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Regulation of virulence and antimicrobial peptide resistance in Pseudomonas aeruginosa Gooderham, William James 2008

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     REGULATION OF VIRULENCE AND ANTIMICROBIAL PEPTIDE RESISTANCE IN PSEUDOMONAS AERUGINOSA   by  WILLIAM JAMES GOODERHAM  B.Sc., The University of British Columbia, 2005    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE   in   THE FACULTY OF GRADUATE STUDIES  (Microbiology and Immunology)    THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)        July, 2008   © William James Gooderham  ii ABSTRACT  Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. These P. aeruginosa infections can be extremely difficult to treat due to the high intrinsic antibiotic resistance and broad repertoire of virulence factors, both of which are highly regulated. It was demonstrated here that the psrA gene, encoding a transcriptional regulator, was up-regulated in response to sub-inhibitory concentrations of antimicrobial peptides. Compared to wild-type and the complemented mutant, a P. aeruginosa PAO1 psrA::Tn5 mutant displayed intrinsic super-susceptibility to polymyxin B, a last resort antimicrobial used against multi-drug resistant infections, and indolicidin, a bovine neutrophil antimicrobial peptide; this super-susceptibility phenotype correlated with increased outer membrane permeability. The psrA mutant was also defective in simple biofilm formation, rapid attachment, and normal swarming motility, phenotypes that could be complemented by the cloned psrA gene. The role of PsrA in global gene regulation was studied by comparing the psrA mutant to wild-type by microarray analysis, demonstrating that 178 genes were up- or down-regulated by greater than 2-fold (P ≤0.05). Dysregulated genes included those encoding known PsrA targets, the type III secretion apparatus and effectors, adhesion and motility genes and a variety of metabolic, energy metabolism and outer membrane permeability genes. This indicates that PsrA is a central regulator of antimicrobial peptide resistance and virulence. P. aeruginosa containing a mutation in the PhoQ sensor kinase-encoding gene was highly attenuated for persistence in a rat chronic lung infection model. In addition, the polymyxin B hyper-resistant phoQ mutant displayed reduced type IV pili-dependent twitching motility and was less cytotoxic towards human bronchial epithelial cells, indicating that the virulence defect observed could be due at least in part to these phenotypes. Using microarrays it was further demonstrated that PhoQ regulates a large number of genes that are PhoP-independent and that the phoQ mutation leads to up-regulation of PhoP- and PmrA- regulated genes as well as other genes consistent with its virulence phenotypes.  iii TABLE OF CONTENTS ABSTRACT.........................................................................................................................ii TABLE OF CONTENTS ...................................................................................................iii LIST OF TABLES ..............................................................................................................v LIST OF FIGURES ...........................................................................................................vi LIST OF ABBREVIATIONS ...........................................................................................vii ACKNOWLEDGEMENTS .............................................................................................viii CO-AUTHORSHIP STATEMENT .................................................................................. ix CHAPTER 1 – Introduction ...............................................................................................1 Pseudomonas aeruginosa ............................................................................................................ 1 P. aeruginosa virulence ............................................................................................................... 1 P. aeruginosa regulators ............................................................................................................. 4 Cationic antimicrobial peptides (host defense peptides)............................................................ 5 P. aeruginosa resistance to antimicrobial peptides .................................................................... 7 Goals of this study..................................................................................................................... 10 REFERENCES ......................................................................................................................... 12 CHAPTER 2 – PsrA of Pseudomonas aeruginosa............................................................ 16 INTRODUCTION .................................................................................................................... 16 MATERIALS AND METHODS.............................................................................................. 18 Bacterial strains and growth conditions. ...........................................................................................18 Genetic manipulations........................................................................................................................18 Mobilizing the UW-psrA transposon mutation into a new PAO1 background. ................................19 Genetic complementation of psrA. .....................................................................................................19 Killing curves......................................................................................................................................20 Outer membrane permeabilization assays.........................................................................................21 Biofilm and attachment assays...........................................................................................................21 Motility assays. ...................................................................................................................................22 Growth curves. ...................................................................................................................................22 Microarray analysis. ..........................................................................................................................23 Real-time qPCR..................................................................................................................................24 RESULTS ................................................................................................................................. 24 Activation of psrA transcription in response to antimicrobial peptides. ...........................................24 Contribution of psrA to intrinsic antimicrobial peptide and polymyxin B resistance.......................25 The psrA mutation affected the permeabilization of the outer membrane........................................26 Contribution of psrA to biofilm formation and attachment. .............................................................26 Requirement for PsrA for normal swarming.....................................................................................27 Microarray analysis. ..........................................................................................................................28  iv Additional mutant phenotypic analyses.............................................................................................28 DISCUSSION............................................................................................................................ 