a c-di-GMP diguanylate cyclase, to control the level of cellular c-di-GMP in regulating biofilm formation and swarming motility (133, 169). As the cbrA mutant also exhibits a severe swarming defect and a hyperbiofilm phenotype, it will be of interest to examine whether CbrA plays a role in regulating the level of cellular c-di-GMP. Regardless, numerous other mutations were identified that either exhibited a reciprocal relationship between swarming and biofilm formation or showed similar effects on the two processes (190, 276) (Chapter 2), so it seems likely that control of these processes is multideterminant. The antibiotic resistance phenotypes observed for the cbrA mutant were not observed for the cbrB mutant. Intriguingly, these results mirror what has been observed for other twocomponent regulators, where the sensor kinase and response regulator have different phenotypes (e.g., phoQ is constitutively resistant to polymyxin and aminoglycosides, while phoP is null) (157). Using RT-qPCR, substantial changes were confirmed in the cbrA mutant, but in contrast in the cbrB mutant, no significant changes were observed in the transcriptional expression of the oprH-phoPQ operon, the pmrAB operon, or the LPS modification operon (arn operon). Thus, the upregulation of these operons in the cbrA mutant can be concluded to play a major role in the resistance of this mutant to the majority of the antibiotics tested, including polymyxin B, colistin, and tobramycin. Currently I am investigating the mechanism of potential cross talk between CbrA and PhoQ. The involvement of CbrA and CbrB in a number of important adaptation-related processes in P. aeruginosa suggests that these regulators contribute in maintaining the overall physiological balance of the bacterium. By enabling the bacteria to utilize a variety of organic compounds as carbon sources and to undergo swarming motility, while sustaining an optimal 73  level of biofilm production, cytotoxicity, and antibiotic resistance, CbrA optimizes the efficiency of the bacteria to adapt to various environments.  74  Chapter 4: Requirement for P. aeruginosa CbrA for full virulence in a murine acute lung infection model 4.1  Introduction In Chapter 3, I showed that the sensor kinase CbrA played an important role in regulating  various virulence- and virulence-related processes in P. aeruginosa (275). Mutation in the cbrA gene resulted in a mutant that was completely unable to swarm but exhibited increased biofilm production. The cbrA mutant also exhibited increased resistance to a variety of clinically significant antibiotics, including polymyxin B, ciprofloxacin, and tobramycin. Furthermore, in vitro cytotoxicity experiments revealed that the cbrA mutant exhibited increased cytotoxicity toward HBE cells (275). These results led to the examination, with the assistance of Laure Janot and Ashley Hilchie, of the in vivo virulence of the cbrA mutant in two mouse models: the acute lung infection model and the peritoneal infection model. As shown here, the cbrA mutant exhibited 10-fold and 104-fold reduction in virulence in the acute lung infection model and the peritoneal infection model, respectively. Importantly, in a rat chronic lung persistence model, performed in collaboration with Dr Roger Levesque and Irina Kukavica-Ibrulj, no significant difference was found in the ability of the WT and the cbrA mutant to grow and persist in vivo. This result suggested that the decreased in vivo virulence observed for the cbrA mutant was not simply due to any metabolic deficiencies which might otherwise lead to a decreased growth rate in vivo (e.g. consistent with its observed in vitro growth defects in certain media),. To understand the role of CbrA in mediating in vivo virulence in P. aeruginosa, its effect on phagocytosis was investigated and it was revealed that there was significantly enhanced uptake of the cbrA mutant compared to the WT by both macrophages and neutrophils in vitro. Furthermore, the data presented here indicate that CbrA regulated in vivo virulence of P. aeruginosa independent of CbrB. 4.2  Materials and methods  4.2.1 Tissue culture, bacterial strains and growth conditions PA14 WT, the cbrA deletion mutant, the cbrA complemented strain (harbouring pUCP18::cbrAB+), and the cbrB transposon mutant (275) were routinely grown in LB broth. For plasmid or transposon maintenance, antibiotics were added to growth media at the indicated concentrations: 15 µg/ml gentamicin and 500 µg/ml carbenicillin.  75  Venous blood was collected from healthy adult donors in Vacutainer collection tubes containing sodium heparin as an anticoagulant (BD Biosciences) in accordance with the ethical approval guidelines of the UBC Research Ethics Board. PBMC were isolated as previously described (174, 182). To obtained human monocyte-derived macrophages (MDM), isolated PBMCs were resuspended in serum-free RPMI 1640 media and 2 X 106 cells/well were seeded into 24-well tissue culture plates (Corning Life Science, Corning, NY) to rest for 1 h and then the medium replaced with complete RPMI medium [RPMI 1640 medium (Life Technologies, Invitrogen) supplemented with 10% (v/v) heated-inactivated FBS, 2 mM L-glutamine and 1 mM sodium pyruvate]. Cells were cultured for 7 days in 100% humidity and 5% CO 2 at 37oC. On days 2 and 6, cells were washed and replaced with complete RPMI containing 10 ng/ml macrophage colony-stimulating factor (M-CSF) (Research Diagnostic, Concord, MA). Murine bone marrow-derived macrophages (BMDM) were prepared by culturing bone marrow cells of C57BL/6 mice for 7 days at 1 X 106 cells/ml in DMEM-high glucose with 20% FCS, 2 mM L-glutamine and 1 mM sodium pyruvate (all from Invitrogen) and supplemented with 25 ng/ml recombinant M-CSF (eBioscience). On day 7, after incubation with PBS for 30 min at 4oC, cells were detached with a cell scraper, washed by centrifugation and counted. Human neutrophils and serum were isolated as previously described (22, 174), resuspended in HBSS and used immediately for opsonization or treatments. The SV40-transformed, immortalized HBE cell line 16HBE14o- was cultured as described in Chapter 3 Materials and Methods. 4.2.2 Mice models of peritoneal and acute lung infection All mouse experiments were conducted in accordance with the Animal Care Ethics Approval and Guidelines of the University of British Columbia. C57BL/6 or CD-1 female mice (from Center for Modeling Disease, UBC) were maintained under specific pathogen-free conditions. Mice were 6 weeks of age and weight matched in all experiments. For the peritoneal infection model, C57BL/6 mice were injected with cyclophosphamide (150 mg/kg, IP, Sigma) 4 and 2 days before infection with a fresh culture of P. aeruginosa by the intraperitoneal (IP) route at different concentrations (60 CFU/200-l of the PA14 WT and cbrA complemented strains; 60 CFU, 500 CFU, 103 CFU, 104 CFU, and 106 CFU/200-l of the cbrA mutant). At 18 h post-infection, mice were euthanized by CO2 inhalation. Blood was collected by cardiac puncture and the peritoneal lavage was collected by installation, washing  76  and withdrawal of 3 ml sterile saline. Bacterial numbers in the blood and peritoneal lavage were measured by serial dilution and plating on LB-agar plates. In the acute lung infection model, CD-1 mice were anaesthetized via inhalation of aerosolized isoflurane (2 to 5 %) mixed with oxygen and were infected by intranasal administration with a fresh culture of P. aeruginosa at 106 CFU/20-µl for the PA14 WT and the cbrA complemented strains, and 107 CFU/20µl for the cbrA mutant. At 18 h post-infection, mice were euthanized by lethal pentobarbital injection (120 mg/kg, IP). The bronchoalveolar lavage (BAL) was collected by cannulating the trachea and washing the lung 3 times with 600 µl of saline solution. Bacterial numbers in the BAL were measured by serial dilution and plating on LB-agar plates. 4.2.3 Rat model of chronic lung infection Agar beads were prepared according to a modification of a previously described method (35, 134). The cbrA mutant (tagged with gentamicin resistance) and the PA14 WT were grown separately in Trypic Soy Broth (TSB). Overnight cultures were sedimented by centrifugation at 7.2 X 103 rpm for 2 min, washed twice with 500 µl of PBS, resuspended in 1 ml PBS, and added to 9 ml of 2% agar, prewarmed to 48°C. A mixture of equal amounts of WT and mutant was added to 200 ml heavy mineral oil at 48°C with rapid stirring on a magnetic stirrer in a water bath for 5 min at room temperature, followed by a 10 min period without stirring. The oil-agar mixture was centrifuged at 104 rpm for 20 min to sediment the beads and washed twice with phosphate buffered saline (PBS). The preparations, containing beads of 100 µm to 200 µm in diameter were used as inocula for animal experiments. The number of bacteria in the beads was determined by homogenizing the bacterial bead suspension and plating 10-fold serial dilutions on Mueller Hinton agar (MHA) and MHA supplemented with 35 µg/ml gentamicin. Male Sprague–Dawley rats of approximately 500 g in weight were used according to the ethics committee for animal treatment of Laval University. The animals were anaesthetized using Isofluorane (2% of respiratory volume) and inoculated by intubation using a venous catheter 18G and syringe (1 cc Tuberculin) with 120 µl of a suspension of agar beads-embedded bacteria containing approximately 5 X 107 CFU/injection. After 7 days, the lungs were removed from sacrificed rats, and homogenized tissues were plated in triplicates on appropriate media. The in vivo competitive index (CI) was determined as the CFU output (in vivo) ratio of the cbrA mutant in comparison to PA14WT strain, divided by the CFU input ratio of mutant to WT (15, 103). The injections of approximately 120 µl of each bacterial mixture were 77  administrated to 6 animals. After 7 days of infection, the bacterial counts were performed from infected rat lungs using MHA for total bacterial number of PA14 WT and mutant or MHA with 35 µg/ml gentamicin for mutant selection. The presented CIs were calculated as the geometric mean for all animals in the same group. Statistical analyses were performed with GraphPad Prism 5 software using the Mann-Whitney U test. 4.2.4 Cytotoxicity assays To examine the effects of carbon source on cytotoxicity of PA14 WT and the cbrA mutant, interaction assays of 16HBE14o- cells with P. aeruginosa were performed as previously described in Chapter 3, Materials and Methods, with minor modifications. On the day of the experiment, the epithelial cells were washed and rested for at least 1 h in DMEM supplemented with 0.1% (wt/vol) of glucose or succinate. Glucose-free DMEM was used as MEM without glucose was not available. Mid-log cultures of P. aeruginosa were washed, resuspended in supplemented DMEM. The bacteria were then added to the rested HBE at an MOI of 2 and incubated at 37oC with 5% CO2. Cytotoxicity toward human isolated PBMCs was examined by incubating PBMCs with P. aeruginosa (MOI 1) for 4 h at 37oC. At various post-infection time points, supernatants from the samples were obtained and tested for LDH release. Three independent experiments were performed for each assay. 4.2.5 Growth curves P. aeruginosa PA14 WT and mutants were grown overnight in DMEM supplemented with 0.1% (wt/vol) of glucose or 25 mM succinate. Growth curves were generated as described in Chapter 2, Materials and Methods, with minor modifications. P. aeruginosa were grown at 37oC with 5% CO2 and growth was assessed by determining the OD600-nm every h for 9 h. 4.2.6 In vitro phagocytosis assays (gentamicin protection assay) In vitro phagocytosis assays of PA14 WT and mutants by MDM were performed as previously described (7) with modifications. Briefly, mid-log cultures of P. aeruginosa were washed with complete RPMI media and resuspended in 1 ml of the media. MDM at 1.5 X 10 5 cells/ml were mixed with the resuspended bacteria at an MOI of 10 and incubated for 1 h at 37oC. Subsequently, cells were washed with complete RPMI and incubated with 400 µg/ml gentamicin for 30 min at 37oC to kill the extracellular and attached bacteria. After the gentamicin treatment, MDM cells were washed and selectively lysed with 0.1% Triton X-100. Lysates were plated onto LB agar and incubated overnight at 37oC. The next day, colonies were counted and 78  relative phagocytosis was determined by CFU counts. Three independent experiments with duplicates in each experiment were performed for each bacterial strain. 4.2.7 In vitro macrophage and neutrophil killing assays In vitro killing of PA14 WT and mutants by stimulated MDM were performed as previously described (278) with minor changes. To activate MDM, IFN-γ (20 ng/ml), in addition to M-CSF, was added on day 6 of culturing. Briefly, mid-log cultures of P. aeruginosa were washed with complete RPMI media and resuspended in 1 ml of the medium. MDM at 1.5 X 105 cells/ml were incubated with the resuspended bacteria at an MOI of 1 for 1 h at 37oC. Subsequently, MDM were washed with complete RPMI and incubated with 400 µg/ml gentamicin for 2 h at 37oC to kill extracellular bacteria. After the gentamicin treatment, MDM were either lysed and plated for viable intracellular bacteria (T0), or replaced with fresh medium and incubated for an additional 3 (T3) and 6 (T6) hours at 37oC. At T3 and T6, MDM were lysed with 0.1% Triton X-100. Lysates were plated onto LB agar, incubated overnight at 37oC and residual bacterial colonies were counted. Three independent experiments with duplicates for each experiment were performed for each bacterial strain. An in vitro neutrophil killing assay was adapted from the protocol of Mishra et al (171). Briefly, mid-log cultures of P. aeruginosa were washed and opsonized with 10% fresh human serum for 20 min at 37oC while rotating the samples end-over-end (93). Opsonized bacteria were added to freshly isolated neutrophils (2 X 106 cells/ml) at an MOI of 10 followed by incubation at 37oC for 30 min, rotating end-over-end. Subsequently, neutrophils were washed with HBSS and incubated with 400 µg/ml gentamicin for 30 min at 37oC to kill bacteria. After 30 min, neutrophils were either lysed and plated for viable intracellular bacteria (T0), or replaced with fresh medium and incubated for an additional 0.5 (T0.5) and 1 (T1) hour at 37oC. At T0.5 and T1, neutrophils were lysed with 0.1% Triton X-100. Lysates were plated onto LB agar and incubated overnight at 37oC. The next day, colonies were counted and relative phagocytosis was determined by the CFU counts. At least 3 independent experiments with duplicates in each experiment were performed for each bacterial strain.  79  4.3  Results  4.3.1 Lack of effect of varying carbon sources on cytotoxicity of the cbrA mutant toward HBE cells Previous study of the ability of the PA14 cbrA deletion mutant to infect and destroy a monolayer of cultured 16HBE14o- epithelial cells revealed that the cbrA mutant displayed 3.5and 2.2-fold greater cytotoxicity compared to the PA14 WT at 4 and 6 hours post-infection, respectively (275). Based on the known role of the CbrA sensor kinase in carbon metabolism of P. aeruginosa, I investigated whether the ability of the cbrA mutant to cause killing of the epithelial cells was associated with its ability to grow/metabolize particular carbon sources during infection. I confirmed, by growth studies, that when glucose was provided as a sole carbon source, the cbrA mutant grew weakly while the mutant grew like the WT in succinate (data not shown). In this study, MEM (a medium with glucose already incorporated) was replaced by glucose-free DMEM medium supplemented with equimolar succinate or glucose. Fig. 4.1 shows that substitution of the infection media with DMEM supplemented with succinate or glucose did not affect the increased cytotoxicity phenotype of the cbrA mutant. DMEM supplemented with other carbon sources, including glycerol or mannitol gave similar cytotoxicity results (data not shown). While DMEM is almost the same as MEM, it has more amino acids and vitamins. Therefore, it was not unexpected to observe a slight overall improvement in cytotoxicity for P. aeruginosa in DMEM compared to MEM. These results indicated that the ability of the cbrA mutant to kill epithelial cells is not dependent on the carbon source or its ability to support rapid growth.  80  4 hours 100 90 MEM  Cytotoxicity (%)  80 70  DMEM + glucose  60 DMEM + succinate  50 40 30 20 10 0 PA14WT  cbrA mutant cbrA complemented  6 hours 100  90  MEM  Cytotoxicity (%)  80  DMEM + glucose  70  DMEM + succinate  60 50 40 30 20  10 0 PA14WT  cbrA mutant cbrA complemented  Figure 4.1 In vitro cytotoxicity toward HBE cells. The abilities of the PA14 WT, the cbrA mutant and the cbrA complemented strains to induce cell damage under various conditions were determined by monitoring the release of intracellular LDH into the supernatant from HBE cells. Bacteria were co-cultured with the cells and LDH release was monitored at the time points indicated. Each result represents the mean of 3 independent biological repeats, each assayed in triplicate. 4.3.2 Similar cytotoxicity towards PBMCs of PA14 WT and the cbrA mutant The ability of the cbrA mutant to display enhanced cytotoxicity toward 16HBE14o- cells led us to investigate whether the mutant also exhibit enhanced ability to kill other host cell types, such as immune cells. Here, the relative in vitro cytotoxicity of the cbrA mutant toward human 81  PBMCs was examined. PBMCs comprise the circulating mononuclear cells, and include monocytes, NK-cells, T-cells and B-cells. As shown in Fig. 4.2, after 4 hours of incubation at 37oC, the cbrA mutant was only slightly more cytotoxic (~1.5 fold greater cytotoxicity compared to PA14 WT) toward PBMCs. The insignificant difference (P ˃ 0.05) in cytotoxicity toward PBMCs for the cbrA mutant and PA14 WT suggested that the enhanced cytotoxicity observed toward HBE cells might have been a cell-type specific phenomenon. 