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Global regulation of the Lon protease of Pseudomonas aeruginosa and its influence on ciprofloxacin resistance… Breidenstein, Elena Bernadette Monika 2012

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GLOBAL REGULATION OF THE LON PROTEASE OF PSEUDOMONAS AERUGINOSA AND ITS INFLUENCE ON CIPROFLOXACIN RESISTANCE AND VIRULENCE  by ELENA BERNADETTE MONIKA BREIDENSTEIN DIPLOM, Eberhard Karls Universitӓt Tübingen, Germany, 2006  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) APRIL 2012  Elena Bernadette Monika Breidenstein, 2012  Abstract This thesis focuses on investigating global regulation, ciprofloxacin resistance and virulence by the ATP-dependent Lon protease of Pseudomonas aeruginosa. Screening a P. aeruginosa PA14 mutant library for non-essential genes involved in altered ciprofloxacin susceptibility identified more than 100 genes (35 involved in intrinsic and 79 in mutational resistance). The identification of known and novel genes involved in resistance through mutant library screening provided new insights into the ciprofloxacin resistome. These mutants provide insights into adaptive resistance mechanisms and the clinical phenomenon of creeping baselines. The ATP-dependent Lon protease, which showed a 4-8 fold increase in ciprofloxacin susceptibility upon mutation, was chosen for more detailed studies. This study showed that Lon protease regulated antibiotic resistance and virulence properties despite the fact that it is not a traditional regulator. The ciprofloxacin susceptible phenotype of a lon mutant could be complemented. Furthermore, the lon mutant was identified to influence cytotoxicity, adhesion, anaerobic growth and metabolism as well as impact on global regulation. Microarray analysis showed that Lon is at the top of a transcriptional hierarchy dysregulating around 200 genes. Proteomic profiling showed that Lon appeared to be involved in cleaving GroEL, Hfq and KatA amongst others. Furthermore, mechanistic studies on the involvement of Lon protease in the SOS response under sub-inhibitory concentrations of ciprofloxacin revealed that SOS response was less induced in the lon mutant which could be explained by the action of Lon on the key SOS regulator RecA. Lon protease influences virulence in vivo as shown in a lettuce leaf model, an amoeba assay as well as a rat model of chronic infection. The alterations in virulence-related processes in vitro in a lon mutant were also paralleled by defective virulence in vivo. Antibiotic resistance and motility phenotypes were also investigated for other proteases and mutations in pfpI, clpP and clpS had distinct, but overlapping phenotypes cf. the lon mutant. Overall, my results suggest that while the Lon protease is not a traditional regulator, it is still involved in a multitude of cellular processes highlighting its importance for the bacterial cell. Thus, it would be a good target for therapy.  ii  Preface Part of the work presented in this thesis has already been published and the corresponding publications are listed by chapters below and copyright permission has been granted. Other studies have not yet been published and are in the process of submission. Some experiments carried out in collaboration are crucial for the story developed in this thesis, and are thus included here. In each case these are clearly labelled as collaborative research and the individual who performed the work is acknowledged.  Chapter 1:  Fernandez L., E.B.M. Breidenstein and R.E.W. Hancock. 2012. Importance of adaptive and stepwise changes in the rise and spread of antimicrobial resistance. In: P.L. Keen and M.H.M.M. Montforts (eds). Antimicrobial Resistance in the Environment, pp. 43-71. WileyBlackwell.  Schurek K.N., E.B.M. Breidenstein and R.E.W. Hancock. 2011. Pseudomonas aeruginosa: a persistent pathogen in cystic fibrosis and hospital-associated infections. In: T.J. Dougherty and M.J. Pucci (eds). Antibiotic Drug Discovery and Development, pp. 679-715. Springer Publishing Company. Breidenstein E.B.M., C. de la Fuente-Núñez and R.E.W. Hancock. 2011. Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol. 19(8):419-426. Review  Breidenstein E.B.M., L. Fernandez and R.E.W. Hancock. 2011. Creeping Baselines and Adaptive Resistance to Antibiotics. Drug Resistance Updates. Feb;14(1):1-21. Review The introduction is mostly taken from review articles and book chapters written by Dr. K.N. Schurek, Dr. L. Fernandez, C. de la Fuente-Núñez, Dr. R.E.W. Hancock and me. All manuscripts were edited by Dr. R.E.W. Hancock. The sections written by me are used directly in this introduction and sections written by other authors are either not mentioned or rewritten for the iii  purpose of this thesis. The sections of the two review articles and two book chapters were combined and expanded to give a complete background introduction for this thesis.  Chapter 3:  Breidenstein E.B.M, B.K. Khaira, I. Wiegand, J. Overhage and R.E.W. Hancock. 2008. Complex ciprofloxacin resistome revealed by screening a Pseudomonas aeruginosa mutant library for altered susceptibility. Antimicrob Agents Chemother 52:4486-91. Chapter 3 was mainly taken from the manuscript on the ciprofloxacin resistome for which I performed all the experiments. Dr. I. Wiegand and Dr. J. Overhage gave guidance and helped with the experimental setup. The figure of the “Distribution of genes around the PAO1 genome“ was created by B.K. Khaira. Dr. R.E.W. Hancock supervised the research, provided suggestions as to direction and edited the manuscript. Additional experiments on the fleS mutant were performed by me, except for the cytotoxicity assay that was performed by S. Gellatly.  Chapter 4:  Breidenstein E.B.M., M. Bains and R.E.W. Hancock. 2012. Involvement of the Lon protease in the SOS response triggered by ciprofloxacin in Pseudomonas aeruginosa PAO1. Antimicrob Agents Chemother doi:10.1128/AAC.06014-11. Chapter 4 was taken from the manuscript that was recently accepted. I performed all the experiments presented in this chapter, except the microarray experiment which was done with the help of M. Bains. Dr. R.E.W. Hancock supervised the research, provided suggestions as to direction and edited the manuscript.  iv  Chapter 5: Experiments in this chapter were all conducted by me, except for the microarray experiments that were done with the help of M. Bains. For the SILAC experiment, I worked with Dr. L.J. Foster (UBC, Vancouver, Canada) who trained me on performing these methods.  Chapter 6:  Breidenstein, E.B.M., P.K. Taylor, Irena Kukavica-Ibrulj, S.N. Gellatly, J. Overhage, R.C. Levesque and R.E.W. Hancock. Involvement of the Lon protease in the pathogenesis of P. aeruginosa. In preparation. This chapter derived mainly from work in collaboration with several people. I was directly involved in the adhesion/invasion assays towards HBEs, the lettuce leaf virulence model as well as all experiments carried out with mucin, such as motility, antibiotic resistance and biofilm. S.N. Gellatly and P.K. Taylor were involved in performing the cytotoxicity assay, Dr. J. Overhage (KIT, Karlsruhe, Germany) performed the amoeba virulence model and Dr. R.C. Levesque (University Laval, Quebec, Canada) was involved with the chronic lung infection model in rats. Dr. R.E.W. Hancock supervised the research, provided suggestions as to direction and edited the manuscript.  Chapter 7:  Breidenstein E.B.M., L. Fernandez, D. Song and R.E.W. Hancock. 2011. Analysis of the role played by intracellular proteases in the antibiotic resistance, motility, and biofilm formation of Pseudomonas aeruginosa. Antimicrob Agents Chemother 56:1128-32. Chapter 7 is a draft of a manuscript that was recently accepted. This project on intracellular proteases was originally started by an honour thesis student D. Song who was supervised by me and Dr. L. Fernandez. Dr. L. Fernandez and I repeated these experiments (swarming, swimming, twitching, biofilm formation and MIC) and also performed additional experiments. v  I was directly involved in the RT-qPCR and PCR experiments, Congo red assay, growth curve, clpP and pfpI cloning as well as the MIC determination and the nitrocefin assay. The manuscript was largely written by Dr. L. Fernandez and thus for the purpose of this thesis, the manuscript was rewritten. Dr. R.E.W. Hancock supervised the research, provided suggestions as to direction and edited the manuscript. Dr. L. Fernandez, D. Song and I share first authorship.  vi  Table of Contents    Abstract.......................................................................................................................................... ii Preface........................................................................................................................................... iii Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations .................................................................................................................. xv Acknowledgements ................................................................................................................... xvii Dedication ................................................................................................................................. xviii 1.   Introduction ........................................................................................................................... 1  1.1  P. aeruginosa ................................................................................................................... 1  1.1.1  Pathogenesis and virulence factors ............................................................................ 1  1.1.2   P. aeruginosa as a “Superbug” .................................................................................. 6   1.1.3   Antibiotic resistance in P. aeruginosa ....................................................................... 6   1.1.4   Correlation between antibiotic resistance and virulence ......................................... 14   1.1.5   P. aeruginosa hospital-associated acute infections ................................................. 15   1.1.6   Chronic P. aeruginosa infections in CF .................................................................. 16   1.1.7   Treatment options for P. aeruginosa infections in patients with CF....................... 17   1.1.8   The challenge of treating the “Superbug” P. aeruginosa ........................................ 20   1.2  Ciprofloxacin ................................................................................................................. 21  1.2.1  Structure and activity ............................................................................................... 21  1.2.2   Mechanisms of fluoroquinolone resistance ............................................................. 22   1.3  Lon protease................................................................................................................... 23  1.4  Rationale and objectives ................................................................................................ 25  1.4.1  Hypothesis ............................................................................................................... 25   2.   1.4.2   Overall aim .............................................................................................................. 25   1.4.3   Objectives ................................................................................................................ 25  Material and methods ......................................................................................................... 27  2.1  Bacterial strains and growth conditions ......................................................................... 27  2.2  Mutant library agar screen ............................................................................................. 28  2.3  Primer design ................................................................................................................. 29  vii  2.4  2.5  2.6  2.7  2.8  2.9  2.10  2.11  2.12  2.13  2.14  2.15  2.16  2.17  2.18  2.19  2.20  2.21  2.22  2.23  2.24  2.25  2.26  2.27  2.28  2.29  2.30  2.31  2.32  2.33  2.34  2.35  2.36  2.37  2.38  3.   PCR amplification ......................................................................................................... 29  Complementation of lon ................................................................................................ 29  Overexpression studies .................................................................................................. 29  Minimal inhibitory concentration (MIC) ....................................................................... 30  Growth experiments....................................................................................................... 30  Killing assay .................................................................................................................. 30  UV irradiation assay ...................................................................................................... 31  Heat shock ..................................................................................................................... 31  Microarray analysis ....................................................................................................... 31  Reverse transcription reaction ....................................................................................... 32  Real-time PCR (qPCR) .................................................................................................. 32  Anaerobic growth .......................................................................................................... 33  Cellular respiration assay WST1 ................................................................................... 33  Swarming motility assay................................................................................................ 33  Swimming motility assay .............................................................................................. 34  Twitching motility assay................................................................................................ 34  Measurement of rapid attachment ................................................................................. 34  Measurement of mature biofilm formation.................................................................... 34  Congo red assay ............................................................................................................. 35  Nitrocefin assay ............................................................................................................. 35  Morphological determination ........................................................................................ 35  Lactate dehydrogenase (LDH) – release assay .............................................................. 35  Adhesion assay .............................................................................................................. 36  Lettuce leaf virulence model ......................................................................................... 36  Amoeba virulence model ............................................................................................... 37  Competitive index (CI) determination ........................................................................... 37  Mucin ............................................................................................................................. 38  Construction of a lon deletion mutant in an auxotrophic background........................... 38  Extraction of the cytoplasmic fraction ........................................................................... 39  SILAC: stable isotope labelling by amino acids in cell culture..................................... 39  Whole cell preparation ................................................................................................... 40  Protein gel ...................................................................................................................... 40  Western blot ................................................................................................................... 41  Measurement of oxidative stress ................................................................................... 41  Pathway analysis ............................................................................................................ 41  A complex ciprofloxacin resistome revealed by screening a P. aeruginosa mutant library for altered susceptibility....................................................................................... 42  3.1  Introduction.................................................................................................................... 42  3.2  Results and discussion ................................................................................................... 42  3.2.1  Ciprofloxacin library screen .................................................................................... 42  3.2.2   Functional classes involved in altered ciprofloxacin susceptibility ........................ 46   3.2.3   FleS plays part in antibiotic resistance and virulence.............................................. 49   3.3   Concluding remarks ....................................................................................................... 53   viii  4.   Involvement of the Lon protease in the SOS response triggered by ciprofloxacin in P. aeruginosa PAO1 ............................................................................................................... 55  4.1  Introduction.................................................................................................................... 55  4.2  Results............................................................................................................................ 56  4.2.1  The lon mutant displays enhanced susceptibility to ciprofloxacin .......................... 56  4.2.2   Altered gene expression in P. aeruginosa wild type strain PAO1 and lon mutant under sub-inhibitory concentrations of ciprofloxacin.............................................. 57   4.2.3   Role of the Lon protease in the presence of ciprofloxacin ...................................... 59   4.2.4   The lon mutant demonstrates increased sensitivity to UV irradiation..................... 60   4.2.5   Dysregulation of lon during lethal UV light and ciprofloxacin exposure ............... 61   4.2.6   Phenotypic effects of sulA (PA3008) and lexA (3007) overexpression................... 61   4.2.7   Effect of sulA overexpression on transcriptional regulation.................................... 64   4.2.8   Effect of ciprofloxacin on protein expression in the lon mutant ............................. 65   4.3  5.   Regulation by the Lon protease ......................................................................................... 70  5.1  Introduction.................................................................................................................... 70  5.2  Results............................................................................................................................ 71  5.2.1  Transcriptional regulation........................................................................................ 71  5.2.2   Anaerobic growth and anaerobic biofilm formation ............................................... 80   5.2.3   WST1 assay ............................................................................................................. 82   5.2.4   Overexpression of lon leads to a similar swarming defect as a mutation in lon ..... 83   5.2.5   Upregulation of the lon gene under certain stress conditions .................................. 84   5.2.6   Post-translational regulation: SILAC (stable isotope labelling of amino acids in cell culture) ..................................................................................................................... 85   5.3  6.   Discussion ...................................................................................................................... 67  Discussion ...................................................................................................................... 88  Involvement of the Lon protease in the pathogenesis of P. aeruginosa ......................... 94  6.1  Introduction.................................................................................................................... 94  6.2  Results............................................................................................................................ 96  6.2.1  Reduced cytotoxicity of the lon mutant towards human bronchial epithelial cells . 96  6.2.2   Lon mutants show impaired virulence in the lettuce leaf model ............................. 98   6.2.3   Lon alters virulence towards amoeba ...................................................................... 99   6.2.4   A chronic lung infection model demonstrates that a mutation in lon leads to a major defect in growth and maintenance in vivo ............................................................. 102   6.2.5   Mucin alteration of motility, ciprofloxacin resistance and biofilm formation in P. aeruginosa ............................................................................................................. 103   6.3   Discussion .................................................................................................................... 106  ix  7.   Role of other intracellular proteases in motility, biofilm formation and antibiotic resistance of P. aeruginosa .............................................................................................. 111  7.1  Introduction.................................................................................................................. 111  7.2  Results.......................................................................................................................... 112  7.2.1  Several protease mutants exhibit a motility defect ................................................ 112  7.2.2   Altered biofilm formation of the swarming-deficient protease mutants ............... 114   7.2.3   Mutations in intracellular proteases affect antibiotic resistance ............................ 116   7.2.4   clpP and lon are not located in an operon ............................................................. 117   7.2.5   clpP and lon do not regulate each other................................................................. 119   7.3  8.   Discussion .................................................................................................................... 119  Conclusions and future directions ................................................................................... 122  References .................................................................................................................................. 127 Appendix .................................................................................................................................... 154   x  List of Tables Table 1.1. Comparison between the 3 major types of antibiotic resistance. .................................. 7 Table 1.2. Empiric therapy for the treatment of P. aeruginosa infections in patients with cystic fibrosis........................................................................................................................................... 18 Table 2.1. P. aeruginosa strains and plasmids used in this study. ............................................... 27 Table 4.1. Selected P. aeruginosa genes differentially expressed in the lon mutant in mid-log phase microarray under sub-inhibitory (one half MIC) concentrations of ciprofloxacin. ............ 58 Table 4.2. Expression of selected genes involved in SOS response and DNA damage repair under various conditions. .............................................................................................................. 59 Table 4.3. Expression of the lon gene in response to lethal concentrations of ciprofloxacin and UV light compared to the wild type. ............................................................................................. 61 Table 4.4. Effects on SOS transcriptional regulation of the sulA overexpressing strain compared to the vector control after sub-inhibitory ciprofloxacin induction. ............................................... 65 Table 5.1. Selected P. aeruginosa genes differentially expressed in the lon mutant in midlogarithmic phase microarray. ...................................................................................................... 73 Table 5.2. Selected P. aeruginosa genes differentially expressed in the lon mutant in stationary phase microarray. .......................................................................................................................... 75 Table 5.3. Selected P. aeruginosa genes differentially expressed in the lon mutant under swarming conditions. .................................................................................................................... 76 Table 5.4. Expression of the lon gene under wild type swarming vs. swimming conditions as assessed by RT-qPCR. .................................................................................................................. 77 Table 5.5. Impact of downstream regulators of lon on the other regulators measured by RTqPCR. ............................................................................................................................................ 79 Table 5.6. Effects of stress conditions on lon gene expression. ................................................... 85 Table 7.1. P. aeruginosa PA14 transposon mutants used in this study. .................................... 112 Table 7.2. MICs (µg/ml) of selected mutants to various antibiotics. ......................................... 116 Table 7.3. Expression level of lon in the clpP mutant and of clpP in the lon mutant. ............... 119   xi  List of Figures Figure 1.1. Development of a P. aeruginosa biofilm. ................................................................... 5 Figure 1.2. Chemical structure of ciprofloxacin. ......................................................................... 22 Figure 1.3. Mechanisms of fluoroquinolone resistance in Gram-negative bacteria include: ...... 23 Figure 3.1. Distribution of genes around the PAO1 genome identified in the ciprofloxacin library screen which exhibit increased or decreased susceptibility upon mutation. ..................... 44 Figure 3.2. Growth curve of selected mutants with a slightly impaired ability to grow compared to the wild type PA14. .................................................................................................................. 45 Figure 3.3. Distribution of the genes involved in decreased (A) and increased (B) ciprofloxacin susceptibility according to their PseudoCAP functional class...................................................... 47 Figure 3.4. Analysis of the mutants that showed an increase in resistance to 3 antibiotics, representing different antibiotic classes. ....................................................................................... 49 Figure 3.5. The effect of a fleS mutation on killing by 0.1 µg/ml of ciprofloxacin. .................... 50 Figure 3.6. Impact of a fleS mutation on motility (A: swarming and swimming) and biofilm formation (B). ............................................................................................................................... 51 Figure 3.7. Cytotoxicity of the fleS mutant in comparison to the wild type and complemented strain 7.5 hours post-infection....................................................................................................... 52 Figure 4.1. Survival rates of the lon mutant compared to the wild type and complemented strain in the presence of 0.1 µg/ml ciprofloxacin. .................................................................................. 57 Figure 4.2. UV-mediated killing of P. aeruginosa after 10 seconds of UV light exposure. ....... 60 Figure 4.3. Effect of lexA and sulA overexpression on various types of motility and filamentation. ................................................................................................................................ 64 Figure 4.4. Induction of the P. aeruginosa RecA protein by sub-inhibitory concentrations of ciprofloxacin in the wild type, complemented lon strain and lon mutant. .................................... 66 Figure 4.5. Proposed model for the involvement of the Lon protease in the DNA damage response......................................................................................................................................... 68 Figure 5.1. Overview of the experiments conducted in this chapter regarding global regulation. ....................................................................................................................................................... 71 Figure 5.2. Transcriptional hierarchy of the Lon protease, including psrA and rpoS. ................. 79  xii  Figure 5.3. Anaerobic growth of mutants in regulators involved in anaerobic growth, lon mutant, complemented lon strain and wild type H103. ............................................................................. 81 Figure 5.4. Biofilm formation of the wild type H103 and the lon mutant under anaerobic relative to aerobic conditions. .................................................................................................................... 82 Figure 5.5. Anaerobic respiration analysis of the wild type H103, lon mutant and complemented strain using the WST1 assay. ........................................................................................................ 83 Figure 5.6. Swarming motility of the wild type, vector pBBR1 (control), lon overexpressing strain (pBBR1::lon+) and lon mutant. .......................................................................................... 84 Figure 5.7. One representative mass spectra for peptides identified from SILAC experiment. .. 86 Figure 5.8. Ability of the lon mutant, the complemented strain and wild type to withstand oxidative stress (100 mM paraquat (upper disc) and 30% H2O2 (lower disc)). ............................ 87 Figure 6.1. Cytotoxicity of the lon mutant and the wild type H103 at 10 and 18 hours postinfection. ....................................................................................................................................... 96 Figure 6.2. HBEs infected with P. aeruginosa wild type, lon mutant and complemented strain. 97 Figure 6.3. Romaine lettuce leaves infected with P. aeruginosa wild type, complemented lon strain and lon mutant. .................................................................................................................... 98 Figure 6.4. Amoeba virulence model. ........................................................................................ 101 Figure 6.5. In vivo competitive index assay of the lon mutant and the complemented strain. .. 103 Figure 6.6. Motility of the wild type PAO1 and the lon mutant in the presence and absence of mucin........................................................................................................................................... 104 Figure 6.7. Increase in ciprofloxacin resistance promoted by 0.5% mucin and surfing motility promoted by 0.1 and 0.5% mucin. .............................................................................................. 105 Figure 6.8. Mature biofilm formation with increasing concentrations of mucin. ...................... 106 Figure 7.1. Motility phenotypes of P. aeruginosa mutants in intracellular proteases: .............. 113 Figure 7.2. Growth curve of the clpP mutant compared to the wild type PA14........................ 114 Figure 7.3. Analysis of mature biofilm formation (A) and Congo red assay (B) of PA14 wild type, PA14 transposon insertion mutant clpP and clpP complemented strain (clpPc). ............. 115 Figure 7.4. Qualitative nitrocefin assay. .................................................................................... 117  xiii  Figure 7.5. RT-PCR to determine if clpP and lon are located in an operon. ............................. 118   xiv  List of Abbreviations AAA AAC AMP ATP AZLI CAZ cDNA cf. CF CFTR CFU CI CIP DNA ESBL Exo FDA GM h HBEs HSL ICU IPTG KAN LB LDH LES LPS MBLs MCS MFS mg MH MIC min ml mM MOI mRNA MRSA MW µg NHSN NNIS  ATPases associated with various cellular activities aminoglycoside acetyltransferase ampicillin adenosine triphosphate inhaled aztreonam and lysine ceftazidime complementary deoxyribonucleic acid compare cystic fibrosis cystic fibrosis transmembrane regulator colony forming units competitive index ciprofloxcin deoxyribonucleic acid extended spectrum β-lactamase exotoxin food and drug administration gentamicin hour human bronchial epithelial cells homoserin lactone intensive care unit isopropyl β-D-1 thiogalactopyranoside kanamycin Luria Bertani broth lactate dehydrogenase Liverpool epidemic strain lipopolysaccharide metallo--lactamases multiple cloning site major facilitator superfamily milligram Mueller Hinton broth minimal inhibitory concentration minute milliliters millimolar multiplicity of infection messenger ribonucleic acid methicillin resistant Staphylococcus aureus molecular weight microgram national healthcare safety network national nosocomial infections surveillance xv  nm OD ORFs PAGE PBS PCR PQS PVDF QS RNA RND RT-qPCR SCVs SDS s SILAC TLR TOB TOBI UV UW VAP  nanometer optical density open reading frames polyacrylamide gel electrophoresis phosphate buffered saline polymerase chain reaction Pseudomonas quorum-sensing signal polyvinylidene fluoride quorum-sensing ribonucleic acid resistance nodulation cell division reverse transcriptase quantitative PCR small colony variants sodium dodecyl sulphate second stable isotope labelling of amino acids in cell culture Toll-like-receptor tobramycin inhaled tobramycin Ultraviolet University of Washington ventilator associated pneumonia  xvi  Acknowledgements First of all, I would like to thank my supervisor, Dr. Robert E.W. Hancock, for his advice and support during this study as well as for giving me the opportunity for being part of his lab. The support I received from him over the last years made this thesis possible and furthermore prepared me for the next stage in my career. I owe many thanks to my committee members, Dr. Yossef Av-Gay, Dr. Leonard Foster and Dr. Steven Hallam who guided me throughout the years and gave important advice. Special thanks go to my collaborators Dr. Roger Levesque (University Laval, Quebec, Canada), Dr. Joerg Overhage (KIT, Karlsruhe, Germany) and Dr. Leonard Foster (University of British Columbia, Vancouver, Canada) who helped me gaining fruitful experiments for this thesis and for their discussions. Importantly, a big thank you goes to the current and past members of the Hancock lab for an always amazing lab atmosphere, for their friendship and support. Special thanks to Manjeet Bains, Reza Falsafi, Dr. Lucia Fernandez and Dr. Irith Wiegand for sharing good times in the office. I am very grateful to Dr. Bettina Bommarius, Dr. Lucia Fernandez and Dr. Ashley Hilchie for proofreading and editing my thesis. Thank you so much Bettina, Lucia and Ashley! I acknowledge funding from a Canadian Cystic Fibrosis Scholarship and a training award from BC Proteomic Network. Many thanks to all my friends, especially from St. John’s College, who always gave me support and encouragement. I particularly want to express my appreciation to Susana Zoghbi and Dr. Nicolas Saquet for their invaluable support. Finally, and most importantly, I would like to thank my dear parents who always provided understanding, support, care and inspiration throughout my studies.  xvii  Dedication  To my parents  xviii  1. Introduction 1.1  P. aeruginosa Pseudomonas aeruginosa is a versatile Gram-negative bacterium that exhibits a single  polar flagellum and belongs to the Pseudomonadaceae family. P. aeruginosa has been associated with disease since the 19th century; however, its role in acute and chronic infections was not elucidated until mid-20th century. This bacterium is now recognized as a major opportunistic human pathogen, and is the third most common cause of nosocomial infections. It can cause pneumonia, urinary-tract infections and bacteremia, as well as morbidity and mortality in cystic fibrosis (CF) patients due to chronic infections that eventually lead to lung damage and respiratory failure. Furthermore, P. aeruginosa is found in many natural environments (such as soils and marshes) as well as causing infections in animals and plants (202). Pseudomonas infections are difficult to eradicate due to its high intrinsic resistance and its ability to develop resistance to common antibiotics through adaptation and mutation. P. aeruginosa is now referred to as a “Superbug” because of its multi-drug resistance. P. aeruginosa possesses a large genome (239) that is predicted to encode 5570 open reading frames (ORFs). The genome encodes a high proportion of regulatory genes (9.3%) which coordinate gene expression and diverse virulence/resistance processes, as well as many genes involved in catabolism, transport, efflux of organic compounds and chemotaxis. Furthermore, Pseudomonas exhibits a large number of two-component regulatory systems. Taken together, all of these genes play a role in the pathogenesis of this bacterium. Consequently, the large number of regulatory genes could explain the high adaptability of this microorganism to different conditions. Correspondingly this high versatility may contribute to the difficulties we experience in controlling Pseudomonas infections. 