30 REFERENCES ......................................................................................................................... 49 CHAPTER 3 – PhoQ of Pseudomonas aeruginosa........................................................... 54 INTRODUCTION .................................................................................................................... 54 MATERIALS AND METHODS.............................................................................................. 56 Tissue culture, bacterial strains, primers, and growth conditions. ...................................................56 Competitive index (CI) determination. ..............................................................................................56 RNA extraction, cDNA synthesis and hybridization to DNA microarrays. ......................................58 Analysis of DNA Microarrays............................................................................................................59 Real-Time quantitative PCR (RT-qPCR). .........................................................................................59 Cytotoxicity assays. ............................................................................................................................60 Minimal inhibitory concentration determination (MICs). ................................................................61 Twitching motility analysis. ...............................................................................................................61 Lettuce leaf model of infection. ..........................................................................................................61 RESULTS ................................................................................................................................. 62 PhoQ mutants were highly attenuated for virulence in a model of chronic lung infection...............62 PhoQ mutants demonstrated reduced cytotoxicity toward human bronchial epithelial cells...........63 PhoQ mutants were impaired in twitching motility...........................................................................63 Reduced lettuce virulence of phoQ mutants.......................................................................................64 Analysis of the altered transcriptome in the phoQ mutant................................................................64 DISCUSSION............................................................................................................................ 67 REFERENCES ......................................................................................................................... 83 CHAPTER 4 – Concluding remarks ................................................................................ 88 INTRODUCTION .................................................................................................................... 88 PsrA........................................................................................................................................... 88 PhoQ.......................................................................................................................................... 90 FUTURE RESEARCH DIRECTIONS.................................................................................... 90 REFERENCES ......................................................................................................................... 93  APPENDIX I……………………………………………………..…………………………94  APPENDIX II……………………...….……….………….….……………………………..98    v LIST OF TABLES    CHAPTER 2 – PsrA of Pseudomonas aeruginosa  Table 2.1  P. aeruginosa strains and plasmids used in this study……………………… 36  Table 2.2 Known PsrA targets significantly dysregulated in psrA mutants as determined using microarray……………………………………………………………. 37  Table 2.3 Type III secretion, adhesion (tad), motility, and type II secreted genes significantly dysregulated in psrA mutants as determined using microarray. 38  Table 2.4 Other known genes significantly dysregulated in psrA mutants as determined using microarray……………..……………………………………………... 39    CHAPTER 3 – PhoQ of Pseudomonas aeruginosa  Table 3.1 P. aeruginosa strains and plasmids used in this study……………..……….. 73  Table 3.2 Competitive index (CI) analysis of P. aeruginosa mutant strains grown with the wild type PAO1 strain after 7 days of in vivo passage in the rat lung….. 73  Table 3.3 Microarray analysis of genes significantly dysregulated in the phoQ mutant relative to wild-type……………………………………………….………... 74  Table 3.4 qPCR gene expression analysis of select genes in phoQ mutants relative to wild-type……………………………………………………………….....… 80   vi LIST OF FIGURES   CHAPTER 1 – Introduction  Figure 1.1 A model for the P. aeruginosa PhoP-PhoQ and PmrA-PmrB regulatory networks in resistance to cationic antimicrobial peptides……………………. 9   CHAPTER 2 – PsrA of Pseudomonas aeruginosa  Figure 2.1 Intrinsic polymyxin B and antimicrobial peptide super-susceptibility in psrA mutants………………………………………………………..…………….. 41  Figure 2.2 PsrA mutation affects outer membrane permeability to peptides…..………. 42  Figure 2.3 Defects in biofilm formation and attachment in psrA mutants…………..…. 43  Figure 2.4 Swarming motility defect in psrA mutants…………………………….….... 45  Figure 2.5 Peptide susceptibility, swarming and biofilm analysis of PA14 mutants in selected genes transcriptionally downregulated in psrA mutants………...… 46  Figure 2.6 Mutants in the B-band O antigen biosynthetic operon showed altered outer membrane permeability to indolicidin antimicrobial peptides…………...… 48   CHAPTER 3 – PhoQ of Pseudomonas aeruginosa  Figure 3.1 PhoQ mutants displayed reduced in vitro cytotoxicity towards human bronchial epithelial cells………...………………………………………….. 81  Figure 3.2 PhoQ mutants displayed reduced twitching motility…………………....….. 81  Figure 3.3 PhoQ mutants were attenuated for virulence in lettuce leaves………..….… 82  Figure 3.4 Pigmentation differences of mid-logarithmic phase phoQ mutant and wild- type liquid cultures………………………………………………………….. 82   vii LIST OF ABBREVIATIONS    °C – degrees Celsius ATP – adenosine triphosphate CCCP – carbonyl cyanide m-chlorophenol hydrozone CFU – colony forming unit CI – competitive index DNA – deoxyribonucleic acid EDTA – ethylene diamine tetra acetic acid FBS – fetal bovine serum HEPES – 2-(4-(2-hydroxyethyl)-1-piperazinyl) ethanesulfonic acid LB – Luria-Bertani LDH – lactate dehydrogenase LPS – lipopolysaccharide MEM – minimal essential medium MIC – minimal inhibitory concentration NPN – 1-N-phenylnapthylamine ORF – open reading frame PA – Pseudomonas aeruginosa locus of identity PAGE – polyacrylamide gel electrophoresis PBS – phosphate buffered saline PCR – polymerase chain reaction RNA – ribonucleic acid RPM – revolutions per minute SDS – sodium dodecylsulphate TSB – tryptic soy broth UW – University of Washington WT – wild-type   viii ACKNOWLEDGEMENTS   I want to first and foremost thank Bob Hancock, my supervisor, for his support, guidance, and unmatched generosity over these years. He is a truly a towering role model for everyone in our lab and I am privileged to have had the opportunity of being associated with this wonderful lab group, both past and present. Indeed, I could not have asked for anything more than to have had the chance to initially learn about Pseudomonas and lab techniques from Shawn Lewenza, Joe McPhee, Joerg Overhage, and Manjeet Bains. Also, Susan Farmer and Barbara Sherman provided great organizational help with various applications. I would also like to thank the members of my supervisory committee, Rachel Fernandez and Erin Gaynor, for their suggestions and insights that have helped improve my thesis. Finally, I wish to salute my labmates for their friendship and thank my family for their constant love and support.   