60  Cytotoxicity (%)  50 40 30 20 10  0  PA14WT  cbrA mutant  Figure 4.2 In vitro cytotoxicity toward human PBMCs. The abilities of the PA14 WT and the cbrA mutant to induce cell damage were determined by monitoring the release of intracellular LDH into the supernatant from the PBMCs. Bacteria were co-cultured with the cells and LDH release was monitored at 4 h post-infection. Each result represents the mean of 3 independent biological repeats, each assayed in triplicate. 4.3.3  Reduced virulence of the cbrA mutant in mouse models of acute lung infection To examine the role of the CbrA sensor kinase in in vivo virulence of P. aeruginosa  strain PA14, my collaborator Dr. Laure Janot inoculated a sublethal dose of PA14 WT or cbrA mutant into the lungs of mice. In this lung infection model, P. aeruginosa were administered to the mice intranasally, where small droplets (20 μl) of bacterial suspensions were placed upon the nares such that the bacteria were aspirated into the lungs as the mice breathed. The viable bacterial load in the BAL was assayed at 18 hours post-infection. This model of infection is typically used to mimic acute pneumonia caused by P. aeruginosa. As shown in Fig. 4.3, administration of same inoculum (106 CFU/20 μl) for the PA14 WT or cbrA mutant gave significantly different results in bacterial recovery in both the BAL and lungs of mice after 18 hours of infection. For mice administered with the PA14 WT, after 18 hours, mice showed signs of distress, including lack of grooming, hunched postures, and mild changes in breathing rates. The average bacterial count recovered from the BAL for the PA14 WT in a single trial was 103 82  CFU/ml. For mice administered with the cbrA mutant, at the same inoculum as PA14 WT, mice did not show any signs of distress and the mutant was not detectable in the BAL after 18 hours. When the experiment was repeated, increasing the inoculum of the cbrA mutant to 107 CFU/20 μl, mice showed similar signs of distress and level of bacterial recovery from the BAL as did mice infected with 106 CFU/20 μl of PA14 WT. These results showed that the cbrA mutant was less virulent than the PA14 WT in a mice model of acute lung infection in that 10 times more of the cbrA mutant was required to cause a similar level of infection in the lungs of mice as the PA14 WT.  Figure 4.3 Bacterial load in BAL of mice infected with P. aeruginosa. CD-1 mice were inoculated via intranasal route with PA14 WT at 106 CFU/20 µl, or cbrA mutant at 106 CFU/20 µl or 107 CFU/20 µl. After 18 h post-infection, the mice were sacrificed and their BALs were collected for CFU determinations. 4.3.4 Reduced virulence of the cbrA mutant in a mouse model of peritoneal infection Nosocomial peritonitis is caused by exogenous pathogenic bacteria, including P. aeruginosa, that gain access to the abdominal cavity through a variety of routes, including during abdominal surgery (13). In the study, with the assistance of Laure Janot, the ability of PA14 WT and the cbrA mutant to promote growth in the peritoneum, as well as spread to disseminated site (e.g. the blood), was examined using a mouse model of peritoneal infection. Since the P. aeruginosa WT strains are readily cleared by healthy mice, mice were rendered neutropenic with the chemotherapeutic agent cyclophosphamide. In this infection model, various concentrations of PA14 WT or cbrA mutant were injected into the peritoneum (IP) of the immunosuppressed mice. 83  Viable bacteria in the peritoneal lavage and the blood after 18 hours of infection were determined. As shown in Fig. 4.4, with an injection of 60 CFU/200 μl of the PA14WT, after 18 hours, the bacterial counts increased to approximately 103 CFU/ml in the blood and 102 CFU/ml in the peritoneal lavage. Mice infected with PA14 WT showed signs of distress, including lack of grooming and reduced mobility. In contrast, mice infected with the cbrA mutant at 60 CFU/200 μl showed no signs of distress and the mutant could not be detected in the blood or peritoneal lavage. When the experiment was repeated by injecting mice IP with 5 X 102, 103, 104 or 106 CFU of the cbrA mutant, they were able to clear the cbrA mutant at doses up to 105 CFU. However, at a dose of 106 CFU of the cbrA mutant, mice showed signs of substantial distress after 18 hours of infection and after euthanasia, the recovered bacterial counts for the cbrA mutant were similar to those for mice infected with 60 CFU of the PA14 WT. The infectivity of the cbrA mutant could be restored to the WT level by complementation. Thus the cbrA mutant was approximately 104-fold less virulent than the PA14 WT in a mice model of peritoneal infection, indicating CbrA most likely plays a crucial role in the ability of PA14 to grow in the peritoneum and/or disseminate to the blood. Previously I showed that CbrA mediated in vitro cytotoxicity toward HBE cells via its cognate response regulator CbrB (Chapter 3) (275). In contrast, CbrA demonstrated attenuated in vivo virulence but this did not occur in conjunction with CbrB. As shown in Fig. 4.4, mice infected with 60 CFU/200 µl of the cbrB mutant showed similar bacterial recovery in the peritoneal lavage and blood (and similar levels of distress) as mice infection with 60 CFU/200 µl of the PA14 WT.  84  Blood  PA14WT 60  cbrA 60  Peritoneal lavage  cbrB 60  PA14WT 60  cbrA 60  cbrB 60  Peritoneal lavage  PA14WT 60  cbrA 500  cbrA 103  cbrA 104  Figure 4.4 Bacterial load in the peritoneal lavage and blood of mice infected with P. aeruginosa. C57BL/6 mice were injected with cyclophosphamide via the IP route 4 and 2 days prior to the infection. On the day of the infection, the mice were injected via IP route with 60 CFU PA14 WT or cbrA complemented strains, or at various amounts (60, 500, 103, 104, or 106 CFU) of the cbrA mutant. After 18 h post-infection, the mice were sacrificed and their blood and peritoneal lavage was collected for CFU determination.  85  4.3.5 Lack of effect of the cbrA mutant on in vivo competitive growth The significant reduction in in vivo virulence for the cbrA mutant compared to the PA14 WT in both mice models of acute lung infection and peritoneal infection led to an investigation of whether the attenuated virulence observed for the cbrA mutant was due to the mutant’s metabolic deficiency (e.g. poor ability to grow in vivo). To study this, in collabroation with Irina Kukavica-Ibrulj we examined the in vivo competitive growth between strains PA14 WT and cbrA mutant in a rat model of chronic lung infection. Equal ratios of each strain were mixed and encapsulated in agar beads and the mixture was inoculated into the rat lung. Bacteria were enumerated from the lung at day 7 post-infection and the CI calculated. Embedding P. aeruginosa in agar beads was essential to create a chronic lung infection that to some extent mimics the chronic lung infections caused by P. aeruginosa in CF patients. The purpose of embedding P. aeruginosa within the beads was to retain the bacteria physically in the airways and avoid physical elimination by the host (e.g. mucociliary clearance). As shown in Fig. 4.5, after 7 days post-infection, the cbrA mutant had no significant difference in bacterial counts from the lung with a mean CI of 0.66. These results suggested that the cbrA mutant was able to grow and persist in the lungs as well as the PA14 WT.  0.88 0.49  Trial 1  Trial 2  0.66  Trials 1 and 2  Figure 4.5 In vivo CI analysis of cbrA mutant in a rat model of chronic lung infection in competition with the PA14 WT strain. Equal ratios of the WT and mutant were embedded in agarose beads and delivered to the rat lungs via intubation. After 7 days post-infection, rats were sacrificed and lungs were recovered for CFU determinations. Each data point represents the CI for a single animal in each group. The geometric means of the CIs from each trial and both trials are represented as a solid bar.  86  4.3.6 Enhanced uptake of the cbrA mutant by macrophages The cbrA mutant exhibited attenuated virulence in both in vivo mouse infection models studied. The reduced in vivo virulence observed here in contrast to the increased virulence of the cbrA mutant toward HBE cells (Chapter 3) stimulated investigation of possible mechanisms for this difference. There are many differences between the in vitro and in vivo models, but an obvious difference would the absence of host defence mechanisms, particularly phagocytes, in in vitro models. To investigate whether the cbrA mutant was more susceptible to uptake by phagocytes than the WT strain, Olga Pena and I incubated the PA14 WT and cbrA mutant with human macrophages and looked for uptake by killing extracellular bacteria with gentamicin (e.g. a gentamicin survival assay). In this assay, following the infection of macrophages with P. aeruginosa, the macrophages were thoroughly washed get remove bacteria that were not taken up by the macrophages. However, washing alone was not sufficient to remove all extracellular bacteria. Therefore, following the washing step, the macrophages were incubated in the presence of gentamicin. Due to the large number of bacteria (1.5 X 106 cells/ml) initially added to the macrophages, and the fact that the cbrA deletion mutant harboured a gentamicin resistance cassette, macrophages were exposed to a high concentration (400 µg/ml) gentamicin for a sufficient amount of time (at least 30 min). Compared with the PA14 WT, there was an average of 3.6-fold increase in uptake of the cbrA mutant by human macrophages (Fig. 4.6). The level of uptake for the cbrA mutant could be restored to the WT level by introducing the WT cbrAB genes into the mutant. Similarly, significant increases were observed in the uptake by murine BMDM of the cbrA mutant compared to the WT strain (data not shown). Consistent with its unaltered virulence, a PA14 cbrB mutant exhibited a WT level of uptake by macrophages (data not shown; collaboration with Laure Janot).  87  % Phagocytosis relative to PA14WT  450% 400% 350% 300% 250% 200% 150% 100% 50% 0%  PA14WT  cbrA mutant  cbrA complemented  Figure 4.6 Phagocytosis of P. aeruginosa by human macrophages. PA14 WT, cbrA mutant and cbrA complemented strains were incubated with macrophages for 1 h. Subsequently, extracellular and attached bacteria were killed by treatment with gentamicin, macrophages were lysed with Triton X-100 and lysates plated to enumerate viable intracellular bacteria. Results are expressed as the percentage of bacteria that survived relative to the PA14 WT. 4.3.7 Enhanced clearance of the cbrA mutant by macrophages and neutrophils The enhanced uptake of the cbrA mutant compared to the PA14 WT led me to examine, in collaboration with Olga Pena, whether the cbrA mutant was also more sensitive to macrophage-mediated killing than PA14 WT. Mid-log phase cultures of PA14 WT and cbrA mutant were added at MOI of 1 to IFNγ-stimulated human macrophages and incubated at 37oC. Subsequently, cells were washed and incubated with gentamicin for 2 hours. For this experiment, the gentamicin treatment was increased from 30 min to 2 hours since, following the gentamicin treatment, we needed to incubate the macrophages for another 3 to 6 hours; hence it was necessary to extend the time of gentamicin treatment to ensure complete killing of the extracellular bacteria (both free-floating and adhered). We confirmed this by the absence of bacterial colonies observed on agar plates after plating the extracellular medium following the gentamicin treatment. After the gentamicin treatment, macrophages were either lysed and plated for viable intracellular bacteria (T0), or replaced with fresh medium and incubated for an additional 3 (T3) and 6 (T6) hours. At T3 and T6, macrophages were lysed and viable bacteria were plated for counting. Results shown in Table 4-1 were the average intracellular bacterial counts for PA14 WT and cbrA mutant at each time point while results shown in Fig. 4.7A represent percent bacterial survival (e.g. the percentage of viable bacteria recovered from the macrophages at each time point relative to the initial inoculum). At T0, the intracellular bacterial counts for the cbrA mutant was on average 9.5-fold higher than that of the PA14 WT, consistent 88  with previous phagocytosis results. Between T0 and T3, there was little to no bacterial killing with similar bacterial counts at T0 and T3 for both the WT and mutant strains. The reduction in bacterial counts for both strains at T6 suggested both strains were being cleared by the macrophages. About 10% of the cbrA mutant added to the macrophages was cleared at T6 relative to T0 while only about 1% of the PA14 WT was cleared. Extending the incubation period beyond 6 hours led to an increase in the bacterial CFU counts, indicating that P. aeruginosa were replicating intracellularly, perhaps due to inactivation of the macrophages, or had exited the macrophages to replicate extracellularly. The bacterial counts in the cbrA complemented strain at all time points were the same as the WT level. Similar results were observed with murine BMDM (data not shown). In addition to macrophages, one of the primary cell-mediated defences against P. aeruginosa is the neutrophils. Therefore, the ability of the PA14 WT and cbrA mutant to resist killing by neutrophils was also examined in collaboration with Olga Pena. Due to difficulties in obtaining neutrophils from mice, the neutrophil-killing assays were only performed using human isolated neutrophils. Therefore PA14 WT and cbrA mutant, opsonized with fresh serum, were incubated with freshly isolated human neutrophils and a similar assay as the macrophage killing assay was performed. Due to the shorter life-span and greater sensitivity to bacterial induced damage of neutrophils cf. macrophages, the incubation time of neutrophils with P. aeruginosa was reduced to 30 min and the incubation time of gentamicin-treated neutrophils in fresh media was followed for only 1 hour. To compensate for the reduced incubation periods both the concentration of neutrophils used and the MOI of P. aeruginosa were increased. As shown in Table 4-1 and Fig. 4.7B, the average bacterial cell counts at T0 revealed that uptake of the cbrA mutant by neutrophils was 13-fold greater than the uptake of PA14 WT. As early as 30 min of incubation in fresh media, a decrease in bacterial cell counts was observed for both the PA14 WT and cbrA mutant. Beyond 1 hour, the bacterial count for both strains started to increase. Similar to the macrophages-killing results, the significantly enhanced uptake of the cbrA mutant compared to the PA14 WT by neutrophils was followed by a greater percentage of killing of the cbrA mutant by neutrophils than that of the PA14 WT.  89  A  30% PA14WT  Percent survival  25%  cbrA mutant cbrA complemented  20% 15%  10% 5% 0% 0  B.  3  Time (h)  6  70% PA14WT 60% cbrA mutant  Percent survival  50%  cbrA complemented  40% 30% 20%  10% 0% 0  0.5  1  Time (h)  Figure 4.7 Killing of P. aeruginosa by phagocytes. Uptake and intracellular killing of PA14 WT, cbrA mutant, and cbrA complemented strains by (A) macrophages and (B) neutrophils were determined. P. aeruginosa were incubated with macrophages or neutrophils for 1 h or 30 min, respectively. Following gentamicin treatment, phagocytes were either lysed and plated for viable intracellular bacteria, or replaced with fresh media and incubated for the indicated times. Results are expressed as the percentage of bacteria recovered from the phagocytes at each time point relative to the initial inoculation amount.  90  Table 4-1 Average intracellular bacterial counts for PA14 WT and cbrA mutant recovered from macrophages or neutrophils at various time points. Values are represented as average values from 3 independent experiments. Average CFU/mL of intracellular P. aeruginosa recovered from macrophages Time (h) 0 3 6 PA14WT 3900 ± 900 3900 ± 1350 2400 ± 600 cbrA mutant 37050 ± 2850 35700 ±4950 23342 ± 4500 cbrA complemented 8400 ± 1350 7500 ± 3300 3750 ± 1950 Average CFU/mL of intracellular P. aeruginosa recovered from neutrophils Time (h) 0 0.5 1 PA14WT 112500 ± 12500 67500 ± 27500 60750 ± 22500 cbrA mutant 1450000 ± 120000 830000 ± 200000 785000 ± 125000 cbrA complemented 122500 ± 5000 110000 ± 30000 63750 ± 20000  4.4  Discussion In this study, the role of the sensor kinase CbrA in in vivo virulence was examined.  Deletion of the cbrA gene resulted in a P. aeruginosa mutant strain that was significantly attenuated in virulence compared to the WT in both mice models of acute lung infection and peritoneal infection. As the CbrAB TCS plays an important role in regulating the expression of several catabolic pathways and utilization of a variety of organic compounds as the sole carbon source in P. aeruginosa, we investigated whether the reduced virulence of the cbrA mutant was simply due to a reduced ability to grow in vivo. An in vivo competitive analysis between PA14 WT and cbrA mutant revealed the cbrA mutant had a mean CI value of 0.66, which is within the range of error of this analysis and suggests minimal attenuation of in vivo growth. Clearly any minor change observed was insufficient to explain the large (10- to 104-fold) reduction in virulence for the cbrA mutant compared to the WT in the acute lung infection and the peritoneal infection models. Despite the common use of cell lines to predict what is happening in vivo, they are obvious simplifications of the situation in animals. For example, cell lines are usually immortalized (e.g. 16HBE14o-) and these cells lines do not therefore maintain normal epithelial cell properties and growth characteristics. While primary cells are more representative of normal physiology, thus a better representative model of the in vivo state, these still lack the structural complexity of intact organs. Moreover, in vitro results often correlate poorly with in vivo results due the complex physiological environment in vivo that cannot be replicated precisely in an in vitro model. One substantial difference is the lack of host defences in in vitro models. 