1.1.1 Pathogenesis and virulence factors P. aeruginosa is the third leading cause of nosocomial infections in North America, causing more than 160,000 infections annually in the United States of America. The infections can be acute or chronic, and will be discussed individually in later sections. Once an infection is 1  manifested, Pseudomonas is very difficult to eradicate due to its high intrinsic and adaptive antibiotic resistance mechanisms (22) and range of virulence factors. These virulence factors include secreted toxins (e.g., exotoxin A, exoenzyme S, U, T and/or Y), elastase and other proteases, lipases and phospholipases, lipopolysaccharide (LPS), pili and flagella. All of these virulence factors can be responsible for host tissue damage and several mediate invasion upon bacterial colonization. A number of secretion systems are present in P. aeruginosa, and at least 4 of them play a role in virulence (Type I, II, III and VI), thereby allowing the bacterium to direct virulence factors to the host. AprA, an alkaline protease virulence factor, is involved in Pseudomonas infections (161). AprA is secreted through a type I extracellular secretion system. The Type II secretion system is involved in secreting exoproteins (e.g., LasA and LasB), lipases (e.g., LipA and LipC) as well as ExotoxinA (ToxA) into the extracellular milieu. These secreted factors are proposed to be involved in causing infections in immunocompromised patients. For example, LasB is involved in degrading elastin and surfactant protein D (SP-D), which are involved in lung elasticity and bacterial clearance, respectively (179). Therefore, immune function and lung tissue become compromised due to the secretion of LasB and other virulence factors. The Type III secretion system is distinguished from the previous two because the toxic proteins are secreted directly into the cytosol of the host cell due to direct contact between the bacterium and the host cell. Bacterium/host contact is mediated by a needle-like filament (91). The exoproteins (e.g., ExoU, ExoT, ExoY and ExoS), which are responsible for lung tissue damage, are transported through the assembled needle. Notably, the Type III secretion system involves around 40 genes, including those involved in needle formation (71, 268). The Type VI secretion system is also thought to be involved in the virulence of P. aeruginosa; however, this system has not been well studied. Consequently, we only have a limited knowledge of the mechanism by which it contributes to Pseudomonas virulence (142). The single polar flagellum contributes to P. aeruginosa virulence as well and is involved in swimming (in an aqueous environment) and swarming (viscous surface) motility (126), attachment, invasion and biofilm formation, as well as mediation of the inflammatory immune response. More than 40 genes participate in flagellar synthesis in P. aeruginosa, however; the main regulatory proteins are RpoN, FleQ, FleR and FliA (41). Interestingly, P. aeruginosa isolates from chronic CF patients are often non-motile due to the loss of the flagellum (246). Due 2  to the dual role of the flagellum as a virulence factor and as an activator of the host immunity, the flagellum contributes to bacterial clearance mechanisms because it stimulates phagocytic cells. However, CF isolates undergo genetic changes, including increased alginate production, which allows the bacteria to escape detection and clearance by phagocytic cells (198). Moreover, CF isolates often lose or down-regulate flagellum synthesis, which contributes to impaired inflammatory responses and leads to the evasion of the host immune system. Amiel et al. (3) showed that P. aeruginosa strains expressing a nonfunctional flagellum are resistant to phagocytosis. Therefore, the lack of motility may also contribute to P. aeruginosa survival in CF patients. Type IV pili is another virulence factor and consist of the type IVa pilin protein, which is encoded by pilA in P. aeruginosa (90). Approximately 50 genes directly or indirectly impact type IV pili synthesis. Type IV pili are important for motility (e.g., swarming and twitching) as well as cell adhesion and biofilm formation (123). Twitching motility allows bacteria to pull themselves along surfaces through an extension and retraction mechanism (18). Therefore, it seems intuitive that loss of pili leads to reduced P. aeruginosa virulence. This conclusion is supported by findings from Zolfaghar et al. (276) who showed that a pilA mutant exhibits a reduced ability to invade corneal epithelial cells. More importantly, pili are the major mediators of P. aeruginosa adherence which allows the pathogen to adhere to many cell types (86). P. aeruginosa can form complex organized structures called biofilms. Biofilms represent a form of social behaviour that involves the formation of microbial aggregates on surfaces, including epithelia and medical devices (32, 118). Initiation of biofilm formation is mediated by flagella motility that allows planktonic (free-swimming) cells to approach a surface where they can attach using flagella and/or type IV pili as described above (123). Subsequently, the bacteria attach more strongly and then grow into a heterogeneous mushroom-shaped structure (Figure 1.1) which is stabilized by the structural polysaccharides Pel and Psl, as well as alginate and possibly DNA. Biofilm formation is a complex process that is partly controlled by quorumsensing (QS) signals. QS is a cell-to-cell-communication mechanism whereby a certain threshold of bacteria are required in order to produce a sufficiently strong signal. Once this threshold is reached, signals can be transmitted to neighbouring cells, thereby initiating dramatic changes in gene expression (162, 220). P. aeruginosa has several QS signalling molecules including two homoserine lactone (HSL) autoinducer molecules, namely 3-oxododecanoyl homoserine lactone 3  (Las-system) and N-butyryl homoserine lactone (Rhl-system). Furthermore, it also produces the Pseudomonas quinolone signal (PQS) 3,4-dihydroxy-2-heptylquinoline (45, 228). The HSL regulatory systems, LasRI and RhlRI are regulated at the transcriptional level by the regulators LasR and RhlR, where the RhlRI system is subordinate to the LasRI system (23). This hierarchical QS system controls more than 200 genes, including several of the virulence factors described above. Compared to planktonic cells, cells growing in biofilms possess a distinct transcriptome and are much more resistant to many antimicrobials (up to 1000 fold) (38, 104, 183, 260). Antimicrobial resistance occurs in several ways. The broad dysregulation of many genes can lead to the upregulation of efflux pumps, enzymes, various regulatory proteins and other products identified through genomic resistome studies. Also, differential access to nutrients within the biofilm leads to differential metabolic activity, whereby cells in the outer layer are metabolically active and cells in the inner part of the biofilm grow more slowly. Some antibiotics only work on growing cells (e.g., most β-lactams, aminoglycosides and ciprofloxacin), while a few (e.g., polymyxins) preferentially kill poorly growing bacteria. Therefore these antibiotics would affect different regions of the biofilm (192). Although the extracellular matrix has been suggested to act as a barrier to antibiotic penetration, it might also act to concentrate extracellular enzymes, such as secreted β-lactamases, near the bacterial surface. Biofilms are also known to have a greater fraction of so-called persisters than planktonic cells. Persisters represent slowly growing or non-dividing cells that can easily withstand stressful conditions, such as antibiotic pressure. Little is known about the mechanisms underlying the development of persister cells in P. aeruginosa biofilms; however, it is known that spoT, relA, dksA, rpoS, dinG, spuC, algR and pilH mutations can affect persistence (144). Apart from being more resistant to antibiotic treatment, biofilms can also evade the host defences due to the fact that the biofilm polysaccharide matrix protects the cells from the components of the immune system (105). Importantly, P. aeruginosa readily forms biofilms on mechanical ventilators and catheters, making P. aeruginosa particularly problematic for patients requiring these devices. Furthermore, additional evidence suggests that P. aeruginosa can form biofilms in burn wounds and in the lungs of cystic fibrosis patients (13, 38, 225).  4  Figure 1.1. Development of a P. aeruginosa biofilm. Stage 1: Cells are planktonic and free-swimming and exhibit antimicrobial susceptibility. Stage 2: Planktonic cells initiate attachment to a surface via their type IV pili and flagellum. Stage 3: Small aggregative communities begin to form and QS signals begin to accumulate. Stage 4: A critical threshold of QS signals is reached. Microcolonies become encased in an extracellular matrix. Cells enter a slower phase of growth, adapt and become highly resistant to antimicrobials. Individual cells and small microcolonies dissociate from the mature biofilm initiating further biofilm development (76). Figure 1.1 was drawn by E.B.M. Breidenstein (218). Another major virulence factor is lipopolysaccharide (LPS) which is present in the outer membrane of P. aeruginosa. LPS mediates bacterial endotoxicity and host immune responses, both of which are dependent on the structure of the LPS and the clinical condition of the patient. LPS consists of 3 components: lipid A, core polysaccharide and variable O-Antigen. Each of these components have their own role in pathogenesis. For example, lipid A plays a major role in activating the host innate immune system by binding to a receptor on the host cell surface that consists of Toll-like-receptor 4 (TLR-4) and specific co-receptors (60). Due to different acetylation states (penta-, hexa-, or hepta-acylated (61)) of lipid A, differences in activation occur. Hexa-acylated lipid A from P. aeruginosa shows a strong inflammatory response and this type is often isolated from patients with chronic lung infections that exhibit severe lung damage. The core polysaccharide has been proposed to be the bacterial ligand for wild type cystic fibrosis transmembrane receptor (CFTR), thereby triggering the internalization of the bacteria by lung epithelial cells (153, 199). Attached to the core polysaccharide is the O-antigen, which consists 5  of a variable number of repeated tri- to penta-saccharides. The variable O-antigen side chain impacts on serotype specific host immunity and is important for protective immunity. Strains lacking the O-antigen are commonly referred to as “rough”, whereas O-antigen-producing strains are known as “smooth”. Rough strains are less likely to cause a systemic infection (88), but are predominant in the lung of CF patients (88, 197). LPS vaccines have been studied in clinical trials as a means to treat Pseudomonas infections in CF patients; however, none of these vaccines have been successful to date, which may be due to antigenic variation (49, 89, 137, 194). 1.1.2  P. aeruginosa as a “Superbug” P. aeruginosa is often resistant to multiple antibiotics and consequently has joined the  ranks of “Superbugs” due to its enormous capacity to engender resistance. The pathogen demonstrates high intrinsic resistance to almost all antibiotics due to its low outer membrane permeability, which is coupled with acquired and adaptive resistance mechanisms. The European Antimicrobial Resistance Surveillance System reported that 18% of P. aeruginosa isolates were multi-drug resistant (231). High-level resistance has been noted for piperacillin, ceftazidime, fluoroquinolones, aminoglycosides and carbapenems. Only low levels of resistance have been demonstrated for colistin and polymyxin; therefore the latter two antibiotics are referred to as “last hope” antibiotics (145). The National Nosocomial Infections Surveillance System (NNIS) reported that, over the last few years, resistance rates and the number of infections are increasing (77) thus highlighting that P. aeruginosa is a major problem in Western society. 1.1.3  Antibiotic resistance in P. aeruginosa 1.1.3.1 Mechanisms of intrinsic resistance P. aeruginosa is intrinsically resistant to most antibiotics. In comparison to most other  Gram-negative bacterial species, wild type P. aeruginosa exhibits reduced susceptibility to antibiotics. The low outer membrane permeability of P. aeruginosa, which is 12-100 times less than that of Escherichia coli (87), is the main mechanism underlying antibiotic resistance. The outer membrane of Gram-negative bacteria acts as a selective barrier to antibiotic uptake (180) and has been compared to a molecular sieve in which the uptake of most hydrophilic molecules 6  is size-dependent due to diffusion through water-filled channels composed of porin molecules. P. aeruginosa has a large exclusion limit due to the limited number of large channels of its major porin OprF (most OprF channels being very small). Other antibiotic uptake pathways are mediated via similar small size channels of other porins, such as OprB and OprD. The latter promotes the specific uptake of carbapenems. Furthermore, other uptake pathways include selfpromoted uptake of polycationic antibiotics (described below) and uptake of hydrophobic molecules through the outer membrane bilayer. While low outer membrane permeability plays a decisive role in reducing the rate of drug uptake, equilibration of hydrophilic molecules across the outer membrane is still managed in a matter of seconds. Thus, the high intrinsic resistance of this pathogen is absolutely dependent on other intrinsic and adaptive secondary mechanisms, such as rapid efflux (146, 147) due to the intrinsic or induced expression of efflux pumps, particularly the Resistance Nodulation Cell Division (RND) systems MexAB-OprM and MexXY-OprM, and AmpC β-lactamase production (159) that take advantage of the reduced flow of antibiotic across the outer membrane (Table 1.1). Table 1.1. Comparison between the 3 major types of antibiotic resistance. Type of resistance Acquisition  Intrinsic  Not acquired, part of the genetic make-up of the strain or species Characteristics Inheritable Stable Not easily reversible Independent of environment  Acquired  Adaptive  Mutation Horizontal transfer  Changes in gene expression triggered by environmental factors or presence of antimicrobials Not inheritable Transient Generally reverts upon removal of inducing signal Dependent on environment  Inheritable Stable Not easily reversible Independent of environment  The table was taken with modifications from the review article written by L. Fernandez, E.B.M. Breidenstein and R.E.W. Hancock (63). Recent studies screening comprehensive mutant libraries for mutants that possess altered susceptibility to antibiotics, including the studies described in Chapter 3, identified new candidate mechanisms involved in the intrinsic resistome of P. aeruginosa (2, 19, 21, 50, 62, 219). The results of these studies support the participation of dozens of genes from different 7  functional classes. The wide range of newly identified mutations form part of very complex resistomes. For example, a large number of energy metabolism mutants affect aminoglycoside resistance, including mutations in the nuo, nos, nqr and cytochrome genes (219). Another study revealed that ampG (PA4393) is required for the induction of AmpC β-lactamase, showing that a mutation in this gene reduced susceptibility to β-lactams by 8 fold (275). An overlap between genes dysregulated upon treatment with sub-inhibitory or lethal exposure to antibiotics and genes involved in antibiotic resistance, has been observed. This finding indicates that P. aeruginosa adaptively activates defence (resistance) mechanisms to combat the inhibitory effects of antibiotics (20, 21, 121, 219). These studies collectively demonstrate that high intrinsic resistance of P. aeruginosa to antibiotics is ultimately due to the combination of several mechanisms acting simultaneously. The underlying mechanisms likely play a significant role in clinical outcome.  1.1.3.2 Mechanisms of acquired resistance: horizontal gene transfer and mutational resistance In addition to its high intrinsic resistance, P. aeruginosa can become even less susceptible to antimicrobials due to the acquisition of inheritable traits. The two types of acquired resistance include horizontal gene transfer of genetic elements and mutational resistance (Table 1.1). DNA elements, including plasmids, transposons, integrons, prophages and resistance islands, can harbour antibiotic resistance genes, and can be acquired by conjugation, transformation or transduction, which can increase antibiotic resistance. It can even cause multidrug resistance, if the DNA element contains multiple resistance cassettes. Such horizontal transfers mainly affect aminoglycoside and β-lactam resistance in P. aeruginosa, but they can also affect several other classes of antibiotics. For example, aminoglycoside modifying enzymes located on mobile genetic elements can inactivate aminoglycosides, thereby leading to various chemical modifications of the aminoglycoside that reduce the affinity for the 30S ribosomal subunit, which is the main aminoglycoside target (253). In addition to the inducible chromosomal AmpC β-lactamase, some P. aeruginosa strains acquire plasmids encoding new βlactamases that confer resistance to penicillins and cephalosporins (213). Of great concern is the 8  proliferation of plasmid-mediated extended-spectrum β-lactamases (ESBLs), which were originally described in the Enterobacteriaceae, and metallo-β-lactamases (MBLs) that inactivate carbapenems (24), subsequently conferring resistance. A second form of acquired resistance is mutational resistance. The spontaneous mutation frequency varies among antibiotics, however, resistance frequencies range from 10-6 to 10-9. The mutation rate can further increase under certain conditions such as in the presence of DNAdamaging agents or during biofilm growth. For example, the mutation frequency for meropenem increased 10 fold when the culture was pre-incubated with sub-inhibitory concentrations of ciprofloxacin (245). Likewise, a >100 fold increase in mutation frequency leading to ciprofloxacin resistance was observed in biofilms compared to free-living cells (54). This may be due to the downregulation of antioxidants during biofilm growth which then lead to increased DNA damage. Mutation frequencies are increased by as much as 70 fold or more in hypermutator strains (262), as they contain mutations in genes involved in DNA repair efficiency. These strains can acquire resistance to several different antibiotics. Examples of strong hypermutators are mutL and mutS, which are commonly found in patients with CF (185). However, it is noteworthy that a variety of weaker mutators have been found by mutant library screens (262), which could also play a role in the early stages of CF lung infections (117). “Breakthrough” mutations make P. aeruginosa untreatable by antibiotics and include those mutations that lead to overexpression of efflux pumps, reduced antibiotic uptake, contribution to hyperproduction of β-lactamases and alteration of antibiotic targets. For example, an important mutational mechanism is the derepression of the efflux pumps MexAB-OprM and MexCD-OprJ due to mutations in genes mexR and nfxB, respectively (236). Furthermore, overexpression of MexXY-OprM due to a mutation in mexZ leads to aminoglycoside, fluoroquinolone and cefepime resistance in clinical strains of P. aeruginosa (175). Mutations in the specific porin OprD reduce the uptake of the antibiotic imipenem and therefore lead to clinical resistance (257), whereas mutations in either mexT or mexS (nfxC) are known to reduce the expression of OprD as well as increase expression of the efflux pump MexEF-OprN leading to imipenem and multiple antibiotic resistance (229). Hyperproduction of β-lactamases occurs upon mutation of an effector of the ampC β-lactamase, AmpD, which controls activity of the AmpR regulator. Mutations in target enzymes can also lead to clinically meaningful resistance  9  (102), e.g. mutations in gyrA and gyrB (gyrase) as well as parC and parE (topoisomerase IV) reduce fluoroquinolone binding affinity, leading to clinical resistance (56). Recent screening studies employing the P. aeruginosa PA14 comprehensive Harvard library (148), including those described in Chapter 3, have shown that many additional mutations can also lead to increased resistance, although, in most cases, the observed increases are modest (around twofold) and might easily be missed in a clinical setting (2, 21, 50, 62, 219). It has been proposed that low-level resistance might evolve to high-level resistance in a stepwise manner, a phenomenon referred to as creeping baselines (63). The accumulation of several mutations with modest changes in minimal inhibitory concentration (MIC) can, over time, result in stepwise increases in resistance, ultimately leading to high-level clinical resistance. Indeed, single mutations in either galU (central intermediary metabolism), nuoG (energy metabolism), mexZ (transcriptional regulator) or rplY (adaptation) exhibited only a twofold increase in resistance to tobramycin, but a quadruple mutant had a major, 16 fold, increase in resistance (58). These screening studies define the resistome, which collectively represents all mutations that can lead to antimicrobial resistance. A wide variety of mutations constitute the aminoglycoside, β-lactam, fluoroquinolone, tetracycline and sulphonamide resistomes. For example, the P. aeruginosa aminoglycoside resistome (50, 219) involves 150 different genes from many functional categories including those related to energy metabolism, DNA replication and repair and LPS- biosynthesis. In summary, mutations in a large number of unrelated genes can give rise to acquired resistance to different antibiotics.  1.1.3.3 Adaptive resistance Adaptive resistance is inducible and dependent on the continuing presence of an antibiotic or another environmental stimulus. Although it was first observed in 1966 (9), no important connection to a clinical outcome was established for adaptive resistance, in contrast to intrinsic and acquired resistance, therefore this phenomenon did not receive much attention. Subsequently, it was shown that tetracycline induces plasmid-mediated tetracycline resistance, biofilms demonstrate broad spectrum non-mutational resistance, and that in P. aeruginosa the inducibility of chromosomal -lactamase limits the efficacy of several -lactam antibiotics that are effective in other bacterial species. 10  The advent of the genomic era has enabled a broader understanding of the complex phenomenon of adaptive resistance. A number of triggering factors are now recognized to induce adaptive resistance. These factors include antibiotics, biocides, polyamines, pH, anaerobiosis, cations, and carbon sources, as well as social activities such as biofilm formation and swarming (reviewed in (63)). These triggering factors modulate the expression of many genes leading to effects on efflux pumps, the cell envelope and antibiotic-degrading enzymes. The importance of adaptive resistance in P. aeruginosa is consistent with the large repertoire of regulatory genes (9.4% of all genes) in its genome (239). Environmental cues and sub-inhibitory concentrations of antibiotics lead to defined changes in the gene expression profile of P. aeruginosa, which allows the bacterium to withstand subsequent exposures to lethal concentrations of the inducing antibiotic as well as related antibiotics. An important feature of adaptive resistance is that once the inducing factor or condition is removed, the organism reverts to wild type susceptibility. This may explain the observation that in vitro efficacy does not predict success in vivo in P. aeruginosa therapy (65), as this likely cannot reflect the development of adaptive resistance within the host. This is of particular concern in clinical settings where P. aeruginosa grows as a biofilm (e.g., CF, catheterassociated infections and ventilator-associated pneumonia). Polycationic antimicrobials such as aminoglycosides, polymyxins and cationic antimicrobial peptides pass across the outer membrane by self-promoted uptake which involves an interaction between the polycations and divalent cation binding sites on LPS to competitively displace these cations. This causes localized disruption that allows the polycation to cross the membrane (87). The arnBCADTEF operon mediates the addition of 4-aminoarabinose to Lipid A of LPS, which blocks self-promoted uptake leading to resistance. Adaptive resistance to polymyxins and host cationic antimicrobial peptides can be mediated by low concentrations of divalent cations (Mg2+ and Ca2+), thereby leading to the activation of 2 two-component regulatory systems, PhoPQ and PmrAB, and consequent induction of the arn operon; however, antimicrobial peptides or polymyxins are more likely the physiological triggers (166). The arn operon can also be induced in biofilms by extracellular DNA, which creates a cation-limited environment (173). Recently a new two-component regulatory system, ParRS, was identified. ParRS mediates the upregulation of the arn operon and adaptive resistance in the presence of the antipseudomonal drug colistin and certain antimicrobial peptides (64). 11  A major adaptive mechanism in P. aeruginosa is the induction of a chromosomallyencoded β-lactamase (encoded by the ampC gene) by pre-exposure to β-lactam antibiotics, which can cause enzymatic inactivation of several -lactam antibiotics. Clinical failure of ceftazidime, anti-pseudomonal penicillins, and cefotaxime therapy strongly correlates with ampC dysregulation, because these drugs strongly upregulate the ampC gene. Conversely, some lactam antibiotics, including the newest fourth generation cephalosporins, cefepime and cefpirome, show weaker or no upregulation of ampC (215). Recently, Lee et al. (140) demonstrated that the two-component regulator AmgRS, which is involved in adaptive membrane stress response, is activated after tobramycin exposure. Similarly, Kindrachuk et al. (121) demonstrated that the heat shock stress response, which leads to low-level resistance to aminoglycosides, is controlled through the heat shock sigma factor RpoH and the ATP-dependent protease AsrA. The heat shock response allows the cell to repair the proteins damaged by aminoglycosides. In contrast, the Lon protease, which controls the DNA stress response and fluoroquinolone susceptibility, is upregulated by aminoglycosides (157), however no connection to adaptive aminoglycoside resistance was observed. Another mechanism resulting from bacterial exposure to sub-inhibitory concentrations of antibiotics is the overexpression of genes encoding efflux pumps. For example, aminoglycosides induce the expression of the MexXY efflux pump (100). Consequently, the antibiotic is more rapidly effluxed and the bacterium adaptively becomes more resistant. Moreover, efflux pumps often mediate multi-drug resistance. Swarming is a special form of motility that differs from flagella-mediated swimming on liquid and semi-solid media and pili-mediated twitching on solid surfaces. Swarming motility is a social behaviour distinct from biofilm formation that involves hundreds of genes (189). Swarming is thought to be relevant to the movement of P. aeruginosa through mucosal layers, since conditions that trigger swarming (e.g., intermediate viscosity, and amino acids as a poor nitrogen source) exist in the lung (108). Compared to planktonic cultures, swarming colonies exhibit greater resistance to antibiotics (134, 189) and have a higher expression level of virulence-related factors. The mechanisms leading to adaptive resistance in swarming cells are only starting to be understood (26) although they appear to involve Lon and CbrA-mediated PhoPQ dysregulation (271).  12  Adaptive resistance has long term consequences. If cells are not completely eradicated, re-growth can be observed once the treatment is stopped (192). This is particularly relevant in the clinic where biofilm formation has been observed, e.g., in the lungs of CF patients (13). A related problem is the high rate of persister cells in CF and biofilms (144, 174). 1.1.3.4 Stepwise increase in resistance: creeping baseline MICs It has been well documented that clinically-relevant pathogens have, throughout the years, become increasingly resistant to antibiotics currently available on the market. Thus, the initial susceptibility, observed when an antibiotic was first introduced in the market, relatively quickly gave way to phenotypes with varying degrees of resistance. One example would be the rise of resistance to the aminoglycoside tobramycin in P. aeruginosa. Originally, Pseudomonas strains were susceptible to tobramycin (MIC < 2 μg/ml) (44), but resistance increased steadily after tobramycin became frequently prescribed to patients. Now physicians frequently are isolating cultures from patients with an MIC of 16 μg/ml. Moreover, strains with an MIC > 128 μg/ml are occasionally isolated (156). These highly resistant strains represent a serious threat to patients, because they are often resistant to more than one antibiotic. Multi-drug resistance is a serious health concern due to lack of therapeutic options. It was originally thought that clinically relevant increases in antibiotic resistance only happened suddenly through a “breakthrough” mutation that takes the organism from a treatable point to an untreatable point. However, it is becoming increasingly clear that the “baseline” MIC actually shows a stepwise rise over time, gradually eroding the efficiency of therapy. Stepwise mutations that cause small changes in MIC and adaptive resistance are good candidates to play a role in this MIC creep. This phenomenon has been clearly demonstrated by Steinkraus et al. (234) in vancomycin-susceptible, methicillin-resistant Staphylococcus aureus (MRSA) isolates. A stepwise increase in MIC of vancomycin in the years 2001-2005 was observed in MRSA isolates, whose geometric mean MIC for vancomycin shifted from 0.62 μg/ml to 0.94 μg/ml. While this change is small, it is of significant concern because it can lead to a further increase in antibiotic resistance over time. Insufficient attention has been paid to these small changes, which often remain unnoticed in clinical screenings. However, over time these subtle increases in the 13  MIC may contribute, in a combinatorial fashion, to the high-level resistance that leads to clinical failure. The phenomenon of low-level antibiotic resistance and its impact on high-level antibiotic resistance has been extensively reviewed by Baquero (8). Baquero clearly describes how several compounds can select for low-level resistance and how these phenotypes may lead to high-level resistance. An example of this phenomenon would be the accumulation of mutations over time in a given strain. In this regard, even though a single mutation may cause only a subtle increase in the MIC, the occurrence of additional mutations in the same strain would result in a more dramatic change. For example as mentioned above, El’Garch et al. (58) showed that mutation in either galU, nuoG, mexZ or rplY led to twofold increases in resistance to aminoglycosides; with a MIC for tobramycin of 1 μg/ml for all the single mutants cf. 0.5 μg/ml for the wild type. Importantly, greater increases in resistance were observed in double, triple and quadruple mutants. However, the galU/nuoG/mexZ/rplY quadruple mutant exhibited a tobramycin MIC of 8 μg/ml, thus clearly demonstrating the additive effect of low level resistance mutations and hence their potential to contribute to stepwise resistance. Although our understanding of the mechanisms involved in low-level antibiotic resistance is in its infancy, several resistome studies suggest that they are far more complex than originally anticipated (2, 21, 50, 219). Moreover, while certain mutations lead to low-level resistance to one antibiotic class, others result in low-level resistance to multiple antibiotic classes. 1.1.4  Correlation between antibiotic resistance and virulence Interestingly, a number of bacterial regulatory genes play a role in both antibiotic  resistance and virulence. Virulence genes can impact on bacterial motility, biofilm formation, cytotoxicity and the production of virulence factors such as toxins, proteases and lipases. Many genes (e.g. mexS (nfxC), mexGHI-ompD, gacAS, retS, ladS, algR, rsmA, lon, crc, psrA, cbrA and phoQ) are known to affect both antibiotic resistance and virulence (80). For example, psrA mutants are supersusceptible to polymyxin B and indolicidin (78), while crc mutants are more susceptible to β-lactams, aminoglycosides, fosfomycin and rifampin (150). Despite the difference in intrinsic antibiotic resistance between the two mentioned mutants, they all have common defects in virulence properties (i.e., swarming motility and biofilm deficiencies). The 14  motility deficiency in the psrA mutant can be explained by the dysregulation of a variety of genes involved in adhesion and a few in motility (78, 116, 127). CbrA, which is the regulator of carbon and nitrogen metabolism, has been extensively studied and has been shown to play a role in acquired antibiotic resistance (e.g., polymyxin B, colistin, ciprofloxacin and tobramycin), swarming motility, biofilm formation and cytotoxicity towards human bronchial epithelial cells (271). A mutation in phoQ leads to constitutive expression of the arn operon, which confers resistance to aminoglycosides, polymyxin B and antimicrobial peptides (79, 154). Importantly, phoQ mutants have been isolated in the clinic (169). Furthermore, PhoQ is involved in bacterial virulence because a mutation in phoQ leads to a deficiency in swarming, twitching, biofilm formation and cytotoxicity compared to the parental strain. The involvement of phoQ in virulence has also been shown in vivo in a rat model of chronic lung infection as well as a lettuce leaf model (79). Overall, several regulators play a role in regulating both antibiotic resistance and virulence-related properties. Biofilm formation and swarming are considered the most important virulence-related properties. Moreover, it has been suggested that genetic switches between biofilm formation and swarming motility exist (28, 272). However, it is quite clear that more studies need to be conducted in order to elucidate the regulatory network influencing antibiotic resistance and virulence determinants in P. aeruginosa. 1.1.5  P. aeruginosa hospital-associated acute infections P. aeruginosa is capable of causing acute nosocomial infections, which are infections that  occur when the patient is hospitalized. The chances of becoming infected with P. aeruginosa in a hospital setting can be quite significant because Pseudomonas persists on surfaces (e.g. sinks, toilets, etc.), catheters, and mechanical ventilation apparati. Moreover, the immune system of hospitalized patients is often compromised, thereby inhibiting their ability to fight infections. The National Health Care Safety Network (NHSN) determined that 8% of all hospital-associated infections are due to P. aeruginosa (94). Hospital-associated infections include bacteremia, urinary tract infections, as well as ventilator-associated pneumonia. P. aeruginosa is capable of leaving the primary site of infection and invading the bloodstream, where it can cause sepsis (132). Sepsis can however also occur in burn wound infections (which are not considered 15  hospital-acquired), where Pseudomonas is prevalent (6). However, not only is tissue colonization of major concern, but the patients suffering from burn wound infections often require catheters and mechanical ventilation during hospitalization, leading to ventilator-associated pneumonia (VAP). This causes further threat of hospital-acquired infections. 1.1.6 Chronic P. aeruginosa infections in CF CF is an autosomal recessive, multi-organ disorder that occurs at a frequency of 1:2,500 in the Caucasian population (205). CF patients suffer from chronic respiratory infections and lung damage which eventually leads to mortality. Several pathogens are involved in CF lung infections. The most important ones include Staphylococcus aureus and Haemophilus influenzae which are the predominant pathogens found in younger patients. In contrast, Stenotrophomonas maltophilia, Burkholderia cepacia and P. aeruginosa are mainly found in adults. Importantly, P. aeruginosa has also been isolated from patients less than two years old (106). Airway colonization with P. aeruginosa often occurs after the patient’s airway had been colonized by S. aureus: at this point P. aeruginosa becomes the predominant pathogen in the CF lung by replacing S. aureus (101). The nature of this disease is important in understanding why P. aeruginosa dominates as the primary pathogen in CF patients. The defective gene involved in CF was identified in 1989. One in every 25 Caucasians carries a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Importantly, the presence of a defective CFTR protein in individuals with both alleles mutated leads to pathological changes in organs and tissues that express CFTR, including secretory cells and the liver, pancreas and lungs. CFTR regulates chloride, sodium and water transport. A CFTR mutation leads to impaired chloride transport at the apical surface of epithelial cells, and increased sodium absorption by the epithelial sodium channel with the most common mutation being ∆508 (70% of the cases). Therefore, the transepithelial potential difference is higher in CF than normal (>35mV and <30mV in CF patients and healthy individuals, respectively) (205). These abnormalities contribute to thickened and dehydrated mucus secretions that impair mucociliary clearance of bacterial pathogens (37). Consequently, the invading pathogens become trapped in the mucus and adhere to and damage epithelial surfaces. Non-mucoid Pseudomonas strains are then able to undergo phenotypic changes such as a transition to a mucoid form due to 16  overexpression of alginate (83), loss of the LPS O-antigen, causing a switch from a smooth to a rough colony morphology (88), and formation of small colony variants (SCVs). SCVs feature a smaller size than the parental strain, exhibit increased antibiotic resistance, are autoaggregative, and can be hyperpiliated leading to an increase in twitching motility and biofilm formation (92). Another adaptation mechanism involves Pseudomonas biofilm formation in the lungs of patients with CF (13, 225), also impacting antibiotic resistance. Furthermore, CF isolates exhibit a hypermutator phenotype (described in section 1.1.3.2) with an up to 1000 fold increase in mutation frequency. The ability to rapidly mutate gives a survival advantage in harsh environments, such as that of the CF lung. Moreover, unlike other invading pathogens, Pseudomonas can grow in microaerophilic and anaerobic environments. A steep hypoxic gradient has been observed in the thickened mucus of the CF lung and it has been proposed that after initial attachment and colonization of the upper respiratory tract by non-mucoid strains, Pseudomonas proceeds down the oxygen gradient in the lung to the lower respiratory tract where it becomes mucoid (265). The mucoid form of P. aeruginosa is common in CF where it has been reported to occur in 90% of P. aeruginosa lung infections in CF patients compared to only 2% in non-CF lung infections (47). It is noteworthy that CF is associated with chronic, high bacterial load colonization that triggers a prolonged inflammatory response, and this is ultimately responsible for the symptoms accompanying CF lung infections. The chronic progression of lung disease is accompanied by intermittent episodes of acute symptoms, also referred to as acute pulmonary exacerbations. These symptoms include cough, sputum production, loss of lung function and breathing problems. All of these symptoms can lead to respiratory failure and lung damage, which in turn can result in the death of the patient. 1.1.7  Treatment options for P. aeruginosa infections in patients with CF Chronic P. aeruginosa infections are almost impossible to eradicate. Antibiotic treatment  only delays the onset of chronic infections by decreasing the bacterial load in the respiratory tract (242, 254). Therefore, the main goals of antimicrobial therapies are to prevent colonization, eradicate early stage colonization, and/or reduce the bacterial load once the infection is established (249). The treatment regimens, which are summarized in Table 1.2, depend on the 17  disease state. Treatment options include β-lactams, fluoroquinolones, aminoglycosides and colistin which are used alone or in combination, and are administered orally, intravenously or as aerosols (73, 152, 233, 242, 254). Table 1.2. Empiric therapy for the treatment of P. aeruginosa infections in patients with cystic fibrosis. Disease state First isolation of P. aeruginosa (without clinical symptoms) Pulmonary exacerbations  Mild exacerbations Maintenance therapy  Antibiotic  Pediatric dose (> 6 years) 15 mg/kg  Ciprofloxacin + Tobramycin 300 mg or Colistin 150 mg Ceftazidime 50 mg/kg or Piperacillin 100 mg/kg or Imipenem 15-25 mg/kg or Meropenem 40 mg/kg or Aztreonam 50 mg/kg + Tobramycin 3 mg/kg or Amikacin 5-7.5 mg/kg Ciprofloxacin 15 mg/kg  Adult dose  Daily dosing interval 15 mg/kg 12 h 300 mg 150 mg 2g 3g 0.5-1 g 2g 2g  12 h 12 h 8h 6h 6h 8h 8h  3 mg/kg 2g 2g  8h 8h 8h  Tobramycin 300 mg 300 mg or Colistin 150 mg 150 mg ± Ciprofloxacin 10 – 15 mg/kg 0.5-0.75 g  12 h 12 h 12 h  Route Oral  Duration of treatment 3-4 weeks  Inhalation Inhalation IV 2-3 weeks or longer if no signs of IV improvement IV IV IV IV IV Oral  2-3 weeks  Inhalation 28 day on/off Inhalation cycle Oral  2-4 weeks cycled every 3-4 month  The table was taken from K.N. Schurek, E.B.M. Breidenstein and R.E.W. Hancock (218). 