ix CO-AUTHORSHIP STATEMENT   This thesis is submitted in manuscript format and in this section I acknowledge the contribution of a number of co-authors. Unless indicated, all experimental results and manuscript composition are my responsibility.   A version of Chapter 2 has been accepted to the Journal of Bacteriology as:  Gooderham, W. J., M. Bains, J. B. McPhee, I. Wiegand, and R. E.W. Hancock. 2008. Induction by cationic antimicrobial peptides and involvement in intrinsic polymyxin and antimicrobial peptide resistance, biofilm formation and swarming motility of psrA in Pseudomonas aeruginosa.  • M. Bains helped perform microarray experiments and analysis. • J. B. McPhee and I. Wiegand helped with preliminary killing curve experiments • R. E. W. Hancock edited this manuscript.   A version of Chapter 3 is being prepared for publication as:  Gooderham, W. J., J. B. McPhee, F. Sanschagrin, M. Bains, S. Gellatly, C. Cosseau, R. C. Levesque, and R. E.W. Hancock. 2008. Regulation of virulence by the PhoQ sensor kinase in Pseudomonas aeruginosa.  • M. Bains helped perform microarray experiments and analysis. • J. B. McPhee helped design experiments and helped write part of the manuscript. • S. Gellatly and C. Cosseau performed the cytotoxicity experiments. • F. Sanschagrin and R. C. Levesque performed the in vivo experiments and helped write this part of the manuscript. • R. E. W. Hancock edited the manuscript.   1 CHAPTER 1 – Introduction  Pseudomonas aeruginosa  Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium capable of inhabiting diverse soil and water habitats. Not solely a harmless environmental bacterium, P. aeruginosa is also an opportunist capable of infecting plant, insect, nematode, and animal tissues (Rahme et al. 2000). This remarkable versatility and adaptability is governed by a relatively large and complex genome permitting nutritional and metabolic adaptation driven by sophisticated and coordinated regulation of gene expression by a large proportion of regulatory genes (Stover et al. 2000). P. aeruginosa is a major opportunistic pathogen of immunocompromised patients. It is the third most common hospital-acquired pathogen in North America, the most common cause of ventilator-associated pneumonia, and a leading cause of infection and sepsis in burn patients (Fridkin et al. 1999). Multi-determinant virulence and high intrinsic antibiotic resistance are two noteworthy hallmarks of P. aeruginosa infections (Hancock and Speert 2000). P. aeruginosa is also well known as a dominant cause of chronic lung infections and resulting morbidity and mortality in cystic fibrosis, the most common genetic disease in North America (Gibson et al. 2000). Adaptive and mutational resistance is increasingly impacting on treatment success and new antimicrobial therapeutic options are needed for resistant strains, some of which have developed resistance to virtually every antibiotic and have thus become hospital “Superbugs” (Falagas and Bliziotis 2007; Mesaros et al. 2007). P. aeruginosa virulence  P. aeruginosa is capable of causing both chronic and acute infections and appropriately regulates the virulence determinants required for each type of infection  2 (Furukawa et al. 2006). Often refractory to antimicrobial treatment, chronic infections can be microcolony or biofilm-like in nature, such as those present in cystic fibrosis lung infections and on urinary catheters and other indwelling medical devices (Parsek and Singh 2003). Conversely, acute P. aeruginosa infections are typified by bacterial penetration of the host epithelium and systemic spread, such as in severe burn wound infections. An arsenal of virulence factors impacts on the pathogenicity of P. aeruginosa (Lyczak et al. 2000). Included among these are cell-associated factors such as lipopolysaccharide (LPS), flagella and type IV pili, exopolysaccharides such as alginate, and secreted factors including toxins, elastase and other proteases, phospholipases and small molecules such as phenazines, rhamnolipids and cyanide. The single polar flagellum is an important and highly regulated virulence factor for motility and attachment to surfaces. P. aeruginosa isolates from long-term chronic cystic fibrosis infections often lack flagella and it is thought that these non-motile cells are at some stage of a biofilm-like adaptation (Singh et al. 2000). In addition, quorum-sensing, secretion systems and other regulatory systems are important for virulence (Kirisits and Parsek 2006; Venturi 2006). Biofilms are bacterial communities surrounded and infiltrated by an extracellular matrix containing exopolysaccharide, DNA and proteins. P. aeruginosa biofilms are readily found both in natural environments (e.g. attached to rocks in streams and to particulate matter in soil) and health-care settings (e.g. attached to indwelling medical devices). Regardless, whether growing as true biofilms or as microcolonies, P. aeruginosa infections in cystic fibrosis are highly resistant to antibiotics, making eradication impossible after establishment of mature infections (Gibson et al. 2003). P. aeruginosa biofilm formation in vitro is thought to follow three distinct stages: surface attachment, microcolony formation,  3 and differentiation into mature biofilm communities encased in an extracellular matrix (Kirisits and Parsek 2006). Type IV pili-dependent twitching and flagellar-based swimming motility are important for initial surface attachment and niche colonization (O’Toole and Kolter 1998), and many genes associated with these motility appendages are necessary for proper biofilm formation (Kirisits and Parsek 2006). In addition, there is a strong but not obligate correspondence in gene requirements between biofilm formation and swarming motility. In P. aeruginosa, swarming motility (distinct from swimming and twitching motility), is dependent on both flagella and type IV pili appendages and is thought to represent a complex social adaptation to viscous environments (Kohler et al. 2000; Overhage et al. 2008). Type III secretion system-mediated cytotoxicity is a key virulence property of P. aeruginosa (Yahr and Wolfgang 2006). Gene expression of the five type III secretion system operons is modulated by a complex set of regulatory mechanisms and the expression of this secretion machinery is coupled to the secretory activity of three effectors, ExoS, ExoT and ExoY, which possess anti-phagocytic and cytotoxic properties (Yahr and Wolfgang 2006). The two P. aeruginosa type II secretion systems (xcp and hxc) mediate the secretion of a broad range of toxins and other virulence related enzymes including exotoxin A, lipase, and phospholipase C. P. aeruginosa LPS is a prominent virulence factor that mediates virulence, resistance to antibiotics, and induction of host inflammatory and