91  Consequently it was not too surprizing that the cbrA mutant was significantly attenuated in in vivo virulence despite exhibiting enhanced in vitro cytotoxicity toward HBE cells. Furthermore, only a moderate increase (< 2-fold) in in vitro cytotoxicity of the cbrA mutant compared to the WT toward human PBMCs led to the suggestion that the increased cytotoxicity of the cbrA mutant might be somewhat cell-specific. I am currently testing the cytotoxicity of the WT and mutant toward other cell types, including erythrocytes, MDM, and primary CF epithelial cells. Since the metabolic deficiency of the cbrA mutant was insufficient to explain the significant reduction in in vivo virulence for the mutant, I investigated whether CbrA normally plays a role in protecting P. aeruginosa from host defences, particularly clearance by phagocytes. Intracellular killing following engulfment of microbes by phagocytic cells, such as macrophages and neutrophils, plays a major role in host innate defence against P. aeruginosa infections (236). In this study, I showed that the deletion of the cbrA gene resulted in significantly increased uptake by phagocytes when compared to the PA14 WT (Fig. 4.6). As a result of the enhanced uptake of the cbrA mutant, a greater number of this mutant than the WT was also cleared by phagocytes (Table 4-1). Thus, CbrA appears to play an important role in the in vivo virulence of P. aeruginosa by protecting the pathogen from phagocytosis. To gain insight into the mechanism of the enhanced susceptibility of the mutant to uptake by the phagocytic cells, I am currently investigating the gene expression of the cbrA mutant vs. PA14 WT during interaction with the phagocytes. Interestingly, a recent study by Overhage et al. showed that cbrA mutant exhibited hypersensitivity toward amoebae–mediated killing (Overhage, J. et al. unpublished data). In this study, they screened a library of P. aeruginosa transposon mutants for virulence in a Dictyostelium discoideum host system. They showed that compared to the WT, significantly fewer amoebae were required to produce a zone of clearance on a lawn of the cbrA mutant. The Dictyostelium model represents a simple non-mammalian host system to assess the pathogenicity of P. aeruginosa. Moreover, since Dictyostelium amoebae are unicellular organisms that feed phagocytically upon bacteria, such as P. aeruginosa, the model can also be used to study phagocytosis. CbrB is the cytoplasmic response regulator, part of the CbrAB TCS in P. aeruginosa first identified to be involved in regulating the carbon and nitrogen balance of the bacteria (149, 183). Similar to a cbrA mutant, a mutation in the cbrB gene also rendered the bacterium incapable of growing on a variety of organic compounds as the sole carbon source. Previously, it was demonstrated that CbrA mediates the regulation of swarming, biofilm formation, and in vitro 92  cytotoxicity toward HBE cells via CbrB (275). Here, a cbrB mutant was shown to exhibit a WT phenotype in the murine model of peritoneal infection and mutation of the cbrB gene did not affect the phagocytic ability of mammalian macrophages. These results suggest CbrA is mediating in vivo virulence of P. aeruginosa independent of CbrB and thus does not appear to be due to altered swarming, biofilm formation or cytotoxicity. This is consistent with the general observation that CbrA was able to regulate antibiotic resistance independently of CbrB involvement, presumably by interacting with another regulatory system, such as the PhoQ sensor kinase. The PhoPQ system mediates in part the adaptive response to low extracellular Mg2+ concentrations in P. aeruginosa, controlling resistance to aminoglycosides, polymyxin B, and antimicrobial peptides (79). Interestingly, it has been demonstrated that PhoQ regulates in vivo virulence as a phoQ mutant displayed significantly lower virulence than the WT in a neutropenic mouse model (158). During bacterial infections in murine infection models, large amounts of leukocytes are recruited to the sites of infection. The impaired ability of the cbrA mutant to escape engulfment by the phagocytes could play a major role in the inability of the cbrA mutant to sustain an infection at the site of initial inoculation, as well as spread to disseminated sites (in peritoneal infection model). Previously we showed that the cbrA mutant was completely abolished for swarming motility. Serratia liquefaciens swarmer cells exhibited enhanced resistance to engulfment by Tetrahymena sp. (9) so one possibility would be that swarming might play a role in protecting P. aeruginosa from phagocytes. However unaltered virulence of the swarmingdeficient cbrB mutant indicates that altered swarming cannot by itself explain the reduced virulence of the cbrA mutant. Since the in vitro conditions that promote swarming of P. aeruginosa mimic those of the mucous layer that overlay epithelial surfaces, it is possible that the bacteria use this form of motility to rapidly spread and colonize the mucosal surfaces of the body. Therefore, the cbrA mutant swarming defect may contribute to its inability to spread to disseminate to other sites (e.g. the blood and other organs) in the murine peritoneal infection model. This may work synergistically with the phagocytic defect since phagocytes would be able to deal more effectively with localized rather than disseminated organisms.  93  Chapter 5: Rapid surface motility promoted by mucin in P. aeruginosa 5.1  Introduction Due to the difficulty of studying motility in a living host, the in vitro characteristics of  this form of motility studies has been used to suggest swarming as a likely mode of motility utilized by P. aeruginosa to colonize the lung. This is largely based on the conditions that promote swarming motility in vitro (semi-solid agar and weak N source), which mimic the use of amino acids as an N source and the viscous environment in the lung, especially for chronic (mucoid) infections in CF patients where the lung environment is characterized by the production of copious amounts of mucous. An obvious issue, however, is that under in vivo conditions, the gel-like properties of the mucous layer are contributed largely by the production of mucin, a major component of the respiratory mucous. Here the motility of P. aeruginosa was examined under in vitro conditions that mimicked, as close as possible, the conditions in the CF lung. For this purpose a motility assay medium was used based on the synthetic CF sputum medium (SCFM), developed by Palmer et al to mimic the nutritional composition of the CF sputum (192), with no added NH4Cl but with added mucin and DNA. Addition of mucin at concentrations as low as 0.05% wt/vol to SCFM swimming agar (e.g. with 0.3% wt/vol agar), resulted in P. aeruginosa demonstrating accelerated motility on the surface of the agar. In the presence of mucin, the surface motility colonies of both P. aeruginosa strains PA14 and PAO1 appeared circular with bright green centers surrounded by thicker white edges. This form of mucin promoted motility was shown to be dependent on intact flagella but not type IV pili. While quorum sensing (QS) was demonstrated to be important, QS-regulated production of rhamnolipids by P. aeruginosa was not required for this form of surface propagation. Microscopic analysis of cells taken from the motility edge revealed that cells were piled up with the majority of bacterial cells lacking flagella. In contrast, bacterial cells at the center of the motility zone had flagella. Overall, the genetic and phenotypic data led me to suggest that mucin might be promoting a new form of surface motility that I propose should be named "Surfing" motility. 5.2  Materials and methods  5.2.1 Bacterial strains and growth conditions P. aeruginosa strain PA14 WT and PA14 transposon mutants were obtained from Harvard University (150). P. aeruginosa strain PAO1 WT and PAO1 transposon mutants were 94  obtained from the University of Washington (112). Cultures were routinely grown in LB broth, MSCFM (SCFM (192) without NH4Cl), BM2 minimal medium, or BM2-swarming medium comprising BM2 with 0.1% (wt/vol) CAA (PA14) or 0.5% (wt/vol) CAA (PAO1) substituted for 7 mM (NH4)2SO4; glucose was used as the usual C source for minimal media. Swimming and twitching were assayed on LB plates containing 0.3% (wt/vol) and 1% (wt/vol) agar, respectively. Swarming was assayed on BM2-swarming medium with 0.5% (wt/vol) agar. Gentamicin at 15 µg/ml, kanamycin at 200 µg/ml, or tetracycline at 50 µg/ml was added to growth media for transposon maintenance. 5.2.2 Mucin-promoted motility assays The surface motility of P. aeruginosa was examined on MSCFM or BM2 plates containing 0.2-0.3% (wt/vol) agar and 0.05-1.0% (wt/vol) sterilized porcine stomach mucin (Sigma). To sterilize mucin, dry powdered mucin was placed in a flask, covered with 95% ethyl alcohol and heated at 70C for 24 h (172). Sterile mucin was obtained by evaporating off the alcohol. When necessary, herring sperm DNA (1.4 mg/mL; Sigma) was also added to the mucincontaining MSCFM, or mucin-containing BM2 plates (24). After mucin and DNA were added, the ingredients were stirred for approximately 15 min to dissolve. The plates were dried for 1 h and spotted with 1 µl of mid-log phase cultures and incubated at 37C for 13-15 h. The resultant colonies were analyzed by measuring the agar plate coverage. To test the effects of the carbon source, glucose was replaced by equimolar amounts of the carbon source of interest, with aspartate provided as the nitrogen source. To test the effects of the nitrogen source, total free amino acids were replaced with an equimolar amount of a single amino acid. Since the surface motility zones were easily measured at 0.4% (wt/vol) mucin, and we were able to obtain consistent results at this mucin concentration, we screened all flagellar, type IV pili, rhamnolipid, quorum sensing mutants using this mucin concentration. I also used 0.4% wt/vol mucin for microscopic and genetic analyses. To measure the motility zones as a function of time, recording was initiated at the 5th hour as this was approximately the time when motility colonies on all of the plates could be clearly observed and measured. 5.2.3 Growth curves P. aeruginosa cells were grown overnight in MSCFM and diluted in fresh MSCFM with 0-0.8% (wt/vol) mucin. Growth curve experiments were performed as discussed in Chapter 2 Materials and Methods. 95  5.2.4 Electron microscopy Carbon-coated grids were gently placed on the edge or center of the motility colonies growing on swarming or mucin-containing MSCFM plates. After 2 min, the grids were removed and washed with distilled water and stained with 1% uranyl acetate. The negatively stained cells were visualized with a Hitachi H-7600 Transmission Electron Microscope (UBC Bioimaging Facility). 5.2.5 Light microscopy For microscopic examination of surface motility zone progression, 1 µl of mid-log P. aeruginosa cultures were spotted onto the center of a thin layer of MSCFM with 0.4% (wt/vol) mucin and 0.3% (wt/vol) agar that coated the bottom of a 35 mm high µ-Dish (Ibidi).The plates were incubated at 37C for 1 h (time for the motility zone to form) before images were taken. The medium was kept from drying out by placing wetted Whatman filter papers on the wall of the dish and keeping the lids of the plates closed. The plate was maintained at 37C while images were taken at 60 X magnification using an Olympus FV-100 Inverted Confocal microscope. 5.2.6 RT-qPCR Total RNA from P. aeruginosa strain PA14 was harvested under the following conditions: i) the leading edge of the surface motility colonies on MSCFM with 0.4% (wt/vol) mucin; ii) cells swimming within the agar from MSCFM-swimming plates containing 0.3% (wt/vol) agar incubated for 20 h at 37C. Subsequent RNA isolation, treatment, conversion to cDNA, and RT-qPCR were performed as previously described in Chapter 3 Materials and Methods. Fold-change was determined using the comparative Ct method by comparison to the rpoD housekeeping gene. 5.3  Results  5.3.1 Mucin promoted the same surface motility pattern for strains PA14 and PAO1 At a low-percentage agar (0.2%- 0.35% wt/vol), P. aeruginosa swims in the submerged water-filled spaces of the agar using its single polar flagellum resulting in the formation of a halo within the agar layer after overnight incubation at 37 oC (Fig. 5.1A). Interestingly, when mucin was added to “swim” agar, in addition to swimming, P. aeruginosa was observed to move relatively rapidly across the surface of the agar. The surface motility zones could be observed when mucin was added at concentrations as low as 0.05% wt/vol and the diameter of the surface motility zone increased as the concentration of added mucin increased (up to 1% wt/vol mucin 96  tested; Fig. 5.1D shows an example at 0.4% wt/vol mucin). Moreover, the addition of mucin to 0.5% wt/vol agar (0.5% agar normally promotes swarming motility of P. aeruginosa) changed the surface motility pattern from dendritic to circular although the diameter of the motility colony remained similar (data not shown). The same surface motility patterns were observed when mucin was spread onto an agar slab. In the presence of mucin, the surface motility colonies of both P. aeruginosa strains PA14 and PAO1 appeared circular with a green center surrounded by a thick white edge. This motility pattern somewhat resembles the solar flare-like colonial swarming pattern of strain PAO1 (Fig. 5.1C), but differ from the dendritic swarming colony of strain PA14 (Fig. 5.1B). To better mimic the nutritional composition of the CF sputum, we replaced the typical BM2 minimal medium in the swim plates with MSCFM where ammonium was excluded. When mucin was added to MSCFM, virtually identical surface motility colonies were observed (data not shown). The same motility phenotype was also observed when physiological amounts of DNA (1.4 mg/mL) were added to the mucin-MSCFM plates (data not shown).  Figure 5.1 Swimming (A), Swarming (B, C) and mucin-promoted (D) motilities of P. aeruginosa. Motilities were examined on plates containing 0.3% [wt/vol] agar (swim), 0.5% [wt/vol] agar (swarm), or 0.3% [wt/vol] agar with 0.4% [wt/vol] mucin.  97  5.3.2 Mucin promoted rapid surface motility When P. aeruginosa strain PA14 was spotted onto MSCFM swim plates with mucin (0.05% -1% wt/vol) and incubated overnight at 37oC, the resultant surface motility zones were always greater, in diameter, than the swimming zones observed in the same plate. This led me to examine the rate of surface colonization at varying concentrations of mucin. Table 5-1 shows the average diameters of the motility zones at varying concentrations of mucin while Fig. 5.2 shows example images of the expanding surface motility zones in the presence of 0.4% (wt/vol) mucin taken every hour from 5 to 13 hours. After 13 hours of incubation at 37oC, swimming was found to be the slowest of the 3 forms of motility with a resultant average motility zone diameter of 14 mm while the swarming zone was 30.4 mm and the mucin-promoted surface motility zone ranged from 21.5-38.1 mm in 0.1%-0.8% (wt/vol) mucin. Table 5-1 also showed the average changes in diameter of the motility zones after each hour. For all 3 forms of motilities, the rate of motility increased as time progressed. The rates of swimming motility between the 5 th and 13th hour increased from 0.5 mm/h to 1.5 mm/h and swarming increased from 0 mm/h to 6.4 mm/h. 5h  10h  6h  7h  11h  12h  8h  9h  13h  Figure 5.2 Progression of P. aeruginosa PA14 surface motility zones over time. Mid-log cultures of PA14 WT were spotted onto plates comprised of MSCFM with 0.3% (wt/vol) agar and 0.4% (wt/vol) mucin. Plates were incubated at 37 oC and pictures were taken every hour from the 5th to 13th hour. 5.3.3 Increasing the concentration of mucin did not significantly enhance growth of P. aeruginosa As shown in Table 5-1, as the concentration of mucin added to the plates increased, the surface motility zone increased. Therefore, I investigated whether the increased motility zones were due to enhanced growth of the bacteria in the presence of mucin. P. aeruginosa were grown in liquid MSCFM with 0, 0.1, 0.4, 0.6, or 0.8% wt/vol mucin shaking at 37 oC and aliquots were 98  plated every hour for colony counting. Growth appeared the same at all concentrations of mucin tested (data not shown). Table 5-1 Average diameters of motility zones, and changes in diameter over time, of P. aeruginosa strain PA14 motilities in 0.3% (wt/vol) agar (swimming), 0.5% (wt/vol) agar (swarming) and 0.3% (wt/vol) agar with varying concentrations of mucin (surfing). Motility zones were recorded every h from the 5th to 13th h while incubating at 37oC. Results are displayed as means ± SD of triplicates and are representative of 3 independent experiments. Average diameter of motility zones (mm) Time (h) Swim Swarm* 0.1% mucin 0.4% mucin 0.6% mucin 0.8% mucin 4.0 ± 0.1 4.0 ± 0.1 7.0 ± 0.2 8.0 ± 0.2 8.5 ± 0.2 11.0 ± 0.2 5 4.5 ± 0.1 4.0 ± 0.1 7.5 ± 0.1 9.1 ± 0.2 9.8 ± 0.1 12.3 ± 0.1 6 5.5 ± 0.1 4.0 ± 0.1 8.0 ± 0.1 10.5 ± 0.1 11.5 ± 0.1 14.0 ± 0.1 7 6.5 ± 0.2 6.0 ± 0.2 9.0 ± 0.2 12.5 ± 0.2 13.9 ± 0.2 16.5 ± 0.1 8 8.0 ± 0.2 9.0 ± 0.1 11.0 ± 0.2 14.9 ± 0.1 17.0 ± 0.2 19.3 ± 0.2 9 9.5 ± 0.2 13.0 ± 0.2 13.0 ± 0.1 18.0 ± 0.2 20.5 ± 0.1 23.3 ± 0.2 10 11.0 ± 0.3 18.0 ± 0.2 15.0 ± 0.1 22.2 ± 0.1 25.0 ± 0.2 27.9 ± 0.2 11 12.5 ± 0.2 24.0 ± 0.1 18.0 ± 0.2 26.5 ± 0.1 29.8 ± 0.1 32.7 ± 0.3 12 14.0 ± 0.3 30.4 ± 0.2 21.5 ± 0.1 31.0 ± 0.1 35.0 ± 0.2 38.1 ± 0.3 13 Average Rate of change in diameter of motility zones (mm/h) 0.5 ± 0.1 0.0 ± 0.1 0.5 ± 0.2 1.1 ± 0.3 1.3 ± 0.2 1.3 ± 0.2 5 to 6 1.0 ± 0.1 0.0 ± 0.1 0.5 ± 0.1 1.4 ± 0.2 1.7 ± 0.1 1.7 ± 0.1 6 to 7 1.0 ± 0.2 2.0 ± 0.1 1.0 ± 0.2 2.0 ± 0.2 2.4 ± 0.2 2.5 ± 0.1 7 to 8 1.5 ± 0.3 3.0 ± 0.2 1.0 ± 0.3 2.4 ± 0.2 3.1 ± 0.3 2.8 ± 0.2 8 to 9 1.5 ± 0.3 4.0 ± 0.2 2.0 ± 0.2 3.1 ± 0.2 3.5 ± 0.2 4.0 ± 0.3 9 to 10 1.5 ± 0.4 5.0 ± 0.3 2.0 ± 0.1 4.2 ± 0.2 4.5 ± 0.2 4.6 ± 0.3 10 to 11 1.5 ± 0.4 6.0 ± 0.2 3.0 ± 0.2 4.3 ± 0.1 4.8 ± 0.2 4.8 ± 0.4 11 to 12 1.5 ± 0.4 6.4 ± 0.2 3.5 ± 0.2 4.5 ± 0.1 5.2 ± 0.2 5.4 ± 0.5 12 to 13 *For swarming, the same two tendrils on either side of the point of inoculation were measured at every time point. 