1.1.7.1 Antibiotic treatment for initial infections with P. aeruginosa Undoubtedly, preventing chronic infections represents a better treatment option than eradicating established infections. Prevention may be possible if treatment is initiated immediately after the pathogen is first isolated from the airways of the patient. At that point the bacterial load would be low, and, more importantly, the isolates would be non-mucoid, thereby making them more susceptible to antibiotic therapy. This early treatment results in a better life expectancy by delaying the onset of chronic infections (181, 210). Successful eradication is defined by at least 3 consecutive negative cultures that are spaced at least one month apart. Importantly, some patients who receive treatment remain culture negative for P. aeruginosa after 18  several years (73). Different antimicrobial treatment regimens exist for eradication of early stage infections, including intravenous ciprofloxacin, or a combination of an oral and an aerosolized antibiotic (73, 152, 233, 254). Valerius et al. (254) and Frederiksen et al. (73) used a combination therapy of oral ciprofloxacin and aerosolized colistin in their studies. In this regard, antibiotics were administered twice daily over 3 weeks and disease progression in the patients was monitored over 27 month. The data clearly showed that 80% of patients treated with antibiotics did not develop a chronic infection, whereas only 20% of the control group (without antimicrobial therapy) failed to develop a chronic infection. This finding confirmed the assumption that early eradication strategies are invaluable for preventing chronic lung infections. 1.1.7.2 Treatment strategies for established chronic infections Established chronic infections are difficult to treat due to the high bacterial load in the lung, in addition to the significant morphological changes that make the pathogen highly resistant to antimicrobial agents. Thus, the goal for treating chronic infections is to reduce the bacterial load, ultimately increasing the quality of life and life expectancy of the patients. Maintenance therapy and treatment of acute exacerbations are the two main objectives for antimicrobial therapy in chronic CF infections (48). The goal of maintenance therapy is to reduce the bacterial load in the lung, and to maintain overall lung function. Chronic infections are treated with long-term antibiotic therapies that are associated with significant side effects. These side effects generally include: loss of hearing, increased cough, and alterations in the patient`s voice. Moreover, these therapies select for antibiotic resistant strains. However, the side effects are reduced if an on/off cycle is used (203). Cheer et al. (31) investigated the effect of an on/off cycle with inhaled tobramycin (TOBI, Chirom Corporation, Emeryville, CA, USA) on the lung function of patients with CF. In this regard, patients were treated twice daily with 300 mg TOBI for 28 days, followed by a break without antibiotics for 28 days. This on/off cycle of inhaled tobramycin reduced the bacterial load of P. aeruginosa in the lower respiratory tract, and drastically improved the lung function of CF patients. Additionally, aerosolized tobramycin is less toxic than tobramycin administered intravenously. Recently, Gilead Science developed an aerosolized formulation of the monobactam aztreonam and lysine (AZLI) (165). Clinical trials demonstrated the efficacy of 19  AZLI as an additional therapy to TOBI. AZLI improved lung function and was well tolerated by the patients. However, AZLI was not approved after its phase III clinical trial by the Food and Drug Administration (FDA) (Sept 2008), therefore additional studies over a longer time period are required before approval by the FDA will be granted. Another treatment option for CF patients is orally administered ciprofloxacin, which improves lung function. Although P. aeruginosa isolates from CF patients can be multi-drug resistant, cross-resistance between major classes of antibiotics does not occur frequently. Therefore combination therapy is beneficial and has been shown to reduce the bacterial load in the CF lung. A study by Shawar et al. (222) examined cross-resistance (tobramycin, amikacin, gentamicin, aztreonam, ceftazidime, ticarcillin and ciprofloxacin) for 1200 P. aeruginosa isolates from CF patients. The study revealed that although cross-resistance between aminoglycosides occurred, no cross-resistance between aminoglycosides and β-lactams was evident. Consequently, a good therapeutic approach would be a combination treatment regime of β-lactam and aminoglycoside antibiotics, which could be delivered orally, intravenously or in aerosol form. Another study showed that the macrolide antibiotic azithromycin might be an useful option for antibiotic treatment. Azithromycin administered orally 3 times per week for 24 weeks improved CF lung functions although some mild side effects were observed, including nausea, diarrhea and wheezing (214). As a last resort, isolates resistant to the commonly used antibiotics are treated with aerosolized colistin which exhibits a high activity against multi-drug resistant strains. Importantly, colistin resistance rarely occurs (212). Frequent acute exacerbations along with a strong inflammatory response are observed in patients with chronic lung infections due to the presence of a high bacterial load. In this case, antibiotics are not used to reduce the bacterial load, but rather to reduce the inflammatory response caused by Pseudomonas. In cases of mild exacerbations, oral ciprofloxacin is administered (29) whereas in case of moderate exacerbations, a combination of tobramycin and β-lactam antibiotics is given intravenously for two to three weeks (226). 1.1.8  The challenge of treating the “Superbug” P. aeruginosa P. aeruginosa resistance to currently available antibiotics rises every year, even when  combination therapies are given to the patient. Consequently, there is an urgent need to discover 20  novel therapeutic strategies to combat the infections caused by this “Superbug”. Optimal antibiotic dosages administered to patients clearly need to be well defined since the inappropriate use of antibiotics contributes to the rise of adaptive, stepwise, and breakthrough resistance. Once breakthrough resistance occurs, the chance of finding an effective treatment option decreases considerably. Indeed, there are multi-drug resistant P. aeruginosa strains for which no effective antibiotic is available, and these strains are likely to become more common over time. Ultimately, further research will unravel the complexities of antibiotic resistance in this recalcitrant organism, which may guide the development of new and more efficacious antimicrobial agents or strategies that minimize drug resistance. Since differences between in vitro and in vivo antibiotic efficiency exist, more in vivo studies need to be undertaken to reevaluate the determinants of in vivo efficiency of certain antibiotics. These studies would help to improve the use of antibiotics in human infections.  1.2  Ciprofloxacin  1.2.1  Structure and activity Ciprofloxacin,  or  1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-  quinoline-carboxylic acid, is a second-generation fluoroquinolone that was patented in 1983 and approved by the FDA in 1987 (158, 264). The first quinolone antibiotic, nalidixic acid, was approved in the 1960s’, however, nalidixic acid was only moderately effective against some Gram-negative bacteria, rapidly leading to resistance, and was not active against P. aeruginosa (196). The addition of fluorine at the C-6 position of the basic quinolone structure in ciprofloxacin significantly increased the antimicrobial properties and enabled its use in treatment of P. aeruginosa infections. Ciprofloxacin is a very potent compound with an average MIC of 0.1 µg/ml for PAO1 (19). The chemical structure of ciprofloxacin is shown in Figure 1.2.  21  Figure 1.2. Chemical structure of ciprofloxacin. Fluoroquinolones are DNA damaging agents that act by targeting the essential bacterial enzymes DNA gyrase and topoisomerase IV, interferring with DNA synthesis (55). DNA gyrase and topoisomerase IV are essential to keep DNA in the appropriate topological state for replication and transcription (10). DNA gyrase, which is encoded by the genes gyrA and gyrB, is involved in introducing negative supercoils into the DNA, thereby removing topological stress (115). On the other hand, topoisomerase IV, encoded by parC and parE, is essential for unwinding DNA following DNA replication (55). Ciprofloxacin and other quinolones target these enzymes, block DNA synthesis and cell growth ultimately leading to cell death (55, 93). Typically, upon DNA damage, bacteria initiate a DNA repair response, called the SOS response, which involves at least 15 genes in P. aeruginosa (34). However, a failure in the DNA repair system leads to error-prone repair which results in either mutagenesis or cell death. Interestingly, several studies have shown that ciprofloxacin can induce the expression of genes involved in the SOS response under both sub-inhibitory and inhibitory concentrations (20, 34). The induction of the SOS response is strongly dependent on the dose of ciprofloxacin and the duration of exposure as high concentrations become lethal over time. The upregulation of the SOS response leads to efficient DNA repair, thereby rendering the cell less susceptible to further antibiotic challenge. 1.2.2  Mechanisms of fluoroquinolone resistance The overuse of fluoroquinolones has led to an increase in quinolone resistance in P.  aeruginosa (267). Resistance to fluoroquinolones is often mediated by mutations in genes coding for target enzymes, efflux pump overexpression, or mutations that lead to decreased permeability (103) (Figure 1.3). 22  1  3  2 Figure 1.3. Mechanisms of fluoroquinolone resistance in Gram-negative bacteria include: (1) reduced permeability; (2) mutations in target enzymes; (3) overexpression of efflux pumps. As mentioned in section 1.1.3, fluoroquinolone resistance can occur by mutations in the target genes gyrA and/or parC. These mutations reduce the binding affinity of the fluoroquinolone for the target and therefore increase resistance. Clinical isolates are more resistant to fluoroquinolones if they have multiple mutations in the target enzymes. For example, high-level fluoroquinolone resistance has been observed in clinical isolates of P. aeruginosa that have two mutations in gyrA (Thr83Ile and Asp87Gly) and an additional mutation in parC (Ser80Leu) (139, 178). Other mechanisms leading to fluoroquinolone resistance include decreased permeability and increased drug efflux (129). The overexpression of efflux pumps is achieved by mutations in the respective regulators. For example, a mutation in the regulator mexZ, which acts as a repressor, leads to the overexpression of the MexAB-OprM and MexXYOprM efflux pumps. Similarly, a mutation in the regulator nfxB, leads to the overexpression of MexCD-OprJ and, therefore, to quinolone resistance (95, 122). Despite our current knowledge of ciprofloxacin resistance mechanisms, it is likely that many more genes and resistance mechanisms play a role in fluoroquinolone resistance, which can have an impact in a clinical setting. Indeed, the characterization of the ciprofloxacin resistome is one objective of this thesis. 1.3  Lon protease The Lon protease is found in many prokaryotes as well as in eukaryotic mitochondria;  however, it has been best studied in E. coli (81). Lon is an 87 kDa adenosine triphosphate (ATP)dependent cytoplasmic serine protease that associates into hexameric rings in Gram-negative bacteria. The Lon protease belongs to the AAA+ protein family (ATPases associated with various 23  cellular activities) with a Ser679-Lys722 dyad, responsible for catalytic activity (16). Lon proteases are divided into two subfamilies: LonA (E. coli) and LonB (Archaeoglobus fulgidus). LonA members contain an N-terminal domain for substrate recognition, an ATP-binding domain and a proteolytically active C-terminal domain, whereas members of the LonB family lack the Nterminal domain and have instead a membrane domain acting as an anchor (211). The P. aeruginosa Lon protease belongs to the LonA family. Furthermore, Lon bears the group name for self-compartmentalized or chambered proteases and consists of a homosubunit complex, indicating that the ATPase and proteolytic active sites are within the same polypeptide chain (27). The crystal structure of the P domain of the Lon protease of E. coli is shown in Supplementary Figure 1. The Lon protease is involved in protein quality control and regulation of biological processes. Thus, Lon is the principle protease that, together with the caseinolytic ClpP protease, is responsible for 70-80% of energy-dependent protein degradation in E. coli (164). Lon degrades unstable and misfolded proteins by translocation of the unfolded proteins into the proteolytic chamber of the protease, where peptide bond cleavage occurs. The P. aeruginosa Lon protease has 84% sequence similarity to the E. coli Lon protease (www.pseudomonas.com). Studies on the E. coli Lon protease show that Lon degrades the antitermination protein (N protein) of phage λ, and that Lon is involved in the lysogenic switch of λ, i.e., lon mutants favour lysis over lysogeny (124, 163). Furthermore, lon expression is induced in heat shock conditions as the Lon protease is required to unfold misfolded proteins and plays a role in their subsequent degradation. Some examples of Lon target proteins are SulA (celldivision inhibitor) (170) and RcsA (transcriptional activator for capsule synthesis) (248). Furthermore, Lon influences certain short-lived regulatory proteins and it is known that such proteases are essential for virulence gene regulation (27). Virulence gene regulation includes the regulation of type III secretion systems as well as invasion proteins of Yersinia, Salmonella and P. syringae. Lon mutants show filamentation, sensitivity to UV light and DNA damage, as well as fluoroquinolone susceptibility in E. coli (269). The lon gene is a non-essential gene; however, lon overexpression is lethal in E. coli, which highlights the importance of regulating lon expression (33). Lon has not been well studied in P. aeruginosa, but it appears to have the appropriate characteristics to be a central player in the complex adaptations of this organism. To date, it is known that lon mutants in P. aeruginosa show ciprofloxacin susceptibility, filamentation, 24  motility defects, and biofilm deficiency (19, 157). Lon is also a negative regulator of the QS system (244). 1.4  Rationale and objectives Drug resistant clinical isolates of P. aeruginosa are on the rise. Consequently, treating  Pseudomonas infections is increasingly challenging. Moreover, the antibiotic resistance mechanisms active in an infected host are not fully understood. For example, in the case of ciprofloxacin, we do know that mutations in target enzymes, decreased permeability and overexpression of efflux pumps are resistance mechanisms. Nevertheless, it is important to elucidate other genes and possible mechanisms that can lead to ciprofloxacin resistance as this might help find new therapeutic targets. In this thesis, Chapter 3 focuses on the identification of novel non-essential genes leading to increased or decreased ciprofloxacin resistance and the following chapters highlight the role of the Lon protease in both antibiotic resistance and virulence. 1.4.1  Hypothesis By modulating the stability of certain key regulators, the Lon protease influences both  resistance to ciprofloxacin and key virulence determinants of P. aeruginosa. 1.4.2 Overall aim The overall aim of this thesis was to investigate the mechanisms by which the Lon protease influences ciprofloxacin resistance and Pseudomonas virulence. 1.4.3  Objectives  1. To elucidate novel non-essential genes involved in altered susceptibility to ciprofloxacin in vitro. 2. To analyze the influence of the Lon protease in determining intrinsic ciprofloxacin resistance. 25  3. To investigate the global regulation of the Lon protease (transcriptome and proteome). 4. To investigate the role of the Lon protease in pathogenesis using in vitro and in vivo models. 5. To determine if other intracellular proteases also impact virulence-related properties and antibiotic resistance.  26  2. Material and methods 2.1  Bacterial strains and growth conditions The bacterial strains used in this study are described in Table 2.1 and are mainly  transposon mutants. Strains were stored at -70°C until use and were grown in Luria-Bertani Broth (Fisher), Mueller-Hinton Broth (BD) or BM2-media (62 mM potassium phosphate buffer pH 7.0, 7 mM (NH4)2SO4, 2 mM MgSO4, 10 µM FeSO4 and 0.4% (w/v) glucose) at 37°C. Antibiotics used in this study were largely purchased from Sigma-Aldrich. Table 2.1. P. aeruginosa strains and plasmids used in this study. Strain or plasmid  Description and characteristics  References  Strains P. aeruginosa H103 (WT) lon (PA1803) mutant recA (PA3617) mutant  Wild type P. aeruginosa PAO1, strain H103 PA01 lon::mini-Tn5-luxCDABE, 74_D9, Tetr PA01 recA::mini-Tn5-luxCDABE, 69_C12, Tetr  Lab collection (143) (143)  recG (PA5345) mutant  PA01 recG::mini-Tn5-luxCDABE, 16_G12Tetr  ruvA (PA0966) mutant  PA01 ruvA::mini-Tn5-luxCDABE, 67_B1, Tetr  (143) (143)  fleS (PA1098) mutant fliC (PA1092) mutant lexA (PA3007) mutant sulA (PA3008) mutant pfpI (PA0355) mutant clpS (PA2621) mutant  PA01 fleS::mini-Tn5-luxCDABE, 73_E3, Tetr PA01 fliC::mini-Tn5-luxCDABE, 130_B11, Tetr PA3007::ISphoA, Tetr, derived from UW-WT PA3008::IslacZ, Tetr, derived from UW-WT PA0355:: ISphoA, Tetr, derived from UW-WT PA2621:: ISphoA, Tetr, derived from UW-WT PAO1 lon::mini-Tn5-luxCDABE (pBBR1MCS4::lon+), Cbr PAO1 fleS::mini-Tn5-luxCDABE (pUCP23::fleSR+), Gmr PAO1 pBBR1MCS4::sulA+, Cbr PAO1 pBBR1MCS4::lexA+, Cbr PAO1 pBBR1MCS4::lon+, Cbr Wild type P. aeruginosa PA14 Harvard library PA14: PAMr_nr_mas_04_1:A9, Gmr  lonc strain fleSc strain sulA + strain lexA + strain lon + strain PA14 (WT) lon (PA1803) mutant  (143) (143) (110) (110) (110) (110) This study This study This study This study This study Lab collection (148) 27  Strains fleS (PA1098) mutant clpP (PA1801) mutant clpX (PA1802) mutant clpS (PA2621) mutant pfpI (PA0355) mutant clpPc strain pfpIc strain H399 lon:H399 E. coli TOP10 DH5α  Description and characteristics Harvard library PA14: PAMr_nr_mas_10_3:C5, Gmr Harvard library PA14: PAMr_nr_mas_12_4:E7, Gmr Harvard library PA14: PAMr_nr_mas_08_1_F11, Gmr Harvard library PA14: PAMr_nr_mas_06_2:F9, Gmr Harvard library PA14: PAMr_nr_mas_12_1:C6, Gmr PA14 clpP-:pBBR1MCS4::clpP+, Cbr PA14 pfpI-:pBBR1MCS3::pfpI+, Tetr PAO1 auxotroph for leucine and lysine lon deletion in H399 background  References (148) (148) (148) (148) (148) This study This study Lab collection This study  Invitrogen F– mcrA (mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 recA1 ara139 (ara-leu)7697 galU galK rpsL (Strr) endA1 nupG F–80lacZM15 (lacZYA-argF)U169 deoR recA1 Invitrogen endA1 hsdR17(rK– mK+) supE44– thi-1 gyrA96 relA  Plasmids Invitrogen pCR-Blunt II-TOPO PCR cloning vector, Kanr r (128) pBBR1MCS3 Broad-host-range cloning vector, Tet (128) pBBR1MCS4 Broad-host-range cloning vector, Ampr r (221) pUCP18 Broad-host-range cloning vector, Amp r (221) pUCP23 Broad-host-range cloning vector, Gm r (98) pEX18 Suicide plasmid, Amp On a special note, the PA14 Harvard mutant library (148) was used for screening purposes. 2.2  Mutant library agar screen The comprehensive PA14 transposon mutant library (148) was screened for mutants  showing intrinsic and/or mutational resistance. Each individual mutant was stored in a separate well of a 96 well plate. Screening was performed with a 96 well replicator and mutants were taken out of the original 96 well plate and inoculated into Mueller-Hinton broth, grown overnight at 37°C and diluted 1:100 the next day. The freshly diluted cultures were spotted (approximately 3 x 104 cells) onto Mueller-Hinton agar plates containing 0.025 or 0.2 µg/ml ciprofloxacin (Sigma-Aldrich) to test for intrinsic and mutational resistance, respectively. Increased and  28  decreased susceptibility was defined by a lack of growth on the low concentration or growth on the high concentration respectively after 24 and 48 hours incubation at 37°C. 2.3  Primer design The primer 3 program (http://frodo.wi.mit.edu/primer3) and the ABI Primer Express  program (v2.0) were used to design primers for conventional PCR and RT-qPCR, respectively. 2.4  PCR amplification Gene amplification was performed in 20 µl reactions using either Taq-polymerase  (Invitrogen) or Phusion polymerase (Finnzyme). P. aeruginosa genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). 2.5  Complementation of lon To complement the lon::lux mutant, the lon gene (PA1803) was PCR-amplified from the  genomic DNA of the PAO1 Pseudomonas strain. The fragment was then cloned into TopoBlunt (Invitrogen), cut with HindIII and XbaI (Invitrogen), and ligated into the broad-host-range vector pBBR1MCS4 (128). This construct was then transformed into E. coli DH5α cells and subsequently into the P. aeruginosa lux mutant:74_D9 (143), resulting in the complemented Pseudomonas strain PAO1 lon::mini-Tn5-luxCDABE pBBR1MCS4::lon+ (designated lonc). A similar procedure produced the fleSR:pUCP24, pfpI:pBBR1MCS3 and clpP:pUCP19 complemented strains. 2.6  Overexpression studies The low-copy vector pBBR1MCS4 contains a lac promoter upstream of the multiple  cloning site (MCS) (128). In overexpression studies, the genes lexA (PA3007), sulA homolog (PA3008) and lon (PA1803) were PCR-amplified and cloned into TopoBlunt. Cloning was followed by a restriction digestion with HindIII and XbaI, ligation into pBBR1MCS4 and 29  transformation into competent E. coli DH5α cells. Plasmids were then isolated, transformed into wild type P. aeruginosa PAO1, and verified by PCR to ensure that the products were of appropriate size. Experiments performed include RNA isolation, growth curve, MIC, swarming, swimming, twitching and biofilm formation. 2.7  Minimal inhibitory concentration (MIC) The standard broth microdilution method was used to measure the MIC of ciprofloxacin,  polymyxin B, tobramycin, aztreonam, ceftazidime, piperacillin and imipenem against Pseudomonas (261). The first concentration where no growth was observed after incubation at 37°C for 24 and 48 hours was recorded as the MIC for the strains. 2.8  Growth experiments Bacterial growth was measured over time in flasks or in 96 well plates. Briefly, overnight  cultures were diluted 1:100 in BM2 or LB broth and absorbance (600 nm or 620 nm) was measured every 30 or 20 minutes over a time period of 8 hours in experiments conducted in flasks or 96 well plates (TECAN Spectrafluor Plus plate reader), respectively. 2.9  Killing assay P. aeruginosa cells were grown to mid-log phase (OD600 0.5) in LB medium, pelleted by  centrifugation (3,000 x g, 10 minutes), and resuspended in 1 x BM2 salts. Ciprofloxacin at 1 x MIC was added to the samples, which were then shaken at 37°C. Aliquots were taken at the indicated times up to 90 minutes after antibiotic addition, serially diluted and plated on LB agar. The surviving cells were grown overnight at 37°C and the Colony Forming Units (CFU) were counted the next day. All experiments were repeated at least 3 times.  30  2.10  UV irradiation assay Overnight cultures of wild type P. aeruginosa (PAO1), lon mutant and complemented lon  mutant were diluted 1:10 in LB broth, and grown for 4 hours at 37°C under shaking conditions. After 4 hours, the cultures were again diluted 1:10 and transferred to an empty Petri dish for UV irradiation. UV irradiation was performed using a Chemi genius2 Bio imaging system from Syngene, which contains 10 UV lamps (total radiant energy is 12 watt). The samples were exposed to UV light for 10 seconds at an approximate distance of 0.5 inch. Cultures were then serially diluted, plated on LB agar and incubated overnight at 37°C. The CFUs were counted the next day. 2.11  Heat shock P. aeruginosa wild type cultures were grown to mid-logarithmic phase at 37°C. The  cultures were then incubated at 42°C for 20 minutes. Cells were subsequently collected by centrifugation and RNA was isolated from the cells using the RNeasy Mini RNA isolation kit (Qiagen) and prepared for RT-qPCR experiments as described in section 2.14. 2.12  Microarray analysis Each microarray experiment was performed, with technical assistance from Manjeet  Bains, on 3-5 independent cultures of wild type PAO1 and the lon mutant. Cultures were grown either to mid-logarithmic phase (OD600 0.5) or early stationary phase (OD600 1.8) in the presence or absence of sub-inhibitory concentrations of ciprofloxacin (0.05 and 0.0125 µg/ml for wild type and mutant cells, respectively). RNA was extracted using the RNeasy Midi RNA isolation kit (Qiagen). RNA samples were treated with the DNA-free kit for 90 minutes (Ambion) to remove any contaminating genomic DNA, and then Superase Inhibitor (Ambion) was added. RNA (10 µg) was reverse transcribed into cDNA, which was labelled with Cy3 or Cy5 dyes (Amersham Pharmacia Biotech). Labeled sample pairs were applied to a spotted microarray provided by the Craig Venter Institute, hybridized overnight, scanned on a ScanArray Express  31  scanner, and analyzed using Imagene software and ArrayPipe version 1.7. Only statistically significant changes (p<0.05) were used for further analyses as measured with the Student’s t test. 2.13  Reverse transcription reaction DNase treated RNA (1-10 µg) was mixed with random primers (Invitrogen) and  incubated at 70°C for 10 minutes, followed by incubation at 25°C for 10 minutes. RT buffer (1x), 10 mM DTT, 10 mM dNTPs, 30 U SUPERase-IN and 1500 U Superscript II reverse transcriptase were added to the RNA. Samples were then incubated at 37°C for 1 hour, 42°C for 3 hours, and finally 72°C for 10 minutes. Residual RNA was removed from the cDNA by incubating with 1 N NaOH for 15 minutes at 65°C. NaOH was then neutralized with 1 N HCl. The prepared samples were used for real-time PCR. 2.14  Real-time PCR (qPCR) The ABI Prism 7000 sequence detection system and the SYBR green dye (Applied  Biosystems) were used to perform real-time PCR to validate the microarray data and analyze transcriptional changes under stress conditions. Primers were designed using ABI Primer Express v2.0 and the sequence of the gene of interest. cDNA was prepared as described in section 2.13, diluted (1:100) in nuclease-free water (Ambion), and mixed (2.5 µl) with 1 x SYBR Master Mix (Applied Biosystems) and 10 µM of each primer. Samples were loaded, in triplicates, in 96 well plates and analyzed with the ABI Prism 7000 sequence detection system. The following cycling parameters were used: 50°C for 2 minutes, 95°C for 2 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds, followed by 95°C for 15 seconds, 60°C for 20 seconds and 95°C for 15 seconds. The raw data was analyzed using Sequence Detection software, and relative fold changes (FC) were calculated using the following formula where CT denotes the gene expression values: ∆CTtest = CT test – CT housekeeping gene; ∆CTcontrol = CT control – CT housekeeping gene; ∆∆CT = ∆CTtest - ∆CTcontrol; 32  FC = 2-∆∆CT The following genes were used as housekeeping genes for normalization: rpsL (PA4268), anr (PA1544) and PA2018 (efflux transporter). 2.15  Anaerobic growth Bacterial cultures, supplemented with 15 mM KNO3, were grown overnight at 37°C in an  anaerobic jar containing a GasPak Plus hydrogen and carbon dioxide generator envelope (BD). Cell suspensions were then prepared to a 0.5 McFarland Standard (1.5 x 108 cfu/ml), diluted 1:20, and anaerobic growth was monitored at OD600 over a time period of 12 - 24 hours. 2.16  Cellular respiration assay WST1 Bacterial cultures grown overnight under aerobic or anaerobic conditions (as described in  section 2.1 and 2.15) were diluted (1:5) and transferred to 96 well microtiter plates. 10 μl WST1{4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene  disulfonate}  (Roche) was added to each well and absorbance (OD450 and OD900) was measured at the indicated time points. The respiration rate was defined as the ∆OD (OD450 minus OD900). The absorbance measured at 450 nm indicated the conversion of WST1 to the yellow-orange product formazan, whereas the absorbance at 900 nm represents a reference wavelength to correct for the background noise. The intensity was a measure of the respiration rate. 2.17  Swarming motility assay Swarming motility was measured by inoculating a bacterial culture (1 µl) on a swarming  plate (modified BM2 + 0.5% agar). In the modified BM2 medium, ammonium sulfate was substituted with 0.5% casamino acids (PAO1) or 0.1% casamino acids (PA14) (Fisher). The swarming behaviour (dentritic colonial appearance) of mutant strains was compared to the wild type strain after 20 hours of incubation at 37°C.  33  2.18  Swimming motility assay Swimming motility was evaluated using BM2 plates containing 0.3% agar. Briefly,  bacterial cultures inoculated on swimming plates were incubated for 20 hours at 37°C. The resulting swim zone diameters (halo of cells inside the agar) were then measured and the average diameter was determined. 2.19  Twitching motility assay The ability of certain Pseudomonas mutants to twitch on solid surfaces was evaluated by  stabbing an overnight culture (1 µl) through an LB agar plate containing 1.5% agar. Twitching zones (movement of bacteria across surfaces) were measured after 24 hours incubation at 37°C. 2.20  Measurement of rapid attachment The abiotic solid surface assay was used to measure rapid bacterial attachment. Overnight  PAO1 and PA14 cultures were diluted (1:100) in LB broth or BM2 broth containing 0.5% casamino acids, respectively. Cultures were transferred to 96 well polystyrene microtiter plates, which were incubated for 1 hour at 37°C. Crystal violet (Sigma-Aldrich) was used to stain the attached cells, and the absorbance (595 nm) was measured on a Microtiter plate reader (Bio-Tek Instruments). 2.21  Measurement of mature biofilm formation The abiotic solid surface assay was used to measure rapid bacterial attachment. Overnight  PAO1 and PA14 cultures were diluted (1:100) in LB broth or BM2 broth containing 0.5% casamino acids, respectively. Cultures were transferred to 96 well polystyrene microtiter plates, which were incubated for 20 hours at 37°C. Crystal violet (Sigma-Aldrich) was used to stain the attached cells, and the absorbance (595 nm) was measured on a Microtiter plate reader (Bio-Tek Instruments).  34  2.22  Congo red assay The Congo red assay was performed to quantify the matrix production of the biofilm  because of the ability of Congo red to bind the polysaccharides (Pel and Psl). Bacteria grown overnight in Tryptone broth (BD) were diluted to an OD600 of 0.025. Aliquots of the bacterial dilution (1 µl and 5 µl) were spotted on a Congo red plate containing Tryptone, agar, Congo red (BDH), and Coomassie brilliant blue (Sigma-Aldrich). Plates were incubated for 24 hours at 37°C, followed by 48 – 72 hours at room temperature before visual examination of Congo red binding. 2.23  Nitrocefin assay The cells used for the quantitative nitrocefin assay were grown as described by Alvarez-  Ortega et al. (2). β-lactamase activity was quantified by using nitrocefin as a β-lactamase substrate and the protein content of each sample was measured by using the Quibit Protein Assay Kit and the Qubit fluorometer (Invitrogen). In case of the qualitative nitrocefin assay, colonies were spread on discs containing nitrocefin and a red colour change indicated the production of βlactamase for the respective strains. 2.24  Morphological determination The morphological shape of bacterial samples was observed on a Zeiss Axioscope (Zeiss  Canada) or a Nikon Eclipse (Canada) microscope. Bacterial samples were spotted onto microscope slides (VWR), heat fixed and stained with crystal violet (BD) or Diff-Quik (Siemens) to look for cell length and attachment to HBEs. 2.25  Lactate dehydrogenase (LDH) – release assay The LDH release assay was used to quantify bacterial induced killing of the human  bronchial epithelial cell line 16HBE14o-, kindly provided by Dr. D. Gruenert (University of California, USA). HBE cells were grown at 37°C + 5% CO2 in Minimum Essential Media 35  (Invitrogen) supplemented with 2% fetal bovine serum and 2 mM L-glutamine (Invitrogen) until 90% confluency was reached. Bacteria (wild type, lon and fleS mutant and complemented strain) were grown in LB to mid-logarithmic phase, washed with PBS and resuspended in MEM medium. This was followed by a multiplicity of infection (MOI) of 50 bacteria per cell with incubation at 37°C + 5% CO2. At 7.5, 10 and 18 hours post-infection, the supernatant was collected and lactate dehydrogenase (LDH) levels were determined using a cytotoxicity detection kit (Roche) as per the manufacturers’ instructions. The absorbance at 490 nm was measured after 30 minutes, whereby vehicle - and treated (2% Triton X-100) - cells served as the negative and positive control, respectively. Percent cytotoxicity was calculated using the formula: % cytotoxicity = 100 x [(E – N)/ (P – N)], where E, N, and P denote experimental absorbance, vehicle treatment absorbance, and 2% Triton X-100 absorbance, respectively. The assay was performed by Shaan Gellatly and Patrick Taylor. 2.26  Adhesion assay HBEs were grown as mentioned in section 2.22 and infected (MOI=50) with mid-  logarithmic phase bacterial cultures. Samples were incubated at 37°C + 5% CO2 for 3 hours at which point the monolayers were washed with PBS (Phosphate Buffered Saline), and the cells were fixed with 4% formaldehyde in PBS for microscopic analyses. 2.27  Lettuce leaf virulence model Romaine lettuce leaves were used to determine the ability of P. aeruginosa wild type,  complemented strain and lon mutant to infect lettuce leaves. Lettuce leaves were rinsed with dH20, washed with 0.1% bleach for 5-10 minutes, washed again with dH20 and dried. Bacteria (1 x 106) were stabbed into the midrib section of the lettuce stems. MgSO4 (10 mM) served as a negative control. The top and bottom of the leaves were monitored over 5 days to assess the symptoms of infection. It was important to compare leaves of the same size as the infection spreaded faster in larger leaves compared to smaller leaves.  36  2.28  Amoeba virulence model Dictyostelium discoideum (amoeba) was used as another host model system, in  collaboration with Dr. Joerg Overhage (KIT, Karlsruhe, Germany), to assess bacterial virulence. Briefly, amoebae were cultured at 23°C until confluent growth was achieved, then detached from the flasks, washed and diluted. For the assay, overnight wild type and lon mutant bacterial cultures were diluted in PBS and spread on agar plates. D. discoideum was then spotted at different concentrations onto the plates. Incubation took place for 3-5 days at 23°C and visible plaque formation on the agar plates was determined. The amoebae concentration needed to form a plaque was an indication of relative susceptibility to infection in this model. 2.29  Competitive index (CI) determination For in vitro CI assays, overnight cultures of the lon mutant were mixed (1:1) with the  wild type PAO1 or the complemented lon strain. Bacterial mixtures were incubated, under shaking conditions, for 8 hours at 37°C. Bacterial mixtures were then plated for CFU counts with the addition of appropriate antibiotics. CFU counts were determined after 18 hours incubation at 37°C. The rat chronic lung infection model, performed in collaboration with Dr. Roger Levesque (University Laval, Quebec, Canada), was used to measure the CI in vivo. In this experiment, the bacteria were enmeshed in agar beads (131). Cultures of the wild type, lon mutant and complemented strain were grown in Tryptic soy broth (BD) overnight at 37°C and diluted to a final concentration of 1 x 1010 cfu/ml. The 1:1 mixture of wild type or complemented strain and lon mutant was mixed with agarose in PBS at 48°C. The agarose-broth mixture was added to heavy mineral oil and stirred on a magnetic stirrer in a water bath. The mixture was then cooled to 0°C on ice at which point the agarose beads were washed with sodium deoxycholate and PBS, and incubated on ice, while the remaining PBS was removed. Male Sprague-Dawley rats were anaesthetized using isofluorane and intubated with 120 µl of the agarose bead suspension (containing 105 bacteria). The animals were sacrificed at 7 days postinfection and the lungs were removed, homogenized and plated (with addition of antibiotics) to 37  determine CFU counts. The CFU counts represented the total number of bacteria present in the lungs of the rats. The CI (141) is defined as the CFU output ratio of mutant compared to the wild type, divided by the CFU input ratio of mutant compared to the wild type. The in vivo experiment was performed on 5 rats, and the final CI was calculated as the geometric mean for all animals. The Mann Whitney test was used to determine statistical significance between the mutant and the wild type. 2.30  Mucin To mimic the CF lung environment, mucin (Sigma-Aldrich), a glycopeptide from the  mucous layer of the lung, was added at various concentrations (0.1, 0.5 and 1%) to 0.4% agar and BM2 motility plates (with and without ciprofloxacin). Mid-logarithmic bacterial cultures (1 µl) were inoculated on the agar plates. Motility and antibiotic resistance were assayed after 20 hours of incubation. For the biofilm formation, mucin was added to the biofilm media and mature biofilm formation was measured after 20 hours of incubation at 37°C (as described in section 2.21). 2.31  Construction of a lon deletion mutant in an auxotrophic background A complete lon knockout was constructed using a 3-step-fusion PCR (133) in which the  lon gene was replaced with the gentamicin cassette from the plasmid pPS858. Separate PCR reactions amplified the up- and downstream regions of the lon gene as well as the gentamicin cassette. Importantly, the primers exhibited an overlapping sequence, so that the 3 pieces could be fused together. After obtaining the knockout construct, this fragment was ligated into the suicide vector pEX18Amp which was conjugated into an auxotrophic (for leucine and lysine) strain H399. Selection on sucrose promoted homologous recombination and loss of the cointegrant. Colony PCR as well as sequencing confirmed the correct construct. This strain was used for the proteomic approach using stable isotope labelling by amino acids in cell culture (SILAC).  