innate immune responses (Pier 2007). P. aeruginosa contains a typical Gram-negative LPS structure composed of lipid A, core, and O-antigen. P. aeruginosa produces two types of LPS, B-band (O-antigen) and A-band (polymer of D-rhamnose), each attached to separate core/lipid A molecules (Pier 2007). The  4 lipid A (contains an N- and O-acylated diglucosamine bisphosphate backbone) can be modified with additional chemical groups and fatty acids. Environmental growth conditions (e.g. Mg2+ levels) are known to affect the degree of acylation and presence of lipid A modifications. For example, LPS of isolates from cystic fibrosis patients predominantly contains lipid A with aminoarabinose modifications (Ernst et al. 1999) and lacks O-antigen moieties (Pier 2007). Lipid A plays an important role in the pathogenesis of many Gram- negative bacterial infections by acting as a signature of such infections, activating the innate immune system, triggering the synthesis of host defense peptides, cytokines, clotting factors, and other immunostimulatory molecules (Pier 2007). P. aeruginosa regulators  Bacterial survival in dynamic environmental conditions requires the capability to sense and quickly respond to many different stimuli. Often, these physiological responses are at the level of gene expression and are accomplished by regulatory proteins. One class of regulators, the bacterial two-component regulatory systems, are designed to sense diverse stimuli and enact a rapid and precise adaptive physiological response that can involve altered transcription of a substantial number of genes. The P. aeruginosa genome encodes one of the largest complements of regulatory proteins at just under 10% of all genes and a major subset are genes encoding 64 sensor kinases and 72 response regulators (Stover et al. 2000; Rodrigue et al. 2000). Two-component regulatory systems classically comprise an inner membrane- spanning sensor histidine kinase and a cytoplasmic response regulator (Stock et al. 2000). The functional mode of both sensor kinase and response regulator is determined by reversible phosphotransfer reactions and ensuing protein conformational changes (Stock et  5 al. 2000). In the archetypical system, the sensor kinase contains an N-terminal periplasmic input domain that detects a specific stimulus and a C-terminal cytoplasmic transmitter domain that binds ATP and has histidine kinase activity. The cognate response regulator contains a conserved receiver domain and a variable output domain that often binds DNA. Classically, a membrane-bound dimeric sensor kinase detects an environmental stimulus/ligand in the periplasm via its input domain, and then undergoes trans- autophosphorylation at a conserved histidine residue in its transmitter domain. This phosphoryl group is then transferred to (and catalyzed by) the conserved aspartate- containing receiver domain of the response regulator. Phosphorylation of the response regulator receiver domain often modifies the activity of the output domain, of which there are many types. Frequently, the output domain is a helix-turn-helix DNA binding domain and so phosphorylation of the receiver domain changes the response regulator’s affinity for specific DNA elements so as to modify gene expression and initiate the corresponding cellular response. Dephosphorylation of the response regulator by the sensor kinase serves to return the system to its pre-activation state. Cationic antimicrobial peptides (host defense peptides)  The last 40 years have seen only three new classes of antibiotics enter medicine (lipopeptides, oxazolidinones, and streptogramins), all geared towards Gram-positive bacterial infections. A lack of new antibiotics, particularly for treatment of Gram-negative infections, combined with emerging multi-drug resistance issues demands that new antimicrobial strategies be explored for treating these infections (Chopra et al. 2008). Cationic antimicrobial peptides (host defense peptides) are presently forming the foundation of a new class of antimicrobial compounds for clinical use (Marr et al. 2006).  6 Cationic antimicrobial peptides have a pivotal role in preventing infections by microbial pathogens in many organisms (Zasloff 2002). To date more than 600 peptides in virtually all species of life have been described that not only kill pathogenic microorganisms, including Gram–positive and Gram–negative bacteria, viruses, protozoa, and fungi, but also to play a central role in recruiting and promoting elements of the innate immune system (Finlay and Hancock 2004; Brown and Hancock 2006). This enormous peptide diversity is achieved through several structural classes, whereby all peptides, regardless of class, share a net positive charge and around 50% hydrophobic residues, which confers the ability to fold into an amphiphilic conformation upon interaction with bacterial membranes (Jenssen et al. 2006). Although interaction with the cytoplasmic membrane is obligatory and some peptides are able to perforate membranes at their minimal effective concentrations, a number of peptides have been shown to translocate across the membrane and act on gene expression and on cytoplasmic targets including macromolecular synthesis, enzyme inhibition, stimulation of autolysis, and cell division (Brogden 2006; Hale and Hancock 2007). Indeed, many severe bacterial infections require systemic antibacterial drug administration to quickly halt and limit the spread of infection; in this case endotoxaemia/sepsis is a common and dangerous complication of systemic therapy in individuals with bacteremia. One substantial advantage of peptides over conventional antibiotics is that they have dual ability to neutralize sepsis/endotoxemia and to participate in diverse roles in modulating mammalian innate immunity (Brown and Hancock 2006). Indeed, one of the most important roles described is an ability to stimulate the innate immune response while simultaneously dampening the potentially harmful inflammatory  7 response (Finlay and Hancock 2004). Other motivations for therapeutic antimicrobial use include a diversity of potential applications that could include use as a single antimicrobial or in combination with other antibiotics for synergistic effects in order to overcome barriers resistant bacteria have created against currently used antibiotics (Zasloff 2002). Indeed, peptides are not hindered by some of the resistance mechanisms that are placing currently used antibiotics in jeopardy, as excellent activity is seen against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant P. aeruginosa. The most potent antimicrobial peptides can have an unusually broad spectrum of activity, killing most Gram- negative and Gram-positive bacteria, while this spectrum can extend to fungi and even viruses (Jenssen et al. 2006). Peptide killing of bacteria is extremely rapid and can involve multiple bacterial cellular targets and often minimal inhibitory concentrations and minimal bactericidal concentrations coincide within 2-fold, indicating that killing is generally bactericidal (a highly desirable mode of action). P. aeruginosa resistance to antimicrobial peptides  The major reasons for the high intrinsic resistance of P. aeruginosa to multiple classes of antibiotics are its low outer membrane permeability coupled with active antibiotic efflux systems (Hancock and Speert 2000). Furthermore, in addition to classical mutational or acquired antibiotic resistance, P. aeruginosa resistance can be triggered by environmental factors and is therefore a subset of adaptive resistance. For example, sub-inhibitory concentrations of antibiotics have a variety of effects on bacteria and can themselves induce resistance to subsequent exposure to otherwise lethal concentrations of antibiotics (Linares et al. 2006; Davies et al. 2006). As discussed below, P. aeruginosa is known to regulate resistance to antimicrobial peptides through adaptive resistance (Gilleland and Farley 1982).  