5.3.4 Mucin-promoted surface motility was dependent on flagella expression but did not require type IV pili First, it was determined using mutants whether P. aeruginosa required its cellular appendages (e.g. flagella, type IV pili) to move on the surface of the mucin plates. As shown in Fig. 5.3A and 5.4A, mutants with transposon insertions in genes involved in flagella biosynthesis, including fliC, fliQ, fliD, fleR/S, flgB, and flgC, were significantly impaired in this form of motility. This suggested the necessity of an intact flagellum for rapid surface motility on mucin plates. P. aeruginosa mutants with transposons inserted in genes encoding its stator complex were of particular interest. The stator complex is the stationary element of the bacterial motor providing energy to turn the flagellum and therefore propel the cell through its 99  environment (250). The stator complex is comprised of 4 integral membrane proteins, MotAB and MotCD. MotAB and MotCD are to some extent functionally redundant for swimming; therefore deletion of any given stator does not significantly impair P. aeruginosa swimming motility. Nevertheless, while a motAB mutant can swarm on 0.5% (wt/vol) agar, a motCD mutant is swarming defective at this concentration (250). A motABmotCD double mutant is able neither to swim nor swarm. Fig. 5.3A shows that the PA14 motAB, motCD mutants exhibited comparable surface motility on the mucin plates compared for PA14 WT while a small reduction in surface motility was observed for the motABmotCD quadruple mutant. The results for the PA14 mutants with deletions in motAB, motCD, and motABmotCD were confirmed using the corresponding mutants and complemented strains provided to me by Dr. O’Toole (250). These data indicate that either flagella rotation is not required for mucin mediated motility or, more likely that an alternative to the conventional stator complexes drives flagella rotation in this type of motility. Mutants with transposon insertions in genes involved in the assembly of type IV pili exhibited surface motility zones on mucin plates comparable to that of the WT (Fig. 5.3B and 5.4B). Moreover a fliCpilA double mutant (defective in flagella and type IV pili, (176)) that was reported to be able to utilize a type of motility termed sliding, was completely abolished in surface motility on mucin plates (Fig. 5.4C) as expected given its flagella deficiency.  100  A. % Fold change relative to PA14WT  100% 90% 80% 70% 60% 50% 40% 30% 20%  * *  *  *  *  *  *  10% 0%  160%  % Fold change relative to PA14WT  B.  140% 120% 100%  80% 60%  40% 20% 0%  180%  % Fold change relative to PA14WT  C.  *  160% 140% 120%  100% 80% 60%  *  *  *  40% 20% 0%  Figure 5.3 Percentage fold changes of surface coverage of PA14 flagella (A) type IV pili (B) and quorum sensing (C) mutants compared to PA14 WT on MSCFM with 0.4% mucin and 0.3% agar. Motility zones were measured after incubation at 37oC for 13 h. Results shown are means ± SD for at least 3 independent experiments with duplicates for each experiment. Asterisks indicate a statistically significant difference (P ≤ 0.05) between the mutants and WT as determined by Student’s t test.  101  A.  PA14WT  C.  PA14WT  fliC B.  WT  fliCpilA  chpB D.  rhlI-  PA14WT  rhlI-  PA14WT  +C4-HSL  +C4-HSL  Figure 5.4 Example images of mucin-promoted surface motilities of PA14 WT and a fliC flagella mutant (A), chpB type IV pili mutant (B), fliCpilA flagella and type IV pili double mutant (C), and rhlI quorum sensing mutant with and without addition of C4HSL (D). P. aeruginosa were spotted onto MSCFM plates with 0.3% agar and 0.4% mucin and incubated at 37oC for 13 h. For surface motility restoration, 10 µM of C4-HSL was added. 5.3.5 Mucin-promoted surface motility was dependent on cell-to-cell signalling P. aeruginosa possesses 3 intertwined QS systems (las, rhl, pqs) and 1 orphan autoinducer receptor (qscR). Previously it was demonstrated that in addition to controlling the expression of a number of extracellular virulence factors, the las and rhl cell-to-cell signalling systems were also required for swarming in P. aeruginosa (130). As shown in Fig. 5.3C, PA14 mutants with transposon insertions in rhlR, rhlI and lasI were significantly impaired in surface motility in the presence of mucin (no PA14 transposon mutant was available for lasR) compared to the PA14 WT. Since transposon mutants in the genes rhlR, rhlI, lasR, lasI were available in the PAO1 mutant library (112), these mutants were also tested for their ability to propagate on the surface of mucin plates compared to the PAO1 WT and similar defects were observed with the PAO1 mutants as for the PA14 mutants (data not shown). The addition of the cognate signals C4-HSL and 3-oxo-C12-HSL restored the surface motility zones in the rhlI and lasI mutants, respectively, to WT levels confirming the involvement of QS in this surface propagation (Fig. 5.4D). Mutations in genes involved in synthesis of PQS did not affect this motility. However, a 102  qscR transposon mutant exhibited significantly increased surface propagation relative to the PA14 WT. Previous studies showed that the QS control repressor (QscR) transiently represses the expression of several genes activated by LasR or RhlR (39, 140), thus provided a plausible basis for the mutant effects on the surface motility. Previously it was demonstrated that P. aeruginosa synthesizes rhamnolipids that act as biosurfactants to promote swarming on a semi-solid surface. Rhamnolipid production is mainly controlled by the rhl system which regulates the expression of the rhlAB operon, encoding rhamnosyltransferase involved in rhamnolipid biosynthesis (128). Here I examined whether rhamnolipid production is required for surface propagation in the presence of mucin. PA14 mutants with transposon insertions in genes involved in the rhamnolipid biosynthesis pathway, including rhlA and rhlB, exhibited similar surface motilities in the presence of mucin compared to the PA14 WT (data not shown). These results were confirmed in the PAO1 mutants with insertions in rhlA or rhlB genes. 5.3.6  P. aeruginosa at the edge of mucin-promoted surface motility zone lacked flagella Transmission electron microscopy (TEM) was used to examine the morphology of P.  aeruginosa strain PA14 taken from the edge and the center of the mucin promoted surface motility zone. The method employed involved placing carbon-coated grids directly onto the surface of the motility colonies and then staining the cells with 1% uranyl acetate. The negatively stained cells were visualized using a TEM. As shown in Fig. 5.5A, the cells on the edge of the mucin-promoted surface motility zone were elongated compared to cells taken from the center of the motility zone. Interestingly, examination of cells on the edge of the motility zone revealed the lack of flagella for the majority of the cells analyzed, although a few bacterial cells still had flagella attached. In contrast flagella were clearly observed on the bacterial cells from the center (Fig. 5.5B). Moreover, while cells taken from swarming tendrils were highly organized (e.g. cells were aligned one next to another in the same direction as the moving tendril), the orientation of the surfing cells was apparently random with bacterial cells overlaying each other (Fig. 5.5C).  103  A.  0.4% mucin edge  center  B.  edge  center  C.  0.4% mucin  0.5% agar  edge  edge  Figure 5.5 Electron microscopy images of P. aeruginosa strain PA14 WT from motility colonies on 0.5% agar or 0.4% mucin. P. aeruginosa were taken directly from the leading edge or center of mucin-promoted surface motility zone (A, B) or the leading edge of swarming motility zone (C). The cells were stained with 1% uranyl acetate and observed using a transmission electron microscope. 5.3.7 Promotion of mucin-mediated motility by amino acids and inhibition by ammonium Since swarming motility is highly influenced by the medium composition, the effects of different carbon and nitrogen sources on the ability of P. aeruginosa to propagate on the mucin plates were investigated. To examine the effects of different carbon sources on mucin promoted 104  motility, lactate was excluded and glucose from the MSCFM medium replaced with equimolar amounts of each of the following carbon sources: α-ketoglutarate, succinate, fumarate, citrate, malate, glycerol, or mannitol (Fig. 5.6A). P. aeruginosa exhibited a statistically significant increase in mucin-promoted surface motility when glucose was replaced with citrate and a decrease in surface motility with succinate, even though these carbon sources supported similar growth (data not shown) indicating that growth differences could not explain these results. In collaboration with Alicia Parayno the effects of individual amino acids serving as the sole nitrogen sources on the surface motility were also examined, replacing the total free amino acids in the normal MSCFM with equimolar amounts of single amino acids. When provided as the sole nitrogen source, we observed that many of the amino acids tested were able to strongly support this form of surface motility (Fig. 5.6B). Replacement with glycine, methionine, valine, tryptophan, glutamine, isoleucine, and ornithine gave significantly weaker mucin-stimulated surface motility phenotypes, but the motility zones in these cases still demonstrated more than 50% of the surface coverage compared to the presence of the total free amino acids in MSCFM. Growth of P. aeruginosa was greatly impaired in methionine, valine, tryptophan, isoleucine, glycine, or ornithine as the sole nitrogen sources and moderately impaired when glutamine was used (Fig. 5.7A). Although the majority of amino acids that supported strong surface motility exhibited only little to moderate growth impairment (data not shown), leucine, phenylalanine, and threonine each caused significant growth impairments when provided as sole nitrogen sources, but supported strong surface motilities (Fig. 5.6B and 5.7A). One possibility is that there were less bacterial cells in the motility zones with leucine, phenylalanine or threonine than on normal MSCFM plates. To test this, surface motility colonies grown on threonine, leucine, phenylalanine, or normal MSCFM were resuspended in buffer and serial plating performed. After growth overnight, the resultant bacterial cell counts revealed significantly lower numbers of bacterial cells from the surfing colonies grown on leucine, phenylalanine, or threonine compared to normal MSCFM (Fig. 5.7B). In addition to amino acids, surface propagation was significantly impaired by about 50% when ammonium chloride was provided as the sole nitrogen source (Fig. 5.6B).  105  200%  A.  *  180%  % Fold change relative to Glucose  160% 140% 120% 100%  *  80% 60% 40% 20% 0%  200%  B.  % Fold change relative to total amino acids  180% 160% 140% 120% 100% * 80%  *  *  *  *  *  * *  60% 40% 20% 0%  Figure 5.6 Surface motility of PA14 WT on mucin plates supplemented with various carbon and nitrogen sources. P. aeruginosa were spotted onto MSCFM agar-mucin plates supplemented (A) glucose (control) or an alternative carbon source, or (B) total amino acids (control) or alternative nitrogen source and incubated at 37 oC for 13 h. Results shown are means ± SD for at least 3 independent experiments with duplicates for each experiment. * indicates a statistically significant difference (P ≤ 0.05) between the tested condition and control as determined by Student’s t test.  106  A. 1.5  Control Thr Gly  1.0  OD620  Val Met Ile  0.5  Leu Phe 0.0 0.0  5.0  7.5 10.0 Time (h)  12.5  15.0  Trp Gln  100%  % Fold change relative to total amino acids  B.  Orn 2.5  90% 80%  *  70% 60%  *  *  50% 40% 30% 20% 10% 0%  Figure 5.7 Growth and bacterial cell counts of PA14 WT using various amino acids as sole nitrogen sources. (A) P. aeruginosa were grown in liquid MSCFM supplemented with total amino acids (control) or a single amino acids serving as sole N source. Growth was measured at 37oC using a TECAN Spectrofluor Plus. (B) P. aeruginosa were spotted onto MSCFM agar-mucin plates supplemented with total amino acids (control) or single sole nitrogen source (as indicated) and incubated at 37oC for 13 h. The entire surface motility colony was resuspended in 1 X PBS and serial plated on LB agar plates. Results shown are means ± SD for at least 3 independent experiments with duplicates for each experiment. * indicates a statistically significant difference (P ≤ 0.05) between the sole nitrogen source and control as determined by Student’s t test. 5.3.8 P. aeruginosa in the mucin-mediated motility zone upregulated the expression of certain genes involved in virulence and resistance RT-qPCR was used to examine the expression of selected genes involved in virulence and antibiotic resistance in P. aeruginosa. The gene expression of P. aeruginosa taken from the surface motility zone with 0.4% mucin was compared to that of bacteria swimming in the same 107  medium without mucin. Table 5-2 shows the results. It was evident that P. aeruginosa in the mucin-mediated motility zone downregulated the expression of the type 3 secretion system (T3SS) (the fold change for pcrV was -10.2) and T3SS secreted factors (fold changes of exoT and exoY were -3.5 and -6.8). In contrast, genes encoding the Xcp type 2 secretion system (T2SS) and its relevant extracellular virulence factors including elastase, exotoxin A, and lipase, were upregulated compared to swimming cells. Genes encoding the biosynthesis of phenazine compounds were downregulated while genes involved in pyoverdine and pyochelin biosynthesis (iron sequestration) were upregulated (Table 5-2). Interestingly, certain genes involved in antibiotic resistance/susceptibility were hugely upregulated. For example, the expression of phoP and arnB were upregulated with fold changes of 182 and 189, respectively. Table 5-2 Expression of selected genes in P. aeruginosa strain PA14 taken from the surface motility zone with 0.3% agar and 0.4% mucin compared to bacteria swimming in 0.3% agar without mucin, as determined using RT-qPCR. Gene Name Fold change Gene Name Fold change ID ID Type III secretion system Pyochelin biosynthesis PA0044 exoT -3.5 ± 1.1 PA4221 fptA 4.3 ± 1.5 PA1706 pcrV -10.2 ± 0.9 PA4224 pchG 3.6 ± 1.1 PA1708 popB -9.0 ± 1.1 PA4226 pchE 8.2 ± 1.6 PA1709 popD -6.0 ± 1.4 PA4228 pchD 4.5 ± 1.3 PA1710 exsC -5.9 ± 1.1 PA4229 pchC 6.6 ± 1.7 PA1711 exsE -4.2 ± 1.3 Phenazine biosynthesis PA1713 exsA -6.4 ± 1.1 PA1900 phzB2 -3.3 ± 1.1 PA1717 pscD -10.2 ± 1.2 PA4209 phzM -4.6 ± 1.4 PA1721 pscH -12.3 ± 1.5 PA4210 phzA1 -4.9 ± 1.5 PA2191 exoY -6.8 ± 1.9 PA4211 phzB1 -5.4 ± 1.8 PA4213 phzD1 -3.9 ± 1.1 Type II secretion system PA1148 toxA 3.1 ± 1.1 PA4216 phzG1 -6.3 ± 1.5 PA3096 xcpY 4.6 ± 1.4 Adaptation and Resistance PA3104 xcpP 7.1 ± 1.3 PA1178 oprH 1173.0 ± 21.5 PA3724 lasB 72.6 ± 2.5 PA1179 phoP 181.9 ± 6.8 PA4813 lipC 5.1 ± 1.5 PA1797 4.2 ± 1.9 PA3552 arnB 188.8 ± 15.7 Pyoverdine biosynthesis PA2385 pvdQ 5.1 ± 1.3 PA4777 pmrB 3.2 ± 1.4 PA2399 pvdD 3.3 ± 1.1 PA2426 pvdS 6.1 ± 1.6  108  5.3.9 Mucin promotion of surface motility may involve lubricant-like action As described above, rhamnolipid production was not required for this form of surface motility on mucin plates. This led to the possibility that mucin might be serving as surrogate for a surfactant to reduce surface tension and promote this form of motility. It has been demonstrated that soluble mucin is both viscous and lubricative (142, 274). For example, the addition of porcine gastric mucin enhances the viscosity and wettability of solutions (194). Basically, the viscosity enhancing property of mucin allows mucin to bind water, reducing the free water content in the environment and thus increasing the ability of bacteria to slide across the surface. To examine whether the lubricant/wetting properties of mucin affected the surface motility, I substituted mucin with various wetting agents. Substitution of mucin with Tween20 promoted rapid surface propagation of P. aeruginosa (Fig. 5.8A). The replacement with Tween20 did not result in an identical pattern to that of mucin and gave more inconsistent surface motility patterns, with variable surface coverage between different trials, and the edges of the Tween20-mediated motility colony were also thinner than the rest of the motility colony, in contrast to the consistent surface coverage and thick edges of mucin mediated motility zones. A. Tween20  0%  0.005%  0.0025%  0.01%  B. Carboxymethylcellulose (CMC)  0%  0.2%  0.4%  0.8%  1.0%  Figure 5.8 Surface propagation of P. aeruginosa strain PA14 WT on wetting or viscosity enhancing agents. Cultures of P. aeruginosa were spotted onto MSCFM plates with 0.3% (wt/vol) agar and varying concentrations of (A) Tween80 or (B) CMC. Images were taken after incubation at 37oC for 13 h.  109  Also flagellar mutants were demonstrated to be able to spread on Tween-containing plates (data not shown). These results were overall consistent with the suggestion that the enhancement of surface wetness by mucin facilitated the ability of bacterial cells to expand on the agar, but suggested that other factors might be involved. Mucin was also replaced with carboxymethyl cellulose (CMC) that has both lubricative and viscous properties, to examine its effect on surface motility. As shown in Fig. 5.8B, CMC promoted surface propagation, but higher concentrations of CMC were required cf. mucin. At 1% (wt/vol), CMC only promoted about 50% of the surface coverage promoted by 0.4% (wt/vol) mucin. Overall it was concluded that a unique combination of wetness and viscosity conferred by mucin likely contributed to its effects on surface motility. 