38  2.32  Extraction of the cytoplasmic fraction To obtain cytoplasmic bacterial fractions, the membrane-cytoplasmic fraction was first  separated from the periplasm. Auxotrophic bacterial cultures were grown overnight in 10 ml BM2 minimal medium containing leucine and lysine. Parent strain bacteria H399 were grown in the presence of normal isotopic abundance (“light”) amino acids, whereas the lon mutant was grown in the presence of deuterium labelled amino acid lysine (“heavy (D4)”). The labelled cells were pelleted and washed with washing buffer (50 mM Tris (pH 8.0), 0.2 M MgCl2). After washing, the HALT protease inhibitor (Sigma-Aldrich) was added to the samples, which were incubated at 30°C for 30 minutes, 5 minutes in ice-chilled water, and 15 minutes at room temperature. The cells were collected by centrifugation and the pellet (membrane-cytoplasmic fraction) was washed in 50 mM Tris, resuspended, and treated with 100 µg/ml DNase I for 20 minutes at 4°C. Protease inhibitors were added and the cell suspension was sonicated (6 x 10 seconds with 10 seconds intervals between sonication) with a probe-sonicator to break apart the cells (Fisher). To remove the unbroken cells, the suspension was pelleted and the supernatant was collected by centrifugation for 1 hour at 100,000 x g using an ultra-centrifuge (Beckman Coulter). The supernatant (cytoplasmic fraction) was used for the SILAC experiment. 2.33  SILAC: stable isotope labelling by amino acids in cell culture SILAC can be used to determine differences in protein expression between two bacterial  strains or two different conditions (187). The cytoplasmic fractions of the lon mutant and its parent strain labelled with heavy and light amino acids, respectively, were used in this study. The Bradford protein assay (Oz Bioscience) was used to quantify protein concentrations. The parent and the lon mutant supernatants (50 to 100 µg/ml) were mixed together, and the In-solutiondigest plus sodium deoxycholate was performed. Briefly, samples containing 3% deoxycholate were boiled for 5 minutes, DTT (dithiothreitol), iodoacetamide and LysC (endoproteinase which hydrolyzes the carboxyl side of lysine) were added, and incubated at 37°C overnight. After this digestion, peptides were purified using the C18 (desalted column) STAGE-Tip purification method. Briefly, the column and the peptide sample were prepared for binding by adding methanol to the column and diluting the sample in sample buffer containing 3% acetonitrile, 1% 39  trifluoroacetic acid, 0.5% acetic acid. The sample was loaded onto the column and eluted with a buffer containing 0.5% acetic acid, 80% acetonitrile in water. To obtain better fractionation, the sample was separated with the OFFGEL Fractionator (Agilent) through isoelectric focussing of peptides, and the different fractions were purified a second time using the C18 STAGE-Tip purification method before analysis by mass spectrometry (MS). The raw data files were collected and analyzed by MaxQuant (40) in several steps: (1) MaxQuant was used first to detect peak features and quantify peptides; (2) Mascot was used to associate fragment spectra with particular amino acid compositions; (3) the final identification step identified peptides based on the known sequences of Pseudomonas proteins, assembled them into protein sequences, and calculated ratios of proteins in the two strains. 2.34  Whole cell preparation Bacteria strains were grown overnight in the presence or absence of sub-inhibitory  concentrations of ciprofloxacin and 200 µl of the cultures were pelleted. The pellets were lysed with lysis buffer containing Triton X-100 (Fisher) for 30 minutes at room temperature. After centrifugation, the supernatants were used to measure the protein concentration of the samples with the Qubit fluorometer (Invitrogen) to ensure that same amount of proteins were loaded on the SDS gel for analysis. After denaturing at 100°C, samples were loaded on a polyacrylamide gel. 2.35  Protein gel Whole bacterial cells were denatured by boiling at 100°C for 5 minutes in 2 x protein  solubilisation buffer (0.5 M Tris pH 6.8, Glycerol, 10% SDS, 0.5 M EDTA, dH20). Denatured samples were then separated on a 12% polyacrylamide gel. Gels were stained overnight in Coomassie Blue, and destained the following morning using a destaining solution (with a 3:1 ratio of ethanol to acetic acid in dH20).  40  2.36  Western blot Western blotting was carried out with whole cell lysates of bacterial strains. A total of 20  µg protein was loaded per lane. After separating the samples on a 12% polyacrylamide gel, the protein bands were transferred to a polyvinylidene fluoride membrane (PVDF) (Amersham) using a tank blot, blocked for 1 hour in TBS (20 mM Tris, pH 7.4, 150 mM NaCl) + 5% skimmed milk powder and 1% BSA, and incubated with a rabbit polyclonal antibody against E. coli RecA protein (1:1000, Abcam) overnight at 4°C in TBS plus 5% skimmed milk powder. Membranes were washed for 8 x 10 minutes in TBST (TBS + 0.1% Tween-20) and then incubated with anti-rabbit IgG HRP-linked (1:3000 Cell Signaling Technology) antibody (secondary antibody) for 30 minutes in TBS + 5% skimmed milk powder. Washing the membranes for 5 x 10 minutes in TBST allowed for the removal of unbound secondary antibody. The ECL Kit (Amersham) was used to develop protein bands. 2.37  Measurement of oxidative stress  Bacterial cultures were grown to mid-logarithmic phase and spread on an agar plate. Filter discs containing 100 mM paraquat and 30% H2O2 were placed on the agar plates and incubated for 24 hours at 37°C. The clearing zone around the disc indicated the inhibition of bacterial growth which was an indication if the strain can withstand oxidative stress. 2.38  Pathway analysis “PseudoCyc” on the P. aeruginosa website (www.pseudomonas.com) was used to group  identified genes that are involved in the same pathway. “PseudoCyc” enables microarray data to be clustered; thereby allowing the user to identify key pathways that are affected by a specific treatment (e.g., ciprofloxacin), or by a specific mutation (e.g. lon).  41  3. A complex ciprofloxacin resistome revealed by screening a P. aeruginosa mutant library for altered susceptibility 3.1  Introduction Mutations in genes encoding DNA-gyrase and topoisomerase IV, which are both  essential enzymes, lead to resistance to ciprofloxacin (55). Furthermore, mutations in several non-essential genes have been observed with altered susceptibility (19, 50) and a more detailed analysis of genes that show a change in susceptibility is necessary in order to understand the complete ciprofloxacin resistome and provide insights into the mechanism of action. The resistome would reveal potential genetic contributors to stepwise increases in ciprofloxacin resistance. In addition, sub-inhibitory concentrations of fluoroquinolones play an important role in resistance development in P. aeruginosa cultures pre-treated with sub-inhibitory concentrations of ciprofloxacin which develop an adaptive resistance phenotype (19). It has been proposed that antibiotics rarely have a simple mechanism of action. Microarray studies indicate that they lead to the upregulation of dozens to hundreds of genes at or around the MIC (20, 34, 149). It has been suggested that some of these genes might reflect target inhibition, others would represent the induction of cellular stress pathways and some are involved in defensive measures by the bacterium to resist the action of antibiotics. In light of antibiotic action it seems likely that there should be many more genes that are involved in increased or decreased susceptibility in bacteria than have been previously supposed, and some of these might have clinical relevance. To reveal changes in susceptibility compared to the wild type, it was necessary to screen a P. aeruginosa mutant library (148). Chapter 3 and 4 focuses on ciprofloxacin resistance of P. aeruginosa and the involvement of Lon protease in this process. 3.2  Results and discussion  3.2.1  Ciprofloxacin library screen To investigate the extent of the ciprofloxacin resistome, a comprehensive PA14  transposon mutant library was screened for mutants that showed either an increased (reflecting 42  intrinsic resistance) or a decreased (reflecting mutational resistance) susceptibility to ciprofloxacin (148). Mutants identified in the agar screen were confirmed to show a changed susceptibility through MIC value determination by broth dilution methods (261) and changes as low as twofold were taken into account. I acknowledge that twofold changes in MIC values are generally considered within the error of the standard assessment protocols. However, only results for those mutants for which I could consistently confirm changes in at least 3 independent measurements are shown. The first ciprofloxacin screen at 0.025 µg/ml which was used to screen for increased susceptibility (wild type has an MIC at around 0.1 µg/ml) yielded a total of 62 mutants with the inability to grow in the plate assay, leading to 28 confirmed mutants with an increase in susceptibility measured with the MIC broth dilution method. An additional 7 mutants were identified by specifically measuring the MIC of ciprofloxacin for mutants from the same operon as mutants that appeared in the screen, accounting for a total of 35 mutants. Mutants with mutations located in an operon might exhibit polar effects. The constellation of genes involved in intrinsic resistance is demonstrated in Figure 3.1, indicating that they were spread throughout the chromosome. Although the high-throughput nature of this screen did not permit complementation of each mutant, I confirmed the increased susceptibility phenotype with several independent isolates from the PAO1 lux mutant library (143) as well as multiple mutants from 5 operons. Interestingly, prior microarray analysis revealed that 10 of the genes giving rise to an increase in susceptibility upon transposon mutation, were also dysregulated after exposure to 0.3 x or 1 x MIC of ciprofloxacin in the wild type strain (20). This overlap between global gene expression and resistance determinants highlights the fact that Pseudomonas can activate certain defence mechanisms in order to combat the bactericidal activity of ciprofloxacin. The clpP and fleS mutants, despite showing only twofold altered susceptibility, could be successfully complemented.  43  Figure 3.1. Distribution of genes around the PAO1 genome identified in the ciprofloxacin library screen which exhibit increased or decreased susceptibility upon mutation. This genome image was generated by B.K. Khaira using CGView (238). The majority of these mutants demonstrated only twofold changes in susceptibility, although the cell division gene ftsK was 8 fold more susceptible and lon showed an increase in susceptibility of 4-8 fold, with the latter result indicating that the Lon protease is very important for ciprofloxacin intrinsic resistance. Among the ciprofloxacin mutants with increased susceptibility, the number of mutants that were involved in DNA replication and repair was 44  noteworthy, such as the Holliday junction helicase ruvA, the ATP-dependent RNA helicase recG, the recombinase xerD, and the site-specific recombinase sss. Not surprisingly, some of the mutants showed somewhat slower growth compared to the wild type strain (Figure 3.2). However, all of these mutants remained more susceptible even after 48 hours, while several other slower-growing mutants tested were not more susceptible to ciprofloxacin, indicating that slow growth did not cause increased ciprofloxacin susceptibility per se. In addition, mutations in the major intrinsic multidrug efflux pump MexAB-OprM were observed that led to increased susceptibility.  Figure 3.2. Growth curve of selected mutants with a slightly impaired ability to grow compared to the wild type PA14. Absorbance of the strains was measured at 620 nm every 20 minutes over a time period of 10 hours in the TECAN Spectrafluor Plus plate reader. Library screening for mutants that showed at least a twofold decrease in susceptibility to ciprofloxacin identified 46 mutants. A further 13 from adjacent genes in operons, and 20 phagerelated mutants could be confirmed resulting in a final total of 79 mutants with a decrease in susceptibility (Figure 3.1). It is worth noting that such a high-throughput approach is only applicable for genes for which the complete loss of the protein is practical (i.e. non-essential genes). Indeed, nearly all tested mutants with decreased susceptibility were able to grow as well as the wild type. We were able to identify previously known genes such as the MexCD-OprJ efflux regulator nfxB (95), mutators mutS and mutL (186), and the phage-related mutants (20), and observed multiple mutants in 9 operons including the nuoD dehydrogenase operon. We also 45  observed mutations in several iron transport genes, consistent with, but not proving, the recent views suggesting roles for free radicals in antibiotic killing (57, 125). Among the genes giving rise to decreased susceptibility upon transposon mutation, we found 32 that were differentially expressed in response to ciprofloxacin (20). It should be noted that while the library was quite comprehensive, it was not complete; e.g. a mutation in the mexS gene that regulates the MexEFOprN efflux operon was not available. 3.2.2  Functional classes involved in altered ciprofloxacin susceptibility The ciprofloxacin resistome is not only large (comprising over 100 genes leading to  altered susceptibility), but also diverse in that a variety of genes involved in different cellular functions had an impact on ciprofloxacin resistance. As mentioned above, most of these showed only a twofold change and many of the genes were distinct from those traditionally found to be involved in antibiotic resistance. Indeed, many genes from different functional classes, as categorized according to www.pseudomonas.com, were involved. The main functional classes affected were transport of small molecules, membrane proteins, energy metabolism, phage proteins, DNA replication and recombination, cell division and hypothetical proteins. The respective % of genes involved in each functional class is shown in Figure 3.3 that distinguishes between genes leading to decreased (A) and increased (B) susceptibility upon mutation. A lot of genes belonged to the category of hypothetical or unknown, indicating that a large portion of genes which are involved in altered ciprofloxacin susceptibility still need to be categorized further and no correlation to a functional class could be made for these genes.  46  A  B  Figure 3.3. Distribution of the genes involved in decreased (A) and increased (B) ciprofloxacin susceptibility according to their PseudoCAP functional class. 47  Some genes, when inactivated, exhibited not only an altered susceptibility to ciprofloxacin, but also to different classes of other antibiotics, as shown by other researchers (2, 50, 219, 262). For instance, inactivation of ftsK led to a supersusceptibility to ciprofloxacin, levofloxacin, ceftazidime, imipenem, meropenem, ertapenem and cefotaxime. Furthermore, the major intrinsic efflux pump MexAB-OprM, which is known to be involved in ciprofloxacin resistance, was also demonstrated by other researchers to have increased susceptibility to a variety of tested antibiotics upon mutation (β-lactams, tetracycline and sulfonamide). It is therefore clear that the antibiotic resistance mechanisms are not specific as a significant overlap exists (Figure 3.4). One other example is dealing with the overlap in resistance that occurred during different antibiotic screens for mutants in the genes mutS and mutL, which are known hypermutators. Generally, a mutator phenotype is related to a disruption in DNA repair genes. If either one of these two genes is mutated, the spontaneous mutation rate increased dramatically, as demonstrated by studies in our lab. Wiegand et al. (262) could show that the spontaneous mutation frequency for the mutS and mutT mutants increased 71- and 23.6 fold respectively allowing them to accumulate secondary mutations which resulted in acquisition of high resistance to several antibiotics.  48  Figure 3.4. Analysis of the mutants that showed an increase in resistance to 3 antibiotics, representing different antibiotic classes. The overlap between the different antibiotics is shown in the figure and examples are given. The abbreviations are as follows: CAZ, ceftazidime, CIP, ciprofloxacin and TOB, tobramycin. The figure represents the number of mutants found in several screenings (2, 21, 50, 219) and was taken from the book chapter of L. Fernandez, E.B.M. Breidenstein and R.E.W. Hancock (WileyBlackwell, accepted). The substantial variety of identified genes that, when mutated, lead to minor changes in the MIC indicated that it is highly possible that these mutations might have an additive effect. This phenomenon whereby independent low-level resistance mutations lead to additive changes in overall resistance was previously demonstrated by El’Garch et al. for the aminoglycosides (58), and I propose it might also be true for fluoroquinolones. 3.2.3 FleS plays part in antibiotic resistance and virulence The sensor FleS (PA1098), together with the regulator FleR (PA1099), is part of a twocomponent regulatory system and is known to be involved in the regulation of flagellar biosynthesis (206). In the mutant library screens (PA14 and mini-Tn5-lux PAO1) fleS mutants led to altered ciprofloxacin susceptibility. Complementation with the cloned fleSR genes restored the wild type phenotype. To further confirm the ciprofloxacin susceptibility observed for the fleS mutant, I sought to determine the rate of killing of the mutant in the PAO1 background. In agreement with the observed MIC values, the supersusceptibility phenotype of the fleS mutant 49  was reflected by the kinetics of killing. After an initial 30 minutes of incubation with 1 x MIC (0.1 µg/ml) of ciprofloxacin, the fleS mutant showed a significantly lower ability to survive the exposure to ciprofloxacin compared to the wild type, the complemented strain and a fliC mutant that was used as a control to test if the loss of flagella had an impact on ciprofloxacin susceptibility. After 60 minutes of incubation, the wild type, fliC and complemented strain exhibited survival levels of around 1%, whereas the fleS mutant had a survival level of only 0.01% (Figure 3.5).  Figure 3.5. The effect of a fleS mutation on killing by 0.1 µg/ml of ciprofloxacin. Cultures of P. aeruginosa wild type strain H103, the fleS mutant, the complemented mutant (fleS c) and the fliC mutant were grown to mid-log phase, washed and then exposed to 0.1 μg/ml ciprofloxacin (1 x MIC). Colony forming units were counted over a time period of 90 minutes. One out of 3 representative experiments is shown. The effect of fleS on motility (swarming, swimming and twitching) and biofilm formation was further investigated since fleS is involved in flagellar biosynthesis. For a fleS mutant, we could clearly determine a defect in swarming motility, a major defect in swimming, as well as a decrease in mature biofilm formation as compared to the wild type and the complemented strain (Figure 3.6). All of them showed a statistically significant difference as determined by Student`s t test (p≤0.0001). Twitching motility was not affected.  50  A  * **  B  **  Figure 3.6. Impact of a fleS mutation on motility (A: swarming and swimming) and biofilm formation (B). Swarming and swimming motility was investigated by inoculating 1 µl on a swarming plate (modified BM2 + 0.5% agar) or swimming plate (BM2 + 0.3% agar). In the modified BM2 medium, ammonium sulfate was substituted with 0.5% casamino acids. The swarming and swimming behaviour of the mutant was compared to the wild type and complemented strain (fleSc) after 20 hours of incubation at 37°C. The results represent the average and standard deviation of 4 independent experiments. A statistically significant difference (measured with Student’s t test) could be observed (p≤0.0001). For biofilm formation, cells were incubated in 96 well microtiter plates containing LB for 20 hours at 37°C. Biofilm formation was measured by crystal violet staining of the adherent cells. The fleS mutant produced statistically significant less mature biofilm compared to the wild type as determined by Student`s t test (p≤0.0001). The ability of a fleS mutant to swarm was reduced by 40% compared to the wild type H103 and the complemented mutant. Even more pronounced was the defect in swimming; the 51  fleS mutant could not swim and only the point of inoculation could be observed on the swimming plates. Swimming motility is solely dependent on the single polar flagellum of P. aeruginosa and this observation was consistent with the demonstration that FleR regulated flagella production and swimming motility (206). However, twitching motility, dependent on type IV pili, was not affected in the mutant. The fleS mutant also exhibited a major defect in mature biofilm formation, with only 40% biofilm formation compared to the wild type. These factors all indicated that a mutation in fleS may lead to a less virulent phenotype compared to the wild type Therefore, in collaboration with Shaan Gellatly, we went on to investigate to what degree the mutant was capable of causing destruction to epithelial cells by determining the cytotoxicity of the wild type, mutant and complemented strain. To measure cytotoxicity, the amount of lactate dehydrogenase released from the epithelial cells was determined by measuring absorbance, and percent cytotoxicity was calculated as indicated in section 2.25. Our results clearly indicated that after 7.5 hours, the wild type and the complemented fleS strain showed greater cytotoxicity compared to the fleS mutant (Figure 3.7).  *  Figure 3.7. Cytotoxicity of the fleS mutant in comparison to the wild type and complemented strain 7.5 hours post-infection. The abilities of the wild type H103, the complemented fleS strain and fleS mutant to induce cell damage were determined by measuring the release of lactate dehydrogenases (LDH) from the HBEs. The results represent the average and standard deviation of two technical repeats, which show the same trend than other experiments. A statistically significant difference was observed at 7.5 hours post-infection with p=0.05 for the fleS mutant compared to the wild type as measured with the Student’s t test. This was expressed as percentage of the amount of LDH releasesd by the Triton X-100 control. The cytotoxicity assay was performed by Shaan Gellatly. Therefore, it is clear that FleS has a role in both antibiotic resistance as well as in virulence-related properties. 52  3.3  Concluding remarks The resistome comprises all genes that, when mutated, give rise to altered susceptibility.  The present chapter indicated for the first time that the resistome for ciprofloxacin in P. aeruginosa is very large, comprising more than 100 genes. It is important to note that, while we have not demonstrated that these mechanisms are clinically relevant, they do indicate the enormous gene pool that can influence susceptibility to this antibiotic class. This might become important in understanding two complex clinical phenomena, namely MIC creep (where the background level of intrinsic resistance to a given antibiotic rises over time in the population of clinical isolates) (8) and adaptive resistance (where the level of resistance is affected by environmental factors such as growth in vivo or exposure to sub-inhibitory concentrations of antibiotics) (63). MIC creep previously has been attributed to the accumulation of mutations over time, and differs from more obvious clinical resistance, which is caused by “breakthrough” mutations that cause very large changes in susceptibility (e.g. DNA gyrase or efflux pump overexpression mutants). Adaptive resistance has also been proposed to represent a complex phenomenon in which multiple genes that influence gene expression can combine to induce resistance. Indeed, no fewer than 43 of the genes giving rise to altered ciprofloxacin susceptibility were included in the list of those that are differentially expressed in P. aeruginosa in the presence of ciprofloxacin, which is known to promote adaptive resistance to itself (20). This broad screening provided much food for thought and it will be essential in future studies to follow up these observations with detailed studies to determine if these candidate mutants are indeed relevant to clinically meaningful antibiotic resistance. At the same time, different authors also investigated the mechanisms involved in lowlevel resistance to other antibiotics and suggested that the mechanisms are far more complex than originally anticipated. Interestingly, all of these studies confirm the view that non-classical antibiotic resistance genes participate in resistance, an idea that has developed in recent years. Schurek et al. (219) identified 135 genes leading to a twofold or greater tobramycin resistance phenotype upon mutation. These genes are thought to contribute to the gradual lowlevel increase in tobramycin resistance and include the following functional classes: energy metabolism, DNA replication and repair and lipopolysaccharide biosynthesis. Alvarez-Ortega et al. (2) and Doetsch et al. (50) revealed that far more genes than originally thought play a role in 53  increased β-lactam resistance upon mutation. The main affected functional classes were efflux and LPS biosynthesis. Overall, the identification of hundreds of mutated genes contributing to antibiotic resistance provides a new perspective on resistance and in my case specifically to fluoroquinolone resistance. The large number of mutations leading to low-level antibiotic resistance might provide an explanation for the clinical phenomenon of MIC creep, and ultimately lead to high-level antibiotic resistance. Indeed, such mutations might readily occur in environmental situations, and while significant, be easily overlooked.  54  4. Involvement of the Lon protease in the SOS response triggered by ciprofloxacin in P. aeruginosa PAO1 4.1  Introduction As previously stated, Lon is an ATP-dependent cytoplasmic serine protease that  associates into hexameric rings in Gram-negative bacteria (27). Studies carried out in E. coli showed that the targets of the Lon protease include SulA (170), RcsA (248) and the N-protein of phage lambda (163). Furthermore, E. coli lon mutants demonstrated characteristics like filamentation, increased sensitivity to ultraviolet (UV) light and DNA damage, as well as fluoroquinolone supersusceptibility (269). Even though the role of the P. aeruginosa Lon protease is still not fully understood, this protease appears to have the appropriate characteristics to be a central player in the complex adaptations of this organism as it has been demonstrated to play a role in virulence-related properties and antibiotic resistance. Indeed, we recently determined that the P. aeruginosa lon mutants show ciprofloxacin supersusceptibility (19, 21). It is of great importance to understand how the Lon protease affects ciprofloxacin susceptibility, due to its relevance to treatment of Pseudomonas infections. The SOS response is known to be an important transcriptional response to environmental stress and has been characterized best in E. coli. The E. coli error-prone repair system is comprised of 43 genes (39), whereas to date only 15 genes have been identified in the P. aeruginosa genome to be directly involved in the SOS response (34) and 33 genes in B. subtilis (4). The SOS regulon is LexA-controlled and consists of recA, sulA, lexA, recN, recX and dinG, amongst others. All these genes share a consensus binding sequence for LexA that has high homology to the one found in E. coli (34). This consensus sequence consists of 16 nucleotides: CTGTATAAATAACAG (the underlined nucleotides are 100% conserved). During normal growth, the SOS response genes are negatively regulated by the autorepressor LexA, which binds to the consensus sequence of the SOS box. Therefore, depending on how strong the binding is, no or only slight transcription of the SOS response genes occurs. However, upon DNA damage (ciprofloxacin treatment, UV light exposure, mitomycin C, etc.) RecA forms filaments on the single stranded DNA and at the same time stimulates autoproteolysis of the transcriptional repressor LexA (25). As a result, all of the other SOS repair genes are transcribed 55  in order to help overcome the DNA damage. Once the damage has been repaired, LexA binds again to the SOS box, thereby repressing the expression of the genes involved in the SOS response. To date it can only be speculated as to why, for example, recA and recX are LexAregulated in P. aeruginosa whereas others like uvr are not (34), despite being LexA-regulated in other bacteria (4, 39). The speculation points towards differences in the adaptation of pathogens to different environmental niches. In this study, I investigated the mechanisms by which the Lon protease participates in the regulation of ciprofloxacin resistance in P. aeruginosa. My results indicated that the SOS response triggered by DNA-damaging agents is suppressed in a lon mutant compared to the wild type, explaining the increased sensitivity to fluoroquinolones and UV light in the absence of Lon. 4.2  Results  4.2.1 The lon mutant displays enhanced susceptibility to ciprofloxacin In agreement with our previous data (19), MIC measurements showed that the lon miniTn5-luxCDABE (transposon) mutant displayed 4-8 fold increased susceptibility to ciprofloxacin compared to the wild type and the complemented strain. The supersusceptibility phenotype was also evident when susceptibility was analyzed by means of killing curves. After 10 minutes incubation in the presence of bactericidal concentrations of ciprofloxacin (0.1 µg/ml), the lon mutant, the complemented strain and the wild type showed survival rates of 10, 70 and 60%, respectively. The same trend was observed when survival was assessed at 2 x MIC. The increased susceptibility of the mutant compared to the wild type was even more pronounced at later time points in that after 45 minutes, only 0.1% of the lon mutant cells survived exposure to ciprofloxacin, whereas the wild type and the complemented strain showed survival rates of around 5-10% (Figure 4.1).  56  Figure 4.1. Survival rates of the lon mutant compared to the wild type and complemented strain in the presence of 0.1 µg/ml ciprofloxacin. Cultures of P. aeruginosa wild type strain H103, the lon mutant and the complemented mutant (lonc) were grown to mid-log phase, washed and then exposed to 0.1 μg/ml ciprofloxacin (1 x MIC). Colony forming units were counted over a time period of 90 minutes. One out of 3 representative experiments is shown. 4.2.2 Altered gene expression in P. aeruginosa wild type strain PAO1 and lon mutant under sub-inhibitory concentrations of ciprofloxacin To understand the molecular basis for the ciprofloxacin supersusceptibility phenotype displayed by lon mutants, gene expression analysis in the presence of sub-inhibitory concentrations of ciprofloxacin via microarray was used to identify any adaptive mechanisms. In the presence of ciprofloxacin, the microarray showed that differential expression of 230 genes between the mutant and the wild type (~ 4% of all P. aeruginosa genes) occurred using a cut-off of greater than twofold change and a P-value of p<0.05 (see Table 4.1 for selected genes and Supplementary Table S A.1 for a complete list of dysregulated genes). Genes involved in the SOS response (e.g., recA, lexA, sulA, recN, prtR and prtN) and heat shock genes (e.g., dnaK, hslV, hslU and groES) are known to be upregulated by ciprofloxacin in the wild type (20). Here we demonstrated that all of these genes except groES were expressed at lower levels in the lon mutant under the conditions utilized (Table 4.1, 4.2). Furthermore, 13 out of the 15 LexAregulated genes identified by Cirz et al. (34) appeared to be expressed less in the lon mutant in the presence of sub-inhibitory concentrations of ciprofloxacin (Table 4.1 and Supplementary 57  Table S A.1), further emphasizing that the SOS damage response is majorly impacted by a mutation in lon. Notably, the microarray showed that in the presence of sub-inhibitory concentrations of ciprofloxacin Lon is a repressor of the phenylalanine degradation pathway and is likely affecting anthranilate synthesis, based on the dysregulation of the following genes: phhA, hpc, hpd, hmgA, fahA, trpG, trpD and trpC in the lon mutant. Table 4.1. Selected P. aeruginosa genes differentially expressed in the lon mutant in mid-log phase microarray under sub-inhibitory (one half MIC) concentrations of ciprofloxacin. Mid log phase PA number  Gene name  PA0069 PA0610 prtN PA0611 prtR PA0625 PA0649 trpG PA0650 trpD PA0651 trpC PA0669 PA0670 PA0671 sulA2 PA0865 hpd PA0872 phhA PA0922 PA2008 fahA PA2009 hmgA PA2288 PA3007 lexA PA3413 PA3414 PA3616 recX PA4761 dnaK PA5053 hslV 1 a negative fold upregulation. The response.  Gene description  Fold change1  P value  Conserved hypothetical protein -5.5 0.00 Transcriptional regulator PrtN -12.7 0.00 Transcriptional regulator PrtR -3.0 0.00 Hypothetical protein -5.6 0.00 Anthranilate synthase component II -3.2 0.00 Anthranilate phosphoribosyltransferase -3.5 0.01 Indole-3-glycerol-phosphate synthase -2.4 0.00 Probable DNA polymerase alpha chain -3.1 0.00 Hypothetical protein -5.3 0.00 Hypothetical protein -5.6 0.00 4-hydroxyphenylpyruvate dioxygenase 2.8 0.00 Phenylalanine-4-hydroxylase 2.8 0.00 Hypothetical protein -2.4 0.00 Fumarylacetoacetase 3.0 0.00 Homogentisate 1,2-dioxygenase 8.1 0.00 Hypothetical protein -2.1 0.00 Repressor protein LexA -2.2 0.01 Hypothetical protein -2.4 0.00 Hypothetical protein -2.7 0.00 Conserved hypothetical protein -2.2 0.00 DnaK protein -2.1 0.01 Heat shock protein -2.4 0.00 change represents downregulation whereas a positive change represents genes highlighted in bold play a major role in the SOS- and heat shock  58  4.2.3  Role of the Lon protease in the presence of ciprofloxacin To further investigate if some of the dysregulated genes mentioned above were  responsible for the supersusceptible phenotype of the lon mutant, I carried out additional transcriptional analyses. Expression levels of selected genes were determined by RT-qPCR, comparing the lon mutant to the wild type, both with and without sub-inhibitory ciprofloxacin concentrations. Investigation of the gene expression levels demonstrated that the induction of genes involved in the SOS response and DNA repair were indeed significantly impaired in the lon mutant, as this mutant showed only a minor or no response in the presence of ciprofloxacin, cf. the situation in the wild type. For example, recA, a major SOS response gene, was upregulated 6.1 fold in the wild type in the presence of ciprofloxacin; however, the mutant showed just a 2.4 fold increase in the transcription of this gene. The differential induction of the expression of genes involved in the SOS response and DNA repair was also shown for lexA, sulA, recA, prtN, and prtR, as well as the entire RecA/PrtR/PrtN-regulated phage pyocin region, e.g. PA0625 (Table 4.2), which is known to be involved in ciprofloxacin susceptibility (20). Table 4.2. Expression of selected genes involved in SOS response and DNA damage repair under various conditions. PA number  Gene Gene name description  Fold change WT sub-cipro vs.  lon- vs. WT  WT PA0610  prtN  PA0611  prtR  PA0625 PA3007  lexA  PA3008  sulA  PA3617  recA  PA4763  recN  lon- sub-cipro vs. lon- sub-cipro vs. lon-  WT sub-cipro  Transcriptional regulator PrtN Transcriptional regulator PrtR Hypothetical protein Repressor protein LexA Hypothetical protein RecA protein  11.08 ± 2.85  2.40 ± 0.14  0.77 ± 0.29  0.15 ± 0.03  4.06 ± 0.72  1.65 ± 0.36  0.84 ± 0.03  0.33 ± 0.00  28.40 ± 8.13  2.95 ± 0.16  1.30 ± 0.30  0.16 ± 0.05  9.01 ± 0.81  0.99 ± 0.22  3.27 ± 0.76  0.35 ± 0.05  9.35 ± 1.77  1.38 ± 0.34  3.42 ± 0.46  0.50 ± 0.09  6.07 ± 1.68  1.29 ± 0.06  2.35 ± 0.65  0.50 ± 0.00  DNA repair protein RecN  14.85 ± 2.35  0.83± 0.25  5.44 ± 1.85  0.29 ± 0.06  Fold changes are represented as an average of 3 biological repeats with standard deviation. The data was consistent with the observation that sub-inhibitory ciprofloxacin concentrations caused DNA damage in the wild type, eliciting an SOS response in an attempt to 59  repair this damage, but that the lon mutant was substantially deficient in the induction of this response. We thus suggest that this limited inducibility of the SOS DNA repair responses in the lon mutant explained its observed supersusceptible phenotype to the DNA-damaging agent ciprofloxacin, consistent with previous observations that mutants in the recA, recN and recG genes showed increased supersusceptibility to ciprofloxacin and/or UV light (19, 119). 4.2.4 The lon mutant demonstrates increased sensitivity to UV irradiation Like ciprofloxacin, UV light is a DNA-damaging agent and, therefore, I predicted that the lon mutant would be more sensitive to UV killing than the wild type, as previously shown for lon mutants of E. coli and P. fluorescens Pf-5 (81, 82, 259). For the P. aeruginosa lon mutant, a 20 fold increase in susceptibility to UV irradiation was observed after 10 seconds of UV light exposure. This susceptibility phenotype could be restored by complementation. A mini-Tn5luxCDABE recA mutant (143) was included as a control of the experimental conditions, confirming the known susceptibility of recA mutants to UV damage (119) (Figure 4.2).  Figure 4.2. UV-mediated killing of P. aeruginosa after 10 seconds of UV light exposure. Cultures of P. aeruginosa wild type strain H103, lon mutant, complemented mutant (lonc) and recA mutant were grown to mid-log phase, diluted and then exposed to UV light for 10 seconds. Colony forming units were counted. One out of 3 representative experiments is shown.  60  4.2.5  Dysregulation of lon during lethal UV light and ciprofloxacin exposure To determine if the Lon protease was important in controlling the expression level of the  SOS response genes by suppressing lethality (associated with overexpression of the SOS response genes), the PAO1 wild type strain was treated with either a lethal dose of UV light (30 seconds exposure) or 4 x MIC (0.4 g/ml) of ciprofloxacin. The results showed a moderate downregulation of lon; 0.25 ± 0.16 and 0.42 ± 0.20 respectively (Table 4.3). Table 4.3. Expression of the lon gene in response to lethal concentrations of ciprofloxacin and UV light compared to the wild type. Condition  UV light (30 s) Ciprofloxacin (4 x MIC) 4.2.6  Gene name  Fold change  lon lon  0.25± 0.16 0.42 ± 0.20  Phenotypic effects of sulA (PA3008) and lexA (3007) overexpression LexA is the primary repressor of the SOS DNA damage response and a target of the  RecA coprotease (151). SulA acts downstream of LexA and RecA in the SOS response to repress cell division while damage is being repaired (107). In E. coli SulA has been shown to be a direct substrate of Lon protease (170). If Lon was acting directly on either LexA or SulA in P. aeruginosa, then rendering Lon inactive through mutation would increase their activity, which I attempted to mimic by overexpressing the cloned genes. Overexpression of sulA (PA3008) and lexA (PA3007) was achieved by constitutive expression under the control of the lacZ promoter (constitutive in Pseudomonas) using the low-copy number vector pBBR1MCS4. The expression level was checked by RT-qPCR and revealed a 31 fold upregulation for pBBR1MCS4:sulA compared to the vector alone and 101 fold in the case of lexA. Overexpression of neither sulA nor lexA resulted in any major growth defect using the TECAN Spectrafluor Plus for measurements. Cells overexpressing LexA showed a normal swarming behaviour like the wild type and a lexA mutant. In contrast, overexpression of sulA led to a similar swarming deficiency to that exhibited by a lon mutant, whereas a sulA mutant had a normal swarming phenotype (Figure 4.3 A, E). The same observations were made for the other types of motility affected by Lon, namely swimming (mediated by flagella in an aqueous environment) and twitching (on solid surfaces 61  and interfaces mediated by type IV pili) (Figure 4.3 B, C). No major changes in ciprofloxacin MIC and biofilm formation, another form of social behaviour mediated by flagella and type IV pili, (Figure 4.3 D), were noted for either construct compared to the control. As expected, like a lon mutant, the strain overexpressing sulA formed long filaments, since SulA is a cell division inhibitor (Figure 4.3 F-H). This is consistent with the explanation that Lon also degrades SulA in P. aeruginosa and that lon mutants likely accumulate SulA. Accumulation of SulA in cells would lead to the formation of long filaments and such cells might demonstrate motility defects. However, SulA accumulation clearly did not affect other Lon dependent phenotypes, including biofilm formation and ciprofloxacin MIC.  