8 During infections in health-care settings, P. aeruginosa is likely exposed to endogenous β-defensin and cathelicidin antimicrobial (host defense) peptides at epithelial surfaces and also to polymyxins (peptide antibiotics) when therapeutically administered. Owing to a severe limitation of therapeutic options for multidrug-resistant P. aeruginosa infections, polymyxin peptides have re-emerged as effective drugs for combating these formidable infections (Li et al. 2006; Zavascki et al. 2007). Adaptive resistance to cationic antimicrobial peptides and polymyxin B is known to occur in response to limiting extracellular concentrations of divalent Mg2+ and Ca2+ cations, but this mechanism is not likely a clinically meaningful observation as the body contains 1-2 mM divalent cations. However as noted below peptides themselves are able to induce peptide resistance mechanisms through two-component systems. The PhoP-PhoQ two-component system mediates in part the adaptive response to limiting (µM) extracellular Mg2+ concentrations and concurrent resistance to polymyxin B and antimicrobial peptides (Fig. 1.1; Macfarlane et al. 1999; Ernst et al. 1999; Macfarlane et al. 2000). Another two-component system, PmrA-PmrB, also responds to low Mg2+ signals and regulates resistance to polymyxin B and antimicrobial peptides (Fig. 1.1; McPhee et al., 2003; Moskowitz et al. 2004). In response to low Mg2+, both PhoP and PmrA response regulators positively regulate the arnBCADTEF operon which encodes a pathway for addition of 4-aminoarabinose (positively charged amino sugar residue) to lipid A (Moskowitz et al. 2004; McPhee et al. 2003), causing resistance by reducing the net negative charge of LPS and limiting its interaction with polycationic antibiotics such as polymyxin B and other cationic peptides. Moreover, the addition of aminoarabinaose to lipid A appears to be specific for P. aeruginosa cystic fibrosis isolates (Ernst et al. 2007).  9  Figure 1.1. A model for the P. aeruginosa PhoP-PhoQ and PmrA-PmrB regulatory networks in resistance to cationic antimicrobial peptides. Mg2+ limitation leads to the activation (phosphorylation) of the PhoP and PmrA response regulators which positively autoregulate the transcription of their respective operons, as well as the arnBCADTEF operon. The ArnBCADTEF pathway modifies Lipid A with aminoarabinaose, reducing the net negative charge on LPS, and consequently decreasing self promoted uptake across the outer membrane, increasing resistance to polymyxins and cationic antimicrobial peptides. Conversely, under high Mg2+ conditions, PhoP and PmrA are dephosphorylated and presumed inactive (not shown). However, during growth in high Mg2+ plus subinhibitory antimicrobial peptides, an unidentified regulatory system is proposed to promote activation of arnBCADTEF and pmrAB operons, consequently increasing resistance to antimicrobial peptides. Other genes regulated by PmrA and PhoP are not shown.  Interestingly, both pmrAB and arnBCADTEF operons were shown to be strongly induced by a variety of cationic antimicrobial peptides, including polymyxins, indolicidin, human LL-37, and other synthetic peptides (McPhee et al. 2003), in stark contrast to Salmonella where direct binding of peptides to PhoQ mediates peptide resistance (Bader et al. 2005). In P. aeruginosa the induction of arnBCADTEF was partially dependent on PmrA-PmrB (McPhee et al. 2003). Transposon mutations in both pmrAB and arnBCADTEF operons result in super-susceptibility to polymyxin B and antimicrobial peptides such as  10 improved variants of indolicidin, an endogenous bovine neutrophil host defense (antimicrobial) peptide (Lewenza et al. 2005). Peptide-induced activation of the pmrAB operon appeared to be independent of both the PhoP-PhoQ and PmrA-PmrB systems, which likely indicates that another protein regulator responds to cationic peptides to promote resistance (Fig. 1.1). Interestingly, a phoQ mutant displays super-resistance to polymyxin B and antimicrobial peptides when grown in high Mg2+, a constitutive resistance phenotype, in contrast to wild-type cells which are normally sensitive to antimicrobial peptides in high Mg2+ (Macfarlane et al. 1999). The role of PhoP in this resistance appears to be significant as overexpression of the PhoP response regulator in a phoP mutant gives phoQ-like constitutive polymyxin B resistance (Macfarlane et al. 1999). These findings indicate that in non-inducing high Mg2+ conditions, PhoQ dephosphorylates PhoP (inactivating it), and conversely in the phoQ mutant this activity is lost and possibly some other protein/mechanism allows PhoP to be activated through phosphorylation. To note, a phoP mutant displays wild-type polymyxin B resistance in low Mg2+ media (due to the activity overlap of PmrAB) and remains sensitive when grown under high Mg2+ conditions (in contrast to phoQ; Macfarlane et al. 1999; McPhee et al. 2003). Goals of this study  The overall theme of these studies encompassed P. aeruginosa regulation of antimicrobial peptide resistance and virulence. A starting point was to examine how P. aeruginosa regulates resistance to polymyxin B and antimicrobial peptides under non- resistance-inducing high (mM) Mg2+ conditions. Preliminary microarray experiments were used to gain insight into how P. aeruginosa regulates its transcriptome in response to sub-  11 inhibitory concentrations of antimicrobial peptides. These results showed the psrA gene (encoding a transcriptional regulator) was transcriptionally up-regulated in response to peptides. Second, with collaborators, we found that the phoQ mutant is highly attenuated for virulence in a rat model of chronic lung infection. Therefore, the major hypotheses and goals included:  1. PsrA regulates resistance to antimicrobial peptides and other virulence processes. Goals were to phenotypically characterize these processes and the corresponding gene expression governing them. 2. PhoQ is a regulator of virulence and the PhoQ regulon contributes to virulence and antimicrobial peptide resistance. The goal here was to investigate this phenotype by making a new phoQ mutant, performing microarray analysis, and further characterize other phenotypes of this mutant.  12 REFERENCES  Bader, M. W., S. Sanowar, M. E. Daley, A. R. Schneider, U. Cho, W. Xu, R. E. Klevit, H. Le Moual, and S. I. Miller. 2005. Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 122:461-72.  Brogden, K. A. 2005. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3:238-50.  Brown, K. L., and R. E. W. Hancock. 2006. Cationic host defense (antimicrobial) peptides. Curr Opin Immunol 18:24-30.  Chopra, I., C. Schofield, M. Everett, A. O'Neill, K. Miller, M. Wilcox, J. M. Frere, M. Dawson, L. Czaplewski, U. Urleb, and P. Courvalin. 2008. Treatment of health-care- associated infections caused by Gram-negative bacteria: a consensus statement. Lancet Infect Dis 8:133-9.  Davies, J., G. B. Spiegelman, and G. Yim. 2006. The world of subinhibitory antibiotic concentrations. Curr Opin Microbiol 9:445-53.  