5.3.10 Regulation of mucin-mediated surface motility To gain insight as to how the mucin-mediated surface motility was regulated in P. aeruginosa, all of the known and putative TCS sensor kinase and response regulator mutants available in the PA14 transposon mutant library were screened (150) (127 in total) for motility on MSCFM plates with 0.4% mucin. As shown in Table 5-3, a total of 27 mutants were identified that were either significantly impaired or exhibited significantly increased surface motility on the MSCFM-mucin plates. The surface motility of these mutants were confirmed by testing the corresponding mutants from the PAO1 mutant library (112). As the flagellum was shown to be required for this form of surface motility, the 27 mutants were tested for swimming motility (Table 5-3). The fleR and fleS mutants, which were unable to mediate mucin-promoted motility, were also unable to swim. Mutants with transposon insertions in genes such as rcsB, wspE, PA5017, and PA2571 exhibited moderate defects in both mucin-mediated motility and swimming. Interestingly, mutants that exhibited increased motility on the mucin plates (e.g. envZ, rocR, rocA1 roxS, roxR) exhibited WT swimming motility. Moreover, cbrA, cbrB, ntrB, retS, PA2882 and PA1611 mutants exhibited moderate to major motility defects on the mucin plates, but exhibited only slight to no swimming defects. Since the type IV pili was demonstrated to be dispensable for mucin-mediated motility, it was not surprising to see that the majority of the mutants (except PA1611, PA2571 and ntrB) exhibiting normal twitching motility. To confirm that the mucin-promoted surface motility defects observed in the regulatory mutants were not contributed to by growth impairment, growth curves in MSCFM were performed. Most of the mutants, except PA5017, cbrA and cbrB, exhibited WT growth (Table 5-3). While the growth impairment of PA5017 may contribute to its moderate motility defect on the mucin 110  plates, it seemed unlikely for the cbrA or cbrB mutants since these mutants exhibited almost no motility on the mucin plates. The ability of the 27 TCS mutants to swarm was also examined. Mutants that exhibited moderate to major motility defects on mucin plates also exhibited impairments in swarming. However, with the exception of the gacA, envZ and PA4398 mutants, the majority of the mutants that exhibited increased mucin-promoted surface motility exhibited WT swarming motility. Table 5-3 Characteristics of TCS sensor kinases or response regulators involved in surface motility on MSCFM-mucin plates. (Some screening performed with Alicia Paranyo). Regulatory mutant  PA0034 PA0756 PA0757 PA0928 PA1098 PA1099 PA14_59770 PA1458 PA1611 PA2072 PA2571 PA2586 PA2687 PA2882 PA3704 PA3947 PA3948 PA4032 PA4398 PA4493 PA4494 PA4725 PA4726 PA4856 PA5017 PA5124 PA5199  gacS fleS fleR rcsB  gacA pfeS wspE rocR rocA1  roxR roxS cbrA cbrB retS ntrB envZ  % Fold change relative to PA14 WT 0.4% mucin Swarm Swimming Twitching 147.5% 107.4% 99.7% 100.6% 177.7% 101.2% 98.3% 96.0% 178.2% 118.5% 99.3% 97.4% 20.2% 3.0% 68.1% 90.2% 11.7% 3.0% 0.0% 90.7% 14.6% 3.0% 0.0% 99.8% 32.8% 3.4% 43.1% 92.2% 39.5% 12.0% 9.1% 98.1% 43.1% 71.9% 99.0% 31.8% 21.4% 11.0% 50.6% 95.3% 52.1% 9.0% 73.5% 10.2% 182.5% 228.0% 105.4% 94.3% 150.7% 118.0% 98.8% 97.2% 45.9% 25.5% 81.8% 101.0% 54.3% 60.8% 76.2% 78.2% 146.3% 83.8% 100.2% 102.8% 152.3% 115.4% 101.0% 103.5% 145.2% 115.4% 98.0% 101.3% 143.1% 38.0% 96.2% 108.5% 154.7% 101.2% 90.1% 92.4% 146.1% 106.1% 88.5% 90.7% 27.7% 3.0% 97.1% 98.3% 21.6% 3.0% 98.3% 99.2% 64.7% 55.6% 98.9% 99.8% 53.0% 3.2% 31.4% 82.1% 63.8% 3.0% 90.5% 30.9% 185.0% 130.1% 96.3% 98.3%  Growth defect? no no no moderate no no no no no no no no no no no no no no no no no moderate moderate no yes no no  111  5.3.11 Bacteria at the edge of the mucin-mediated motility zone appeared to stack The addition of mucin promoted the formation of a thick edge surrounding the motility colony. Based on the TEM observations, it was suspected that the thick edge might result from cells piling up and/or stacking at the edge of the motility zone, much as they would within the centre of a normal bacterial colony. To confirm this, with the technical assistance of Lindsay Heller, a mucin-promoted motility colony was observed using a light microscope after spotting P. aeruginosa onto a thin layer of 0.3% (wt/vol) agar containing MSCFM and 0.4% (wt/vol) mucin. The agar was spread evenly onto a coverslip at the bottom of a small Petri dish. To keep the agar from drying out, a wet chamber (placed wetted Whatman paper around the wall of the plate) was created, the lids of the plate kept closed throughout the entire experiment and the length of time of the experiment minimized to at most 2 hours. Under the light microscope, the leading edge of the surface motility zone appeared to be densely packed with cells that were mostly immotile with a couple moving cells (Fig. 5.9A). The center of the motility colony, however, was occupied by rapidly moving cells (Fig. 5.9B). I noticed that at the center of the motility zone, bacterial cells were moving randomly (in all directions) while closer to the edge, the majority of cells appeared to be moving towards the edge. After 1 hour incubation at 37oC, there was formation of a thin edge. After 2 hours, along with an increased motility zone diameter, the edge became clearly thicker and wider (Fig. 5.9C). The depths of the edge and the center of the motility zones at 1 and 2 hours were estimated using a light microscope. On average the depths at the edge were 80 µm and 160 µm at 1 and 2 hours post-incubation, respectively, while the depths of the center were approximately 8 µm and 20 µm at 1 and 2 hours post-incubation, respectively. Thus, the edge became wider and deeper as time progressed. Interestingly, just beyond the thick immotile edge at the outside, a small number of moving bacterial cells could be observed (data not shown).  112  Figure 5.9 Light microscopy images of PA14 WT surface motility zones on MSCFMmucin plates. P. aeruginosa were spotted onto thin MSCFM-mucin agar slab and observed under the microscope at 37oC. Pictures of the leading edge (A) and center (B) of the surfing motility zone were taken after 2 h incubation at 37 oC. Pictures comparing the motility edge (C) were taken at 1 h and 2 h incubation at 37oC. Note: Elongated wavy cells shown in (B) represent single rapidly moving cells. 5.4  Discussion The ability of a spectrum of microorganisms, and notably P. aeruginosa, to colonize the  lungs of CF patients is associated with subsequent lung function deterioration and health decline. Motility of these microorganisms has been shown to aid in the initial colonization process (59, 100). The CF lung environment is characterized by the production of copious amounts of mucous (sputum), which covers the surface of epithelial cells. Mucin, being a major component of respiratory secretions, typically at concentrations of around 0.5-1% (wt/vol) (105, 122, 198), gives mucous its gel-like properties and is regarded as an important molecule in the initial 113  colonization by P. aeruginosa of the airways of CF patients (198). Due to the current limitations of our ability to observe bacterial motility in a host, I constructed an in vitro model to study motility. This in vitro motility model utilized the typical carbon, nitrogen and energy sources for growth of P. aeruginosa in a version of SCFM lacking NH4+ (192). DNA added at 1.4 mg/mL, the concentration found in the CF sputum (34), did not affect mucin-mediated motility. The use of 0.3% agar provided a surface for easy observation of the motility of P. aeruginosa while the water content in the plate mimicked the water content of the sputum. Under these conditions, the addition of mucin led to both P. aeruginosa strains, PAO1 and PA14, exhibiting rapid motility across the surface of the agar resulting in the formation of large green colonies surrounded by thick white edges that somewhat resembled the swarming pattern of strain PAO1 (without the solar flare like tendrils) but differed from that of strain PA14. P. aeruginosa is well-known for its ability to swim, twitch, swarm and slide. Swimming is mediated by rotation of the bacterium’s single polar, monotrichous flagellum. Independent of the flagellum, twitching requires type IV pilus filaments that extend from the cell body to adhere to a surface and then retract, thus "dragging" the bacterium forward. Swarming motility requires both functional flagella and type IV pili (although one study suggested that swarming does not require functional pili), while sliding motility occurs in the absence of flagella and type IV pili. Mucin-promoted surface motility is independent of type IV pili suggesting that mucin is not promoting twitching motility. The ability of a fliCpilA double mutant to slide but not to propagate on a mucin-containing plate indicated that sliding is separate from this form of surface motility. Also, the requirement for intact flagella, but not particular stator flagellar functions, clearly distinguishes this form of surface motility from swimming and conventional swarming motility, and many other differences in the required genes were observed between swarming and mucin mediated motility, including nutritional requirements, the phenotypes of many mutants and rhamnolipid dependence. Based on these results, I suggest that mucin is promoting a new form of surface motility rather than just a highly modified form of swarming motility. As there are so many distinguishing features of this form of surface motility, I suggest the name “surfing motility” based to the wetting properties of mucin and the appearance of the wave-like motility front as the motility zone progressed in time. It was demonstrated that QS plays an important role in promoting surfing motility, since mutations in the rhl and las systems significantly impaired this mucin-promoted surface motility and surfing could be rescued with the cognate homoserine lactones. Although swarming has been shown to be dependent on QS for the production of the biosurfactant rhamnolipids, 114  swimming and twitching motilities are independent of QS. Dependence on rhamnolipid production for sliding motility was indicated by Murray et al. where they showed that a fliCpilArhlA mutant was unable to slide (176). Here, it was demonstrated that mutations in the genes involved in rhamnolipid biosynthesis, rhlA and rhlB, did not impair the ability of P. aeruginosa to undergo surfing motility on the mucin plates. Thus rhamnolipid production was not required for this surface motility, likely because mucin was acting as an alternative surfactant to rhamnolipid or otherwise promoting surfactant activity. In addition to flagella, type IV pili and rhamnolipids, classical swarming motility is also dependent on specific carbon and nitrogen sources. Kohler et al. showed that while glucose promoted swarming when provided as the sole carbon source, glycerol and succinate did not (128). Also swarming motility was shown to be completely inhibited when ammonium chloride was provided as the sole nitrogen source while aspartate, glutamate and histidine promoted strong swarming phenotypes (128). In contrast, carbon and nitrogen source substitutions in the mucin-containing plates revealed that surfing motility was less nutritionally restricted than swarming and it was demonstrated here that surfing motility occurred with a wide variety of carbon and nitrogen sources. Specifically, amino acids, including arginine, asparagine and leucine, promoted strong surface motility on mucin plates, while these individual amino acids completely abolished swarming when provided as sole N-source. Examination of P. aeruginosa cells from edge of the mucin motility zones paradoxically revealed a lack of flagella for the majority of the cells analyzed, although a few bacterial cells still had flagella attached. This is in contrast to P. aeruginosa at the edge of a swarm zone where the majority of bacteria cells possess two polar flagella (128). Moreover, while swarmer cells are highly organized (e.g. cells are aligned one next to another in the same direction as the moving tendril), the orientation of the bacterial cells on the edge of the mucin motility zones appeared random with bacterial cells overlaying each other. Interestingly, the bacterial cells from the edge of the mucin motility zones resembled cells in a biofilm as bacteria lose their flagella and cells are piled on top of each other inside a growing biofilm. Moreover, various aspects, such as QS, shown to be important in formation of the mucin motility colony, also plays important roles in biofilm formation (125, 186). I am currently testing normal swimming mutants defective in biofilm production as well as hyperbiofilm formers for their ability to undergo surface motility on MSCFM-mucin plates. Previously Overhage et al. performed microarrays on P. aeruginosa from the leading edge of a swarm compared to bacteria growing in identical medium under swimming conditions 115  (189). Their study led them to identify major changes in gene expression patterns in swarming cells. In particular they demonstrated that swarmer cells overexpressed a large number of virulence-related genes including those encoding the T3SS and its effectors, those encoding extracellular proteases, and those associated with iron transport (189). Analysis by RT-qPCR of gene expression of P. aeruginosa taken from the mucin-mediated surface motility colony revealed, in contrast to swarming cells, that genes encoding the T3SS and its effectors and those involved in phenazine biosynthesis were downregulated. In contrast, genes encoding for the T2SS and the exoproducts that this system secretes were upregulated. The important role of these virulence factors in P. aeruginosa pathogenesis has been demonstrated in various in vivo respiratory infection models (80, 115, 123). Significant upregulation of the oprH, pmr and arn operons, which are not dysregulated in swarmer cells, was also observed. Activation of these operons is known to induce bacterial resistance to polymyxins and cationic antimicrobial peptides in response to low-Mg2+ conditions by controlling the addition of aminoarabinose to lipid A, thereby reducing the net negative charge of LPS. Consequently the reduced net negative charge on the bacterial cell surface limits its interaction with increases resistance to polycationic peptide antimicrobials (168, 184). The increased expression of virulence and resistance associated genes in these cells was not simply due to the presence of mucin, as no dysregulation of these genes was observed in P. aeruginosa grown in liquid MSCFM with and without 0.4% mucin (data not shown). Overall this investigation has provided evidence for a new form of surface motility, termed surfing, in P. aeruginosa and evidence is presented that is consistent with it being a new complex adaptation in P. aeruginosa. Future studies will permit more in depth understanding of the surface motility promoted by mucin in P. aeruginosa.  116  Chapter 6: Concluding chapter 6.1  Introduction P. aeruginosa is an opportunistic human pathogen that can cause a number of serious  acute and chronic infections in patients with impaired immunity and mucosal defences. In addition to its arsenal of virulence factors, metabolic versatility, intrinsic resistance to antibiotics and ability to form biofilms, the motility of P. aeruginosa also contributes to the ability of the pathogen to cause severe infections. Motility facilitates the pathogen to attach to surfaces, colonize host tissues, facilitate colony spread, evade toxic substances, and accelerate biomass production. At the start of this thesis research it was known that P. aeruginosa was unusual in that it was capable of multiple types of motilities, including swimming, twitching and swarming. The latter is characterized by rapid and coordinated group movement of cells over a semisolid surface resulting from intercellular interactions and morphological differentiation. A role for swarming motility during in vivo infection was suggested based on a screen of P. aeruginosa transposon mutants attenuated for virulence in a rat chronic pulmonary infection model, which mapped to genes required for swarming motility (201). A microarray analysis revealed that swarmer cells upregulated expression of genes encoding virulence factors. Swarmer cells were also demonstrated to exhibit adaptive resistance toward a variety of antibiotics (189). Moreover, the semi-viscous conditions that promote swarming in vitro are believed to mimic those of the mucous layer that overlays epithelial surfaces such as those of the lung, a major site of P. aeruginosa infection. The current data provide strong support for the concept that swarming is a complex adaptation triggered in response to specific environmental stimuli. Although implicated in the pathogenesis of P. aeruginosa, at the start of this thesis swarming was still poorly understood, particularly the regulatory mechanisms that promote swarming differentiation. The research addressed in this thesis sheds light on the intricate, sophisticated regulation of the complex adaptation process of swarming. A detailed study of a sensor kinase CbrA unravelling its role in swarming, biofilm formation, antibiotic resistance, in vitro cytotoxicity, and in vivo virulence provided evidence for the interrelationship of a variety of complex adaptations, and the existence of genetic switches that control these processes. Furthermore, application of mucin, which is physiologically encountered at mucosal surfaces in the host, provided an unanticipated window into a new form of surface motility, termed here surfing motility, with many features that distinguish it from swarming motility.  