A **  **  B *  B **  62  C  **  **  D  **  E  63  F  G  H  Figure 4.3. Effect of lexA and sulA overexpression on various types of motility and filamentation. Cultures of P. aeruginosa overexpressing either lexA or the sulA homolog (PA3008) were grown to mid-log phase, and then inoculated onto swarming, swimming or twitching plates. Motility was compared to the wild type and a vector alone control. For biofilm formation, cells were incubated in 96 well microtiter plates containing LB for 20 hours at 37°C. Biofilm formation was measured by crystal violet staining of the adherent cells. Bacterial samples were spotted onto microscope slides, heat fixed and stained with crystal violet to determine cell length with the microscope. Swarming motility (A, E), swimming (B), twitching (C) and biofilm formation (D) were compared to the wild type. Microscope analysis of wild type (F), sulA overexpressing strain (G) and lon mutant (H) are shown. The bars represent the average and standard deviation of 3 independent experiments. A statistically significant difference was observed for the overexpressing strain of sulA at the indicated cases with p≤0.003 (Student’s t test). 4.2.7  Effect of sulA overexpression on transcriptional regulation As shown above, disruption of lon led to a lower level expression of the SOS response  genes in the presence of ciprofloxacin compared to the wild type. To investigate how Lon amplifies the SOS response and, therefore, affects ciprofloxacin susceptibility, transcriptional regulation was investigated for the strain overexpressing sulA. If Lon acted on the SOS response at the transcriptional level through sulA in the presence of DNA damage, I hypothesized that the genes involved in the SOS response should be expressed to a lesser extent in the sulA overexpressing strain similar to the effect I demonstrated for a disruption of lon. However, the strain overexpressing sulA showed a similar transcriptional profile for the genes recA and lexA as did the wild type control in the presence of sub-inhibitory concentrations of ciprofloxacin. Interestingly, sulA was less induced in the overexpressing strain, indicating the autoregulation of sulA levels in the presence of DNA damage (Table 4.4). 64  Table 4.4. Effects on SOS transcriptional regulation of the sulA overexpressing strain compared to the vector control after sub-inhibitory ciprofloxacin induction.  . 4.2.8  Vector control +/ vector pBBR1sulA+/ pBBR1sulA control 4.0 ± 1.5 5.0 ± 1.9 recA 5.9 ± 0.8 11.0 ± 4.1 lexA 4.3 ± 1.3 1.4 ± 0.9 sulA + indicates the induction with sub-inhibitory concentrations of ciprofloxacin Effect of ciprofloxacin on protein expression in the lon mutant Since no apparent difference in transcriptional regulation was observed for the sulA  overexpressing strain, I hypothesized that Lon protease influences protein accumulation in the presence of ciprofloxacin. Whole cell lysates were used to determine if Lon impacted on the major protein of the SOS response (RecA) under DNA-damaging conditions, such as subinhibitory concentrations of ciprofloxacin. SDS-polyacrylamide gel electrophoresis and Western Blot analysis demonstrated that expression of RecA (37 kDa) was induced under sub-inhibitory concentrations of ciprofloxacin in the wild type and the complemented lon strain; however, it failed to be expressed in the lon mutant, thus highlighting that RecA production was dependent on the Lon protease. Clearly, a different band pattern was evident between the lon mutant and the wild type under inducing conditions, and the Western Blot analysis using RecA antibody revealed a strong band for the wild type and complemented strain, but not for the lon mutant (Figure 4.4). However, it is possible that RecA is somewhat expressed in the lon mutant under DNA damaging conditions, but not sufficiently to be seen in the Western Blot. Under noninducing conditions, no RecA band was visible in any of the strains tested, as RecA only gets accumulated upon DNA damage.  65  A  B  RecA  0.3 x MIC cipro -  +  -  +  -  +  B  RecA  0.3 x MIC cipro  -  +  -  +  -  +  Figure 4.4. Induction of the P. aeruginosa RecA protein by sub-inhibitory concentrations of ciprofloxacin in the wild type, complemented lon strain and lon mutant. (A) Coomassie brilliant blue stained SDS-PAGE gel and (B) Immunoblot of the identical gel are shown. Bacteria strains were grown overnight in the presence or absence of sub-inhibitory concentrations of ciprofloxacin and 200 µl of the cultures were pelleted and lysed. After centrifugation, the supernatants were used to measure the protein concentration of the samples with the Qubit fluorometer to ensure that same amount of proteins were loaded on the SDS gel for analysis. After denaturing at 100°C, samples were loaded on a polyacrylamide gel, stained overnight in Coomassie Blue, and destained the following morning (A). For Western Blotting, the samples were separated on a polyacrylamide gel and protein bands were transferred to a PVDF membrane, incubated with a polyclonal antibody against RecA and a secondary antibody. Protein bands were developed with the ECL Kit. One out of 3 repeats is shown. 66  4.3  Discussion This study demonstrated for the first time that the ATP-dependent Lon protease  (PA1803) plays a major role in regulating the SOS response and DNA repair in P. aeruginosa. Studies on the ATP-dependent Lon protease in different bacteria have shown its involvement in such diverse processes as cell division (19, 217), flagella biosynthesis (235), capsule synthesis (248), UV tolerance (259), motility (157) and antibiotic resistance (19). However, the phenotype of the lon mutants varies between different bacterial species. In P. aeruginosa PAO1, lon mutants exhibit a motility defect, show extreme filamentation, greater hemolytic activity, and are supersusceptible to ciprofloxacin (19, 157, 244). Investigating this supersusceptibility was of great interest. Previous studies showed that sub-inhibitory and inhibitory concentrations of ciprofloxacin led to dramatic changes in global transcription. Most striking were the upregulation of a susceptibility determinant, the R2/F2 pyocins (PA0613-PA0648), and the genes involved in DNA repair and SOS response (20). The microarray and RT-qPCR results showed that the Lon protease of P. aeruginosa was important for full induction of the SOS response upon exposure to DNA damaging agents, including ciprofloxacin. Brazas et al. (19) previously showed that preexposure to ciprofloxacin leads to adaptive resistance in the P. aeruginosa wild type PAO1, but failed to induce adaptive resistance in the lon mutant. The authors concluded that the extreme cell elongation phenotype of the mutant contributed to this phenotype. However, here I could provide an alternative explanation namely the weaker induction of the SOS response and DNA repair observed in the lon mutant, while discounting this previous hypothesis. The cell elongation phenotype triggered through the SOS response is controlled by SulA (114), as confirmed here in P. aeruginosa (Figure 4.3). Overexpression of sulA did not recapitulate ciprofloxacin susceptibility, indicating that elongation likely did not explain altered ciprofloxacin susceptibility. The negative and positive regulators of the SOS response, LexA and RecA, respectively, are common in all bacteria species and are highly upregulated in P. aeruginosa in response to environmental stress. While these genes and several other genes involved in the SOS response were upregulated in the wild type after ciprofloxacin treatment to overcome the DNA damage, the present study demonstrated that if lon is mutated, this SOS response was considerably weaker. Some SOS response genes were still upregulated by ~ 3 fold in the lon 67  mutant, but this induction was substantially lower than in the wild type. I propose a model (Figure 4.5) whereby Lon protease inhibits RecA repressors (RecX, RecR, RdgC, etc.) (52, 53, 237, 252), leading to the autoamplification of RecA. In case of inactive Lon, the repressor stays intact and no RecA amplification occurs, leading to supersusceptibility to DNA damaging agents, such as fluoroquinolones and UV light, as damage cannot be repaired. Consistent with this, mutants in genes involved in the SOS response and DNA repair (recA, recN and recG) exhibit an increased susceptibility to ciprofloxacin and UV light (19, 119).  Figure 4.5. Proposed model for the involvement of the Lon protease in the DNA damage response. Under DNA damaging conditions, the Lon protease is necessary to inhibit RecX, RecR, RdgC, etc. which are known repressors of RecA in E. coli. This inhibition leads to autoamplification of RecA and SOS response is induced. However, if the Lon protease is inactive, the repressors inhibit RecA function. Upon DNA damage, the resulting single stranded DNA (ssDNA) gets recognized by RecA, which in turn forms filaments around the ssDNA. At the same time, RecA induces the autocleavage of the repressor LexA enabling the damage repair genes to be transcribed and, consequently, DNA damage is repaired. Interestingly, upon exposure to 30 seconds of UV light and 4 x MIC of ciprofloxacin (representing lethal conditions), the lon gene itself is slightly downregulated. Thus, a possible hypothesis is that the Lon protease plays a role in controlling the expression level of the SOS response genes to perhaps suppress potential lethality associated with overexpression of recA and other SOS response genes, especially to limit the induction of 68  cell death mediated by the phage pyocin operon (20). I could demonstrate that Lon does not act through sulA at the transcriptional level, but likely works at the SulA protein level to regulate cell division. Therefore, I hypothesize that Lon acts through cleavage of one or more of the control elements of the SOS response, for example by impacting on the level of negative regulators of RecA, such as RecX, RecO, RecR or RdgC (52, 53, 237, 252). These studies in E. coli demonstrated that recombination-associated proteins can act as a negative regulator of the major SOS protein, RecA. I demonstrated that protein accumulation of RecA does not occur in the lon mutant upon ciprofloxacin exposure, highlighting that RecA would not be able to cleave the repressor LexA. Furthermore, as RecA expression is much lower in the lon mutant upon DNA-damage, it would bind more poorly to the ssDNA and thus DNA damage could not be repaired. Lon is therefore important for modulating RecA function. Additionally, the Lon protease might be important for cleaving other elements of the SOS response. The ATP-dependent Lon protease investigated in this study controls the DNA stress response and fluoroquinolone susceptibility and is upregulated by aminoglycosides. P. aeruginosa has additional ATP-dependent proteases that exhibit similarity to PA1803. One of these proteases is the recently identified AsrA (aminoglycoside-induced-stress response) ATPdependent protease (PA0779), which is 60% similar to the Lon protease PA1803 (121). AsrA is highly upregulated by bacteriostatic and lethal concentrations of tobramycin and controls the heat shock stress response to tobramycin in Pseudomonas. Therefore, it is becoming clear that ATP-dependent proteases are involved in the regulation and control of specific stress responses in Pseudomonas. The significant role of the Lon protease in virulence-related processes and antibiotic resistance makes it an attractive antimicrobial target to combat P. aeruginosa acute and chronic infections.  69  5. Regulation by the Lon protease 5.1  Introduction Over the past years, high-throughput technology enabled investigations of the genome,  transcriptome and proteome of bacterial pathogens. For instance, after sequencing of the P. aeruginosa genome was completed in the year 2000 (239), mechanisms were created to study global signalling networks in this microorganism by analyzing gene expression. The most commonly used methods for transcriptional profiling are DNA microarray analyses and, more recently, RNA sequencing (68). The latter allows analysis of the transcriptome on a massive scale by using parallel cDNA sequencing, and it is also referred to as “next generation sequencing technology” (190), it is a high-throughput approach and allows for characterization as well as quantification of the transcriptome under various conditions. However, most research available to date has been performed on customized DNA microarrays. Indeed, several studies have investigated the changes in the transcriptomic profile of P. aeruginosa as a result of specific mutations (78, 271) or in response to diverse stimuli, such as exposure to antibiotics (5, 20, 121), copper starvation (70), oxidative stress (30), growth in a biofilm (256) or swarming conditions (250). All these features are important in understanding how Pseudomonas can switch on mechanisms in order to cause infections, become more resistant to toxic compounds or survive under restrictive conditions. Importantly, approximately 10% of all P. aeruginosa genes encode transcriptional regulators (239) which are involved in signalling pathways and control changes in gene expression. Understanding these global responses provides useful insights into the regulatory pathways of this pathogen and often a transcriptional hierarchy can be determined, such as established for quorum-sensing (138, 195). Apart from transcriptome studies, previously developed tools also facilitate the identification of proteins involved in a specific process (120, 266). These techniques include two-dimensional gel electrophoresis followed by mass spectrometry, as well as quantitative proteomic studies, such as iTRAQ (isobaric tag for relative and absolute quantitation) and SILAC (stable isotope labelling of amino acids in cell culture) (187, 204). Thus, these new techniques collectively allow a comparison between transcriptional and post-translational regulation triggered by different stimuli.  70  In this study the involvement of the Lon protease in the global responses of P. aeruginosa at both the transcriptional and the post-translational levels was investigated. The flowchart in Figure 5.1 gives an overview of the global regulation which will be described in this chapter.  Figure 5.1. Overview of the experiments conducted in this chapter regarding global regulation. 5.2  Results  5.2.1  Transcriptional regulation 5.2.1.1 Microarray analyses To understand the role of the Lon protease in the global transcriptional response, gene  expression analyses comparing the lon mutant to the wild type were carried out in midlogarithmic and stationary phase as well as under swarming conditions. Genes were considered to be differentially expressed when they were more than twofold changed in levels and a statistical significance of p<0.05 (measured with Student’s t test), as described by Patterson et al. (193).  71  In particular, we wanted to investigate how the Lon protease contributed to the various phenotypes observed to date in the lon mutant, such as defects in swarming motility and biofilm formation and ask the question as to whether Lon is a major global regulator. To address this goal and to determine what differences occurred between different growth phases, gene expression analyses were carried out as indicated in Figure 5.1. 5.2.1.1.1 Influence of Lon on transcriptional regulation in mid-logarithmic phase The mid-logarithmic phase microarray revealed 275 genes that were differentially regulated in the lon mutant by more than twofold and a P-value of p≤0.05, with 97 upregulated and 178 downregulated genes (Supplementary Table S A.2A). Most of these dysregulated genes showed a two- to fourfold change in expression level (216 genes). A selection of dysregulated genes (particularly genes involved in virulence and anaerobic growth) is shown in Table 5.1. Analysis of the microarray data revealed that many different functional classes were dysregulated in the lon mutant, indicating a broad functional role for the Lon protease in the cell. Interestingly, I observed that genes involved in energy metabolism (nir, nor, nos and nar) were downregulated, which indicates that Lon protease might have a role in anaerobic growth. Also, genes involved in pyochelin biosynthesis which are important for virulence (fptA and pch) were downregulated in the mutant. Surprisingly, only 3 genes involved in the biosynthesis of flagella or type IV pili were dysregulated and no genes related to rhamnolipid production (important for swarming motility). I originally hypothesized that flagella- and pili-related genes would be dysregulated in the lon mutant providing an explanation for the swarming deficient phenotype of the lon mutant. The microarray analysis however showed only 3 pilus genes to be downregulated by twofold in the mutant. This dysregulation is thus likely insufficient to explain the swarming defect of a lon mutant, and other factors would seem to play an important role in controlling this complex adaptation.  72  Table 5.1. Selected P. aeruginosa genes differentially expressed in the lon mutant in midlogarithmic phase microarray. Mid-log phase PA number  Gene name  Gene description  Fold change1  P value  Resistance-Nodulation-Cell Division (RND) multidrug efflux membrane fusion PA0425 2.35 0.00 mexA protein MexA Resistance-Nodulation-Cell Division PA0426 2.51 0.00 mexB (RND) multidrug efflux transporter MexB Major intrinsic multiple antibiotic resistance efflux outer membrane protein PA0427 3.37 0.00 oprM OprM precursor PA0509 nirN Probable c-type cytochrome -3.22 0.01 Probable uroporphyrin-III cPA0510 methyltransferase -5.41 0.01 PA0511 -7.82 0.01 nirJ Heme d1 biosynthesis protein NirJ PA0523 norC Nitric-oxide reductase subunit C -33.60 0.00 PA0524 -5.76 0.00 norB Nitric-oxide reductase subunit B 3-oxoacyl-[acyl-carrier-protein] synthase PA0999 pqsD III -2.17 0.01 PA3391 -8.27 0.00 nosR Regulatory protein NosR PA3392 nosZ Nitrous-oxide reductase precursor -31.22 0.00 PA3875 -4.22 0.01 narG Respiratory nitrate reductase alpha chain PA3876 narK2 Nitrite extrusion protein 2 -5.32 0.03 Fe(III)-pyochelin outer membrane PA4221 fptA receptor precursor -2.01 0.00 PA4224 pchG Pyochelin biosynthetic protein pchg -2.48 0.00 PA4225 -2.14 0.00 pchF Pyochelin synthetase PA4525 pilA Type 4 fimbrial precursor PilA -2.21 0.00 PA5041 pilP Type 4 fimbrial biogenesis protein PilP -2.06 0.00 PA5042 pilO Type 4 fimbrial biogenesis protein PilO -2.28 0.00 1 a negative fold change represents downregulation, whereas a positive change represents upregulation. The genes highlighted in bold are proposed to play a major impact in the global regulation by Lon protease. 5.2.1.1.2 Dysregulation of genes in the lon mutant in stationary phase A similar study was made investigating the influence of the Lon protease during stationary phase. Microarray analysis revealed that, under these conditions, 219 genes were differentially regulated by more than twofold and a P-value of p≤0.05 (Supplementary Table S A.2B). As with the mid-log phase microarray, most of the genes showed a dysregulation of two73  to fourfold (185 genes). For this stationary microarray, in addition to the twofold cutoff analysis, we performed a SOV (skipping outlier values) fold change analysis to compare our results. The SOV fold change excludes single outlier results found in the different repeats and reveals genes with altered expression in most experiments. Through this analysis additional transcriptional regulators were identified that could be also confirmed with RT-qPCR. We found again a dysregulation of genes involved in the denitrification pathway (nir, nor, nos and nar); however, these genes were upregulated in the lon mutant during stationary phase, which is not surprising since metabolism changes dramatically between mid-logarithmic and stationary phase. Furthermore, several transcriptional regulators that could potentially be acting downstream of lon were identified (Table 5.2 and 5.5), including psrA, rpoS and a transcriptional regulator of the denitrification pathway. Most notable was the dysregulation of genes involved in type III secretion, such as the genes exo, psc, pcr and pop (Table 5.2). These genes are relevant for virulence, secretion and cytotoxicity, indicating that the Lon protease could have an effect on these processes. In addition, pyochelin biosynthesis genes (pch) and PA0621, which are known to be involved in ciprofloxacin resistance, were dysregulated in the mutant. However, few flagellum- or pilus-encoding genes or genes involved in rhamnolipid biosynthesis were downregulated in the lon mutant, analagous to the results observed in midlogarithmic phase.  74  Table 5.2. Selected P. aeruginosa genes differentially expressed in the lon mutant in stationary phase microarray. Stationary phase PA number  Gene name  Gene description  Fold change1  P value  PA0044  exoT Exoenzyme T -2.16 0.00 Resistance-Nodulation-Cell Division (RND) multidrug efflux membrane fusion PA0425 2.73 0.00 mexA protein MexA Resistance-Nodulation-Cell Division PA0426 3.62 0.00 mexB (RND) multidrug efflux transporter MexB Major intrinsic multiple antibiotic resistance efflux outer membrane protein PA0427 2.55 0.00 oprM OprM precursor PA0511 3.20 0.00 nirJ Heme d1 biosynthesis protein NirJ PA0512 Conserved hypothetical protein 3.33 0.00 PA0513 Probable transcriptional regulator 3.43 0.00 PA0515 Probable transcriptional regulator 6.42 0.00 PA0621 Conserved hypothetical protein 2.15 0.00 Homologous to beta-keto-acyl-acyl-carrier PA0997 -2.73 0.00 pqsB protein synthase Homologous to beta-keto-acyl-acylPA0998 pqsC carrier protein synthase -2.90 0.00 PA1000 -2.73 0.02 pqsE Quinolone signal response protein PA1078 -2.33 0.00 flgC Flagellar basal-body rod protein FlgC Flagellar basal-body rod modification PA1079 -2.26 0.00 flgD protein FlgD Translocation protein in type III PA1696 pscO secretion -2.10 0.04 PA1704 -14.92 0.00 pcrR Transcriptional regulator protein PcrR PA1705 -3.61 0.00 pcrG Regulator in type III secretion PA1708 popB Translocator protein PopB -2.62 0.00 PA1719 -3.00 0.00 pscF Type III export protein PscF PA1720 -2.31 0.00 pscG Type III export protein PscG PA3392 11.39 0.00 nosZ Nitrous-oxide reductase precursor PA3841 exoS Exoenzyme S -2.39 0.00 Fe(III)-pyochelin outer membrane PA4221 fptA receptor precursor -4.28 0.00 PA4225 pchF Pyochelin synthetase -2.55 0.00 PA4226 -2.74 0.00 pchE Dihydroaeruginoic acid synthetase Still frameshift type 4 fimbrial biogenesis PA4527 -2.13 0.02 pilC protein PilC 1 a negative fold change represents downregulation, whereas a positive change represents upregulation. The genes highlighted in bold were proposed to play a major impact in the global regulation by Lon protease. 75  5.2.1.1.3 Is Lon a direct regulator for swarming motilty? Swarming motility is a complex form of adaptation that is associated with dramatic changes in the growth conditions. Thus, in order to mimic more closely swarming conditions, we set out to perform a microarray comparing the transcriptome of swarming cells of the wild type and the lon mutant. By analyzing swarmer cells, we expected to determine whether flagella and pilus genes were specifically dysregulated in the lon mutant under these conditions, thereby explaining the swarming deficiency of this mutant. Alternatively, the Lon protease itself could be directly involved in the regulatory processes necessary for the switch to a swarming lifestyle. When analyzing the lon mutant compared to the wild type under swarming conditions, only 33 genes were demonstrated with a greater than twofold dysregulation and a P-value of p≤0.05 could be detected (Supplementary Table S A.2C). Of these, 22 genes were upregulated and 11 downregulated and 27 of the genes showed a two- to fourfold dysregulation. As seen before, genes involved in type III secretion were downregulated to a certain degree (Table 5.3). However, neither flagella nor pilus genes were dysregulated which is consistent with the suggestion that Lon itself might play a significant regulatory role in swarming motility, and the results below implicate its effects on PsrA, a known regulator of swarming (78). Table 5.3. Selected P. aeruginosa genes differentially expressed in the lon mutant under swarming conditions. Swarming PA number  PA0044 PA0621 PA1706 PA1708  Gene name  Gene description  Fold change1  exoT Exoenzyme T Hypothetical protein pcrV Type III secretion protein PcrV popB Translocator protein PopB Translocator outer membrane protein PopD PA1709 popD precursor ExsC, exoenzyme S synthesis protein C PA1710 exsC precursor. PA1711 exsE ExsE PA2825 ospR Probable transcriptional regulator PA3841 exoS Exoenzyme S PA3874 narH Respiratory nitrate reductase beta chain 1 a negative fold change represents downregulation, whereas a upregulation.  P value  -2.65 2.35 -3.87 -3.84  0.03 0.04 0.01 0.01  -6.06  0.01  -3.81 0.02 -2.19 0.03 5.02 0.02 -3.24 0.01 -3.42 0.02 positive change represents 76  To further confirm the importance of lon under swarming conditions, RT-qPCR experiments were carried out in order to investigate if lon is dysregulated when comparing swarming versus swimming motility. Indeed, lon showed a 1.9 ± 0.05 fold upregulation under swarming conditions (Table 5.4). Table 5.4. Expression of the lon gene under wild type swarming vs. swimming conditions as assessed by RT-qPCR. Condition  Gene name  Fold change  Swarming vs. swimming  lon  1.9 ± 0.05  The 3 microarrays performed, did not completely answer the question as to why the lon mutant exhibits a swarming motility defect; however one possibility is that the Lon protease itself impacts directly on swarming motility. Nevertheless our microarray results highlighted the possibility that Lon protease is effectively a global regulator, influencing hundreds of genes, depending on the growth phase, consistent with the established dependency of bacterial metabolism on the growth phase. Overall these data clearly establish that the Lon protease is a central regulatory player in P. aeruginosa since its loss results in expression changes in many genes belonging to all functional classes. Interestingly, the transcriptional analyses revealed that the Lon protease could influence anaerobic growth, cytotoxicity and virulence. 5.2.1.2 Transcriptional hierarchy Microarray analysis showed the dysregulation of several transcriptional regulators in the lon mutant during stationary phase. These regulators, which were further confirmed by RTqPCR, include PA0515 (involved in denitrification) (4.6 ± 1.8 fold dysregulated), PA1738 (probable transcriptional regulator) (0.7 ± 0.3), PA3006 (type III secretion regulator PsrA) (4.1 ± 1.6), PA3622 (regulator of stationary phase sigma factor RpoS) (3.9 ± 1.6) and PA3721 (probable transcriptional regulator NalC) (2.2 ± 0.6) and are therefore likely to be downstream of Lon. Transcriptional regulation can be very complex, and different complex behaviours in P. aeruginosa (such as quorum-sensing, swarming motility, biofilm formation and antibiotic resistance) are regulated through the interplay of transcriptional regulators in a cascade.  77  The gene transcriptional hierarchy involving the Lon protease was investigated in this study by measuring the transcriptional level of the regulators in mutants. To do that, I performed RT-qPCR experiments on mutants in these regulators comparing them to the wild type and looking for dysregulation of the genes encoding the other regulators. By analyzing the transcriptional data, I was able to determine which regulator had an impact on another regulator and which regulator was directly regulated by the Lon protease (Table 5.5 and Figure 5.2). The data suggested that the Lon protease stands at the top of a transcriptional hierarchy and negatively regulates psrA (PA3006), as psrA is not dysregulated in any of the other regulatory mutants. However, whether Lon protease was directly or indirectly regulating psrA could not be fully determined, since it is possible that Lon acts through another intermediate regulator, which could have been missed in the microarray. The RT-qPCR data further showed that psrA positively regulates rpoS (PA3622), which was already shown by Gooderham et al. (78) while studying gene expression in a psrA mutant, and it was shown here that an rpoS mutation affected the genes encoding the other identified regulators except for psrA and nalC. Based on similar analyses, rpoS positively regulated PA1738, which in turn positively regulated PA0515, which negatively regulated PA3721. At the end of this regulatory hierarchy a positive feedback loop might exist since PA0515 also upregulated PA1738. Taking all these data together, our findings show that the Lon protease stands on top of a transcriptional hierarchy which includes the known virulence factors psrA and rpoS (Table 5.5 and Figure 5.2) (78, 241).  78  Table 5.5. Impact of downstream regulators of lon on the other regulators measured by RT-qPCR. Mutation in transcriptional regulator  Effect on gene expression of transcriptional regulator  PA1738 PA0515 PA3006 PA3622 PA3721 PA0515 PA1738 PA3006 PA3622 PA3721 PA0515 PA3006 PA1738 PA3622 PA3721 PA0515 PA3622 PA1738 PA3006 PA3721 Gene expression levels led me to propose a transcriptional hierarchy  RT-qPCR  0.3 ± 0.1 1.8 ± 0.9 0.6 ± 0.5 2.9 ± 1.3 0.2 ± 0.1 1.2 ± 0.4 0.4 ± 0.2 0.4 ± 0.2 0.1 ± 0.1 0.3 ± 0.2 0.1 ± 0.1 1.6 ± 1.2 0.1 ± 0.1 0.4 ± 0.1 1.5 ± 0.3 1.3 ± 0.5  Figure 5.2. Transcriptional hierarchy of the Lon protease, including psrA and rpoS. RT-qPCR experiments on mutants in the regulators, which are dysregulated in the lon mutant, were compared to the wild type and dysregulation of the genes encoding the other regulators were investigated. – indicates repressor and + indicates activator. 79  5.2.2  Anaerobic growth and anaerobic biofilm formation Anaerobic growth is important with regards to Pseudomonas and CF lung disease as it is  well known that Pseudomonas encounters a steep hypoxic gradient in the thickened mucus of the CF lung, facing anaerobic growth conditions (265). Given that several genes involved in the denitrification pathway (nar, nir, nor and nos), which influences anaerobic growth (277), were dysregulated in the lon mutant, I performed anaerobic growth studies comparing the lon mutant to the wild type and the complemented strain. In these experiments, 15 mM KNO3 was used as an electron acceptor instead of oxygen. The assays were carried out in two different ways. First, the cultures were grown in flasks containing a tight seal to ensure anaerobic growth conditions, and the absorbance was measured every hour over a time period of 10 hours. Second, cell suspensions were prepared to an OD650 of 0.1 and diluted one to 20 before incubating for 24 hours in an anaerobic jar at 37ºC. The absorbance was then measured and used as an indicator of anaerobic growth. As expected, mutants in genes involved in the denitrification pathway, namely nir, nor and nar, exhibited an anaerobic growth defect (67), however, no anaerobic growth defect could be shown for mutants in nos. On average, these mutants had a moderate 50% reduction in the ability to grow anaerobically compared to the wild type. Overall, all the mentioned mutants only showed anaerobic growth defects, but not under aerobic conditions. In the case of the lon mutant, we were able to demonstrate an anaerobic growth defect, although, this decrease was modest with a 25% reduction of the ability to grow anaerobically compared to the wild type and the complemented strain (Figure 5.3). This difference was statistically significant with a P-value of p=0.04 as measured with the Student’s t test. Overall, this indicated that not only do the genes involved in the denitrification pathway need to be present in the cell in order to grow anaerobically, but also a certain expression level of these genes is important for a full ability to grow anerobically.  80  * *  * **  Figure 5.3. Anaerobic growth of mutants in regulators involved in anaerobic growth, lon mutant, complemented lon strain and wild type H103. Bacterial cultures, supplemented with 15 mM KNO3, were grown overnight at 37°C in an anaerobic jar containing a GasPak Plus hydrogen and carbon dioxide generator envelope. Cell suspensions were then prepared to a 0.5 McFarland Standard, diluted one to 20, and 200 μl were inoculated into a 96 well microtiter plate. The plate was put in an anaerobic jar and anaerobic growth was measured at OD600 after 24 hours. The bars represent the average and standard deviation of two to three independent experiments. A statistically significant difference between the nir (p=0.03), nor (p=0.0005), nar (p=0.006) and lon (p=0.04) mutants compared to the wild type was observed as determinend by Student`s t test. Next, we investigated the ability of the wild type H103 and the lon mutant to form biofilms under anaerobic conditions. In accordance with previous experiments, the lon mutant exhibited a biofilm defect under aerobic conditions when oxygen was used as the electron acceptor (157). Under anaerobic conditions the wild type H103 showed a reduced ability to form mature biofilms, compared to that under aerobic conditions. Thus, under anaerobic conditions only 40% of the bacterial cells were engaged in biofilms compared to 100% under aerobic conditions. The lon mutant, which showed 40% of the wild type cell adherence under aerobic conditions (p=0.002), showed no further reduction in biofilm formation under anaerobic conditions. Therefore, under anaerobic conditions the same number of cells of the wild type H103 and the lon mutant adhered to the the surface of the 96 well microtiter plate (Figure 5.4).  81  **  Figure 5.4. Biofilm formation of the wild type H103 and the lon mutant under anaerobic relative to aerobic conditions. Cells were incubated under aerobic and anaerobic conditions (addition of 15 mM KNO3) in 96 well microtiter plates containing LB for 20 hours at 37°C. Biofilm formation was measured by crystal violet staining of the adherent biofilm. The bars represent the average and standard deviation of 3 independent experiments.The lon mutant showed a reduction in mature biofilm formation under aerobic conditions which was statistically significant (p=0.002) as measured with the Student’s t test, but no further reduction under anaerobic conditions was observed. Of note, none of the mutants with transposon insertions in the genes nir, nor and nar, which were shown to be involved in the denitrification pathway and exhibited an anerobic growth defect, had any deficiency in motility (swarming, swimming and twitching) or biofilm formation compared to the wild type. 5.2.3 WST1 assay To determine if the anaerobic growth defect of the lon mutant was due to an anaerobic respiration defect, anaerobic respiration was measured by using WST1 as a respiration indicator. This assay allows for colorimetric measurements of respiration as dehydrogenases active in growing cells reduce WST1 to a water-soluble coloured formazan, and the intensity of this colour is a measure of the respiration rate. The purpose of anaerobic respiration is to convert biochemical energy from nutrients such as amino acids and fatty acids to cellular energy which can be used for bacterial growth. Here, anaerobic respiration was measured for the lon mutant, the wild type and the complemented strain for a time period of 90 minutes after adding WST1 and it was observed that the lon mutant exhibited a significant defect in anaerobic respiration 82  (Figure 5.5). I concluded that the observed anaerobic growth defect was, at least partially, due to an anaerobic respiration defect in the lon mutant, lacking sufficient energy generation in the absence of oxygen.  Figure 5.5. Anaerobic respiration analysis of the wild type H103, lon mutant and complemented strain using the WST1 assay. Bacterial cultures were grown overnight under anaerobic conditions and, diluted and transferred to 96 well microtiter plates. 10 μl WST1 was added to each well and absorbance (OD450 and OD900) was measured after 20, 60 and 90 minutes. The respiration rate was defined as the ∆OD (OD450 minus OD900). 5.2.4  Overexpression of lon leads to a similar swarming defect as a mutation in lon Overexpression was achieved by constitutive expression of the cloned lon gene behind  the lacZ promoter (constitutive in Pseudomonas) in the low-copy number vector pBBR1MCS4 in wild type PAO1. The expression level was checked by RT-qPCR, which revealed a 18 fold upregulation for pBBR1MCS4::lon+ compared to the vector pBBR1MCS4 alone. No growth defect was observed as monitored with the TECAN Spectrafluor Plus, indicating that this 18 fold overexpression of lon was not lethal in P. aeruginosa (data not shown). Overexpression of lon led to a similar swarming deficiency to that exhibited by a lon mutant (Figure 5.6). However, no swimming deficiency, defect in biofilm formation or change in the MIC to ciprofloxacin was observed for the strain overexpressing lon compared to wild type (data not shown), indicating that balanced amounts of Lon need to be present in the cell in order to maintain normal swarming motility, a complex adaptation in P. aeruginosa.  83  ** **  pBBR1::lon+ pBBR1::lon+  Figure 5.6. Swarming motility of the wild type, vector pBBR1 (control), lon overexpressing strain (pBBR1::lon+) and lon mutant. Swarming motility was investigated by inoculating 1 µl of the respective strain on a swarming plate (modified BM2 + 0.5% agar). In the modified BM2 medium, ammonium sulfate was substituted with 0.5% casamino acids. The swarming behaviour of the mutant and overexpressing strain was compared to the wild type and the vector control after 20 hours of incubation at 37°C. The left graph bars represent the average and standard deviation of 6 independent experiments. A statistically significant difference could be observed (p≤0.0001) as measured with the Student’s t test. On the right side one swarming repeat shows the swarming behaviour. 5.2.5  Upregulation of the lon gene under certain stress conditions As mentioned above, treatment of the PAO1 wild type with a lethal dose of either  ciprofloxacin or UV light led to the downregulation of the lon gene, whereas no dysregulation of the lon gene occurred in sub-inhibitory doses of DNA damaging agents. Furthermore, Marr et al. (157) showed by measuring luminescence of a mini-Tn5lux library of mutants that lon is significantly upregulated by 2.3 and 3.6 fold, in the presence of sub-inhibitory concentrations of gentamicin or tobramycin, respectively, compared to no antibiotic treatment. This observation was further confirmed by measuring gene expression using RT-qPCR. Therefore, I wanted to determine if there are other stress conditions under which lon is dysregulated, which would further demonstrate the importance of the Lon protease during stress. Indeed, I found that lon was also significantly upregulated under lethal concentrations of tobramycin and under heat shock at 42ºC. In contrast, the expression of lon was not changed by exposure to sub-inhibitory concentrations of the peptide indolicidin (Table 5.6). 