Ernst, R. K., S. M. Moskowitz, J. C. Emerson, G. M. Kraig, K. N. Adams, M. D. Harvey, B. Ramsey, D. P. Speert, J. L. Burns, and S. I. Miller. 2007. Unique Lipid A Modifications in Pseudomonas aeruginosa Isolated from the Airways of Patients with Cystic Fibrosis. J Infect Dis 196:1088-92.  Ernst, R. K., E. C. Yi, L. Guo, K. B. Lim, J. L. Burns, M. Hackett, and S. I. Miller. 1999. Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa. Science 286:1561-5.  Falagas, M. E., and I. A. Bliziotis. 2007. Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era? Int J Antimicrob Agents 29:630-6.  Finlay, B. B., and R. E. W. Hancock. 2004. Can innate immunity be enhanced to treat microbial infections? Nat Rev Microbiol 2:497-504.  Fridkin, S. K., C. D. Steward, J. R. Edwards, E. R. Pryor, J. E. McGowan, Jr., L. K. Archibald, R. P. Gaynes, and F. C. Tenover. 1999. Surveillance of antimicrobial use and antimicrobial resistance in United States hospitals: project ICARE phase 2. Project Intensive Care Antimicrobial Resistance Epidemiology (ICARE) hospitals. Clin Infect Dis 29:245-52.  Furukawa, S., S. L. Kuchma, and G. A. O'Toole. 2006. Keeping their options open: acute versus persistent infections. J Bacteriol 188:1211-7.  Gibson, R. L., J. L. Burns, and B. W. Ramsey. 2003. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am J Respir Crit Care Med 168:918-51.   13 Gilleland, H. E., Jr., and L. B. Farley. 1982. Adaptive resistance to polymyxin in Pseudomonas aeruginosa due to an outer membrane impermeability mechanism. Can J Microbiol 28:830-40.  Hale, J. D., and R. E. W. Hancock. 2007. Alternative mechanisms of action of cationic antimicrobial peptides on bacteria. Expert Rev Anti Infect Ther 5:951-9.  Hancock, R. E. W., and D. P. Speert. 2000. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resist Updat 3:247-255.  Jenssen, H., P. Hamill, and R. E. W. Hancock. 2006. Peptide antimicrobial agents. Clin Microbiol Rev 19:491-511.  Kirisits, M. J., and M. R. Parsek. 2006. Does Pseudomonas aeruginosa use intercellular signalling to build biofilm communities? Cell Microbiol 8:1841-9.  Kohler, T., L. K. Curty, F. Barja, C. van Delden, and J. C. Pechere. 2000. Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J Bacteriol 182:5990-6.  Lewenza, S., R. K. Falsafi, G. Winsor, W. J. Gooderham, J. B. McPhee, F. S. Brinkman, and R. E. W. Hancock. 2005. Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes. Genome Res 15:583-9.  Li, J., R. L. Nation, J. D. Turnidge, R. W. Milne, K. Coulthard, C. R. Rayner, and D. L. Paterson. 2006. Colistin: the re-emerging antibiotic for multidrug-resistant Gram- negative bacterial infections. Lancet Infect Dis 6:589-601.  Linares, J. F., I. Gustafsson, F. Baquero, and J. L. Martinez. 2006. Antibiotics as intermicrobial signaling agents instead of weapons. Proc Natl Acad Sci U S A 103:19484-9.  Lyczak, J. B., C. L. Cannon, and G. B. Pier. 2000. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2:1051-60.  Macfarlane, E. L., A. Kwasnicka, M. M. Ochs, and R. E. W. Hancock. 1999. PhoP- PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer-membrane protein OprH and polymyxin B resistance. Mol Microbiol 34:305-16.  Macfarlane, E. L., A. Kwasnicka, and R. E. W. Hancock. 2000. Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides. Microbiology 146:2543-54.  Marr, A. K., W. J. Gooderham, and R. E. W. Hancock. 2006. Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Curr Opin Pharmacol 6:468-72.   14 McPhee, J. B., M. Bains, G. Winsor, S. Lewenza, A. Kwasnicka, M. D. Brazas, F. S. Brinkman, and R. E. W. Hancock. 2006. Contribution of the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to Mg2+-induced gene regulation in Pseudomonas aeruginosa. J Bacteriol 188:3995-4006.  McPhee, J. B., S. Lewenza, and R. E. W. Hancock. 2003. Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol Microbiol 50:205-17.  Mesaros, N., P. Nordmann, P. Plesiat, M. Roussel-Delvallez, J. Van Eldere, Y. Glupczynski, Y. Van Laethem, F. Jacobs, P. Lebecque, A. Malfroot, P. M. Tulkens, and F. Van Bambeke. 2007. Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium. Clin Microbiol Infect 13:560-78.  Moreau-Marquis, S., B. A. Stanton, and G. A. O'Toole. 2008. Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway. Pulm Pharmacol Ther. (In Press)  Moskowitz, S. M., R. K. Ernst, and S. I. Miller. 2004. PmrAB, a two-component regulatory system of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid A. J Bacteriol 186:575-9.  O'Toole, G. A., and R. Kolter. 1998. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30:295-304.  Overhage, J., M. Bains, M. D. Brazas, and R. E. W. Hancock. 2008. Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance. J Bacteriol 190:2671-9.  Parsek, M. R., and P. K. Singh. 2003. Bacterial biofilms: an emerging link to disease pathogenesis. Annu Rev Microbiol 57:677-701.  Pier, G. B. 2007. Pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity. Int J Med Microbiol 297:277-95.  Rahme, L. G., F. M. Ausubel, H. Cao, E. Drenkard, B. C. Goumnerov, G. W. Lau, S. Mahajan-Miklos, J. Plotnikova, M. W. Tan, J. Tsongalis, C. L. Walendziewicz, and R. G. Tompkins. 2000. Plants and animals share functionally common bacterial virulence factors. Proc Natl Acad Sci U S A 97:8815-21  Rodrigue, A., Y. Quentin, A. Lazdunski, V. Mejean, and M. Foglino. 2000. Two- component systems in Pseudomonas aeruginosa: why so many? Trends Microbiol 8:498- 504.   15 Singh, P. K., A. L. Schaefer, M. R. Parsek, T. O. Moninger, M. J. Welsh, and E. P. Greenberg. 2000. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407:762-4.  Stock, A. M., V. L. Robinson, and P. N. Goudreau. 2000. Two-component signal transduction. Annu Rev Biochem 69:183-215.  Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. Hancock, S. Lory, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959-64.  Venturi, V. 2006. Regulation of quorum sensing in Pseudomonas. FEMS Microbiol Rev 30:274-91.  Yahr, T. L., and M. C. Wolfgang. 2006. Transcriptional regulation of the Pseudomonas aeruginosa type III secretion system. Mol Microbiol 62:631-40.  Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415:389-95.  Zavascki, A. P., L. Z. Goldani, J. Li, and R. L. Nation. 2007. Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review. J Antimicrob Chemother 60:1206-15.   16  CHAPTER 2 – PsrA of Pseudomonas aeruginosa*  INTRODUCTION  The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is a dominant pathogen in the lungs of cystic fibrosis patients (Gibson et al. 2003; Rowe et al. 2005) and the third leading cause of severe hospital-acquired infections (Kielhofner et al. 1992; Lyczak et al. 2000). P. aeruginosa infections can be extremely difficult to combat owing to high intrinsic antibiotic resistance and a wide repertoire of virulence factors (Hancock and Speert 2000). Exacerbating these problems is the emergence of multi-drug resistant P. aeruginosa clinical isolates resistant to virtually all antibiotics, infamously designating these strains as ‘superbugs’ (Falagas and Bliziotis 2007). Current treatment of P. aeruginosa infections often involves potent β-lactam antibiotics, aminoglycosides or fluoroquinolones, or a combination thereof, however resistance can nevertheless arise (Mesaros et al. 2007). When multiple drug resistance occurs, such as often found in the late stages of cystic fibrosis lung disease, polymyxins have become a drug of last resort (Li et al. 2006; Zavaski et al. 2007). Thus it is important to understand the basis for resistance in this organism and its interrelationship with pathogenesis. For example, there is a well-known discrepancy discrepancy between in vitro antibiotic susceptibility and the clinical success of particular antibiotics against P. aeruginosa (Flick et al. 1972; Hanccok and Speert 2000; Karlowsky et al. 1997). One basis for this is the induction of resistance mechanisms due to environmental factors, a process termed adaptive resistance  *  A version of this chaper has been accepted for publication. Gooderham, W.J., M. Bains, J. B. McPhee, I. Wiegand, and R. E.W. Hancock. 