117  6.2  Complex regulation of swarming motility in P. aeruginosa Swarming of P. aeruginosa is dependent on flagella and type IV pili as well as the  production of rhamnolipids. Mutations in genes involved in the synthesis or function of flagella, type IV pili or rhamnolipids greatly impair swarming motility (128). A recent screen of 1,200 genes for which mutants are available in the P. aeruginosa PAO1 mini-Tn5-luxCDABE transposon mutant library, identified 36 mutants with altered swarming phenotypes (190). These mutants had Tn5 insertions in genes for the synthesis or function of flagellin and type IV pilus, in genes for the Xcp-related T2SS, and in regulatory, metabolic, chemosensory, and hypothetical genes with unknown functions (190). However, this screen did not lead to a comprehensive understanding of all genes in P. aeruginosa involved in swarming and the multifaceted regulation swarming, since mutants in less than 25% of the genes in the PAO1 genome were available in this library. Therefore, to increase our understanding we screened, for mutants with altered swarming phenotypes, the comprehensive P. aeruginosa strain PA14 transposon mutant library (150), which covers more than 80% of all of the predicted 5,962 PA14 genes. As described in Chapter 2, the screen led to the identification of 233 mutants with alterations in swarming motility. Expanding on earlier observations (128, 190), there were multiple swarming deficient mutants with transposon insertions in flagella and type IV pili biosynthesis, and quorum sensing. Moreover, there were also mutants with transposon insertions in genes from a wide variety of functional classes, including those functioning in metabolism, transport of small molecules, adaptation and protection. This large number of genes found to be involved in this form of motility (4% of the PA14 genome), and the broad variety of functions performed by these genes, clearly indicated that, rather than being a mere form of locomotion, swarming was an alternative growth state. The preliminary broad screen enabled the identification of hundreds of mutants on a single plate. However, due to the nature of this preliminary screen, subsequent confirmatory studies demonstrated certain mutants that were positive in the screen but could not be independently confirmed and certain genes involved in swarming may have been missed. For example, a flgK mutant was previously identified to be swarming deficient (32), but flgK was not identified from the preliminary screen. Therefore, I anticipate that more than 233 genes in P. aeruginosa strain PA14 are involved in swarming motility. Moreover, nearly 30% of the identified swarming-associated genes are currently labelled as hypothetical genes and await future studies that will determine their functions. Among the mutants with genes that had altered swarming motility were 35 mutants with transposon insertions in genes encoding regulators. To my knowledge, the swarming phenotypes 118  of only 8 regulatory mutants had been described previously; therefore, 27 swarming-deficient regulators, 10 of which were two-component sensors or response regulators, were newly identified in this screen. Interestingly, only a few of these regulatory mutants showed significant defects in the production of type IV pili, flagella, or rhamnolipid, suggesting that majority of these regulators control other factors involved in swarming. While 14 of the 35 swarmingassociated regulators are currently of unknown function and have not been related to any phenotype, the remaining regulators have known regulatory roles in various processes, including metabolism, quorum sensing, antibiotic resistance, virulence and biofilm production. The relatively high number of swarming regulators identified in this study reflects the complexity of this motility and implies this form of surface behaviour is tightly regulated. Most but not all of the swarming-negative regulatory mutants showed an enhanced ability to form simple biofilms, a surface-related differentiation process which presumably shares many common features with swarming motility. In contrast, 2 mutants that exhibited hyperswarming phenotypes exhibited biofilm impairment compared to the WT. The results appear to suggest an inverse relationship between the regulation of swarming and biofilm formation where these regulators act as switches between swarming, that might be considered to reflect a virulent lifestyle, and biofilm formation, that reflects a chronic lifestyle. To increase confidence in these conclusions, it would be necessary to investigate in detail a wide variety of mutants that exhibit either hyperswarming or swarming-deficient phenotypes for biofilm production to identify the effectors and regulatory circuits. Swarming has been proposed to influence the early stages of biofilm formation, with actively swarming cells forming flat biofilms and swarming-deficient cells forming structured biofilms (225). Therefore, it would be interesting to determine at which stage of biofilm production these regulators play a role. The reciprocal regulation of biofilm and swarming contrasted with the conclusions of Overhage et al. where they demonstrated a range of P. aeruginosa strain PAO1 mutants with defects in swarming coinciding with deficiencies in biofilm formation (190). This demonstrates that limited library screens can result in general conclusions that do not accurately reflect an overall pattern. Clearly in specific mutants such as Lon (164) there are coordinate rather than reciprocal effects on swarming and biofilm formations, and this was also observed here for e.g. nosR, phoQ, rsaL and mvaT; nevertheless there is a much more obvious overall pattern of reciprocal regulation of the virulent and chronic infectious lifestyles. While P. aeruginosa strain PA14 forms optimal static biofilms in minimal medium supplemented with CAA, strain PAO1 grows more efficiency in rich media, such as LB. 119  Reciprocal relationships between the regulation of swarming and biofilm formation, based on limited studies, have previously been proposed in other studies (33, 169). Particularly, a study by Caiazza et al. revealed that a sadB mutant was able to initiate surface attachment but failed to form biofilm microcolonies. Moreover, the mutant exhibited a hyperswarming phenotype. SadB participated in the inverse regulation of biofilm formation and swarming motility by modulating flagellar rotation reversal and the production of pel exopolysaccharides (33). 6.3  The CbrAB TCS is a global regulator of swarming and various virulence-related processes in P. aeruginosa From the screen of the PA14 transposon mutant library, 8 swarming-associated  regulatory mutants were identified that displayed major swarming deficiencies, with little or no defects in swimming, twitching or rhamnolipid production, that are known to play important roles in metabolism, e.g. carbon (cbrAB), nitrogen (ntrBC), and amino acid metabolism (trpI, oruR, hutC, metR). These metabolic regulators have not been previously investigated for their possible roles in virulence and virulence-related processes in P. aeruginosa. Since it was not possible to study, in great detail, all of the swarming-associated metabolic regulators identified from the screen, I chose to study the CbrAB TCS as an example to illustrate that these metabolic regulators might be involved in substantially more than just metabolic regulation in P. aeruginosa. As described in Chapter 3, a cbrA mutant was completely abolished in swarming motility but exhibited enhanced biofilm formation. The cbrA mutant also exhibited increased resistance toward several common clinical antibiotics, including polymyxin B, tobramycin, and ciprofloxacin. Furthermore, the sensor kinase CbrA appeared to regulate swarming and biofilm formation via its cognate response regulator CbrB. However, a cbrB mutant did not have a resistance phenotype, CbrA likely modulated antibiotic resistance in a manner independent of CbrB. Interestingly, the cbrA and cbrB mutants exhibited some of the common phenotypes exhibited by clinical isolates of P. aeruginosa during chronic airway infection of patients with CF, including the lack of motility, biofilm formation and increased antibiotic resistance. Therefore, I hypothesize that there are clinical isolates of P. aeruginosa with alterations in the CbrAB system. Importantly, the involvement of CbrAB in a number of important adaptationrelated processes in P. aeruginosa indicates that these regulators contribute in maintaining the overall physiological balance of the bacterium. By enabling the bacteria to utilize a variety of organic compounds as carbon sources and to switch between the swarming motility and biofilm  120  behaviours while regulating antibiotic resistance, CbrA optimizes the efficiency of the bacterium to adapt to various environments. 6.4  Requirement of acylhomoserine lactone acylase activity for swarming? The cbrA mutant was completely unable to swarm. The ability of the mutant to swim and  twitch like the WT, and in microarray analysis express relevant genes (e.g. fliC, pilA) at similar levels as that for the WT, indicated the cbrA mutant was not impaired in flagella or type IV pilus biosynthesis. The cbrA mutant also produced WT levels of rhamnolipids suggesting CbrA is likely promoting the expression of additional factors required for swarming motility. Although the cbrA mutant exhibited moderate growth defects in certain swarming media due to its weakened ability to use glucose as sole carbon source, this appeared insufficient to explain the complete abolition in swarming in a cbrA mutant. Microarray analysis of the cbrA mutant under swarming condition provided clues to possible genes CbrA might be regulating to influence swarming. Comparison of the cbrA mutant vs. PA14 WT microarray with a previous swarming vs. non-swarming P. aeruginosa microarray (189) revealed the opposite regulation of a number of genes, including a probable ATP-binding component of the ABC transporter, PA4223, genes involved in pyoverdine and pyochelin biosynthesis; and a gene encoding an ammonium transporter, amtB. These genes were also downregulated in the cbrB mutant, consistent with their control during swarming by the CbrAB two component regulatory system. Of these genes, only the PA4223 and pvdQ mutants displayed major swarming impairments. As discussed in Chapter 3, while the function of PA4223 is unknown, PvdQ has dual functions in pyoverdine biosynthesis and degradation of 3-oxo-C12-HSL (acylase activity). The role of PvdQ in pyoverdine biosynthesis seems unlikely to explain the swarming defects observed for the cbrA and cbrB mutants, since mutations in other genes involved in pyoverdine biosynthesis did not affect swarming deficiency and the addition of iron to the swarm plates did not restore swarming motility of the cbrA or cbrB mutants. However, the role of PvdQ in maintaining a balance of the QS signals in swarming appears more likely to have contributed to the swarming deficiency of these mutants. Another possibility worth investigating is whether the degradation product produced by PvdQ may serve as a signal during swarmer cell differentiation. Intriguingly, I also observed downregulation of PA4223 and pvdQ in the other swarming-deficient regulatory mutants, including metR (276), ntrB and ntrC, (data not shown).  121  6.5  The P. aeruginosa CbrA/CbrB TCS exhibits similarities to the GacS/GacA TCS CrcZ, a small RNA encoded in the P. aeruginosa genome was first identified by  Sonnleitner et al. (233) and its expression was to be controlled by the CbrAB TCS. The crcZ small RNA binds to and sequesters the Crc protein to relieve catabolite repression of a variety of degradative genes (233). In Chapter 3, phenotypic and genetic analyses were performed that provide evidence that the CbrAB TCS was likely modulating swarming and biofilm formation via CrcZ, with at least partial inverse regulation by Crc. From my studies, the participation of Crc in the virulence-related phenotypes was less apparent. While the crc mutant exhibited a substantial decrease in biofilm formation, it did not exhibit significantly increased swarming compared to the WT as expected. Although more investigation is merited, my research on the CbrAB TCS revealed striking similarities to the GacAS TCS, a well-studied system in P. aeruginosa proposed to control the reciprocal expression of acute and chronic virulence determinants (82). In P. aeruginosa, the GacAS TCS directly regulates the transcription of two small regulatory RNAs, RsmZ and RsmY, which in turn jointly antagonize the action of the RNA-binding protein RsmA. RsmA normally negatively controls AHL-dependent QS as well as a number of QS-dependent genes, some of which encode for secondary metabolites and virulence determinants, while positively controlling the expression of lipase and rhamnolipids (256). Although studies have shown that the sole output of the GacAS system is the direct regulation by GacA of transcription of rsmY and rsmZ, it is evident that the CbrAB TCS does not act only through crcZ, since CbrA regulated antibiotic resistance in P. aeruginosa independent of crcZ. It will be important in the future to identify other acute and chronic virulence determinants regulated through the CbrAB/CrcZ/Crc signalling pathway to support its involvement in the reciprocal development of acute or chronic infections in P. aeruginosa. 6.6  Interaction of CbrA with other sensor kinases? TCSs, consisting of sensor kinase and response regulator pairs, have emerged as key  mediators of successful adaptation of P. aeruginosa to its changing environments. In the classical TCS system, the signal transduction involves a cognate sensor-regulator pair. Most, but not all, of the functionally coupled sensor kinase and response regulator genes are encoded adjacent to each other in the genome, forming an operon (213). In turn, this is as though to favour co-expression of the corresponding proteins and decrease the opportunities for cross talk (e.g. non-cognate phosphotransfer events) between non-cognate sensors and regulators. 122  Nevertheless, in addition to cognate sensor-regulator pairs, the P. aeruginosa genome also encode for orphan TCS proteins where a sensor kinase gene is not accompanied by a response regulator gene and vice versa. While studies have shown that certain orphan sensors partner with specific orphan regulators, other orphan sensor or regulators lack prototypical partners and function in unorthodox ways to modify gene expression and cellular behaviour (202). A wellstudied example is the direct interaction of the orphan sensor kinase RetS with the GacS protein. Studies have shown that RetS modulates the phosphorylation state of GacS by initiating a direct interaction between these two sensor kinases promoting the formation of RetS-GacS heterodimers, impacting on the phosphotransfer to the GacA response regulator (81). As described in Chapters 3 and 4, the sensor kinase CbrA appeared to mediate antibiotic resistance and in vivo virulence in mice independent of its cognate response regulator CbrB. At present it is not known how the membrane-bound sensor kinase CbrA accomplishes this. Since CbrA is membrane-bound, it is not likely the sensor is directly regulating gene expression but it is possible it acts by modifying the phosphorylation state of an alternative but unknown response regulator. There is also the possibility that CbrA may be interacting with another sensor kinase to modulate these phenotypes. The 7 two-component sensor kinase/response regulators (phoPQ, PA1243, pmrAB, ompR, and PA4197) identified in the cbrA microarray serve as good candidates for this cross regulation. Intriguingly, the antibiotic resistance and to some extent the in vivo virulence results for the cbrA and cbrB mutants mirror the phoQ mutants. Previous studies have shown that while a phoQ mutant exhibit constitutive resistance to polymyxin and aminoglycosides, a phoP mutant exhibits WT resistance to these antibiotics, and it was suggested this reflects a major role of PhoQ in dephosphorylating PhoP, with another sensor kinase being responsible for PhoP phosphorylation (158). Moreover, a phoQ mutant displayed significantly reduced virulence than the WT in several infection models (79, 158), although PhoQ showed a very substantial growth disadvantage in the chronic rat lung model (79). Thus, the PhoQ appears to be a leading candidate for interaction with the CbrA sensor kinase and may explain a subset of the independent phenotypes. Future studies, including mutation and overexpression of the phoQ gene in the cbrA mutant, bacterial two-hybrid assays, and in vitro phosphorylation assays, may provide evidence to support the interaction between these proteins. In addition to the candidates identified from the cbrA microarray, I propose the possibility that CbrA interacts with another TCS, NtrBC. While CbrA shares 34% identity with sensor domain of NtrB, CbrB shares 45% identity with the response regulator NtrC. In P. aeruginosa, the CbrAB and NtrBC TCSs have been demonstrated to work co-ordinately to 123  control cellular carbon and nitrogen metabolism (149). For example, P. aeruginosa is capable of growing on histidine as a sole source of carbon and/or nitrogen. Both CbrAB and NtrBC can activate the expression of the histidine utilization (hut) operon, depending on whether histidine is the sole source of carbon or of nitrogen. The CbrAB system is indispensable only when histidine is the sole carbon source; however, when histidine is the sole source of nitrogen, expression of hut can be activated by either CbrAB or NtrBC (149). From the swarming screen in Chapter 2, both PA14 ntrB and ntrC transposon mutants exhibited swarming defects while exhibiting WT swimming and twitching. Moreover, the ntrB mutant exhibited increased biofilm formation than the WT and was 4- and 16-fold more resistant than the WT toward tobramycin and ciprofloxacin, respectively (Yeung A.T.Y and R.E.W. Hancock, unpublished data). The ntrC mutant, however, exhibited WT biofilm and antibiotic resistance phenotypes. Currently, I am investigating the transcriptomes of the ntrB and ntrC mutants under swarming conditions. These genetic data would be useful in identifying genes that overlap with the cbrA microarray as well as enabling insight into how TCSs involved in metabolism regulate swarming and other virulence-related processes in P. aeruginosa. 