84  Table 5.6. Effects of stress conditions on lon gene expression. Condition  Gene name  Fold change  2 x MIC tobramycin 42ºC heat shock Sub-indolicidin  lon lon lon  2.9 ± 0.9 2.7 ± 0.6 1.0 ± 0.1  5.2.6 Post-translational regulation: SILAC (stable isotope labelling of amino acids in cell culture) After assessing transcriptional regulation by the Lon protease, its degree of regulation at the protein level was examined. As Lon is an ATP-dependent protease, it is very likely that Lon acts on the regulation of gene expression, through the degradation and cleavage of short lived regulatory proteins (as shown e.g. in the phage  system) as well as cleaving other proteins. To identify the effects of Lon on protein expression and cleavage, SILAC was used in collaboration with Dr. Leonard Foster (UBC, Canada). The SILAC methodology allows labelling strains with different isotopes of amino acids. To take advantage of this, I constructed a lon knockout strain auxotrophic for leucine and lysine, which is necessary for comparison with an auxotrophic parental strain. The two strains were grown in BM2 medium supplemented with essential amino acids, whereby the parent strain was grown in the presence of natural leucine and natural lysine, and the lon mutant in the presence of natural leucine and heavy isotope labelled lysine (LysD4). After the cytoplasmic fraction of each strain was extracted, they were mixed together in a one to one ratio and digested with the protease LysC. The peptides obtained were then purified, fractionated and analyzed in a mass spectrometer. Differences in protein abundance between the parental strain and the lon mutant after the SILAC procedure could be detected by mass spectrometry due to the mass differences of the heavy (H) and light (L) isotopes (Figure 5.7). Four SILAC repeats revealed a number of proteins that seem to be affected by the Lon protease, represented by a protein ratio “H/L” >2, indicating an elevated protein level in the lon mutant. The total number of these identified proteins was however small (Table 5.7). This seemed to indicate that Lon had a moderate, more selective, impact on the proteome. However, the data was likely skewed towards the observation of very abundant proteins, whereas poorly abundant proteins, which could well include some of the most important targets of Lon, would be less evident. 85  Figure 5.7. One representative mass spectra for peptides identified from SILAC experiment. The arrows indicate the mass:charge (m/z) for the light (parental strain) and heavy (lon mutant) forms. Table 5.7. Protein hits of the Lon protease identified by SILAC. PA number  Gene name  Gene description  Functional class  Ratio H/La  PA1673  Hypothetical protein  Hypothetical  3.2/ 11.4/ 3.2  PA1880  Probable oxidoreductase  Putative enzymes  2.4/ 6.2/ 4.2  3-isopropylmalate dehydratase small subunit  Amino acid biosynthesis  5.0/ 2.3/ 2.3  Hypothetical protein  Hypothetical  3.9/ 1.7/ 2.2  Catalase  Adaptation, protection Energy metabolism  1.7/ 4.3/ 2.4  PA3120  leuD  PA4063 PA4236  katA  PA4333  Probable fumarase  PA4385  groEL  PA4944  hfq  PA5078 PA5429  GroEL protein  Chaperone & heat shock proteins  3.9/ 1.7/ 2.6  Hfq  Transcription, RNA processing degradation Hypothetical  1.8/ 3.5/ 1.7  Conserved hypothetical protein Aspartate ammonia-lyase  Amino acid biosynthesis a indicates the protein ratio between the labelled samples of 3 repeats. aspA  4.2/ 1.5/ 2.1  2.3/ 1.6/ 4.0 2.4/ 8.7/ 2.0  86  Differences in protein expression levels between the lon mutant and the parental strain varied from experiment to experiment, so the “Ratio H/L” column in Table 5.7 lists individual results of 3 experiments and indicates that the Lon protease might be involved in cleaving these proteins. Other proteins were only identified in two out of four data sets, and are not included in Table 5.7. However, due to the fact that low abundant proteins are hard to detect, it might be possible that some proteins, and especially regulators, were missed in the different experiments. The proteomic approach showed that the Lon protease is likely involved in cleaving catalase A (PA4236) in P. aeruginosa, which is an important member of the oxidative stress response. Consequently, I sought to determine if a lon mutant exhibited an altered phenotype in response to oxidative stress. However, no difference in its ability to resist oxidative stress (neither with of 30% H2O2 nor 100 mM paraquat) when compared to the wild type PAO1, as indicated by the growth inhibition zones in Figure 5.8, was observed. Lon protease is thus not necessary for the oxidative stress response.  Figure 5.8. Ability of the lon mutant, the complemented strain and wild type to withstand oxidative stress (100 mM paraquat (upper disc) and 30% H2O2 (lower disc)). Bacterial cultures were grown to mid-logarithmic phase and spread on an agar plate. Filter discs containing 100 mM paraquat and 30% H2O2 were placed on the agar plates and incubated for 24 hours at 37°C. The clearing zone around the disc indicates the inhibition of bacterial growth and is an indication if the strain can withstand oxidative stress. One out of 3 repeats is shown.  87  On the other hand, these results indicated that the protein expression level of GroEL, a high abundant heat shock protein in the cell, was increased in a lon mutant compared to the parental strain. This is important as the Lon protease is involved in the stress response and is upregulated upon heat shock that is designed to unfold and/or degrade misfolded proteins. If mutated, the Lon protease could not participate in these tasks. Therefore, the protein expression level of GroEL and other DNA heat shock proteins (which were observed in selected SILAC experiments) might be increased in order to compensate for the malfunction of the Lon protease. Strikingly, Hfq, an RNA-binding protein which is essential for rpoS translation in E. coli (171), showed an elevated protein expression level in the lon mutant compared to the parental strain. Therefore, it is possible that part of the regulation exerted by Lon occurs through this chaperone of small RNAs. Overall, a few potential proteomic targets of the Lon protease could be demonstrated, but more research needs to be done in order to confirm these targets and to assign to the Lon protease a role in protein degradation/cleavage of these identified proteins. 5.3  Discussion For the first time, this study provides data on the global regulation by the P. aeruginosa  ATP-dependent Lon protease, showing its impact both at the transcriptional level as well as at the post-translational level. Investigating the transcriptome has been an important tool in studying the complex P. aeruginosa regulatory network. More recently, proteomic studies using two-dimensional gel electrophoresis revealed differences in the proteome under different conditions. These tools allow understanding the complex regulatory networks that pathogens possess, which can be important for the development of new antimicrobial treatment strategies. To gain further insight into the function of the P. aeruginosa Lon protease, the experiments performed here, aimed to identify its role in transcriptional regulation as well as identifying possible protein targets. The microarray analyses of the lon mutant in mid-logarithmic and stationary phase showed in each case a dysregulation of ~ 200 genes which accounts for approximately 4% of all genes indicating a substantial impact on the transcriptome. Examination of different growth phases led to the observation of differences in the impact of Lon on gene expression, likely because several of the identified genes are known to be considerably growth 88  phase specific, including RpoS (σS), the stationary phase sigma factor (138). RpoS has an important role in the response to stress conditions; for instance rpoS mutants were shown to be less resistant to hydrogen peroxide, high temperature and low pH (35, 113). The microarray data clearly indicated that the Lon protease plays a major role in gene regulation under all growth conditions, since hundreds of genes were dysregulated. The use of the PseudoCyc viewer showed that components of the denitrification pathway were dysregulated in the lon mutant. I therefore hypothesized that due to this dysregulation, the lon mutant would have a potential defect during anaerobic growth conditions. Indeed, lon mutants exhibited an anaerobic growth defect, which could be complemented by introducing the lon gene in trans, albeit not as pronounced as upon mutation of genes involved in the denitrification pathway (67). A possible correlation between the anaerobic growth defect and metabolism was further addressed. Generally, due to the smaller reduction potential of the electron acceptors used during anaerobic growth compared to O2, anaerobic respiration is less efficient than aerobic respiration. The redox potential for O2 is 1.23V, compared to 0.8V in the case of nitrate. Assessing anaerobic respiration allowed us to demonstrate an anaerobic respiration defect for the lon mutant that could in part explain its anaerobic growth defect. Respiration in general produces energy which can be used for cellular growth. If the respiration rate of the mutant is lower than that of the wild type and the complemented strain under anaerobic conditions, less energy will be produced and growth levels would be lower. The microarray analyses also revealed that a significant number of genes involved in pyochelin biosynthesis as well as type III secretion were moderately downregulated in the lon mutant. Previously, it has been shown that exoS plays a role in cytotoxicity and secretion (255), therefore providing the Lon protease a potential role in cytotoxicity. Various systems are regulated hierarchically in P. aeruginosa, such as the QS system (23); therefore, I wanted to determine if there was also a hierarchy for the Lon protease. Several transcriptional regulators appeared to be dysregulated in mid-logarithmic and stationary phase and using RT-qPCR and regulatory mutants, I was able to determine the existence of a transcriptional hierarchy involving the Lon protease in stationary phase. Clearly, these results showed that Lon regulates psrA, which in turn regulates the stationary sigma factor rpoS. Interestingly, psrA mutants share the same swarming deficient phenotype as lon mutants (78).  89  This suggests that the swarming deficiency of the Lon protease is likely to be caused by the dysregulation of psrA. I was not able to completely explain the motility deficient phenotype of the lon mutant, as only a few genes encoding pili and flagella were downregulated. Originally, I hypothesized that genes involved in swarming motility and biofilm formation would be dysregulated in the lon mutant, however this was not observed. A similar result had been previously observed in the cbrA (271) and psrA swarming deficient mutants (78), where no significant dysregulation of flagella and pili genes could be observed. Therefore, the effect of these regulators on swarming motility is likely mediated by a different and possibly more complex mechanism that does not directly involve the obvious players in motility. This could indicate that the Lon protease might be an important regulator of motility and therefore virulence. It is well known that the conditions during swarming motility are different from the ones used for the first microarray experiments (rich medium and liquid cultures). Therefore, a microarray under swarming conditions using a minimal medium was performed, but still did not show pili, flagella or rhamnolipid genes dysregulated. Rhamnolipids are glycolipids which are involved in modulating swarming motility on semisolid surfaces and are required for the flagellum-based movement. Previous results from our laboratory clearly demonstrated that in the complex phenomenon of swarming motility, not only are rhamnolipids, flagella and pili genes important, but also genes involved in metabolism as well as many transcriptional regulators. Overhage et al. (189) showed that genes involved in type III secretion, pyochelin biosynthesis and transcriptional regulators are upregulated under swarming conditions and highlighted that a complex regulatory network is involved in the differentiation process of swarmer cells. Interestingly, some of these genes were downregulated in the lon mutant, suggesting that wild type Lon perhaps plays a role in the upregulation of these genes during swarming. Furthermore, I showed that lon itself is upregulated under swarming conditions, indicating that the Lon protease is a key mediator of swarming motility. The Lon protease regulates a striking diversity of gene sets and has a major role in swarming motility. Transcriptional analyses revealed that the role of the Lon protease must be greatly affected by the growth state of the cell, as several independent repeats showed significant differences in gene expression. A major limitation of these experiments was the difficulty of harvesting the cells at the same cell density, especially due to  90  the fact that lon mutants exhibit a cell filamentation phenotype and, therefore, differences between samples occurred. These studies further emphasized that a certain degree of overexpression of lon did not cause lethality in P. aeruginosa, in contrast to what was previously seen for E. coli (33). Nevertheless, it appears that a specific level of Lon protease needs to be present in the cell to allow swarming motility. Thus, both deletion and overexpression of lon resulted in a loss of swarming motility in P. aeruginosa. However, swimming motility, biofilm formation and susceptibility to ciprofloxacin were not affected by lon overexpression; highlighting that the degree of lon expression largely impacted on swarming, known to be a complex adaptation that is dependent on more than 200 different genes and on cell-to-cell signalling. Interestingly, Takaya et al. (244) described that the Lon protease repressed the expression of lasRI by degrading LasI. These differences in the levels of the quorum-sensing molecules (las and rhl) in a lon mutant or a strain overexpressing lon could explain why swarming is affected. The balance of the quorum-sensing molecules is essential for swarming and is clearly impacted by the Lon protease. Often, an overexpressing strain can compensate for the deletion of a gene; however, for the Lon protease and swarming motility, this was clearly not the case. In addition to swarming conditions, the Lon protease was upregulated in response to heat shock in P. aeruginosa. This was consistent with observations made in E. coli, where the protease La (Lon) was upregulated at high temperatures and categorized as a heat shock gene (251). This upregulation emphasized that an increased amount of the lon gene product must be present in the cell in order to unfold misfolded proteins during the process of heat shock and other stress conditions. In addition to the heat shock response, Marr et al. (157) showed that the Lon protease was upregulated under subinhibitory concentrations of aminoglycosides and we could confirm this also under lethal exposure to the aminoglycoside tobramycin. Taken together, I showed that the Lon protease is an important balance regulator under stress conditions, which most likely is necessary in order to degrade misfolded proteins. Furthermore, another Lon-like protease in P. aeruginosa, AsrA protease, has been shown by our laboratory to be upregulated in response to tobramycin (121); however, differences in the regulation between the two proteases exist and this highlights the importance of the existence of different ATP-dependent proteases. They are clearly not only involved in the stress response, as originally thought, but have distinct functions in protein quality control in pathogens. 91  Pseudomonas can form biofilms in the lungs of CF patients (13). Airway mucus is thought to exhibit anaerobic conditions and, therefore, Pseudomonas encounters an anaerobic environment. For this reason, researchers determined the ability to form biofilms under anaerobic compared to aerobic conditions. Surprisingly, differences between studies were observed. While Field et al. (66) observed a decrease in the number of adhered cells under anaerobic compared to aerobic conditions (1 x 108 compared to 8 x 108 cells), Yoon et al. (273) observed that, under anaerobic conditions, a more robust biofilm was formed that might have resulted from cells with a highly elongated phenotype. Cell elongation (filamentation) appeared to occur under anaerobic conditions to increase the nutrient uptake which could be part of the starvation adaptation. Also of importance is that biofilms after 24 to 48 hours of incubation are usually heterogeneous and contain large anoxic regions as a steep oxygen gradient exists (265). In my case, the performed experiments on the PAO1 wild type, which assessed the formation of biofilms on simple abiotic surfaces, revealed the same observation as those made by Field et al. (66), as a decrease in attachment during anaerobic growth was observed. The reduced biofilm formation under anaerobic conditions is in accordance with the downregulation of genes involved in quorumsensing during anaerobic growth, since quorum sensing signals were proposed to be important for biofilm formation. For example, lasI mutants form structurally different biofilms compared to the wild type (42). However, other studies have shown that quorum-sensing is not essential for biofilm formation, indicating that the role of quorum-sensing in biofilm formation of P. aeruginosa is not entirely clear. Shrout et al. (223) have demonstrated that the nutritional environment plays an important role of the participation of quorum-sensing in biofilm formation. Different carbon sources used in the growth medium resulted in differences in the involvement of quorum-sensing in biofilm formation. For example, quorum-sensing mutants formed similar biofilms to the wild type when glucose or glutamate was used as the carbon source, but showed distinct structural differences under succinate-growth conditions. Taken together, quorumsensing signals play important roles in biofilm formation under certain conditions, whereas under other conditions they do not have an influence. Also, it has to be noted that P. aeruginosa reaches a lower cell density under anaerobic conditions and that might in part explain the decrease in biofilm formation as a lot of changes in gene expression/growth occur in a densitydependent manner. The lon mutant already exhibited a biofilm deficient phenotype under aerobic conditions, and the number of adhered cells was not further reduced under anaerobic conditions. 92  Therefore, my results do not support the hypothesis of Yoon et al. (273) that the formation of the robust anaerobic biofilm was a result of the elongated cell shape. It was mentioned throughout this thesis that one characteristic of the lon mutant is the formation of long filaments and Marr et al. (157) demonstrated that the lon mutant exhibited a biofilm deficient phenotype. Therefore, robust biofilm formation and cell elongation cannot be obligately linked. Also, it has to be mentioned that the nor gene must be partially active in a lon mutant (despite its downregulation in mid-logarithmic phase), because biofilms were still formed under anaerobic conditions. If nor would be completely inactivated then nitric oxide could not be detoxified and therefore no biofilm formation/growth would occur, as is the case with a nor mutant (274). The proteomic studies identified some possible targets of the post-translational regulation exerted by Lon, which seemed to differ from the results of the transcriptional analyses, indicating that the Lon regulon is very complex. Some of the promising hits identified by SILAC include the heat shock protein GroEL (PA4385), Hfq (PA4944) involved in RNA regulation, the catalase KatA (PA4236), the probable oxidoreductase PA1833, as well as several hypothetical proteins. Of special interest was Hfq, as it has been previously shown to be a small RNA binding protein and necessary for virulence in P. aeruginosa (230). Hfq impacts on rpoS expression and RpoS is known to regulate hundreds of genes in stationary phase. Therefore, the Lon protease could act by cleaving Hfq, thereby leading to the dysregulation of several genes, and the lon phenotypes could occur due to the dysregulation of genes in the stationary phase. However, to date we can only speculate and more studies are needed to evaluate whether Hfq is an actual target of the Lon protease. SILAC allowed us to identify some possible protein targets of the Lon protease. However, this technique works largely for highly abundant proteins and, therefore, transcriptional regulators are hard to identify. Additionally, some proteins were only identified in two out of the four biological repeats, and were not described in more detail in this thesis. The discrepancy in the results between the different repeats could be explained either by intrinsic variability between the samples from a biological perspective or due to the low abundance of these proteins which could prevent them from being detected in some of the experiments. Nevertheless, some proteins of interest were found and follow-up studies using His-tagging and overexpression are currently underway to investigate if the identified proteins are actual targets of the Lon protease. 93  6. Involvement of the Lon protease in the pathogenesis of P. aeruginosa 6.1  Introduction To combat infections caused by pathogens, the human body has developed a host defence  mechanism, called the immune system, which is subdivided into innate and adaptive immune responses (176). The innate immune system serves as an immediately available general defence system during the initial establishment of an infection. In this chapter, the main focus is on the innate immune response of the airways (200) as this response is relevant for CF and nosocomial P. aeruginosa infections. Normally, the airway epithelia, which are located at the interface between the environment and the host, serve to restrict uptake of foreign particles and microorganisms (97), playing an important role in the host innate immune response (155) by recognizing invading pathogens through pattern recognition receptors, e.g., TLRs (85, 167, 172) and responding to immediate threats. For example, TLR-5 is found on epithelial cells and recognizes bacteria flagella. Binding to these receptors initiates the innate host immune response leading to the secretion and production of several small cell-signalling protein molecules cytokines and chemokines (TNFα, IL-2, IL-6, IL-8, RANTES, etc.) - in order to recruit cells of the immune system to the site of infection (46, 130, 182). Infections trigger an intense inflammatory response that is necessary to remove the invading pathogens, but these cytokines and chemokines can also, if produced in a prolonged fashion and not appropriately controlled, cause chronic lung inflammation. Generally, pathogens are cleared by binding to mucus in the airway surface liquid and swept away by the mucociliary system or captured by resident alveolar macrophages or recruited immune cells, including neutrophils and macrophages. The innate immune response is further activated when the epithelial layer is perturbed. Although normally susceptible to innate immune mechanisms, under circumstances where it is able to establish an infection, P. aeruginosa, can evade the host defence mechanisms leading to deadly nosocomial infections, especially in immunocompromised patients or patients with a damaged lung epithelium (healthy individuals can deal with this inhaled pathogen) and chronic infections, such as CF. P. aeruginosa can evade the immune system by multiple mechanisms including the production of several proteases, toxins and lipases (Chapter 1), which decrease the function of the immune system by inhibiting the function or integrity of phagocytic cells. Furthermore, 94  toxins and pyocyanin produced by P. aeruginosa can destroy and damage the host tissues inhibiting mucociliary clearance of the pathogen. If the infection is not subsequently controlled by the adaptive immune response, Pseudomonas can establish a chronic infection as can occur in inflammatory lung diseases like cystic fibrosis. Under these circumstances, Pseudomonas can form biofilms in the lung (13). This social behaviour enables the organism to resist clearance by the immune system, since organisms in biofilms are not accessible to phagocytes and bacteria growing in a biofilm are substantially more resistant to antimicrobial agents (84, 111, 168). Thus P. aeruginosa has the ability to cause a persistent infection in part by protecting itself from being cleared by the immune system. In chronically infected patients, P. aeruginosa is highly adaptively resistant to antimicrobial treatment, making eradication very difficult. In CF patients especially, the thickened mucus (due to the absence of CFTR that tends to dehydrate the mucus) does not allow mucociliary clearance, and together with the bacterial inhibition of cilia beating, the invading organisms cannot be transported out of the airway by mucociliary action (227, 265). A major characteristic of the CF lung is the presence of excessive inflammation, which eventually will lead to lung damage and mortality. The inflammation is caused by the inability of patients with a CFTR mutation to control bacterial triggered inflammation (14) leading to excessive overproduction of host proinflammatory cytokines (12, 177). Here, the importance of the Lon protease in the virulence of P. aeruginosa in vitro (cell models) and in vivo infections was investigated, as the Lon protease plays an important role in virulence-related properties, such as motility and biofilm formation. I mimicked the CF lung environment by adding the glycoprotein mucin to the growth media (artificial sputum), as this resembles closer the patient’s lung environment. Consequentely, this provided a better understanding of the importance of the Lon protease during growth in a mucin-rich environment. Overall, the results demonstrated that the Lon protease of P. aeruginosa indeed contributes to pathogenesis in Pseudomonas infections.  95  6.2  Results  6.2.1  Reduced cytotoxicity of the lon mutant towards human bronchial epithelial cells The lon mutant was examined for its ability to infect and destroy a monolayer of human  bronchial epithelial cells (16HBE14o-). Use of an in vitro assay enabled the development of hypotheses regarding what might happen to epithelial cells in the human lung. To measure the cytotoxicity of the wild type H103 and the lon mutant, a lactate dehydrogenase enzyme assay was performed in collaboration with Shaan Gellatly and Patrick Taylor, measuring the amount of cytoplasmic lactate dehydrogenase released by cells into the medium as a criterion for cytotoxicity at 10 and 18 hours post-infection. It was observed that the wild type and the mutant both exhibited cytotoxicity towards HBEs, but interestingly the lon mutant showed a statistically significant (p=0.001) decrease in cytotoxicity from 30 to approximately 20% at 10 hours postinfection compared to the wild type as measured with Student’s t test. However, no difference between the two strains was observed at the 18 hour time point (Figure 6.1). These results demonstrated that the P. aeruginosa lon gene was necessary for wild type levels of cytotoxicity towards HBEs in vitro at early time points and, therefore, I hypothesized that the Lon protease might participate in the initiation and establishment of infections.  *  Figure 6.1. Cytotoxicity of the lon mutant and the wild type H103 at 10 and 18 hours postinfection. The abilities of the wild type H103 and lon mutant to induce cell damage were determined by measuring the release of lactate dehydrogenases (LDH) from the HBEs. The bars represent the average and standard deviation of 3 independent experiments. A statistically significant difference was observed at 10 hours post-infection with p=0.001 for the lon mutant compared to the wild type (measured with Student’s t test). This experiment was performed in collaboration with Shaan Gellatly and Patrick Taylor. 96  Further in vitro assays measuring the invasive and adhesive properties of the abovementioned strains could not detect any major differences between the lon mutant and the wild type. Nonetheless, it must be noted that these other assays are not very sensitive and only major differences would have been detected. Furthermore, Pseudomonas is an extracellular pathogen that is only able to invade epithelial cells to a modest extent (69). Nevertheless, microscopy of bacteria adhered to HBEs revealed that the lon mutant adhered to the HBEs in a filamentous phenotype, whereas the wild type and complemented strain adhered to the HBEs as individual cells (Figure 6.2). This might reduce the surface contact of bacteria and HBEs in the case of the lon mutant. Consequently, toxins might not be as efficiently injected compared to the situation for the wild type, since for Type III secretion a direct contact between the host cell and the bacteria is necessary. This would ultimately lead to reduced damage of epithelial cells.  HBEs  HBEs + lon  HBEs + lonc  HBEs + H103 wild type  Figure 6.2. HBEs infected with P. aeruginosa wild type, lon mutant and complemented strain. HBEs were cocultured with bacteria cells for 3 hours to allow adherence. After 3 hours, unadheared bacteria were removed by washing 3 times with PBS. Remaining bacteria and HBEs were heat fixed and stained with Diff-Quick to allow microscopic analysis. The arrows indicate the bacterial filaments formed by the lon mutant and the single cells of the wild type and complemented strain. 97  6.2.2  Lon mutants show impaired virulence in the lettuce leaf model As shown previously, virulence-related properties were altered in the lon mutant.  Therefore, I hypothesized that virulence in vivo might also be impaired and chose to start investigating this possibility by using a plant infection model, the lettuce leaf model previously described by Gooderham et al. (79). To do that, cultures of the wild type, the lon mutant and complemented strain were stabbed into the midrib of the lettuce leaves and scored the symptoms of infection over a time-period of 5 days. In comparison to the wild type and the complemented strain, it became obvious that the lon mutant was unable to cause the same extent of infection from the point of inoculation outwards. Thus, while the Pseudomonas wild type and complemented lon strain led to severe damage of the midrib as well as to a rotting phenotype (infection indicated by a brown colour), the lon mutant showed a much weaker progression of lettuce leaf infection. The rotten phenotype was, however, still visible in the lon mutant, although the extent of the brown area was much smaller (lesion size of the wild type 18 mm ± 3 mm and of the lon mutant 11mm ± 2 mm) (Figure 6.3). These observations indicated that the P. aeruginosa lon mutant has a defect in virulence, consistent with its impact on virulence-related properties.  WT  lonc  lon-  Figure 6.3. Romaine lettuce leaves infected with P. aeruginosa wild type, complemented lon strain and lon mutant. The lon mutant is attenuated for virulence in the lettuce leaf model. Symptoms of infection were monitored over a time-period of 5 days after the midribs were injected with 1 x 106 cfu of P. aeruginosa. A brown colour change on the ribs indicated the process of infection. This picture was taken 5 days post-infection and represents one biological repeat out of 3 with the same trends. The black line highlights the surface of infection.  98  6.2.3  Lon alters virulence towards amoeba The amoeba, Dictyostelium discoideum, is known to be another host model system to  study bacterial virulence (1). Using the amoeba host model, in collaboration with Dr. Joerg Overhage (KIT, Karlsruhe, Germany) it was determined if the reduced virulence phenotype of the lon mutant observed in other experiments could also be confirmed when virulence was assessed by plaque formation. Macroscopic plaque formation, as a measure of bacterial killing by varying concentrations of amoebae, was evaluated after 3 to 5 days post-infection for all strains tested. It was observed that the PA14 wild type was far more resistant to killing and thus presumptively more virulent than the PAO1 wild type in that, 5000 amoebae were necessary to achieve plaque formation for the PA14 wild type, whereas only 40-60 amoebae were required on average for the PAO1 wild type. The virulence phenotype of the PAO1 and PA14 lon mutants was investigated and a consistent twofold reduction in virulence was observed for the lon mutant in both strain backgrounds (Figure 6.4) in that lower concentrations of amoebae were required to kill the lon mutant bacteria compared to the respective wild type strain.  99  WT-PA14 160  20000 10000  313  5000  625  1250  2500  lon- PA14 160  20000  10000  313  5000 625  1250  2500  100  WT-PAO1 20  240  40  200  60  160  120  80 lon- PA01 20  240  40  200  60  160  80  120  Figure 6.4. Amoeba virulence model. Overnight wild type and lon mutant bacterial cultures were diluted in PBS and spread on agar plates. D. discoideum was then spotted at different concentrations onto the plates. Incubation took place at 23°C for 3-5 days and visible plaque formation on the agar plates was determined. The number of D. discoideum cells per 5 µl inoculum are shown. Arrows indicate the smallest number of amoebae forming a plaque (wild type PA14 and PAO1 as well as respective lon mutants). The experiment was performed in collaboration with Dr. Joerg Overhage (KIT, Karlsruhe, Germany). One out of 3 experiments is shown. 101  6.2.4 A chronic lung infection model demonstrates that a mutation in lon leads to a major defect in growth and maintenance in vivo Based on the above described results, in collaboration with Dr. Roger Levesque (University Laval, Canada) the importance of the Lon protease was investigated in a rat model of chronic infection, chosen due to its relevance to CF. The experiment was carried out by measuring the relative ability to grow in a mixed culture in vivo, also referred to as the competitive index (CI). The CI was first determined in vitro and demonstrated that the wild type and the lon mutant, as well as the lon mutant and the complemented strain grew at approximately the same rate with an in vitro CI of 0.81 ± 0.09 (p = 0.3) and 0.89 ± 0.24 (p = 0.1), respectively, indicating no significant growth difference between the tested strains (Mann Whitney test). In contrast, competitive growth studies in a rat model of chronic infection highlighted the importance of Lon in Pseudomonas chronic lung infections. The lon mutant displayed nearly a 1000 fold reduction in maintenance in vivo when in competition with the wild type strain (CI of 0.0018, p=0.0022) and a 100 fold reduction when compared to the complemented strain (p=0.0013), evaluated 7 days post-infection (Figure 6.5). The P-values were determined with the Mann Whitney test. This represents a major attenuation in virulence of the lon mutant. Furthermore, these studies showed that virulence could be partially restored by complementation with the lon gene in trans; however, likely due to copy number effects affecting levels of expression and/or potential loss of the plasmid during the experiment, a modest attenuation of competitive growth was observed (CI of 0.15, p=0.015).  102  Figure 6.5. In vivo competitive index assay of the lon mutant and the complemented strain. The lon mutant and the complemented strain were grown for 7 days in the rat model of chronic infection in competition with the wild type PAO1. The competitive index was analyzed. Each circle represents the CI for a single animal. The lon mutant had a major attenuation in virulence which was statistically significant as measured with the Mann Whitney test (p=0.0022). The experiment was performed in collaboration with Dr. Roger Levesque (University Laval, Canada). 6.2.5 Mucin alteration of motility, ciprofloxacin resistance and biofilm formation in P. aeruginosa Mucin, a glycopeptide from the mucous layer that covers the airway epithelia, was used to mimic the CF lung environment and was used to investigate its influence on motility, antibiotic resistance and biofilm. Our laboratory has demonstrated that in the presence of mucin Pseudomonas undergoes a unique form of motility, termed surfing motility (A. Yeung et al. 2011. Surfing motility: a novel form of cooperative surface motility in P. aeruginosa, manuscript under review). Surfing motility is another complex adaptation of P. aeruginosa distinct from swarming motility, because surfing motility is flagella-, but not pilus-dependent. Thus surfing motility on BM2 swarming media plates containing 0.4% agar and varying concentrations of mucin (0, 0.1 and 0.5%) was studied. A significant increase in the motility zone at higher concentrations of mucin for the wild type PAO1 strain and to a lesser extent for the lon mutant could be observed. Nevertheless, the lon mutant was highly deficient in both surfing and swarming motility. Indeed, the diameter of the surfing motility zone observed for the wild type  103  PAO1 increased from 11.5 to 46.25 mm when 0.5% mucin was added to the medium. However, in the case of the lon mutant only an increase from 6.25 to 13.6 mm was observed (Figure 6.6).  0% Mucin  0.5% Mucin H103  H103  lon-  lonFigure 6.6. Motility of the wild type PAO1 and the lon mutant in the presence and absence of mucin. P. aeruginosa wild type and lon mutant spotted on medium containing mucin allowed for surfing motility (right), the control without mucin is seen on the left. Mucin was added to the medium in an attempt to more closely mimic the lung environment of cystic fibrosis patients. In addition to the effect of mucin on motility, I also investigated whether this glycopeptide promoted ciprofloxacin resistance. The addition of 0.5% mucin clearly led to an increase in antibiotic resistance for the wild type as well as for the lon mutant. On plates without mucin, the MICs for the wild type and the lon mutant were 0.1 and 0.0125 µg/ml, respectively. However when 0.5% mucin was added, the respective MICs for the two strains doubled to 0.2 and 0.025 µg/ml. In contrast, antibiotic resistance was not changed by the addition of 0.1% mucin, even though this concentration had an impact on the motility zone diameter (Figure 6.7). This clearly demonstrated that mucin promoted ciprofloxacin resistance as well as surfing motility.  104  Figure 6.7. Increase in ciprofloxacin resistance promoted by 0.5% mucin and surfing motility promoted by 0.1 and 0.5% mucin. Bacterial cells were incubated on BM2 plates containing 0.4% agar and varying concentrations of mucin (0, 0.1 and 0.5%) and increasing ciprofloxacin concentrations (0 - 0.2 µg/ml). The motility zone diameter was measured for the different combinations. The graph shows the increase in surfing motility zone due to the addition of mucin as well as the impact of mucin on ciprofloxacin resistance. The bars represent the average and standard deviation of 3 independent experiments. Next, I investigated if mucin also promoted abiotic biofilm formation. No major differences in rapid attachment were observed for either strain when increasing concentrations of mucin were added to the growth medium. However, mucin at a concentration of 0.5% increased the formation of mature biofilms in the lon mutant, but not in the wild type. On average, the biofilm-deficient lon mutant increased its ability to form mature biofilms by up to 180% in the presence of 0.5% mucin compared to no mucin, although it was still deficient in biofilm formation compared to the wild type H103 (Figure 6.8), which was statistically significant as measured with the Student’s t test (p=0.002 and p=0.03, respectively). No differences were observed for either strain when 0.1% mucin was added to the medium, indicating that a certain amount of mucin had to be be present in order to enable the lon mutant to form a biofilm; in each case the biofilm defect could be complemented by introducing the lon gene in trans.  