2008. Induction by cationic antimicrobial peptides and involvement in intrinsic polymyxin and antimicrobial peptide resistance, biofilm formation and swarming motility of psrA in Pseudomonas aeruginosa. J Bacteriol. (in press)   17 that is differentiated from acquired or mutational resistance since it reverts upon removal of the antibiotic or selective pressure. Structurally diverse cationic antimicrobial peptides are part of the innate immune system of complex organisms, and can possess direct antimicrobial activity and/or a profound ability to modulate innate immunity (Jenssen et al. 2006). Directly antimicrobial peptides demonstrate considerable promise against infections by multidrug resistant bacteria (Marr et al. 2006; Hancock and Sahl 2006). However, P. aeruginosa is able to sense the presence of peptide and become adaptively resistant (Glilleland and Farley 1982), for example through peptide-mediated regulation of the arnBCADTEF (pmrHFIKLM; PA3552-9) LPS modification operon, independently of the PmrA-PmrB or PhoP-PhoQ two-component regulatory systems (McPhee et al. 2003; Moskowitz et al. 2004). Virulence is similarly complex, representing a series of complex adaptations to growth in a host organism, including biofilm formation, swarming motility, and quorum sensing. For example, P. aeruginosa motility is important for biofilm formation, virulence, and colonization of different niches (Klausen et al. 2003; O’Toole and Kolter 1998; Parsek and Greenberg 2005). Type IV pili extend and retract to promote twitching motility on solid surfaces, whereas flagella power swimming motility. On the other hand, swarming motility appears to be a coordinated and complex adaptation to low viscosity surfaces, and involves a number of factors that include flagella, type IV pili, quorum sensing, and rhamnolipids etc (Overhage et al. 2007; Overhage et al. 2008). There is considerable overlap in the genes utilized in swarming motility and biofilm formation (Caiazza et al. 2007; Shrout et al. 2006), both of which have been proposed to contribute to disease pathogenesis (Parsek and Singh 2003) and lead to increased resistance to several antibiotics (Overhage et al. 2008).  18  In this study, it was demonstrated that antimicrobial peptides transcriptionally upregulated the expression of psrA, a previously documented Pseudomonas regulator of RpoS and the type III secretion system, but one for which the activating signals were unknown (Kojic and Venturi 2001; Kojic et al. 2002; Shen et al. 2006). Detailed phenotypic studies indicate that PsrA regulated polymyxin and antimicrobial peptide resistance, motility, and biofilm formation. Microarray analysis of the psrA mutant provided insight into the basis for these observed phenotypes. MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains and plasmids used in this study are described in Table 2.1. Cultures were routinely grown in Luria–Bertani (LB) broth containing 1.8% (wt/vol) Difco agar (Becton-Dickinson Co.) when appropriate. The defined medium used was BM2- glucose minimal medium [62 mM potassium phosphate buffer (pH 7), 7 mM (NH4)2SO4, 10 µM FeSO4, 0.4% (w/v) glucose] containing 2 mM (high) MgSO4 concentrations. Antibiotics for selection were used at the following concentrations: tetracycline, 50- 100 µg/ml for P. aeruginosa; ampicillin 100 µg/ml for E. coli; carbenicillin, 500 µg/ml for P. aeruginosa; gentamicin 30 µg/ml for P. aeruginosa and 15 µg/ml for E. coli. Genetic manipulations. Routine molecular biology techniques were performed according to standard protocols (Ausubel 1987). Primers were synthesized by AlphaDNA Inc. (Montreal, QC, Canada). Plasmid DNA was isolated using QIAprep spin miniprep kits (Qiagen Inc., Canada) and agarose gel fragments were purified using a QIAquick gel extraction kit  19 (Qiagen Inc., Canada). T4 DNA ligase was from Invitrogen and restriction endonucleases were from New England Biolabs. Mobilizing the UW-psrA transposon mutation into a new PAO1 background. The UW-psrA mutation (confirmed to be correct by PCR and sequencing the junctions of the transposon mutation) was first transferred into our laboratory wild type P. aeruginosa PAO1 strain H103 as described previously (Choi et al. 2006). Genomic DNA was isolated from the UW-psrA mutant using the hexadecyltrimethyl ammonium bromide (CTAB) method (Ausubel 1987). Approximately one microgram of this DNA (which contained the tetracycline resistance-encoding transposon ISlacZ/hah-Tc insertion in psrA) was electroporated into WT H103. Cells recovered for one hour at 37°C, and then were plated onto LB agar plates containing 100 µg/ml tetracycline. After 18 hours of growth at 37°C, tetracycline resistant transformants were then analyzed by colony PCR using a transposon-specific primer and a custom gene primer together with Taq polymerase (Invitrogen Canada Inc.) to verify that the transposon was correctly inserted into psrA. This new psrA mutant allowed better analysis of motility-related phenotypes (H103 is swarm+ under our conditions, see below) and was therefore used for all experiments reported in this study. Genetic complementation of psrA. Forward and reverse primers for psrA were designed from the P. aeruginosa PA01 genome sequence (www.pseudomonas.com) using Primer3 [http://frodo.wi.mit.edu/cgi- bin/primer3/primer3_www.cgi; (Rozen and Skaletsky 2000)] to clone psrA+ together with 347 bp of upstream DNA with the native promoter and 67 bp downstream DNA. Amplification of psrA from P. aeruginosa wild-type H103 genomic DNA was carried out  20 using high-fidelity Platinum Pfx DNA polymerase (Invitrogen Canada Inc.) with primers PsrA-L 5’-CGGAGCACAGAGAAAGGAGA-3’ and PsrA-R 5’- GACTTGAAGCCGAGTTCCTG-3’. The resulting PCR product was then cleaned (Qiagen PCR Purification Kit) and the amplicons were cloned into pCR-Blunt II-TOPO using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen Inc.) and transformed into One Shot TOP10 cells (Invitrogen Inc.), creating pCR-psrA+. A Nsi1 fragment containing psrA was excised from pCR-psrA+ and subcloned into pUC18mini-Tn7T-Gm, generating pUC18-mini-Tn7T- Gm-psrA+. pUC18mini-Tn7T-Gm-psrA+ was co-electroporated with pTNS2 into the psrA mutant using sucrose electroporation (Choi et al. 2005). Gentamicin resistant transformants were analyzed by colony PCR using primers PglmS-up and PTn7L to determine correct transposon integration of mini-Tn7 into the chromosome as previously described (Choi et al. 2005). Killing curves. Overnight P. aeruginosa cultures were diluted 1/100 into fresh BM2-glucose minimal medium containing 2 mM Mg2+. Upon reaching the mid-logarithmic phase of growth (~0.5 OD600), 1 ml of each culture was pelletted and resuspended in 1 ml 1× BM2 salts (buffer), and diluted 1/10 into pre-warmed 1× BM2 salts. Killing was then initiated by the addition of 1 µg/ml polymyxin B sulphate (Sigma Inc., Oakville, Canada) or 64 µg/ml indolicidin for analysis of intrinsic resistance. Flasks were shaken at 37°C and aliquots were withdrawn at the designated times for assaying survivors by plating diluted 100 µl aliquots onto LB agar plates.    