6.7  Is mucin promoting P. aeruginosa to swarm or to surf? An important environmental factor that determines the mode of motility adopted by  Pseudomonas aeruginosa is the viscosity of the medium, often provided by adjusting agar concentrations in vitro. However the viscous gel-like property of the mucous layer that overlays epithelial surfaces, such as those of the lung, a major site of Pseudomonas infection, is largely due to the glycoprotein mucin. P. aeruginosa is known to swim within 0.3% wt/vol agar and swarm on the surface at 0.5% wt/vol agar with amino acids as a weak nitrogen source. As described in Chapter 5, when physiological concentrations, or as little as 0.05% wt/vol, of mucin was added to swimming agar, in addition to swimming within the agar, P. aeruginosa was observed to undergo highly accelerated motility on the surface of the agar. The surface motility colonies in the presence of mucin appeared to be circular with a bright green center surrounded by a thicker white edge. While intact flagella were required for this surface motility in the presence of mucin, type IV pili and rhamnolipid production were not. Substitution of mucin with other wetting agents indicated that the lubricant properties of mucin might contribute to the surface motility. Based on studies with specific mutants, the QS systems, las and rhl, and the orphan autoinducer receptor, qscR, all played important roles in this form of surface motility, and overall the influence of regulatory mutations was quite different between swarming and 124  mucin mediated surface motility. Transcriptional analysis of cells taken from the motility zone revealed the upregulation of genes involved in virulence and resistance. Based on these results, I suggest that mucin is likely promoting a new or highly modified form of surface motility, which I propose should be termed “surfing”. P. aeruginosa is already known for its ability to swim, twitch and swarm (as well as slide which is opposite to surfing in that it requires a deficiency in flagella and is inhibited by pili (176)). While swimming, twitching and swarming are forms of motilities that depend on the agar concentration, surfing motility was less dependent on the agar concentration but dependent on the presence of mucin (although it could be partially mimicked by substances with analogous physical properties). Specifically, surfing was ruled out as a form of twitching motility as type IV pili is required for twitching but dispensable for surfing Also surfing was phenotypically different from swimming, particularly as it is a form of surface motility. However, due to the similarities (surface motility, rapid colony expansion, requirement for flagella and cell-to-cell signalling) shared by surfing and swarming motilities, more studies will be required in the future to further differentiate these 2 types of surface motilities. Microscopic analysis provided me with strong evidence of differences between surfing and swarming motilities. Studies have shown that cells of P. aeruginosa at the leading edge of the swarming motility zone are elongated and possess 2 polar flagella (128). In contrast, the majority of the cells taken from the edge of the surf colony lacked flagella. Moreover, while swarmer cells were highly organized (e.g. cells were aligned in the same direction as the moving tendril), the orientation of the surfing cells were apparently random with bacterial cells overlaying one another. The TEM observations were supported by analysis of the surfing motility colony under a light microscope. Under the light microscope, the leading edge of the surfing motility zone appeared to be densely packed with cells that were mostly immotile. The surfing motility center, however, was occupied by rapidly moving cells. Moreover, as time progressed, the motility zone diameter expanded and the motility colony edge became noticeably thicker and wider. Based on these data, I propose that motile bacterial cells from the center of the motility zone surf over the edge to get the motility edge (Fig. 6.1). The thick zone of bacteria would consist of bacteria that have surfed over the edge and lose motility or cells that do not make it over and thus become part of the edge. As more cells move away from the center, the edge is expanded outwards. Consequently, as the surfing motility zone expand, more cells are deposited at the edge so that the edge becomes thicker and thicker. Due to limitations of the light microscope, I am currently unable to observe this phenomenon further to provide direct evidence 125  for this ‘surfing’ action of the bacterial cells over the thick edge. In the future, data from experiments, such as single-cell tracking using tagged bacteria, might provide more clues for the differences between swarming and surfing motilities. This thesis has contributed to our understanding of the involvement of TCSs in the regulation of the two complex adaptations- swarming and surfing motilities in P. aeruginosa. Moreover, the involvement of these TCSs in not only motility but also other complex adaptations (e.g. biofilm formation and antibiotic resistance) of the bacteria further support the complex relationship between swarming, surfing, biofilm formation and antibiotic resistance. These studies will assist in determining the basis for regulation of virulence and antibiotic resistance under in vivo conditions that trigger swarming or surfing.  Figure 6.1 Models for the expansion of the surfing motility zone. (A) Motility zone expands outwards due to the accumulation of bacterial cells at the edge. (B) Bacterial cells swim away from the center, upon reaching the motility edge, ‘surf’ over the edge to get to the other side. As a result, the edge is pushed outward and the motility zone expands. Black arrows indicate general direction for majority of the bacterial cells. Red arrows indicate direction of the expanding motility zone.  126  Bibliography 1. Anonymous 1998. 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Immun. 71:53895393.  144  Appendix A Table A-1 List of P. aeruginosa PA14 transposon insertion mutants displaying deficiencies in swarming motility. PAO1 PA14 locus homolog PA14_00120 PA0011 PA14_00460 PA0037 PA14_00700 PA0058 PA14_00750 PA0063 PA14_00780 PA0066 PA14_01340 PA0110 PA14_02230 PA0177  Polar effect possiblea Nb Nb Nb Nb Nb Nb Nb  Gene Name trpI  cheW  PA14_02410 PA14_04410  PA0192 PA0337  Nb Nb  ptsP  PA14_05330 PA14_05340  PA0409 PA0410  Y Y  pilH pilI  PA14_05360  PA0411  Nb  pilJ  PA14_05380 PA14_05450 PA14_05560  PA0412 PA0419 PA0428  Nb Nb Nb  pilK  PA14_06210 PA14_06260  PA0475 PA0479  Nb Nb  PA14_06830 PA14_07170 PA14_07370 PA14_07530 PA14_07700 PA14_07710 PA14_08210 PA14_08500 PA14_54540 PA14_54520 PA14_53520 PA14_53430 PA14_53300 PA14_52730 PA14_52570  PA0524 PA0551 PA0567 PA0577 PA0590 PA0591 PA0633 PA0664 PA0754 PA0755 PA0831 PA0837 PA0848 PA0894 PA0905  Nb Nb Nb Nb Nb Y Nb Nb Nb Y Nb Nb Nb Nb Nb  PA14_52280 PA14_52260  PA0926 PA0928  Nb Y  norB epd dnaG apaH  oruR slyD  rsmA  gacS  Gene Description putative 2-OH-lauroyltransferase trpBA operon transcriptional activator putative protein-disulfide isomerase putative aminopeptidase putative carbonic anhydrases conserved hypothetical protein putative purine-binding chemotaxis protein putative TonB-dependent receptor phosphoenolpyruvate-protein phosphotransferase type IV pilus response regulator PilH type IV pili signal transduction protein PilI type IV pili methyl-accepting chemotaxis transducer PilJ methyltransferase PilK conserved hypothetical protein putative ATP-dependent RNA helicase, DEAD box family putative transcriptional regulator putative transcriptional regulator, LysR family nitric-oxide reductase subunit B D-erythrose 4-phosphate dehydrogenase conserved hypothetical protein putative DNA primase bis(5'-nucleosyl)-tetraphosphatase putative apaG protein putative major tail protein V putative integral membrane protein conserved hypothetical protein probable porin transcriptional regulator OruR peptidyl-prolyl cis-trans isomerase SlyD probable alkyl hydroperoxide reductase hypothetical protein RsmA, regulator of secondary metabolites hypothetical protein sensor/response regulator hybrid  Mutant swarm zone relative to PA14 WT less swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm less swarm no swarm less swarm no swarm no swarm no swarm no swarm no swarm no swarm less swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm less swarm no swarm less swarm less swarm no swarm no swarm no swarm no swarm  145  PA14 locus  PAO1 homolog  Polar effect possiblea  Gene Name  PA14_52210 PA14_51390  PA0932 PA0999  Nb Nb  cysM pqsD  PA14_51360 PA14_50560  PA1001 PA1070  Nb Nb  phnA braG  PA14_50470 PA14_50290 PA14_50280 PA14_50270 PA14_50130 PA14_50110 PA14_49800 PA14_49320 PA14_49280 PA14_49170 PA14_48830 PA14_48280  PA1078 PA1092 PA1093 PA1094 PA1102 PA1103 PA1127 PA1167 PA1171 PA1180 PA1196 PA1237  Nb Nb Nb Nb Y Nb Nb Nb Nb Nb Nb Nb  flgC fliC  PA14_46850 PA14_46440 PA14_46060 PA14_45950 PA14_45810 PA14_45770 PA14_45760 PA14_45720 PA14_45680 PA14_45660 PA14_45590 PA14_45500  PA1347 PA1377 PA1422 PA1431 PA1442 PA1446 PA1447 PA1449 PA1452 PA1453 PA1458 PA1464  Nb Nb Nb Nb Y Y Y Nb Y Y Nb Nb  PA14_45120 PA14_44370 PA14_43420 PA14_41710 PA14_41220 PA14_40900 PA14_40670 PA14_40620 PA14_39070 PA14_38970 PA14_38350  PA1492 PA1544 PA1554 PA1631 PA1767 PA1803 PA1827 PA1843 PA1848 PA1969 PA1976 PA2023  Nb Nb Nb Nb Nb Nb Nb Nb Nb Nb Nb Nb  PA14_37690 PA14_37370  PA2072 PA2098  Nb Nb  PA14_44490  fliD fliG  phoQ  gbuR rsaL fliP fliQ flhB flhA flhF  anr ccoN  lon metH  galU  Gene Description  Mutant swarm zone relative to PA14 WT cysteine synthase B no swarm 3-oxoacyl-[acyl-carrier-protein] synthase no swarm III anthranilate synthase component I no swarm branched-chain amino acid transport less swarm protein BraG flagellar basal-body rod protein FlgC no swarm flagellin type B no swarm hypothetical protein no swarm flagellar capping protein FliD no swarm flagellar motor switch protein FliG no swarm probable flagellar assembly protein no swarm probable oxidoreductase less swarm Hypothetical no swarm probable transglycolase no swarm two-component sensor PhoQ no swarm putative transcriptional regulator no swarm putative multidrug resistance efflux no swarm pump putative transcriptional regulator no swarm putative acetyltransferase no swarm transcriptional regulator no swarm regulatory protein RsaL less swarm putative flagellar protein FliL no swarm flagellar biosynthetic protein FliP no swarm flagellar biosynthetic protein FliQ no swarm flagellar biosynthetic protein FlhB no swarm flagellar biosynthesis protein FlhA no swarm flagellar biosynthesis protein FlhF no swarm putative two-component sensor no swarm putative purine-binding chemotaxis no swarm protein CheW hypothetical protein no swarm less swarm transcriptional regulator Anr cytochrome oxidase subunit (cbb3-type) less swarm putative acyl-CoA dehydrogenase no swarm putative membrane protein less swarm Lon protease no swarm putative short-chain dehydrogenase no swarm methionine synthase no swarm putative MFS transporter no swarm conserved hypothetical protein no swarm putative sensor histidine kinase protein less swarm UTP-glucose-1-phosphate less swarm uridylyltransferase putative sensory box protein no swarm putative esterase no swarm  146  PA14 locus  PAO1 homolog  Polar effect possiblea  Gene Name  Gene Description  PA14_36310 PA14_35550 PA14_34450  PA2195 PA2245 PA2332  Nb Nb Nb  hcnC  PA14_33890 PA14_33650 PA14_33500 PA14_33290 PA14_33010 PA14_32860 PA14_32520 PA14_30840  PA2379 PA2399 PA2413 PA2423 PA2444 PA2455 PA2483 PA2571  Nb Nb Nb Nb Nb Nb Nb Nb  PA14_30660 PA14_30650 PA14_30630  PA2585 PA2586 PA2587  Nb Y Nb  uvrC gacA pqsH  PA14_30290 PA14_30200 PA14_30100 PA14_29830 PA14_29390 PA14_28950 PA14_27580 PA14_27090 PA14_26670  PA2615 PA2622 PA2630 PA2650 PA2685 PA2722 PA2821 PA2863 PA2891  Nb Nb Nb Nb Nb Nb Nb Nb Nb  ftsK cspD  PA14_26640  PA2893  Nb  atuH  PA14_26460  PA2909  Nb  cobK  PA14_25800  PA2957  Nb  PA14_25420 PA14_25110 PA14_24180 PA14_23830 PA14_23470  PA2989 PA3011 PA3091 PA3115 PA3141  Nb Nb Nb Nb Nb  PA14_23170 PA14_22020 PA14_21340 PA14_21320 PA14_21110  PA3175 PA3244 PA3300 PA3301 PA3319  Nb Nb Nb Nb Nb  PA14_20870 PA14_20630  PA3339 PA3359  Nb Nb  hydrogen cyanide synthase HcnC hypothetical protein putative transcriptional regulator, AraC family putative oxidoreductase pyoverdine synthetase D 2-ketoglutarate 4-aminotransferase Hypothetical serine hydroxymethyltransferase hypothetical protein putative flavin-dependent oxidoreductase putative signal transduction histidine kinase excinuclease ABC subunit C response regulator GacA probable FAD-dependent monooxygenase cell division/stress response protein cold-shock protein CspD conserved hypothetical protein putative methyltransferase conserved hypothetical protein conserved hypothetical protein putative glutathione S-transferase lipase modulator protein putative biotin carboxylase/biotin carboxyl carrier protein putative very-long-chain acyl-CoA synthetase ppfam02571, CbiJ, Precorrin-6x reductase putative transcriptional regulator, TetR family conserved hypothetical protein DNA topoisomerase I conserved hypothetical protein pilus assembly protein nucleotide sugar epimerase/dehydratase WbpM putative arginase family protein cell division inhibitor MinD long-chain-fatty-acid--CoA ligase putative lysophospholipase non-hemolytic phospholipase C precursor putative membrane protein conserved hypothetical protein  pvdD pvdH glyA2  lipH atuF  topA fimV wbpM  minD fadD2 plcN  Mutant swarm zone relative to PA14 WT no swarm less swarm less swarm no swarm no swarm no swarm less swarm no swarm no swarm no swarm no swarm less swarm hyperswarm hyperswarm no swarm hyperswarm less swarm no swarm no swarm no swarm hyperswarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm less swarm no swarm no swarm less swarm less swarm less swarm no swarm  147  PA14 locus  PAO1 homolog  Polar effect possiblea  Gene Name  PA14_20290 PA14_20280 PA14_20230  PA3385 PA3386 PA3391  Y Nb Nb  PA14_20110  PA3400  Nb  PA14_19630 PA14_19130 PA14_19120  PA3438 PA3476 PA3477  Nb Nb Nb  folE1 rhlI rhlR  PA14_18960 PA14_17900 PA14_17450  PA3488 PA3587 PA3625  Nb Nb Nb  metR surE  PA14_17410 PA14_17370 PA14_16890  PA3628 PA3631 PA3670  Nb Nb Nb  PA14_16040 PA14_15920  PA3738 PA3749  Nb Nb  PA14_15180  PA3781  Nb  PA14_14850 PA14_14500 PA14_14210 PA14_13510  PA3805 PA3828 PA3850 PA3895  Nb Nb Nb Nb  PA14_12030 PA14_11970  PA4005 PA4010  Nb Nb  PA14_11940 PA14_10440 PA14_10330  PA4012 PA4137 PA4144  Nb Nb Nb  PA14_09970 PA14_09750 PA14_09300  PA4168 PA4186 PA4223  Nb Nb Nb  PA14_55770 PA14_55920 PA14_56070  PA4292 PA4304 PA4315  Nb Nb Nb  PA14_56640 PA14_56730 PA14_57070 PA14_57100  PA4355 PA4362 PA4391 PA4393  Nb Nb Nb Nb  nosR  xerD  pilF  rcpA mvaT  Gene Description  DNA binding-protein conserved hypothetical protein NosR Regulatory protein for N2O reductase putative ABC-type multidrug transport permease GTP cyclohydrolase I precursor autoinducer synthesis protein RhlI acylhomoserine lactone dependent transcriptional regulator hypothetical protein transcriptional regulator MetR putative stationary-phase survival protein SurE putative esterase putative transport permease protein putative auxiliary component of ABC transporter integrase/recombinase XerD putative major facilitator family transporter putative ABC transport system, membrane protein type 4 fimbrial biogenesis protein PilF putative permease hypothetical protein putative transcriptional regulator, LysR family conserved hypothetical protein methylpurine-DNA glycosylase family protein hypothetical protein putative porin putative outer membrane protein precursor putative TonB-dependent receptor putative oxidoreductase putative ATP-binding component of ABC transporter putative probable phosphate transporter putative type II secretion system protein transcriptional regulator MvaT, P16 subunit putative MFS transporter conserved hypothetical protein conserved hypothetical protein putative permease  Mutant swarm zone relative to PA14 WT no swarm less swarm less swarm no swarm no swarm no swarm no swarm no swarm less swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm less swarm no swarm less swarm less swarm no swarm less swarm no swarm no swarm no swarm no swarm  148  PA14 locus  PAO1 homolog  Polar effect possiblea  PA14_57170  PA4398  Y  PA14_57180  PA4399  Nb  PA14_57480 PA14_58050 PA14_58070 PA14_58470 PA14_58560 PA14_60210  PA4423 PA4472 PA4474 PA4505 PA4513 PA4544  Nb Nb Nb Nb Nb Nb  PA14_60960 PA14_61080  PA4607 PA4616  Nb Nb  PA14_61320 PA14_61720 PA14_62130 PA14_62530 PA14_62710 PA14_62870 PA14_62900 PA14_63170 PA14_63370  PA4634 PA4667 PA4694 PA4725 PA4740 PA4752 PA4755 PA4778 PA4796  Nb Nb Nb Nb Nb Nb Nb Nb Nb  PA14_63680 PA14_63710 PA14_63860 PA14_64030 PA14_64170 PA14_64180 PA14_64190 PA14_64590 PA14_64930 PA14_65170 PA14_65320  PA4817 PA4819 PA4830 PA4842 PA4851 PA4852 PA4853 PA4887 PA4916 PA4934 PA4945  Nb Nb Nb Nb Y Y Nb Nb Nb Nb Nb  PA14_65420 PA14_65520 PA14_65990  PA4952 PA4958 PA4990  Nb Nb Nb  PA14_66120 PA14_66140 PA14_66640 PA14_66660 PA14_67090  PA5001 PA5002 PA5042 PA5044 PA5078  Nb Y Nb Y Nb  PA14_67240  PA5091  Nb  Gene Name  pmbA dppD rluD  ilvC cbrA pnp ftsJ greA  fis  rpsR miaA  qacH  pilO pilM  hutG  Gene Description  putative two-component response regulator putative cob(I)alamin adenosyltransferase putative lipoprotein PmbA protein putative tldD protein putative dipeptide ABC transporter Sulfite reductase ribosomal large subunit pseudouridine synthase D conserved hypothetical protein putative C4-dicarboxylate-binding protein conserved hypothetical protein conserved hypothetical protein ketol-acid reductoisomerase two-component sensor CbrA polyribonucleotide nucleotidyltransferase cell division protein FtsJ transcription elongation factor GreA putative transcriptional regulator putative glycine cleavage system H protein conserved hypothetical protein putative glycosyl transferase conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein putative tRNA-dihydrouridine synthase DNA-binding protein Fis putative MFS transporter putative ADP-ribose pyrophosphatase 30S ribosomal protein S18 delta 2-isopentenylpyrophosphate transferase putative GTPase conserved hypothetical protein putative SMR multidrug efflux transporter conserved hypothetical protein conserved hypothetical protein type 4 fimbrial biogenesis protein PilO type 4 fimbrial biogenesis protein PilM periplasmic glucan biosynthesis protein, MdoG N-formylglutamate amidohydrolase  Mutant swarm zone relative to PA14 WT no swarm less swarm less swarm no swarm no swarm no swarm no swarm less swarm no swarm no swarm less swarm no swarm no swarm no swarm less swarm no swarm no swarm no swarm less swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm less swarm no swarm no swarm less swarm no swarm less swarm no swarm no swarm no swarm no swarm no swarm  149  PA14 locus  PAO1 homolog  Polar effect possiblea  Gene Name  PA14_67420  PA5105  Y  hutC  PA14_67670 PA14_67680 PA14_67810 PA14_69090 PA14_69190 PA14_69220 PA14_69470  PA5124 PA5125 PA5134 PA5232 PA5239 PA5241 PA5261  Y Nb Nb Nb Nb Nb Nb  PA14_69560 PA14_70180 PA14_70570 PA14_70850  PA5267 PA5315 PA5345 PA5368  Nb Nb Nb Nb  PA14_71000  PA5376  Nb  PA14_71030  PA5378  Nb  PA14_72000 PA14_72490 PA14_72620  PA5454 PA5493 PA5503  Nb Nb Nb  PA14_73020  PA5536  Y  PA14_73120 PA14_07480 PA14_14350 PA14_15450  PA5545  Nb N/A N/A N/A  PA14_17160 PA14_17750 PA14_23430 PA14_23460 PA14_24360 PA14_30120 PA14_30210 PA14_32960 PA14_48120 PA14_54970 PA14_56280 PA14_56610 PA14_58760 PA14_59060 PA14_59560 PA14_60060 PA14_60290  N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A  Gene Description  histidine utilization genes repressor protein ntrB two-component sensor NtrB ntrC two-component response regulator NtrC putative carboxyl-terminal protease putative membrane fusion protein rho transcription termination factor Rho ppx exopolyphosphatase algR alginate biosynthesis regulatory protein AlgR hcpA secreted protein Hcp rpmG 50S ribosomal protein L33 recG ATP-dependent DNA helicase RecG pstC phosphate ABC transporter, permease protein putative lycine betaine/L-proline ABC transporter, ATP-binding subunit putative glycine betaine/L-proline ABC transporter, periplasmic component rmd oxidoreductase Rmd polA DNA polymerase I putative ATP-binding component of ABC transporter putative C4-type zinc finger protein, DksA/TraR family putative periplasmic transport protein putative reverse transcriptase hypothetical protein merD Mercuric resistence transcriptional repressor protein MerD hypothetical protein hypothetical protein ORF_11 putative heparinase wbpL putative glycosyltransferase L putative serine protease hypothetical protein putative cytoplasmic protease hypothetical protein hypothetical protein probable ABC transporter protein conserved hypothetical protein hypothetical protein pilC type 4 fimbrial biogenesis protein pilC putative DNA binding protein transposase hypothetical protein pilW type 4 fimbrial biogenesis protein PilW  Mutant swarm zone relative to PA14 WT no swarm no swarm no swarm less swarm less swarm no swarm less swarm less swarm no swarm less swarm no swarm no swarm no swarm no swarm no swarm less swarm less swarm less swarm no swarm no swarm no swarm less swarm no swarm no swarm less swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm no swarm less swarm no swarm less swarm no swarm no swarm  150  PA14 locus  PA14_60310 PA14_61380 PA14_62030 PA14_63610 PA14_67070 PA14_69920  PAO1 homolog  Polar effect possiblea  Gene Name  N/A N/A N/A N/A N/A N/A  pilY1  Gene Description  Mutant swarm zone relative to PA14 WT type 4 fimbrial biogenesis protein PilY1 no swarm conserved hypothetical protein no swarm putative paraquat-inducible protein A no swarm hypothetical protein no swarm hypothetical protein no swarm hypothetical protein no swarm  Transposon mutants are organized in ascending order of PAO1 ortholog. The list represents all of the swarming deficient mutants verified on BM2 swarm agar plates. No swarm, surface coverage of mutant relative to PA14 WT is < 25 %; Less swarm, surface coverage of mutants relative to PA14 WT is < 50 %; Hyperswarm, surface coverage of mutant relative to PA14 WT is > 25 %. a Possibility of transposon insertion affecting expression on downstream genes. Y, yes; N, no; N/A, not applicable. b No evidence for polar effect identified from our screens.  151  Appendix B  Figure B.1 Classification of swarming-associated genes according to their predicted functions. Each gene may be represented more than once.  152  Figure B.2 Classification of genes from PA14 genome according to their predicted functions. Each gene may be represented more than once.  153  Figure B.3 Comparison of the functional composition of the swarming-associated genes to the composition of the PA14 genome. Each gene may be represented more than once.  154  Appendix C Table C-1 List of P. aeruginosa genes with expression up- or down-regulated in the cbrA mutant compared to the PA14 WT. Fold Gene Name Function change P-value PA0049 hypothetical protein -2.0311 0.0144 PA0051 phzH potential phenazine-modifying enzyme 2.9015 0.0202 PA0077 icmF1 hypothetical protein -2.304 0.0059 PA0129 gabP gamma-aminobutyrate permease -2.7138 0.0043 PA0149 probable sigma-70 factor, ECF subfamily 2.0006 0.039 PA0159 probable transcriptional regulator -2.0877 0.0257 PA0161 hypothetical protein -2.558 0.0016 PA0192 probable TonB-dependent receptor 2.6847 0.0248 PA0302 spuF polyamine transport protein PotG -2.1286 0.0438 PA0325 probable permease of ABC transporter -2.2531 0.0349 PA0356 hypothetical protein -3.6835 0.0033 PA0447 gcdH glutaryl-CoA dehydrogenase -2.1059 0.0014 PA0482 glcB malate synthase 2.0296 0.0535 PA0510 nirE probable uroporphyrin-III c-methyltransferase 5.4536 0.0217 PA0511 nirJ heme d1 biosynthesis protein NirJ 5.9398 8.7E-05 PA0512 nirH hypothetical protein 3.1707 0.0001 PA0513 nirG probable transcriptional regulator 6.2223 0.0002 PA0514 nirL heme d1 biosynthesis protein NirL 5.9581 0.0004 PA0515 nirD probable transcriptional regulator 5.5383 0.0004 PA0516 nirF heme d1 biosynthesis protein NirF 5.8904 3.2E-05 PA0517 nirC probable c-type cytochrome precursor 8.0915 8.8E-06 PA0518 nirM cytochrome c-551 precursor 4.5102 1.1E-05 PA0519 nirS nitrite reductase precursor 12.4263 2.1E-06 PA0520 nirQ regulatory protein NirQ 6.471 4.5E-06 PA0521 nirO probable cytochrome c oxidase subunit 3.9342 0.0005 PA0522 nirP hypothetical protein 3.9254 0.0047 PA0523 norC nitric-oxide reductase subunit C 12.5408 3.8E-06 PA0524 norB nitric-oxide reductase subunit B 20.5859 8.6E-08 PA0525 norD probable dinitrification protein NorD 4.0052 0.0003 PA0526 hypothetical protein 2.6402 0.0012 PA0567 yqaE hypothetical protein -2.8167 0.0073 PA0577 dnaG DNA primase 2.0784 0.0021 PA0621 hypothetical protein -3.2878 0.0007 PA0663 hypothetical protein -2.2908 0.0005  155  Gene PA0669 PA0677 PA0744 PA0779 PA0796 PA0887 PA0931 PA0949 PA0958 PA0992 PA1053 PA1089 PA1095 PA1126 PA1159 PA1178 PA1179 PA1219 PA1243 PA1280 PA1299 PA1301 PA1323 PA1324 PA1343 PA1414 PA1423 PA1545 PA1555 PA1556 PA1559 PA1560 PA1596 PA1682 PA1746 PA1778 PA1797  Name Function dnaE2 DNA polymerase III subunit alpha hxcW HxcW putative pseudopilin probable enoyl-CoA hydratase/isomerase probable ATP-dependent protease carboxyphosphonoenolpyruvate prpB phosphonomutase acsA acetyl-coenzyme A synthetase pirA ferric enterobactin receptor PirA wrbA Trp repressor binding protein WrbA Basic amino acid, basic peptide and imipenem oprD outer membrane porin OprD precursor cupC1 fimbrial subunit CupC1 slyB hypothetical protein hypothetical protein fliS flagellar protein FliS hypothetical protein probable cold-shock protein PhoP/Q and low Mg2+ inducible outer membrane oprH protein H1 precursor phoP two-component response regulator PhoP hypothetical protein probable sensor/response regulator hybrid cobC hypothetical protein ycgN hypothetical protein probable transmembrane sensor hypothetical protein hypothetical protein hypothetical protein hypothetical protein bdlA probable chemotaxis transducer hypothetical protein ccoP probable cytochrome c ccoO probable cytochrome c oxidase subunit hypothetical protein hypothetical protein htpG heat shock protein 90 probable MFS metabolite transporter hypothetical protein cobA uroporphyrin-III C-methyltransferase hypothetical protein  Fold change 2.1081 -2.041 -2.107 3.0885  P-value 0.0144 0.0107 0.002 0.0003  -2.6746 -2.0483 -2.2746 -2.4424  0.0005 0.0016 0.041 0.0061  -2.0385 2.3516 2.0154 -2.3844 2.0724 -2.0778 -2.3587  0.0011 0.0011 0.0015 0.0016 0.0086 0.0147 0.0353  9.3553 4.1577 2.0402 -2.0879 -3.3204 2.0628 2.0677 -2.5286 -2.5656 4.3834 2.2419 2.5475 2.1654 4.2586 2.0888 5.5015 5.4667 2.2073 -2.1274 2.8784 -2.2021 10.3739  1.6E-06 2.7E-05 0.0171 0.0518 0.0138 0.0151 0.0044 0.0164 0.001 5.7E-05 0.0039 0.0007 0.0042 0.0005 0.0314 2.4E-05 1.4E-05 0.0016 0.0273 0.0002 0.004 1.2E-06 156  Gene PA1847 PA1856 PA1892 PA1894 PA1895 PA1896 PA1897 PA1898 PA1912  Name yhgI ccoN  PA1927 PA1972  metE  PA2019 PA2070 PA2146 PA2176 PA2192 PA2196 PA2197 PA2198 PA2256 PA2273 PA2293 PA2346 PA2356 PA2358 PA2381 PA2385 PA2386  mexX  PA2390 PA2393 PA2397 PA2399 PA2426 PA2468 PA2501 PA2513  qscR feml  yciG  ycnB pvcC soxR  msuD  pvdQ pvdA  pvdE pvdD pvdS foxl antB  Function hypothetical protein probable cytochrome oxidase subunit hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein quorum-sensing control repressor probable sigma-70 factor, ECF subfamily 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase hypothetical protein RND multidrug efflux membrane fusion protein precursor hypothetical protein hypothetical protein hypothetical protein hypothetical protein probable transcriptional regulator hypothetical protein hypothetical protein pyoverdine biosynthesis protein PvcC probable transcriptional regulator hypothetical protein hypothetical protein methanesulfonate sulfonatase MsuD hypothetical protein hypothetical protein PvdQ L-ornithine N5-oxygenase probable ATP-binding/permease fusion ABC transporter probable dipeptidase precursor pyoverdine biosynthesis protein PvdE pyoverdine synthetase D sigma factor PvdS probable sigma-70 factor, ECF subfamily hypothetical protein anthranilate dioxygenase small subunit  Fold change -2.5953 -2.1866 -2.025 -2.1552 -2.4433 -4.0073 -2.7206 -2.0741 2.5722  P-value 0.0073 0.0017 0.0106 0.0011 0.0005 4.2E-05 0.0004 0.0025 0.0007  2.5864 -2.146  0.0407 0.0088  2.7926 -2.2362 2.605 2.0961 -2.0021 2.0651 3.0586 3.7261 -2.8106 2.0956 2.0851 -2.3911 2.0391 7.8228 3.2434 -3.0139 -2.3937  0.0004 0.0105 0.0051 0.0306 0.0018 0.0038 0.0024 0.001 0.0007 0.0322 0.0248 0.0196 0.0026 2.6E-06 0.0001 0.002 0.0011  -2.1668 -2.1717 -3.4386 -2.236 5.4873 4.1088 2.3933 -2.6097  0.0134 0.0527 0.002 0.0061 6.5E-06 6.4E-05 0.0028 0.0028  157  Gene  Name  PA2621 PA2655 PA2750 PA2760 PA2808 PA2830 PA2840 PA2920 PA2931 PA2932  clpS  PA2933 PA2934 PA3011 PA3049 PA3126 PA3157 PA3188 PA3189  oprQ ptrA htpX deaD cifR morB  cif topA rmf ibpA wbpC gltG gltF  PA3190 PA3216 PA3283 PA3284 PA3287 PA3349 PA3351 PA3352 PA3370 PA3391 PA3392 PA3414  gltB  PA3416  pdhB  PA3417 PA3418 PA3423 PA3441  flgM flgN nosR nosZ  ldh ssuF  Function ATP-dependent Clp protease adaptor protein ClpS hypothetical protein hypothetical protein probable outer membrane protein precursor hypothetical protein heat shock protein HtpX probable ATP-dependent RNA helicase probable chemotaxis transducer probable transcriptional regulator morphinone reductase probable major facilitator superfamily (MFS) transporter probable hydrolase DNA topoisomerase I ribosome modulation factor heat-shock protein IbpA probable acetyltransferase probable permease of ABC sugar transporter probable permease of ABC sugar transporter probable binding protein component of ABC sugar transporter hypothetical protein hypothetical protein hypothetical protein hypothetical protein probable chemotaxis protein probable transcriptional regulator hypothetical protein hypothetical protein regulatory protein NosR nitrous-oxide reductase precurser hypothetical protein probable pyruvate dehydrogenase E1 component, beta chain probable pyruvate dehydrogenase E1 component, alpha subunit leucine dehydrogenase probable transcriptional regulator probable molybdopterin-binding protein  Fold change  P-value  2.1079 24.3088 2.4205 -2.1136 2.0544 3.2701 2.2125 2.1365 3.6046 12.3391  0.0268 3.1E-07 0.0237 0.0033 0.0059 0.0001 0.0056 0.0068 0.0002 4.4E-07  20.3542 4.5283 -3.298 2.6184 3.2645 -2.3034 -2.7564 -3.284  4.7E-07 0.0002 0.0007 0.0028 6.2E-05 0.0186 0.0258 0.0009  -2.6502 2.2347 5.4021 3.1421 2.4725 2.1499 2.5076 2.5091 2.933 3.2402 5.267 -2.3018  0.0007 0.0044 5.7E-05 0.0001 0.0185 0.0013 0.0025 0.0013 0.0155 0.0059 3.1E-05 0.0027  2.0906  0.0033  2.0729 3.2264 -2.2598 -2.2456  0.0026 8.8E-05 0.017 0.0564 158  Gene  Name  PA3522 PA3526 PA3535 PA3545 PA3552 PA3553 PA3554 PA3555 PA3556 PA3557 PA3558 PA3559 PA3575 PA3584 PA3588 PA3600 PA3601 PA3739 PA3784 PA3899 PA3911 PA3955 PA4022 PA4063 PA4070 PA4139 PA4140 PA4155 PA4197 PA4217 PA4218  mexF motY eprS algG pmrH pmrF arnA amrJ arnT pmrL pmrM  PA4223 PA4224 PA4225 PA4231 PA4250 PA4340  pchH pchG pchF pchA rpsN  glpD opdR rpl36 ykgM  fecI yhbT exaC2  choS  phzS  Function probable Resistance-Nodulation-Cell Division (RND) efflux transporter probable outer membrane protein precursor probable serine protease alginate-c5-mannuronan-epimerase AlgG hypothetical protein probable glycosyl transferase hypothetical protein hypothetical protein inner membrane L-Ara4N transferase ArnT hypothetical protein hypothetical protein probable nucleotide sugar dehydrogenase hypothetical protein glycerol-3-phosphate dehydrogenase probable porin hypothetical protein hypothetical protein probable sodium/hydrogen antiporter hypothetical protein probable sigma-70 factor, ECF subfamily hypothetical protein hypothetical protein probable aldehyde dehydrogenase hypothetical protein probable transcriptional regulator hypothetical protein hypothetical protein hypothetical protein probable two-component sensor flavin-containing monooxygenase probable transporter probable ATP-binding component of ABC transporter pyochelin biosynthetic protein PchG pyochelin synthetase salicylate biosynthesis isochorismate synthase 30S ribosomal protein S14 hypothetical protein  Fold change  P-value  -2.019 2.2946 -2.1309 2.0846 6.161 3.6112 2.4426 4.494 3.1311 4.5067 2.4548 4.8091 2.3298 2.3586 -2.1308 5.7887 5.4264 -2.0776 -3.4318 2.8811 2.371 -2.105 -2.0319 3.2824 -2.0783 2.2414 2.547 2.448 2.7601 -2.5668 -2.5035  0.0424 0.0015 0.0044 0.0068 6.8E-06 5.2E-05 0.0021 1.6E-05 0.0001 3E-05 0.0028 0.0002 0.0056 0.0154 0.0136 7.6E-06 9.1E-06 0.0346 0.0011 0.0006 0.0047 0.004 0.0048 0.0235 0.0025 0.0017 0.0024 0.0009 0.0004 0.0004 0.0012  -4.1634 -2.3744 -2.5269 -2.4796 -2.0109 -2.0868  8.6E-05 0.0006 0.0005 0.0009 0.0358 0.0451  159  Gene PA4345 PA4352 PA4353 PA4355 PA4356 PA4359 PA4387 PA4467 PA4468 PA4469 PA4470 PA4471 PA4515 PA4542 PA4577 PA4629 PA4659 PA4691 PA4707 PA4708 PA4709 PA4736 PA4752 PA4761 PA4762 PA4773 PA4774 PA4775 PA4776 PA4777 PA4782 PA4791 PA4799 PA4862 PA4876 PA4880  Name  Function hypothetical protein hypothetical protein yajB hypothetical protein probable major facilitator superfamily (MFS) araJ transporter xenB xenobiotic reductase feoA hypothetical protein fxsA hypothetical protein hypothetical protein sodM superoxide dismutase hypothetical protein fumC1 fumarate hydratase fagA hypothetical protein piuC putative hydroxylase clpB ClpB protein hypothetical protein hypothetical protein probable transcriptional regulator hypothetical protein phuU probable permease of ABC transporter phuT hypothetical protein phuS probable hemin degrading factor hypothetical protein ftsJ cell division protein FtsJ dnaK molecular chaperone DnaK grpE heat shock protein GrpE hypothetical protein hypothetical protein hypothetical protein PmrA: two-component regulator system response pmrA regulator PmrA PmrB: two-component regulator system signal pmrB sensor kinase PmrB hypothetical protein hypothetical protein hypothetical protein probable ATP-binding component of ABC transporter osmE osmotically inducible lipoprotein OsmE probable bacterioferritin  Fold change -2.5806 2.649 -3.2934  P-value 0.0022 0.0003 0.0019  2.1164 2.2694 3.7666 3.5119 2.8121 3.5549 5.495 6.2422 7.7982 2.6834 4.8096 2.4297 2.9319 2.095 2.5668 2.0306 4.2018 2.568 -2.3471 2.5949 3.5718 2.9529 3.7248 5.3255 2.7356  0.0507 0.0039 0.0004 0.0017 0.0085 8.9E-05 3.8E-05 2.3E-05 9.1E-05 0.0102 0.0008 0.0167 0.0149 0.0025 0.0505 0.002 6.1E-05 0.0013 0.0228 0.0025 5.3E-05 0.0004 0.0001 2.4E-05 0.0019  3.1231  0.0002  2.0625 4.2428 2.8921 -2.2047  0.0017 0.0001 0.0145 0.013  -2.4016 -2.0019 -2.738  0.0106 0.0161 0.0466 160  Gene PA5053 PA5054 PA5099 PA5100 PA5104 PA5106 PA5172 PA5180 PA5200  Name hslV hslU codB hutU  PA5217 PA5287 PA5315 PA5427 PA5442 PA5460 PA5475 PA5500 PA5526 PA5530 PA5531 PA5553  fbpA amtB rpmG adhA  arcB fdhD ompR  znuC kgtP tonB atpC  Function ATP-dependent protease peptidase subunit ATP-dependent protease ATP-binding subunit probable transporter urocanate hydratase hypothetical protein atrazine chlorohydrolase ornithine carbamoyltransferase, catabolic hypothetical protein two-component response regulator OmpR probable binding protein component of ABC iron transporter ammonium transporter AmtB 50S ribosomal protein L33 alcohol dehydrogenase hypothetical protein hypothetical protein hypothetical protein zinc transport protein ZnuC hypothetical protein probable MFS dicarboxylate transporter TonB protein ATP synthase subunit epsilon  Fold change 4.8979 3.0681 -2.2205 -3.6371 -2.4598 -2.2776 2.3366 -2.3662 2.0163  P-value 9.7E-05 0.0054 0.0042 0.0004 0.0106 0.0011 0.0006 0.0013 0.0023  2.9912 -2.0117 -2.0239 2.0689 -2.7624 2.9871 2.0644 2.1029 -2.2934 -2.2247 2.555 -2.1825  0.05 0.0177 0.025 0.0022 0.0139 0.0001 0.0103 0.0118 0.0303 0.0049 0.0009 0.0014  161  

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