105  **  *  Figure 6.8. Mature biofilm formation with increasing concentrations of mucin. Cells were incubated in 96 well microtiter plates containing LB and varying concentrations of mucin (0 – 0.5%) for 20 hours at 37°C. Biofilm formation was measured by crystal violet staining of the adherent cells. The lon mutant produced more mature biofilm once 0.5% mucin was added to the growth medium compared to no mucin present, which was statistically significant as determined by Student`s t test (p=0.002). However, the wild type H103 still produced more biofilm than the deficient lon mutant which was also statistically significant (p=0.03). The bars represent the average and standard deviation of 3 independent experiments. 6.3  Discussion This study determined if the Lon protease of P. aeruginosa in additon to its altered  motility and biofilm formation also participated in virulence in vivo. To accomplish this goal, several in vivo models were utilized and also the influence of the P. aeruginosa Lon protease on interaction with HBEs was investigated. Clearly, the results demonstrated that the Lon protease of P. aeruginosa played an important role in growth and virulence in vivo. Thus the Lon protease was found to be necessary to cause a wild type virulence phenotype in a lettuce leaf model, normal killing by amoeba and competitive growth in a rat model of chronic lung infection. Several other studies have shown that mutations in the Lon protease in other organisms shared similar phenotypes to those observed here for P. aeruginosa. However, different studies have shown different contributions to virulence, possibly due to intraspecific variation or to different methodology (15, 36, 135, 207, 240, 243, 270). Most studies revealed that the Lon protease plays a critical role in interaction assays with epithelial cells as well as in establishing infections in mice. Moreover, ATPdependent proteases other than the Lon protease such as the ClpXP proteases could also 106  influence virulence (36, 258). The virulence phenotypes of Lon protease mutants have to date been studied most extensively in Salmonella enterica serovar Typhimurium. My results provide new insights about the importance of the Lon protease of P. aeruginosa in virulence. First, it was demonstrated that lon mutants were deficient in cytotoxicity to HBEs compared to the wild type at early time points (10 hours post-infection). The observed deficiency could be explained by the dysregulation of Type III secreted genes that were observed in microarray experiments (Table 5.2 and Table 5.3). Consistent with this, it is known that the Lon protease, alone or in combination with ClpP, can affect Type III secretion in Gram-negative bacteria (109). In the case of the P. aeruginosa lon mutant, a defect was observed at early time points, but not at later time points, consistent with a time dependent effect on cytotoxicity. For example it is possible that genes involved in Type III secretion, which are affected in a lon mutant, played a role in early adherence to HBEs but, at later time points, other secretion and adhesion genes, independent of Lon activity, become more influential leading to an absence of a cytotoxicity defect in the lon mutant. On the other hand, no differences in invasion or adhesion to HBEs could be observed possibly due to the low sensitivity of the methods employed. Interestingly, a phenotypic difference was observed regarding how the lon mutant, wild type and complemented strain adhered to HBEs. The lon mutant adhered as long filamentous chains, which could be explained by its defect in cell division. The importance of the Lon protease in invasion varies in other bacterial species. For instance, a study in Campylobacter jejuni demonstrated lower levels of invasion for a lon mutant, which was consistent with a defect in agglutination (considered to be an indicator of invasion) (36). The results obtained in studies on the invasion of HBEs in S. enterica varied. Thus, while Takaya et al. (243) indicated that the Lon protease negatively regulated the efficiency of invasion and the expression of the invasion genes located on the Salmonella pathogenicity island; Boddicker and Jones (15) observed no differences in invasion. To examine if the Lon protease of P. aeruginosa is necessary for full virulence in vivo, several assays were performed. All resulted in the observation that the Lon protease of P. aeruginosa is required not only for motility-related properties in vitro, but also for virulence in vivo. While the specific mechanisms of reduced virulence in vivo were not examined, it can be speculated that the reduction in virulence-related properties such as motility, biofilm formation and toxicity towards epithelial cells, and the dysregulation of several transcriptional regulators 107  and type III secretion genes in stationary phase, likely reduced virulence. These observations of decreased virulence in P. aeruginosa correlated with observations made for other species, suggesting that the Lon protease has a common role in determining full virulence. For example, lon mutants in S. enterica did not cause lethal systemic disease of mice due to their inability to proliferate within the spleen. Furthermore, lon mutants show a decrease in viability in murine macrophages over a period of 48 hours, consistent with a reduced ability to withstand the bactericidal mechanisms of macrophage killing. Nevertheless, an S. enterica lon mutant was able to cause persistent infection in mice, although it did not multiply as efficiently as the wild type (243). In direct analogy, the P. aeruginosa lon mutant could grow in the chronic rat lung model, but had a severe competitive growth disadvantage. Furthermore, preliminary results from our laboratory indicate that the the lon mutant is less virulent in a mouse model of acute infection compared to the wild type (Dr. Laure Janot). Due to such characteristics, it has been suggested that ATP-dependent proteases might be important targets of antimicrobial therapy. For example, a recent study showed that the S. enterica Lon protease can be inhibited by MG262, a peptidyl boronate (72). MG262 consists of a peptide with a boronic acid modification, both of which are important for inhibiting the proteasome of eukaryotic cells. Similarly, MG262 was demonstrated to be the most potent inhibitor of S. enterica Lon activity amongst the tested commercially-available peptide-based proteasome inhibitors. Other potent inhibitors are known for the ClpP ATP-dependent protease of S. aureus (17), which is also involved in virulence. These ClpP inhibitors are β-lactones and by inhibiting ClpP, the expression of virulence factors decreased drastically. Interestingly, useful observations regarding production of live vaccines in Salmonella were made, wherein immunization with a lon-deficient strain protected mice against the subsequent oral challenge with a virulent Salmonella strain or even a challenge with virulent Listeria monocytogenes (160). To date it has not been demonstrated that the P. aeruginosa Lon protease is a good antimicrobial target as the virulence phenotypes were not known. However, the results of this study indicate that the ATP-dependent P. aeruginosa Lon protease might be a good target as a significant reduction in virulence occurs when the lon-encoding gene is mutated. To date, there are no known inhibitors for the P. aeruginosa Lon protease and further research is required in order to test if MG262 is also a good inhibitor for P. aeruginosa and to identify new potential inhibitors.  108  In addition, further studies should be undertaken to investigate whether a P. aeruginosa lon mutant would be a good candidate for the development of a live vaccine. Another goal of this study was to attempt to mimic the CF lung environment by adding mucin to the growth medium. Mucin has already been demonstrated to serve as an energy and nutrient source, and is required for attachment of Pseudomonas (232). It is likely to mimic the lung environment, but the use of different artificial sputum media showed varying results between studies, indicating that any phenotypes observed are specific to the underlying conditions (75, 191). The results here clearly showed that the addition of mucin at 0.1 and 0.5% promoted an increase in surface motility, recently named “surfing” motility (A. Yeung et al., manuscript submitted). This increase was observed for the P. aeruginosa PAO1 wild type and the lon mutant, even though the latter had a clear defect in surfing motility compared to the wild type. Thus, it can be speculated that mucin in the CF lung allows P. aeruginosa to move and spread faster. The investigation of the speed of surfing motility compared to that of swimming motility (~ 10 - 40 µm s-1) (112) would be valuable . Apart from promoting surface motility, the addition of 0.5, but not 0.1%, mucin led to an increase in antibiotic resistance, specifically ciprofloxacin resistance. This finding is particularly significant since ciprofloxacin is currently used for treatment of P. aeruginosa infections in CF. Furthermore, mucin influences biofilm formation and a study by Landry et al. (136) showed that mucin enhances biofilm formation, particularly when investigated on a surface coated with mucin compared to a glass surface. Indeed, in the Landry study the formation of large cellular aggregates was observed on a mucincoated surface, in contrast to the flat and undifferentiated biofilms seen on uncoated glass surfaces. However, my study did not show an enhanced rapid attachment or mature abiotic biofilm formation in the presence of mucin for the wild type strain, which could be explained by the differences in the conditions used for the experiment (attachment to polystyrene plates compared to a glass surface). Interestingly, the biofilm deficient lon mutant was able to form better biofilms once mucin was added to the growth medium at a concentration of 0.5%. It is likely that lon mutants produce less exopolysaccharides (important for biofilm formation), and that mucin allows for functional complementation leading to enhanced biofilm formation in a lon mutant. On the other hand, the wild type naturally produces copious exopolysaccharides; consequently, mucin does not promote additional biofilm formation in the wild type. These results led me to conclude that mucin promoted biofilm formation in the lon mutant. 109  Interestingly, gene expression studies (75) comparing artificial sputum to normal growth medium revealed an upregulation of QS - signalling genes in the presence of artificial sputum, which might explain the enhancement of complex behaviours, such as surfing motility. Unpublished data (A. Yeung and R.E.W. Hancock) showed that the Clp ATP-dependent proteases (ClpB and ClpS) were upregulated in the presence of mucin, which could potentially highlight the fact that these proteases can compensate for a lon-deficient strain to form biofilms. Overall, this study demonstrated that the Lon protease is important for full virulence in vivo of P. aeruginosa, and that the presence of mucin, which is an important component of the CF lung environment, resulted in an increase in ciprofloxacin resistance and surface motility.  110  7.  Role of other intracellular proteases in motility, biofilm formation and antibiotic resistance of P. aeruginosa  7.1  Introduction Throughout this study I have focused on the influence of the ATP-dependent Lon  protease (PA1803) on virulence-related factors, antibiotic resistance and virulence. It was demonstrated that the Lon protease participates not only in the DNA-damage stress response, but also plays an important role in ciprofloxacin resistance and virulence. Another protease, AsrA, has been recently shown to mediate adaptive resistance to the aminoglycoside tobramycin and have a role in the heat shock stress response (121). As a class, ATP-dependent intracellular proteases are known to be key players in the stress responses via regulation of chaperones and by degrading short lived regulators and misfolded proteins (81). However, it is becoming increasingly clear that these proteases also have more specific roles in antibiotic resistance and virulence. P. aeruginosa produces other proteins characterized as components of ATP-dependent intracellular proteases, including the serine protease complexes ClpXP and ClpAP (clp, caseinolytic protease gene). ClpX and ClpA, also known as translocases, recognize specific peptide sequences (tags) of unfolded proteins (7, 96, 224). Upon recognition, the proteases bind to the tags and translocate the unfolded proteins into the proteolytic chamber of the selfcompartmentalized ClpP protease. ClpP forms the proteolytic part of this heterosubunit complex and degrades the unfolded proteins into small peptides (247). Apart from these proteases, adaptor proteins are present in the cell to bind proteins and transport them to the proteases for degradation (216). One such adaptor protein is ClpS, which interacts with ClpA (51). ClpS recognizes specific substrates, from the so-called N-end rule pathway, that are named N-degrons (containing leucine, tyrosine, phenylalanine or tryptophan residues) (59, 209). Therefore, ClpS binds to these N-degrons and delivers them to the ClpAP complex for degradation. In doing so, it inhibits the degradation of other substrates that are usually recognized by ClpA, as ClpS uses the same N-terminal binding domain of ClpA. In the presence of ClpS, ClpAP degrades N-degrons; in the absence of ClpS, the complex degrades other unfolded proteins (209). Here, in collaboration with Dr. Lucia Fernandez and Diana Song (as indicated in the preface), 10 protease mutants from the PA14 transposon mutant library (148) were screened for alterations in motility-related processes (swarming, swimming and twitching), biofilm formation 111  and antibiotic resistance and using the PA14 lon mutant as a control. It was demonstrated that 3 protease-related proteins, PfpI (PA0355), ClpP (PA1801) and ClpS (PA2621), were involved in motility, biofilm formation and antibiotic resistance. 7.2  Results  7.2.1  Several protease mutants exhibit a motility defect Ten protease mutants from the PA14 transposon mutant library (Table 7.1) were analyzed  for the 3 types of motility exhibited by P. aeruginosa: swarming (complex adaptation), swimming and twitching motility. Table 7.1. P. aeruginosa PA14 transposon mutants used in this study. Mutant from PA14 library PAMr_nr_mas_12_1:C6 PAMr_nr_mas_02_3:G5 PAMr_nr_mas_03_2:C2  PAO1 ortholog (gene name) PA0355 (pfpI) PA0372 PA0459  PAMr_nr_mas_12_4:E7  PA1801 (clpP)  PAMr_nr_mas_08_1:F11  PA1802 (clpX)  PAMr_nr_mas_08_4:C11 PA2620 (clpA) PAMr_nr_mas_06_2:F9 PAMr_nr_mas_11_1:C10 PAMr_nr_mas_11_1:G12 PAMr_nr_mas_04_1:G10  PA2621 (clpS) PA3326 PA3535 PA4576  Product of gene Protease PfpI Probable zinc protease Probable ClpA/B protease ATP binding subunit ATP-dependent Clp protease proteolytic subunit ATP-dependent Clp protease ATP binding subunit ATP-dependent Clp protease ATP binding subunit ATP-dependent Clp protease adaptor protein Probable Clp-family ATP-dependent protease Probable serine protease Probable ATP-dependent protease  Of the tested protease mutants only 3 (pfpI, clpP and clpS) displayed a swarming motility defect, similar to the one observed for a lon mutant. Complementation of the clpP and pfpI genes led to the complete restoration of the swarming deficiency phenotype of the clpP and pfpI mutants, respectively (Figure 7.1). However, complementation of the PA14 clpS mutant could not be achieved, perhaps due to overexpression lethality. Therefore, we examined the swarming phenotype for the University of Washington library (UW) PAO1 clpS (phoAwp08q1A11)  112  mutant (110) in order to verify the deficiency in a second strain background. The PAO1 clpS mutant also showed a significant defect in swarming motility (Figure 7.1). In addition to the swarming deficient phenotype, the clpP mutant showed a substantial loss in the ability to swim in aqueous environments (60%, p≤0.0001), which could be restored by complementation (Figure 7.1). The clpS mutant also showed a slightly reduced ability to swim with approximately 25% decrease in the swimming zone in PA14, but not in PAO1. The third type of motility that P. aeruginosa exhibits on solid surfaces is twitching. Twitching motility assays showed no major defect in any of the tested strains. However, the clpP mutant showed a modest 40% twitching defect (p=0.0003, determined with the Student’s t test) that could be complemented by introducing the wild type clpP gene (Figure 7.1).  E  E ** **  Figure 7.1. Motility phenotypes of P. aeruginosa mutants in intracellular proteases: A) Swarming of PA14 wild type, pfpI and clpS mutants; B) Swarming of PA14 wild type, pfpI and complemented pfpI mutant (pfpIc); C) Swarming of PAO1 wild type, UW- clpS mutant; D) Swarming of PA14 wild type, clpP and complemented clpP mutant (clpPc). The pictures shown are representative of at least 4 independent experiments with the same results. (E) Swimming and twitching motility analysis of PA14 wild type, PA14 transposon insertion mutant clpP and clpP complemented strain (clpPc). The results represent the average percentage of wild type motility and standard deviation of 4 independent experiments. A statistically significant difference between the mutant and the wild type could be observed as determinet by Student`s t test. In the case of swimming motility a P-value of p≤0.0001 was determined and for twitching motility the P-value was p=0.0003. 113  To test the possibility that the motility defects were due to altered growth rates, growth at 37°C was monitored over a time period of 10 hours. Almost all tested strains showed a similar growth rate compared to the wild type; however, a two hour delay in growth rate occurred in the clpP mutant (Figure 7.2). However it seems very unlikely that such a growth delay would completely prevent swarming motility. Nevertheless, the minor impairments of swimming and twitching could be due to this growth defect.  Figure 7.2. Growth curve of the clpP mutant compared to the wild type PA14. Absorbance of the strains was measured at 620 nm every 20 minutes over a time period of 10 hours in the TECAN Spectrafluor Plus plate reader. 7.2.2  Altered biofilm formation of the swarming-deficient protease mutants Biofilms are, like swarming motility, a form of social behaviour and contribute to  antibiotic resistance and virulence. Of all the tested protease mutants, only those that also exhibited a motility defect, namely pfpI, clpP and clpS, showed a difference in biofilm formation. The most significant reduction in formation of mature biofilms was observed for the clpP mutant, where 70% less biofilm occurred compared to the wild type and could be restored to wild type levels after complementation (Figure 7.3 A). This decrease in the ability to form biofilms for the clpP mutant was statistically significant (p=0.0002) as measured with the Student’s t test. clpS and pfpI mutants had a reduction of approximately 65 and 35%, respectively. In case of the pfpI mutant, biofilm formation is a bit more complicated. Thus, most experiments showed a clear decrease in the production of mature biofilms; however, some biological repeats displayed an increase in the ability to form biofilms. Nevertheless, in all cases 114  we observed a complementation of the altered phenotype in the mutant strain carrying plasmid pBBR1MCS3::pfpI (208). To test whether the defect in mature biofilm formation was due to a defect in initial attachment, we investigated the capability of pfpI, clpP and clpS for rapid attachment. However, no significant changes were observed in the rapid attachment assay for the tested mutants. Therefore, it does not seem that the altered mature biofilm phenotype of the mutants is due to differences during the early stages of attachment. The altered biofilm phenotype of the examined mutants raised the question of whether biofilm-related functions, such as extracellular matrix production, were also changed. The matrix of biofilms consists of exopolysaccharides produced by the products of the psl and pel genes. In order to investigate if the biofilm phenotypes of these mutants were caused by an altered production of exopolysaccharides, Congo red binding assays were performed, which allow for the quantification of matrix production due to the ability of Congo red to bind polysaccharides. The 3 biofilm-deficient mutants (pfpI, clpP and clpS) showed a lower Congo red binding ability compared to the wild type that could be restored in the clpP complemented mutant (Figure 7.3 B). Intriguingly, the clpP mutant demonstrated an epigenetic phenomenon whereby discrete spots of the colony were very highly stained. This phenotype was complementable by introduction of the wild type clpP gene. A  B  H103  clpP-  **  clpPc Figure 7.3. Analysis of mature biofilm formation (A) and Congo red assay (B) of PA14 wild type, PA14 transposon insertion mutant clpP and clpP complemented strain (clpPc). The results of the biofilm assay represent the average percentage in relation to wild type biofilm formation and standard deviation of 4 independent experiments. A statistically significant difference between the mutant and the wild type was observed as determined by Student’s t test (p=0.0002). In the Congo red assay, a more intense red tone at the edge of the colony can be observed in the wild type PA14 and the complemented strain while in the clpP mutant an epigenetic phenomenon was evident, whereby dense staining was observed in discrete areas. 115  7.2.3  Mutations in intracellular proteases affect antibiotic resistance Swarming motility, biofilm formation and antibiotic resistance have been proposed to be  intertwined and to share common regulatory pathways. Here, the influence of protease mutants on resistance to clinically important antibiotics such as ciprofloxacin, piperacillin, tobramycin, polymyxin B, imipenem, aztreonam and ceftazidime was investigated. None of the 3 tested mutants had any differences in susceptibility to the aminoglycoside tobramycin or the peptide polymyxin B. However, the selected protease mutants demonstrated an altered antibiotic resistance profile to fluoroquinolones and/or β-lactams. The results demonstrated that the pfpI mutant exhibited a significant 4 fold increase in resistance to ciprofloxacin, whereas the clpP mutant showed a reproducible twofold decrease in resistance to ciprofloxacin. The antibioticresistance phenotype could be complemented in the case of the clpP and pfpI mutants. The clpS mutant showed a two- to fourfold increase in resistance to piperacillin, imipenem, aztreonam and ceftazidime, whereas the lon mutant showed a twofold decrease in resistance to piperacillin (Table 7.2). Table 7.2. MICs (µg/ml) of selected mutants to various antibiotics. Strain  Ciprofloxacin Piperacillin Tobramycin Imipenem Aztreonam Ceftazidime  PA14 (wild type) 0.1 8 2 0.5 4 2 0.4 8 2 pfpI ND ND ND pfpI c 0.05 8 2 ND ND ND 0.1 16 2 1 16 16 clpS 0.05 8 2 clpP ND ND ND clpP c 0.1 8 2 ND ND ND 0.025 4 2 0.5-1 lon ND ND PA01 (wild type) 0.1 4 2 1 4 2 UW-clpS 0.1 16 2 2 8 4 ND: not determined, as additional β-lactam antibiotics were only tested for the clpS mutant. With regards to the increase in MIC towards β-lactam antibiotics, we assessed qualitatively and quantitatively whether the alteration in β-lactam resistance of the clpS mutant was due to an altered β-lactamase production. Visual analysis of β-lactamase production in the mutant appeared to indicate that the clpS mutation resulted in a slight increase in β-lactamase  116  activity, as shown by a colour shift from yellow to red of the nitrocefin present in the discs (Figure 7.4).  clpS-  PA14  Overproducing control strain  Figure 7.4. Qualitative nitrocefin assay. Colonies were spread on discs containing nitrocefin and a red colour change indicated the production of β-lactamase for the respective strain. Nevertheless, these data were not conclusive and we further measured β-lactamase production by analyzing quantitatively the rate of nitrocefin hydrolysis in the clpS mutant compared to the wild type. However, in this case no significant differences were observed between the mutant and the wild type, even when the samples were grown in the presence of sub-inhibitory concentrations of piperacillin, ceftazidime or imipenem (data not shown). Therefore, it seems unlikely that increased β-lactamase production is the explanation for the resistance phenotype of this mutant. 7.2.4  clpP and lon are not located in an operon According to the Pseudomonas database (www.pseudomonas.com), the ClpPX and Lon  proteases, are not located in an operon. On the contrary, several years ago, these proteases were predicted to be in one operon in P. aeruginosa. Due to these controversial data, it was important to determine whether the ClpP and the Lon proteases, which seemed to influence similar virulence-related properties, were located in an operon. To do that, gene transcription analyses were performed by RT-PCR using cDNA as a template, and genomic DNA as a positive control. The primers used permitted the amplification of overlapping fragments (e.g. clpP and lon indicated by the arrows in Figure 7.5), so that it could be determined whether different pairs of genes from this chromosomal region were transcribed together or not. The results indicated that Tig (PA1800), ClpP (PA1801) and ClpX (PA1802) constituted a single transcriptional unit, as PCR products were detected using cDNA as the template. In contrast, when amplifying the 117  overlapping fragments of clpP and lon using cDNA, no PCR product could be observed, indicating that the ClpP protease and the Lon protease (PA1803) are not located in the same operon in P. aeruginosa. However, it looks like the gene encoding the Lon protease forms an operon with hupB (PA1804), coding for a DNA-binding protein. Intriguingly, a weak amplification band could be observed for the overlapping fragments corresponding to clpX and lon. However, it is unlikely that lon is in an operon with clpX, which is clearly cotranscribed with clpP, and our results indicated that lon and clpP are not together in an operon. Furthermore, a putative promoter sequence is predicted to be located within clpX (400bp before its end), which could very likely be the lon promoter. This would explain the amplification of a fragment with the primers ClpX_F and Lon_R. All the PCR combinations and results are shown in Figure 7.5.  www.pseudomonas.com  1kb ladder  1  2  3  4  5  6  7  8  9  10 1 Tif_F/ClpP_R cDNA 2 Tif_F/ClpP_R gen. DNA 3 ClpP_F/ ClpX_R cDNA 4 ClpP_F/ClpX_R gen. DNA 5 ClpX_F/ Lon_R cDNA 6 ClpX_F/ Lon_R gen. DNA 7 Lon_F/ HupB_R cDNA 8 Lon_F/HupB_R gen. DNA 9 ClpP_F/ Lon_R cDNA 10 ClpP_F/ Lon_R gen. DNA  Figure 7.5. RT-PCR to determine if clpP and lon are located in an operon. Amplification was performed by RT-PCR using either genomic DNA (positive control) or cDNA as templates. Different primer combinations were applied and the PCR products were loaded on an agarose gel.  118  7.2.5  clpP and lon do not regulate each other The genes encoding the ClpP and Lon proteases were not located in an operon, indicating  that transcription occurred independently. Furthermore, complementation of the clpP gene successfully restored all of the phenotypes, indicating that the phenotypes which arose upon mutation of clpP were not due to a downstream effect on lon. Additionally, the motility phenotypes of a mutant in the gene clpX (PA1802), which is located between the clpP and the lon genes in P. aeruginosa, were examined and no motility defect was observed for that particular gene (data not shown). However, it might be possible that the ClpP and Lon proteases have an influence on each other by regulating one another. Therefore, the observed motilityrelated phenotypes, which are similar between a clpP and lon mutant, could be due to a dysregulation of the other protease. However, the results in Table 7.3 highlighted that lon and clpP are regulated independently from each other since no dysregulation of lon occurred in the clpP mutant (0.9 ± 0.1) and no dysregulation of clpP occurred in the lon mutant (0.8 ± 0.1) as determined by RT-qPCR. Thus the respective phenotypes of these two protease mutants are independent of one another. Table 7.3. Expression level of lon in the clpP mutant and of clpP in the lon mutant. Fold change clp0.9 ± 0.1 lon clpP 7.3  lon0.8 ± 0.1  Discussion This study provided evidence that intracellular proteases other than Lon and AsrA,  specifically PfpI, ClpP and ClpS, play a role in antibiotic resistance and virulence-related properties in P. aeruginosa. Some of the proteases identified here had been already predicted to have an impact on motility in other species or had been characterized regarding other phenotypes in P. aeruginosa. For instance, PfpI was described to play an antimutator role and, furthermore, it provides protection from stress in P. aeruginosa (208). The study by Rodriguez-Rojas and Blazquez (208) demonstrated that a pfpI mutant had a biofilm defect and exhibited an increased 119  spontaneous-mutation rate. Surprisingly, the authors did not find any increase in antibiotic resistance towards diverse antibiotics, even though hypermutator strains are generally linked to antibiotic resistance. However, in the ciprofloxacin resistome screening (Chapter 3, (21)), it was observed that a mutation in pfpI led to an increase in ciprofloxacin resistance, which was further confirmed here. The discrepancies observed between these results and those described by Rodriguez-Rojas and Blazquez (208) could be due to differences in the protocols used to evaluate resistance. Overall, these results reinforce the notion that intracellular proteases participate in the regulation of resistance to antimicrobial agents. The defects in growth, swarming motility and biofilm formation observed for the clpP mutant had been already demonstrated in P. fluorescens. However, in P. aeruginosa, no previous reports showed that ClpP was involved in motility, although ClpP had been demonstrated to participate in the regulation of alginate production (201). Furthermore, the Congo red assay confirmed that this mutant had a lower ability to bind Congo red, which is consistent with the reduction of biofilm formation. Moreover, the clpP mutant demonstrated an epigenetic phenomenon whereby discrete spots of the colony were very highly stained. The slight impairment in swimming and twitching motility observed in this study could be, to some extent, due to the clpP growth defect. However, it seems unlikely that this growth defect would result in a complete abolishment of swarming motility as the strain grows slower but reaches almost the same OD as the wild type in stationary phase. All the defects observed in the clpP mutant could be complemented with the introduction of the wild type copy of the gene. In contrast, the swarming motility defect of the P. fluorescens mutant could not be fully complemented and it was suggested that this defect was due to a deficiency in production of the cyclic lipopeptide masselotide, which P. aeruginosa does not synthesize (43, 184). Furthermore, we could confirm the already observed decrease in ciprofloxacin resistance for the clpP mutant, which highlights once again the importance of intracellular proteases in antibiotic resistance. The lon mutant is now known to exhibit ciprofloxacin susceptibility as well as a swarming and twitching deficiency in the PAO1 background (19, 157), which I could also demonstrate for the PA14 lon mutant. Interestingly, in the PA14 background, the lon mutant showed a strong increase in biofilm formation, whereas in the PAO1 strain background, the lon mutant was biofilm deficient. The observed discrepancy in biofilm formation between the two strain backgrounds is in accordance with previously published data on the relationship between the two surface120  associated behaviours – swarming and biofilms – in PA14 and PAO1. Thus, these two processes are often coregulated in PAO1 mutant strains (189), whereas there is an inverse regulation in the PA14 background (28, 271). The clpP and lon genes are located in the same region of the P. aeruginosa genome and, according to some prediction tools, they might be cotranscribed. However, the fact that the similar phenotypes of the two mutants were due to a downstream effect of the clpP mutation on lon was ruled out. This emphasized that both intracellular proteases independently play a role in antibiotic resistance and virulence-related properties. ClpS, the adaptor protein for the ClpAP protease complex has not been related to virulence properties before, which was demonstrated here for the first time; however, its importance in β-lactam resistance has been previously published and it was shown that a clpS mutant showed an increase in ceftazidime resistance (2). The present study proved its involvement in resistance to several β-lactams including ceftazidime, piperacillin, aztreonam and imipenem resistance. Thus these data suggested that a certain specificity of the adaptor protein was necessary for motility and antibiotic resistance. Strains carrying disruptions in the genes pfpI, clpS, clpP and lon, all of which encode proteins involved in intracellular protease complexes, were demonstrated here to have altered phenotypes in antibiotic susceptibility, swarming motility and biofilm formation. Previous studies identified that the ATP-dependent proteases, Lon and AsrA, had distinct phenotypic properties (19, 121, 157). However, the patterns of changes varied when these 5 individual ATPdependent proteases were mutated suggesting that these changes are independently determined, likely through processing of one or more regulatory factors by each protease. Overall, this reinforces the notion that virulence and antibiotic susceptibility in P. aeruginosa are regulated in a highly coordinated and interwoven manner, and that these 5 proteases are involved in this coordination. Our results further emphasized the importance of the regulatory functions carried out by intracellular proteases in this pathogen, which goes beyond their expected involvement in the regulation of the stress responses. The specific molecular mechanisms of these proteases need to be investigated more in depth, as this would help to explain the regulation of antibiotic resistance and virulence in P. aeruginosa, which might be useful for improving therapeutic strategies.  121  8.  Conclusions and future directions The emerging problem of multi-drug resistance in P. aeruginosa strains has led to the  classification of this pathogen as a “Superbug”. Therefore, it is becoming increasingly challenging to find new efficacious antibiotics and novel therapeutic strategies, an important goal to combat Pseudomonas infections. This thesis provides relevant information about the complexity of antibiotic resistance, virulence and global regulation in this microorganism. More importantly, my work demonstrates the crucial role of intracellular proteases for the coordination of these processes, making them good candidates as new antimicrobial drug targets, in particular the Lon protease. In this section, I will discuss the relevance of the findings presented in this work within the context of tackling the problems encountered during the treatment of infections caused by this pathogen. Initially, I identified genes involved in ciprofloxacin susceptibility and resistance by performing a screen of the comprehensive PA14 Harvard mutant library of P. aeruginosa (148) and demonstrated that the ciprofloxacin resistome of this microorganism is large and diverse. As a result, a total of 35 genes were shown to be involved in intrinsic resistance, whereas 79 genes were involved in mutational resistance. Within the latter group, a fairly high percentage corresponds to low-level resistance markers. Likewise, studies by other researchers investigating the resistomes of P. aeruginosa to aminoglycosides and β-lactams (2, 50, 219) also observed that the mutation of many genes results in small increases in the MIC. Moreover, overlapping analysis of all these screens revealed that some of the identified genes, upon mutation, lead to a change in MIC towards more than one antibiotic class, thereby indicating a potential mutator or global regulatory function. The mechanisms leading to low-level resistance are not yet fully understood, but recent research (including mine) indicates that these mechanisms are far more complex than originally thought. Until recently, most research focused on the study of acquired resistance genes and the emergence of high-level resistance (11, 74, 99, 263), while little attention was attributed to small (twofold) MIC changes that are often not noticed in clinical settings. Nevertheless, an increasing number of studies suggest that these small changes can accumulate and, over time, lead to high-level resistance. For example, it has been demonstrated in vitro that a double mutant in the gyrase genes is more resistant to fluoroquinolones than the corresponding single mutants (139, 178), and the same result was observed in the case of the 122  aminoglycosides (58). I propose that a similar phenomenon regarding fluoroquinolone resistance may happen in clinical settings, increasing the “baseline” MICs over time in a stepwise manner. Therefore, the investigation of those genes involved in low-level resistance to ciprofloxacin in P. aeruginosa is of great value in order to attain a better understanding of the molecular mechanisms implicated in stepwise resistance. Overall, this broad screen has opened up a new window by indicating that ciprofloxacin resistance in this pathogen is very complex. However, a major limitation of transposon mutant library screenings is that they only allow for the identification of non-essential genes involved in altered susceptibility. In contrast, the participation of essential genes cannot be determined as the loss of the gene would not allow for growth, although some of these genes may also be very important for acquiring antibiotic resistance. In order to evaluate the potential role of essential genes, overexpression libraries are a good choice (188), but are not yet available in P. aeruginosa. The results provided by such studies would complement very nicely the results of the ciprofloxacin resistome analysis presented here. The question as to how each identified mutant is involved in altered susceptibility needs to be addressed in follow-up studies. Indeed, it is possible that some of these mutations occur in clinical isolates, which could be determined by sequencing fluoroquinolone resistant clinical isolates. In the mutant library screen, I identified that a mutation in the gene encoding the Lon protease leads to a 4-8 fold increased susceptibility to ciprofloxacin. Furthermore, lon mutants show defects in motility-related properties (swarming, swimming, twitching and biofilm formation) (157). Studies in other bacteria had already shown that the Lon protease was important for fluoroquinolone resistance and virulence; however, there are important differences between species. Based on all these observations, I hypothesized that the Lon protease influences resistance to ciprofloxacin and key virulence determinants of P. aeruginosa by modulating the stability of certain key regulators. My research identified the mechanistic basis for the involvement of the Lon protease in protecting P. aeruginosa from DNA-damage by exposure to ciprofloxacin and UV-light. Indeed, I show here that the ciprofloxacin related DNA-damage in the lon mutant likely could not be repaired as well compared to the wild type and the complemented strain due to a lesser extent of induction of the SOS response genes. Therefore, I wanted to investigate how the Lon protease amplifies the SOS response. I originally thought one option would be accumulation of the cell 123  division inhibitor SulA, which is known to be degraded by Lon. However, sulA overexpression did not have the same effect on ciprofloxacin resistance or the transcriptional regulation of the SOS response as did the lon mutation, albeit that it did confer similar motility and filamentation phenotypes. I concluded that the Lon protease did not amplify the SOS response at the transcriptional level through SulA. Another possibility would be that Lon has an impact on the SOS response through RecA, since Lon could inhibit RecA repressors; leading to the autoamplification of RecA. Through protein expression analysis I demonstrated that, while the presence of RecA is highly induced upon DNA-damage in the wild type and the complemented strain, no significant changes can be observed in the lon mutant. In summary, I could demonstrate that the Lon protease of P. aeruginosa influences the SOS response at the protein level through regulation of RecA levels, and proposed a model for how this might occur. My work highlights the major regulatory role that Lon protease plays in the SOS response of P. aeruginosa and consequently, in resistance to DNA-damaging agents such as fluoroquinolones and UV-irradiation. During my studies I showed that the Lon protease impacts on global regulation at the transcriptional and post-translational levels, and I identified a Lon-regulated transcriptional hierarchy. I also described new phenotypes for a lon mutant, including defects in anaerobic growth, biofilm formation, metabolism and cytotoxicity. These phenotypes could be partially explained by the microarray data in which genes involved in the denitrification pathway and genes involved in Type III secretion were dysregulated in the mutant, which might have respectively participated in the anaerobic growth and cytotoxicity defects. While I showed that the global regulation at the transcriptional level compromised more than 200 genes; at the proteomic level I was only able to identify with any degree of confidence a few potential Lon targets, including GroEL, KatA, a probable oxidoreductase and Hfq amongst others. The fact that the number of identified proteins was so small might indicate that Lon only had a moderate impact on the proteome. However, the data was likely skewed towards the observation of very abundant proteins, whereas differences in low abundant proteins would be less evident. This is a common limitation of the SILAC methodology used. Nonetheless, I did identify some potential targets of Lon, which should be the subject of follow-up studies aimed at determining whether any of these proteins is directly cleaved or degraded by Lon.  124  The microarray data and the observed motility defects of the lon mutants triggered an investigation of the importance of the Lon protease for virulence of P. aeruginosa. In collaboration with Dr. Joerg Overhage and Dr. Roger Levesque, we could demonstrate that the Lon protease was indeed important for full virulence in this microbe. Thus, lon mutants showed a weaker severity of infection in a lettuce leaf model and decreased competitive ability in a rat model of chronic infection. The latter is particularly relevant as it reflects some of the conditions present during an infection in CF patients. In addition, fewer amoeba were needed to kill the lon mutants than the wild type, indicating that lon mutants were less virulent towards amoeba. Furthermore, data from experiments in which the environment of the CF lung was mimicked in vitro by adding mucin to the growth medium indicated that mucin, found in the CF lung, promoted ciprofloxacin resistance and surfing motility, and that the Lon protease influences these processes. The participation of the ATP-dependent Lon protease in antibiotic resistance and virulence suggests that the design of an inhibitor for this protease would be an attractive new therapeutic option to treat P. aeruginosa infection. Thus, the inhibition of Lon would increase susceptibility to ciprofloxacin and, at the same time, attenuate the virulence of the bacterium, which would be highly beneficial in clinical settings. Further studies should be directed towards identifying inhibitors for these targets and investigating if MG262 and β-lactones, which inhibit the Lon and ClpP proteases from S. enterica and S. aureus, respectively (17, 72), might also be successful inhibitors in P. aeruginosa. Apart from the Lon protease, we observed that other intracellular proteases, some of which are known to work in concert with Lon, are also involved in antibiotic resistance and virulence-related processes. Thus, we demonstrated that PfpI, ClpP and ClpS are involved in antibiotic resistance, motility (swarming, swimming and twitching) and biofilm formation. This suggests that other proteases most likely participate in the virulence of P. aeruginosa and, therefore, in vivo studies involving these protease mutants might be proven useful to obtain more information on the relevance of protease-mediated regulation during infection. Additionally, further research is needed to fully understand the role played by proteases inside the cell, as it has now become clear that they are not only involved in the stress response, as originally thought, but also in global regulation. Also, like Lon, these proteases might be interesting novel antimicrobial targets. Furthermore, creation of a double mutant (clpP and lon) would be 125  beneficial in asking the question if the ciprofloxacin and virulence-related phenotypes can be synergistic when both genes are deleted. Overall, my studies i) identified the ciprofloxacin resistome, ii) helped in answering the question as to how the Lon protease is involved in ciprofloxacin resistance, and iii) demonstrated that Lon has an impact on virulence, indicating that this protease plays a significant role during the infectious process. Other proteases identified in this study further highlight that proteases are not only involved in the stress responses, but play particular roles in antibiotic resistance and virulence-related properties. Therefore I propose here that the development of a drug that inhibits the Lon protease would be beneficial to reduce virulence and antibiotic resistance of P. aeruginosa. 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Microbiol Mol Biol Rev 61:533-616.  153  Appendix Supplementary Table S A.1 (Chapter 4): Complete list of dysregulated genes in the microarray under sub-inhibitory concentrations of ciprofloxacin in the lon mutant compared to the wild type PAO1 under sub-inhibitory concentrations. PA Gene Fold Pnumber Name change ebayes Gene Description PA0040 -2.18 0.00 conserved hypothetical protein PA0066 -2.28 0.00 conserved hypothetical protein PA0069 -5.46 0.00 conserved hypothetical protein PA0085 2.57 0.00 conserved hypothetical protein PA0171 2.01 0.00 hypothetical protein PA0253 -2.66 0.01 probable transcriptional regulator PA0266 gabT 2.30 0.00 4-aminobutyrate aminotransferase PA0277 4.05 0.00 conserved hypothetical protein PA0386 -2.03 0.00 probable oxidase PA0394 -2.01 0.00 conserved hypothetical protein PA0426 mexB 3.30 0.01 RND multidrug efflux transporter MexB PA0447 gcdH 2.42 0.02 glutaryl-CoA dehydrogenase PA0577 dnaG -2.10 0.00 DNA primase PA0604 3.03 0.01 probable binding protein component of ABC transporter PA0606 2.13 0.01 probable permease of ABC transporter PA0610 prtN -12.65 0.00 transcriptional regulator PrtN PA0611 prtR -3.00 0.00 transcriptional regulator PrtR PA0612 -12.87 0.00 hypothetical protein PA0613 -6.51 0.00 hypothetical protein PA0614 -8.25 0.00 hypothetical protein PA0615 -9.09 0.00 hypothetical protein PA0616 -6.57 0.00 hypothetical protein PA0617 -7.24 0.00 probable bacteriophage protein PA0618 -2.21 0.00 probable bacteriophage protein PA0619 -3.50 0.00 probable bacteriophage protein PA0620 -9.93 0.00 probable bacteriophage protein PA0621 -3.07 0.00 conserved hypothetical protein 154  PA number PA0622 PA0623 PA0624 PA0625 PA0626 PA0627 PA0628 PA0629 PA0630 PA0631 PA0632 PA0633 PA0634 PA0635 PA0636 PA0637 PA0638 PA0640 PA0641 PA0642 PA0643 PA0644 PA0645 PA0646 PA0648 PA0649 PA0650 PA0651 PA0654 PA0669  Gene Name  trpG trpD trpC speD  Fold Pchange ebayes -5.10 0.00 -2.06 0.00 -7.63 0.00 -5.61 0.00 -7.32 0.00 -6.30 0.00 -6.95 0.00 -7.79 0.00 -7.20 0.00 -6.46 0.00 -9.07 0.00 -5.46 0.00 -8.14 0.00 -6.25 0.01 -9.30 0.00 -6.93 0.00 -7.61 0.00 -4.05 0.00 -8.94 0.00 -7.71 0.00 -6.42 0.00 -7.98 0.00 -7.42 0.00 -7.25 0.00 -6.98 0.00 -3.18 0.00 -3.46 0.00 -2.43 0.00 -2.11 0.00 -3.08 0.00  Gene Description probable bacteriophage protein probable bacteriophage protein hypothetical protein hypothetical protein hypothetical protein conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein conserved hypothetical protein probable bacteriophage protein probable bacteriophage protein probable bacteriophage protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein anthranilate synthase component II anthranilate phosphoribosyltransferase indole-3-glycerol-phosphate synthase S-adenosylmethionine decarboxylase proenzyme probable DNA polymerase alpha chain 155  PA number PA0670 PA0671 PA0782 PA0783 PA0789 PA0807 PA0808 PA0835 PA0836 PA0855 PA0865 PA0870 PA0871 PA0872 PA0906 PA0907 PA0908 PA0910 PA0911 PA0922 PA0958 PA0962 PA0985 PA1049 PA1069 PA1071 PA1072 PA1074 PA1076 PA1151  Gene Name  putA putP  pta  hpd phhC phhB phhA  oprD  pdxH braF braE braC imm2  Fold Pchange ebayes -5.31 0.00 -5.59 0.00 3.35 0.00 4.74 0.00 2.21 0.00 -2.81 0.00 -3.75 0.00 3.80 0.01 3.76 0.00 2.02 0.00 2.80 0.00 3.11 0.00 3.67 0.00 2.84 0.00 -2.26 0.00 -4.28 0.00 -2.82 0.00 -3.82 0.00 -3.47 0.00 -2.41 0.00 4.42 0.00 2.57 0.01 -4.64 0.00 2.16 0.00 2.60 0.00 2.08 0.00 2.12 0.00 3.38 0.00 4.54 0.00 -2.54 0.00  Gene Description hypothetical protein hypothetical protein proline dehydrogenase PutA sodium/proline symporter PutP probable amino acid permease conserved hypothetical protein hypothetical protein phosphate acetyltransferase probable acetate kinase hypothetical protein 4-hydroxyphenylpyruvate dioxygenase aromatic amino acid aminotransferase pterin-4-alpha-carbinolamine dehydratase phenylalanine-4-hydroxylase probable transcriptional regulator hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein outer membrane porin protein OprD precursor probable dna-binding stress protein probable colicin-like toxin pyridoxine 5-phosphate oxidase hypothetical protein branched-chain amino acid transport protein BraF branched-chain amino acid transport protein BraE branched-chain amino acid transport protein BraC hypothetical protein pyocin S2 immunity protein 156  PA number PA1152 PA1196 PA1288 PA1337 PA1338 PA1339 PA1340 PA1341 PA1342 PA1414 PA1418 PA1419 PA1420 PA1421 PA1498 PA1546 PA1557 PA1647 PA1656 PA1657 PA1658 PA1659 PA1665 PA1673 PA1746 PA1865 PA1866 PA1892 PA1893 PA1894  Gene Name  ansB ggt  gbuA pykF hemN  Fold Pchange ebayes -2.09 0.01 2.41 0.01 5.09 0.00 2.68 0.00 2.76 0.00 2.79 0.00 2.24 0.00 2.21 0.00 5.42 0.00 2.10 0.02 2.09 0.00 2.35 0.00 2.39 0.00 3.75 0.00 2.52 0.01 2.75 0.00 3.36 0.01 2.03 0.00 4.40 0.00 11.03 0.00 3.45 0.00 2.60 0.00 2.06 0.00 3.29 0.00 2.19 0.00 -2.72 0.01 -3.14 0.00 2.33 0.00 2.40 0.00 3.10 0.00  Gene Description hypothetical protein probable transcriptional regulator probable outer membrane protein glutaminase-asparaginase gamma-glutamyltranspeptidase precursor probable ATP-binding component of ABC transporter probable permease of ABC transporter probable permease of ABC transporter probable binding protein component of ABC transporter hypothetical protein probable sodium:solute symport protein probable transporter hypothetical protein agmatinase pyruvate kinase I oxygen-independent coproporphyrinogen III oxidase probable cytochrome oxidase subunit (cbb3-type) probable sulfate transporter hypothetical protein conserved hypothetical protein conserved hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein 157  PA number PA1895 PA1896 PA1897 PA1948 PA2008 PA2009 PA2026 PA2040 PA2041 PA2064 PA2080 PA2119 PA2126 PA2127 PA2193 PA2194 PA2195 PA2227 PA2252 PA2288 PA2321 PA2501 PA2539 PA2624 PA2753 PA2754 PA2760 PA2840 PA2886 PA2951  Gene Name  rbsC fahA hmgA  pcoB kynU  hcnA hcnB hcnC  idh  etfA  Fold Pchange ebayes 2.70 0.00 2.83 0.00 2.48 0.00 2.13 0.00 3.00 0.00 8.12 0.00 3.43 0.00 2.44 0.00 2.09 0.00 -2.00 0.03 2.04 0.00 2.81 0.00 2.18 0.01 3.84 0.00 5.33 0.00 3.21 0.00 2.16 0.00 2.10 0.02 2.78 0.00 -2.14 0.00 -2.04 0.00 3.02 0.00 2.20 0.00 2.24 0.01 2.59 0.01 2.25 0.00 3.63 0.00 -2.41 0.03 2.03 0.03 2.00 0.00  Gene Description hypothetical protein hypothetical protein hypothetical protein membrane protein component of ABC ribose transporter fumarylacetoacetase homogentisate 1,2-dioxygenase conserved hypothetical protein probable glutamine synthetase probable amino acid permease copper resistance protein B precursor hypothetical protein alcohol dehydrogenase (Zn-dependent) conserved hypothetical protein conserved hypothetical protein hydrogen cyanide synthase HcnA hydrogen cyanide synthase HcnB hydrogen cyanide synthase HcnC probable transcriptional regulator probable AGCS sodium/alanine/glycine symporter hypothetical protein gluconokinase hypothetical protein conserved hypothetical protein isocitrate dehydrogenase hypothetical protein conserved hypothetical protein probable outer membrane protein probable ATP-dependent RNA helicase hypothetical protein electron transfer flavoprotein alpha-subunit 158  PA number PA2952 PA3007 PA3021 PA3054 PA3068 PA3126 PA3221 PA3234 PA3278 PA3309 PA3326 PA3337 PA3347 PA3356 PA3413 PA3414 PA3458 PA3562 PA3613 PA3616 PA3661 PA3837 PA3839 PA3850 PA3859 PA3866 PA3876 PA3877 PA3880 PA3905  Gene Name etfB lexA  gdhB ibpA csaA  rfaD  narK2 narK1  Fold Pchange ebayes 2.52 0.00 -2.20 0.01 3.95 0.00 2.04 0.00 2.10 0.00 -3.97 0.00 2.20 0.00 2.36 0.00 3.24 0.02 5.48 0.01 2.41 0.00 3.13 0.01 2.26 0.00 3.26 0.00 -2.39 0.00 -2.69 0.00 2.71 0.01 2.08 0.00 6.09 0.00 -2.18 0.00 2.19 0.00 2.13 0.00 3.07 0.03 2.27 0.00 2.25 0.00 -8.25 0.00 3.01 0.03 4.29 0.00 2.51 0.01 2.37 0.00  Gene Description electron transfer flavoprotein beta-subunit repressor protein LexA hypothetical protein hypothetical protein conserved hypothetical protein heat-shock protein IbpA CsaA protein probable sodium:solute symporter hypothetical protein conserved hypothetical protein probable Clp-family ATP-dependent protease ADP-L-glycero-D-mannoheptose 6-epimerase hypothetical protein conserved hypothetical protein conserved hypothetical protein hypothetical protein probable transcriptional regulator probable phosphotransferase system enzyme I hypothetical protein conserved hypothetical protein hypothetical protein probable permease of ABC transporter probable sodium:sulfate symporter hypothetical protein probable carboxylesterase pyocin protein nitrite extrusion protein 2 nitrite extrusion protein 1 conserved hypothetical protein hypothetical protein 159  PA number PA3906 PA3915 PA3919 PA4022 PA4220 PA4221 PA4224 PA4225 PA4226 PA4230 PA4328 PA4329 PA4348 PA4463 PA4571 PA4579 PA4587 PA4605 PA4606 PA4610 PA4611 PA4625 PA4761 PA4770 PA4771 PA4772 PA4846 PA5025 PA5027 PA5030  Gene Name moaB1  fptA pchG pchF pchE pchB pykA  ccpR  dnaK lldP lldD aroQ1 metY  Fold Pchange ebayes 2.24 0.00 2.71 0.01 2.16 0.01 2.59 0.00 -3.04 0.00 -2.14 0.01 -2.07 0.00 -4.90 0.00 -2.27 0.00 -2.37 0.00 2.73 0.02 2.63 0.00 2.13 0.00 2.08 0.01 3.79 0.01 2.32 0.01 3.42 0.01 2.63 0.00 2.51 0.00 2.04 0.01 6.23 0.00 2.55 0.00 -2.11 0.01 2.94 0.00 2.19 0.00 2.08 0.00 -2.10 0.01 2.52 0.00 3.67 0.01 -2.58 0.00  Gene Description hypothetical protein molybdopterin biosynthetic protein B1 conserved hypothetical protein probable aldehyde dehydrogenase hypothetical protein Fe(III)-pyochelin receptor precursor hypothetical protein pyochelin synthetase dihydroaeruginoic acid synthetase salicylate biosynthesis protein PchB hypothetical protein pyruvate kinase II conserved hypothetical protein conserved hypothetical protein probable cytochrome c hypothetical protein cytochrome c551 peroxidase precursor conserved hypothetical protein conserved hypothetical protein hypothetical protein hypothetical protein hypothetical protein DnaK protein L-lactate permease L-lactate dehydrogenase probable ferredoxin 3-dehydroquinate dehydratase homocysteine synthase hypothetical protein probable MFS transporter 160  PA number PA5053 PA5054 PA5112 PA5171 PA5180 PA5266 PA5296 PA5303 PA5304 PA5312 PA5336 PA5415 PA5436 PA5463 PA5464 PA5470 PA5471 PA5485 PA5486 PA5503 PA5504 PA5510 PA5561 PA5564  Gene Name hslV hslU estA arcA  rep dadA gmk glyA1  atpI gidB  Fold Pchange ebayes -4.99 0.00 -2.30 0.00 2.25 0.00 2.55 0.03 -2.36 0.00 2.07 0.03 -2.03 0.00 2.41 0.00 2.84 0.00 2.03 0.00 -2.13 0.00 2.05 0.03 2.64 0.02 -2.77 0.00 -2.13 0.00 -3.61 0.00 -2.66 0.00 -2.06 0.00 -2.06 0.00 -2.04 0.00 -2.31 0.00 2.03 0.01 -2.76 0.00 -2.11 0.00  Gene Description heat shock protein HslV heat shock protein HslU esterase EstA arginine deiminase conserved hypothetical protein conserved hypothetical protein ATP-dependent DNA helicase Rep conserved hypothetical protein D-amino acid dehydrogenase, small subunit probable aldehyde dehydrogenase guanylate kinase serine hydroxymethyltransferase probable. biotin carboxylase subunit of a transcarboxylase hypothetical protein hypothetical protein probable peptide chain release factor hypothetical protein conserved hypothetical protein conserved hypothetical protein probable ATP-binding component of ABC transporter probable permease of ABC transporter probable transporter ATP synthase protein I glucose inhibited division protein B  161  Supplementary Table S A.2A (Chapter 5): Complete list of dysregulated genes in the microarray under mid-logarithmic phase in the lon mutant compared to the wild type PAO1 PA01 Gene Fold PName Name change ebayes Gene Description PA0047 2.01 0.02 hypothetical protein PA0200 -3.45 0.00 hypothetical protein PA0277 2.49 0.00 conserved hypothetical protein PA0425 mexA 2.35 0.00 Resistance-Nodulation-Cell Division (RND) multidrug efflux membrane fusion protein MexA PA0426 mexB 2.51 0.00 Resistance-Nodulation-Cell Division (RND) multidrug efflux transporter MexB PA0427 oprM 3.37 0.00 Major intrinsic multiple antibiotic resistance efflux outer membrane protein OprM precursor PA0506 -2.00 0.00 probable acyl-CoA dehydrogenase PA0509 nirN -3.22 0.01 probable c-type cytochrome PA0510 -5.41 0.01 probable uroporphyrin-III c-methyltransferase PA0511 nirJ -7.82 0.01 heme d1 biosynthesis protein NirJ PA0512 -6.01 0.01 conserved hypothetical protein PA0513 -5.97 0.01 probable transcriptional regulator PA0514 nirL -5.98 0.00 heme d1 biosynthesis protein NirL PA0515 -6.11 0.00 probable transcriptional regulator PA0516 nirF -6.43 0.00 heme d1 biosynthesis protein NirF PA0517 nirC -4.85 0.00 probable c-type cytochrome precursor PA0518 nirM -6.59 0.00 cytochrome c-551 precursor PA0519 nirS -6.08 0.01 nitrite reductase precursor PA0520 nirQ -5.88 0.00 regulatory protein NirQ PA0522 -2.47 0.03 hypothetical protein PA0523 norC -33.60 0.00 nitric-oxide reductase subunit C PA0524 norB -5.76 0.00 nitric-oxide reductase subunit B PA0525 -15.92 0.00 probable dinitrification protein NorD PA0588 -2.13 0.00 conserved hypothetical protein PA0603 3.25 0.02 probable ATP-binding component of ABC transporter PA0604 4.02 0.07 probable binding protein component of ABC transporter 162  PA01 Name PA0606 PA0613 PA0615 PA0616 PA0617 PA0619 PA0620 PA0622 PA0623 PA0624 PA0625 PA0626 PA0627 PA0630 PA0631 PA0633 PA0634 PA0635 PA0636 PA0637 PA0639 PA0640 PA0713 PA0789 PA0958 PA0999 PA1202 PA1561  Gene Name  oprD pqsD aer  Fold change 2.36 2.16 2.30 2.27 2.88 2.88 2.16 4.37 4.16 3.33 2.23 2.51 2.20 2.24 2.27 3.16 2.38 3.64 2.23 2.61 2.07 2.08 -2.46 2.39 -2.52 -2.17 2.99 -3.44  Pebayes 0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.01 0.01 0.00 0.00  Gene Description probable permease of ABC transporter hypothetical protein hypothetical protein hypothetical protein probable bacteriophage protein probable bacteriophage protein probable bacteriophage protein probable bacteriophage protein probable bacteriophage protein hypothetical protein hypothetical protein hypothetical protein conserved hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein conserved hypothetical protein conserved hypothetical protein probable bacteriophage protein hypothetical protein probable amino acid permease Basic amino acid, basic peptide and imipenem outer membrane porin OprD precursor 3-oxoacyl-[acyl-carrier-protein] synthase III probable hydrolase aerotaxis receptor Aer 163  PA01 Name PA1803 PA2007 PA2008 PA2193 PA2252 PA2253 PA2259 PA2321 PA2322 PA2404 PA2405 PA2409 PA2662 PA2663 PA2664 PA2706 PA2707 PA3042 PA3162 PA3181 PA3182 PA3190 PA3192 PA3194 PA3195 PA3363 PA3364 PA3365  Gene Name lon maiA fahA hcnA ansA ptxS  fhp  rpsA pgl gltR edd gapA amiR amiC  Fold change -2.44 2.11 2.56 2.58 3.10 2.24 -3.45 -2.03 -2.11 2.36 2.46 2.44 -5.98 -8.65 -13.47 -2.10 -2.39 -2.50 2.12 -3.68 -3.54 -2.27 -2.76 -2.17 -3.40 -2.89 -3.11 -4.55  Pebayes 0.01 0.07 0.08 0.01 0.01 0.01 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.06 0.02 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00  Gene Description Lon protease maleylacetoacetate isomerase fumarylacetoacetase hydrogen cyanide synthase HcnA probable AGCS sodium/alanine/glycine symporter L-asparaginase I transcriptional regulator PtxS gluconokinase gluconate permease hypothetical protein hypothetical protein probable permease of ABC transporter conserved hypothetical protein hypothetical protein flavohemoprotein hypothetical protein hypothetical protein hypothetical protein 30S ribosomal protein S1 2-keto-3-deoxy-6-phosphogluconate aldolase 6-phosphogluconolactonase probable binding protein component of ABC sugar transporter two-component response regulator GltR phosphogluconate dehydratase glyceraldehyde 3-phosphate dehydrogenase aliphatic amidase regulator aliphatic amidase expression-regulating protein probable chaperone 164  PA01 Name PA3366 PA3391 PA3392 PA3393 PA3394 PA3395 PA3396 PA3430 PA3432 PA3437 PA3439 PA3531 PA3533 PA3584 PA3813 PA3815 PA3875 PA3876 PA3877 PA3914 PA3915 PA3919 PA3920 PA3972 PA4218 PA4220 PA4221 PA4222  Gene Name amiE nosR nosZ nosD nosF nosY nosL  folX bfrB glpD iscU narG narK2 narK1 moeA1 moaB1  fptA  Fold change -6.71 -8.27 -31.22 -10.78 -4.48 -4.36 -2.98 -2.21 -2.37 -2.11 -2.13 -3.73 -2.80 -2.29 -2.14 -2.70 -4.22 -5.32 -2.05 -2.42 -2.95 -3.66 -2.58 -2.63 -2.27 -2.00 -2.01 -2.39  Pebayes 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.02 0.00 0.00 0.01 0.00 0.00 0.03 0.00 0.01 0.03 0.04 0.01 0.01 0.00 0.01 0.01 0.00 0.02 0.00 0.00  Gene Description aliphatic amidase regulatory protein NosR nitrous-oxide reductase precursor NosD protein NosF protein NosY protein NosL protein probable aldolase hypothetical protein probable short-chain dehydrogenase d-erythro-7,8-dihydroneopterin triphosphate epimerase bacterioferritin conserved hypothetical protein glycerol-3-phosphate dehydrogenase probable iron-binding protein IscU conserved hypothetical protein respiratory nitrate reductase alpha chain nitrite extrusion protein 2 nitrite extrusion protein 1 molybdenum cofactor biosynthetic protein A1 molybdopterin biosynthetic protein B1 conserved hypothetical protein probable metal transporting P-type ATPase probable acyl-CoA dehydrogenase probable transporter hypothetical protein Fe(III)-pyochelin outer membrane receptor precursor probable ATP-binding component of ABC transporter 165  PA01 Name PA4223 PA4224 PA4225 PA4315 PA4358 PA4359 PA4366 PA4370 PA4371 PA4463 PA4523 PA4525 PA4615 PA4658 PA4660 PA4683 PA4918 PA4919 PA4920 PA4921 PA5015 PA5023 PA5041 PA5042 PA5061 PA5172 PA5173 PA5306  Gene Name pchG pchF mvaT  sodB icmP  pilA  phr  pncB1 nadE aceE pilP pilO arcB arcC  Fold change -2.63 -2.48 -2.14 -2.10 2.45 3.21 -2.22 2.76 3.47 -2.32 -2.59 -2.21 -2.17 -2.70 -2.10 -2.20 -2.14 -2.32 -3.97 -2.54 -2.10 -2.37 -2.06 -2.28 -2.23 -2.36 -5.34 -2.09  Pebayes 0.00 0.00 0.00 0.00 0.00 0.03 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.01 0.04 0.00 0.03 0.01 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.01 0.00 0.00  Gene Description probable ATP-binding component of ABC transporter pyochelin biosynthetic protein PchG pyochelin synthetase transcriptional regulator MvaT, P16 subunit probable ferrous iron transport protein conserved hypothetical protein superoxide dismutase Insulin-cleaving metalloproteinase outer membrane protein precursor hypothetical protein conserved hypothetical protein hypothetical protein type 4 fimbrial precursor PilA probable oxidoreductase hypothetical protein deoxyribodipyrimidine photolyase hypothetical protein hypothetical protein nicotinate phosphoribosyltransferase NH3-dependent NAD synthetase hypothetical protein pyruvate dehydrogenase conserved hypothetical protein type 4 fimbrial biogenesis protein PilP type 4 fimbrial biogenesis protein PilO conserved hypothetical protein ornithine carbamoyltransferase, catabolic carbamate kinase conserved hypothetical protein 166  PA01 Name PA5351 PA5435 PA5436 PA5446  Gene Name rubA1  Fold change 2.01 -2.56 -2.28 -4.94  Pebayes 0.04 0.00 0.03 0.00  Gene Description Rubredoxin 1 probable transcarboxylase subunit probable biotin carboxylase subunit of a transcarboxylase hypothetical protein  Supplementary Table S A.2B (Chapter 5): Complete list of dysregulated genes in the microarray under stationary phase in the lon mutant compared to the wild type PAO1 Gene Fold PPA number Name change ebayes Gene description PA0044 exoT -2.16 0.00 exoenzyme T PA0048 2.09 0.00 probable transcriptional regulator PA0049 2.52 0.00 hypothetical protein PA0050 2.25 0.00 hypothetical protein PA0099 2.55 0.01 hypothetical protein PA0112 -2.09 0.04 hypothetical protein PA0131 2.07 0.01 hypothetical protein PA0175 2.29 0.00 probable chemotaxis protein methyltransferase PA0227 -38.04 0.00 probable CoA transferase, subunit B PA0243 -2.21 0.03 probable transcriptional regulator PA0244 2.64 0.01 hypothetical protein PA0323 -2.55 0.01 probable binding protein component of ABC transporter PA0325 -2.18 0.03 probable permease of ABC transporter PA0329 2.06 0.00 conserved hypothetical protein PA0425 mexA 2.73 0.00 Resistance-Nodulation-Cell Division (RND) multidrug efflux membrane fusion protein MexA PA0426 mexB 3.62 0.00 Resistance-Nodulation-Cell Division (RND) multidrug efflux transporter MexB PA0427 oprM 2.55 0.00 Major intrinsic multiple antibiotic resistance efflux outer membrane protein OprM precursor PA0434 -2.37 0.02 hypothetical protein 167  PA number PA0511 PA0512 PA0513 PA0514 PA0515 PA0516 PA0517 PA0518 PA0519 PA0521 PA0525 PA0586 PA0612 PA0613 PA0614 PA0616 PA0617 PA0619 PA0621 PA0622 PA0623 PA0624 PA0625 PA0626 PA0627 PA0629 PA0630 PA0631  Gene Name nirJ  nirL nirF nirC nirM nirS  Fold Pchange ebayes 3.20 0.00 3.33 0.00 3.43 0.00 3.33 0.00 6.42 0.00 2.92 0.00 3.05 0.00 11.51 0.00 7.43 0.00 3.95 0.00 5.17 0.00 2.41 0.03 3.05 0.00 2.30 0.00 2.01 0.00 2.17 0.00 2.99 0.00 2.27 0.00 2.15 0.00 2.58 0.00 3.49 0.00 3.71 0.00 3.05 0.00 2.17 0.00 2.93 0.00 3.03 0.00 3.32 0.00 2.46 0.00  Gene description heme d1 biosynthesis protein NirJ conserved hypothetical protein probable transcriptional regulator heme d1 biosynthesis protein NirL probable transcriptional regulator heme d1 biosynthesis protein NirF probable c-type cytochrome precursor cytochrome c-551 precursor nitrite reductase precursor probable cytochrome c oxidase subunit probable dinitrification protein NorD conserved hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein probable bacteriophage protein probable bacteriophage protein conserved hypothetical protein probable bacteriophage protein probable bacteriophage protein hypothetical protein hypothetical protein hypothetical protein conserved hypothetical protein conserved hypothetical protein hypothetical protein hypothetical protein 168  PA number PA0633 PA0634 PA0636 PA0637 PA0638 PA0639 PA0646 PA0647 PA0648 PA0685 PA0745 PA0782 PA0783 PA0789 PA0852 PA0911 PA0997 PA0998 PA1000 PA1001 PA1078 PA1079 PA1109 PA1142 PA1168 PA1202 PA1217 PA1273  Gene Name  putA putP cbpD pqsB pqsC pqsE phnA flgC flgD  cobB  Fold Pchange ebayes 2.09 0.00 2.02 0.00 2.64 0.00 3.14 0.00 2.66 0.00 2.40 0.00 2.12 0.01 2.54 0.00 2.17 0.00 -2.45 0.02 2.22 0.00 -7.59 0.00 -4.37 0.00 2.13 0.01 2.32 0.04 2.35 0.00 -2.73 0.00 -2.90 0.00 -2.73 0.02 -2.89 0.00 -2.33 0.00 -2.26 0.00 -2.16 0.03 4.37 0.00 2.11 0.05 2.79 0.00 -2.14 0.03 2.09 0.01  Gene description hypothetical protein hypothetical protein hypothetical protein conserved hypothetical protein probable bacteriophage protein conserved hypothetical protein hypothetical protein hypothetical protein hypothetical protein probable type II secretion system protein probable enoyl-CoA hydratase/isomerase proline dehydrogenase PutA sodium/proline symporter PutP probable amino acid permease chitin-binding protein CbpD precursor hypothetical protein Homologous to beta-keto-acyl-acyl-carrier protein synthase Homologous to beta-keto-acyl-acyl-carrier protein synthase Quinolone signal response protein anthranilate synthase component I flagellar basal-body rod protein FlgC flagellar basal-body rod modification protein FlgD probable transcriptional regulator probable transcriptional regulator hypothetical protein probable hydrolase probable 2-isopropylmalate synthase cobyrinic acid a,c-diamide synthase 169  PA number PA1318 PA1319 PA1424 PA1504 PA1565 PA1577 PA1646 PA1696 PA1704 PA1705 PA1708 PA1719 PA1720 PA1721 PA1723 PA1738 PA1803 PA1859 PA1893 PA1999 PA2024 PA2041 PA2065 PA2156 PA2211 PA2249 PA2274 PA2276  Gene Name cyoB cyoC  pscO pcrR pcrG popB pscF pscG pscH pscJ lon  pcoA  bkdB  Fold Pchange ebayes -2.03 0.04 -2.08 0.04 -7.66 0.00 -2.87 0.01 2.01 0.01 -7.40 0.00 3.19 0.00 -2.10 0.04 -14.92 0.00 -3.61 0.00 -2.62 0.00 -3.00 0.00 -2.31 0.00 -2.04 0.04 -2.57 0.01 -2.08 0.01 -5.18 0.00 -2.51 0.02 -4.16 0.02 2.49 0.04 2.19 0.03 -2.11 0.03 3.38 0.00 2.10 0.03 3.23 0.01 3.59 0.03 -2.10 0.04 -13.78 0.00  Gene description cytochrome o ubiquinol oxidase subunit I cytochrome o ubiquinol oxidase subunit III hypothetical protein probable transcriptional regulator probable oxidoreductase hypothetical protein probable chemotaxis transducer translocation protein in type III secretion transcriptional regulator protein PcrR regulator in type III secretion translocator protein PopB type III export protein PscF type III export protein PscG type III export protein PscH type III export protein PscJ probable transcriptional regulator Lon protease probable transcriptional regulator hypothetical protein probable CoA transferase, subunit A probable ring-cleaving dioxygenase probable amino acid permease copper resistance protein A precursor conserved hypothetical protein conserved hypothetical protein branched-chain alpha-keto acid dehydrogenase (lipoamide component) hypothetical protein probable transcriptional regulator 170  PA number PA2302 PA2311 PA2331 PA2359 PA2377 PA2389 PA2428 PA2443 PA2445 PA2512 PA2532 PA2552 PA2553 PA2555 PA2564 PA2590 PA2762 PA2780 PA2781 PA3149 PA3191 PA3201 PA3221 PA3234 PA3235 PA3236 PA3268 PA3332  Gene Name  sdaA gcvP2 antA tpx  wbpH  csaA  Fold Pchange ebayes 2.09 0.00 -2.35 0.02 2.27 0.00 -6.82 0.00 -2.97 0.01 -4.69 0.00 2.71 0.01 2.33 0.00 2.05 0.01 2.67 0.01 2.46 0.00 2.78 0.00 2.08 0.00 2.44 0.04 3.36 0.00 2.31 0.02 3.26 0.00 2.25 0.00 3.09 0.00 -2.39 0.03 -3.21 0.01 -2.49 0.00 2.61 0.00 2.80 0.01 2.40 0.00 3.09 0.00 -2.25 0.03 2.15 0.00  Gene description probable non-ribosomal peptide synthetase hypothetical protein hypothetical protein probable transcriptional regulator hypothetical protein conserved hypothetical protein hypothetical protein L-serine dehydratase glycine cleavage system protein P2 anthranilate dioxygenase large subunit thiol peroxidase probable acyl-CoA dehydrogenase probable acyl-CoA thiolase probable AMP-binding enzyme hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein probable glycosyltransferase WbpH probable two-component sensor conserved hypothetical protein CsaA protein probable sodium:solute symporter conserved hypothetical protein probable glycine betaine-binding protein precursor probable TonB-dependent receptor conserved hypothetical protein 171  PA number PA3361 PA3370 PA3391 PA3392 PA3393 PA3394 PA3395 PA3396 PA3421 PA3432 PA3436 PA3452 PA3502 PA3520 PA3586 PA3641 PA3691 PA3719 PA3721 PA3825 PA3841 PA3899 PA3900 PA3911 PA3922 PA3923 PA3957 PA4022  Gene Name lecB nosR nosZ nosD nosF nosY nosL  mqoA  exoS  Fold Pchange ebayes 2.18 0.01 -2.76 0.00 2.02 0.01 11.39 0.00 5.58 0.00 2.05 0.00 2.00 0.00 2.34 0.00 -2.82 0.01 -2.57 0.01 -7.37 0.00 -2.01 0.00 -2.07 0.04 3.05 0.04 -3.27 0.01 -2.21 0.00 2.35 0.00 7.62 0.01 4.07 0.01 -5.40 0.00 -2.39 0.00 -3.67 0.00 -2.01 0.02 2.25 0.01 3.81 0.00 2.43 0.02 2.10 0.02 2.36 0.00  Gene description fucose-binding lectin PA-IIL hypothetical protein regulatory protein NosR nitrous-oxide reductase precursor NosD protein NosF protein NosY protein NosL protein conserved hypothetical protein hypothetical protein hypothetical protein malate:quinone oxidoreductase hypothetical protein hypothetical protein probable hydrolase probable amino acid permease hypothetical protein hypothetical protein probable transcriptional regulator hypothetical protein exoenzyme S probable sigma-70 factor, ECF subfamily probable transmembrane sensor conserved hypothetical protein conserved hypothetical protein hypothetical protein probable short-chain dehydrogenase probable aldehyde dehydrogenase 172  PA number PA4133 PA4134 PA4139 PA4151 PA4188 PA4189 PA4190 PA4210 PA4218 PA4220 PA4221 PA4222 PA4223 PA4225 PA4226 PA4229 PA4230 PA4273 PA4302 PA4310 PA4527 PA4616 PA4683 PA4687 PA4692 PA4702 PA4710 PA4825  Gene Name  acoB  pqsL phzA1  fptA  pchF pchE pchC pchB rplA pctB pilC  hitA  phuR mgtA  Fold Pchange ebayes 2.51 0.01 2.39 0.00 2.85 0.00 -2.67 0.01 -2.77 0.00 -2.05 0.04 2.17 0.01 -2.06 0.04 -2.88 0.00 -3.92 0.00 -4.28 0.00 -2.03 0.04 -3.08 0.00 -2.55 0.00 -2.74 0.00 -2.58 0.00 -2.56 0.00 -2.07 0.02 -2.51 0.02 -2.47 0.00 -2.13 0.02 -2.37 0.02 -2.17 0.01 -2.14 0.02 -2.11 0.01 2.00 0.01 -2.03 0.04 2.20 0.00  Gene description cytochrome c oxidase subunit (cbb3-type) hypothetical protein hypothetical protein acetoin catabolism protein AcoB conserved hypothetical protein probable aldehyde dehydrogenase probable FAD-dependent monooxygenase probable phenazine biosynthesis protein probable transporter hypothetical protein Fe(III)-pyochelin outer membrane receptor precursor probable ATP-binding component of ABC transporter probable ATP-binding component of ABC transporter pyochelin synthetase dihydroaeruginoic acid synthetase pyochelin biosynthetic protein PchC salicylate biosynthesis protein PchB 50S ribosomal protein L1 probable type II secretion system protein chemotactic transducer PctB still frameshift type 4 fimbrial biogenesis protein PilC probable c4-dicarboxylate-binding protein hypothetical protein ferric iron-binding periplasmic protein HitA conserved hypothetical protein hypothetical protein Haem/Haemoglobin uptake outer membrane receptor PhuR precursor Mg(2+) transport ATPase, P-type 2 173  PA number PA4857 PA4888 PA4913 PA4978 PA4980 PA4990 PA5089 PA5185 PA5220 PA5292 PA5310 PA5355 PA5376 PA5377 PA5378 PA5381 PA5396 PA5397 PA5398 PA5399 PA5400 PA5401 PA5410 PA5411 PA5415 PA5416 PA5417 PA5418  Gene Name  pchP glcD  glyA1 soxB soxD soxA  Fold Pchange ebayes 2.07 0.04 -3.10 0.02 2.60 0.00 -2.17 0.03 -2.41 0.02 11.06 0.00 -2.28 0.01 -2.13 0.03 2.95 0.00 2.01 0.02 3.63 0.00 2.01 0.00 3.32 0.00 2.38 0.00 3.19 0.00 4.50 0.00 8.28 0.00 7.51 0.00 5.35 0.00 2.70 0.00 2.98 0.00 2.08 0.00 11.78 0.00 2.70 0.00 6.53 0.00 6.26 0.00 4.06 0.00 3.40 0.00  Gene description hypothetical protein conserved hypothetical protein probable binding protein component of ABC transporter hypothetical protein probable enoyl-CoA hydratase/isomerase SMR multidrug efflux transporter hypothetical protein conserved hypothetical protein hypothetical protein phosphorylcholine phosphatase conserved hypothetical protein glycolate oxidase subunit GlcD probable ATP-binding component of ABC transporter probable permease of ABC transporter hypothetical protein hypothetical protein hypothetical protein hypothetical protein probable FMN oxidoreductase probable ferredoxin probable electron transfer flavoprotein alpha subunit hypothetical protein probable ring hydroxylating dioxygenase, alpha-subunit probable ferredoxin serine hydroxymethyltransferase sarcosine oxidase beta subunit sarcosine oxidase delta subunit sarcosine oxidase alpha subunit 174  PA number PA5419 PA5420 PA5421 PA5434  Gene Name soxG purU2 fdhA mtr  Fold Pchange ebayes 3.74 0.00 3.07 0.00 5.47 0.00 2.22 0.03  Gene description sarcosine oxidase gamma subunit formyltetrahydrofolate deformylase glutathione-independent formaldehyde dehydrogenase tryptophan permease  Supplementary Table S A.2C (Chapter 5): Complete list of dysregulated genes in the microarray under swarming conditions of the lon mutant compared to the wild type PAO1 Gene Gene Fold PName Name change ebayes Gene Description PA0044 exoT -2.65 0.03 exoenzyme T PA0355 pfpI 2.22 0.04 protease PfpI PA0507 2.81 0.02 probable acyl-CoA dehydrogenase PA0567 3.87 0.02 hypothetical protein PA0618 2.60 0.03 probable bacteriophage protein PA0621 2.35 0.04 hypothetical protein PA0622 4.35 0.02 probable bacteriophage protein PA0633 2.55 0.02 hypothetical protein PA0634 2.66 0.04 hypothetical protein PA0635 2.87 0.02 hypothetical protein PA0636 2.78 0.02 hypothetical protein PA0985 pyoS5 2.42 0.04 pyocin S5 PA1706 pcrV -3.87 0.01 type III secretion protein PcrV PA1708 popB -3.84 0.01 translocator protein PopB PA1709 popD -6.06 0.01 Translocator outer membrane protein PopD precursor PA1710 exsC -3.81 0.02 ExsC, exoenzyme S synthesis protein C precursor. PA1711 exsE -2.19 0.03 ExsE PA1712 exsB -3.69 0.01 exoenzyme S synthesis protein B PA1741 3.69 0.04 hypothetical protein 175  Gene Name PA1803 PA2260 PA2433 PA2550 PA2780 PA2825 PA2826 PA3231 PA3277 PA3692 PA3841 PA3874 PA4625 PA4880  Gene Name lon  ospR  lptF exoS narH cdrA  Fold change -23.74 -2.06 2.25 5.92 2.76 5.02 4.20 2.04 2.32 2.23 -3.24 -3.42 2.24 2.56  Pebayes 0.00 0.04 0.05 0.01 0.01 0.02 0.02 0.04 0.03 0.04 0.01 0.02 0.03 0.04  Gene Description Lon protease hypothetical protein hypothetical protein probable acyl-CoA dehydrogenase hypothetical protein probable transcriptional regulator probable glutathione peroxidase hypothetical protein short chain dehydrogenase probable outer membrane protein precursor exoenzyme S respiratory nitrate reductase beta chain hypothetical protein probable bacterioferritin  176  Supplementary Figure 1: Molecular surface of the proteolytical domain of the Lon protease of E. coli which forms a hexamer. The P domain consists of six α helices and ten β strands. The picture is reproduced with permission from (16).  177  

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