21 Outer membrane permeabilization assays. P. aeruginosa outer membrane barrier function and the efficiency of the self promoted uptake route was determined by the 1-N-phenylnapthylamine (NPN) assay (Loh et al. 1984). Cultures were grown to mid-logarithmic phase in BM2-glucose minimal medium containing 2 mM MgSO4. The cells were then harvested, washed, and resuspended to an OD600 of 0.5 in 5 mM HEPES (pH 7.0), 5 mM glucose, and 5 µM carbonyl cyanide m- chlorophenylhydrazone (CCCP). Two ml of these bacterial suspensions were placed in a quartz cuvette with a magnetic stir bar. NPN (Sigma Inc., Oakville, Canada) was then added to the cuvette at a concentration of 10 µM and the fluorescence (baseline) was measured using a LS-50B fluorescence spectrophotometer (PerkinElmer, Inc.; excitation and emission wavelengths of 350 nm and 420 nm, respectively). Indolicidin peptide was then added to initiate the assay and the increase in fluorescence due to peptide-mediated entry of NPN into the membrane was measured until a stable signal was observed (indicating that additional partitioning of NPN into the membrane had stopped). Biofilm and attachment assays. Static microtitre biofilm assays were generally performed as those previously described (O’Toole and Kolter 1998). Overnight LB cultures were diluted 1/1000 into fresh LB broth, and 100 µl was inoculated into wells of a 96-well polystyrene round bottom microtitre plate (Falcon). For PA14 strains, overnight cultures were diluted 1:500 into BM2-glucose medium containing 2 mM MgSO4 and 0.5% Casamino acids. Plates were then incubated at 37°C without shaking. At the specified time point, media and planktonic cells were dumped and the wells were washed three times with dH2O. Surfaced attached bacteria were then stained  22 with 0.1% (w/v) crystal violet for 20 min, followed by ethanol solubilization of crystal violet for quantification at A600. Rapid attachment was assayed as described previously with slight modification (Ma et al. 2006). Overnight cultures were first diluted 1/100 into fresh LB medium, grown to an OD600 of ~0.5, and 100 µl was added to each well of a 96-well polystyrene microtitre plate (Falcon). Cells were allowed to attach for 30 min at room temperature prior to staining with crystal violet as decribed above. Motility assays. Swimming motility was assayed on BM2-glucose plates containing 0.3% (wt/vol) agar. Swarming was assayed on modified BM2 glucose plates containing 0.5% (wt/vol) agar and with 0.5% (wt/vol) Casamino acids (or 0.1% for PA14 strains) substituted for 7 mM (NH4)2SO4 (Overhage et al. 2007). Swimming and swarming motility were assayed by inoculating 1 µl of mid-logarithmic growth phase liquid cultures grown in BM2-glucose containing 2 mM Mg2+ onto the motility plate, incubating for 16-18 hrs at 37°C, and measuring motility zone diameters. Twitching motility was assessed by toothpick inoculating cells from agar plates into thin LB agar (1%) plates, down to the agar-plastic interface, and measuring the twitch-zone diameter after 24 and 48 h incubation at 37°C. Growth curves. Overnight cultures were grown in BM2-glucose containing 2 mM Mg2+ and 0.1 ml was diluted into 10 ml fresh medium. Flasks were shaken at 37°C and aliquots were withdrawn periodically to determine the cell density as OD600. Similarly, determination of planktonic growth at 37°C in static 96-well polystyrene microtitre plates (simple biofilm conditions) was assayed by monitoring OD600.  23 Microarray analysis. Detailed technical descriptions of microarray analyses were provided previously (McPhee et al. 2006). In overview, for each strain, microarray analysis involved five independent cultures. P. aeruginosa WT and psrA mutant cultures were grown with shaking in BM2-glucose medium plus 2 mM MgSO4 at 37°C for 18 hrs, and then diluted 1 in 100 into fresh medium. Cultures were grown at 37°C with shaking (250 rpm) to the mid- logarithmic phase of growth (OD600=0.5) and then total RNA was isolated using RNeasy midi columns (Qiagen). Contaminating genomic DNA was removed by treatment with a DNA-free kit (Ambion Inc., Austin, TX). RNA was stored at −80°C with 0.2 U/µl of SUPERase-In RNase inhibitor (Ambion Inc., Austin, TX). RNA quality was assessed by agarose gel electrophoresis and spectrophotometrically. RNA was converted to cDNA, hybridized, and analyzed as previously described (McPhee et al. 2006). P. aeruginosa PAO1 microarray slides were provided by The Institute for Genomic Research (TIGR) Pathogenic Functional Genomics Resource Center (http://pfgrc.tigr.org/). Images of slides were quantified using ImaGene 6.0 Standard Edition software (BioDiscovery, Inc., El Segundo, CA). ArrayPipe version 1.7 was used for assessment of slide quality, normalization, detection of differential gene expression, and statistical analysis using available genome annotation from www.pseudomonas.com. Data analysis of DNA microarrays was carried out as previously described (McPhee et al. 2006). The averaging of the five biological replicates were performed to obtain overall fold changes for psrA mutant relative to wild- type and  a two-sided one-sample Student’s t test was applied to determine significant changes in gene expression. Fold changes greater than or equal to 2.0 with a Student’s t test P value of <0.05 were used as the cut-offs for reporting expression changes.  24 Real-time qPCR. Total RNA was isolated from P. aeruginosa grown in BM2-glucose minimal media containing 2 mM Mg2+ using RNeasy midi columns (Qiagen), with or without 2 µg/ml indolicidin, to the mid-logarithmic phase of growth. DNase treatment of RNA samples, cDNA synthesis, and real-time qPCR was carried out as described previously (McPhee et al. 2006): cDNA was diluted 1/1000, and 1 µl was used as template for real-time PCR using 1× SYBR Green PCR Master Mix (Applied Biosystems, Foster, CA) in an ABI Prism 7000 (Applied Biosystems, Foster, CA). Forward and reverse primers were designed internal to psrA using PrimerExpress (Applied Biosystems, Foster, CA). All reactions were normalized to the rpsL gene encoding the 30S ribosomal protein S12. RESULTS Activation of psrA transcription in response to antimicrobial peptides. Preliminary microarray analysis of the P. aeruginosa response to sub-inhibitory indolicidin antimicrobial peptides (2 µg/ml) indicated that the psrA (PA3006) gene was 2.5- fold induced (P <0.05) during growth in the presence of this peptide. This preliminary result utilized cultures grown to mid-logarithmic phase under high Mg2+ (2 mM MgSO4) conditions with or without sub-inhibitory indolicidin, a 13-amino acid endogenous cationic host defense (antimicrobial) peptide from bovine neutrophils (Falla et al. 1996; Selsted et al. 1992). Independent real-time qPCR experiments confirmed that the transcription of psrA was 3.0 ± 0.3 fold up-regulated in the presence of indolicidin, with similar induction by the indolicidin-variant peptide CP11CN (data not shown). Indolicidin was chosen as it is capable of causing strong induction of pmrA-pmrB and arnBCADTEF (aminoarabinaose LPS modification) operons, stronger induction than  25 polymyxin B is capable of (McPhee et al. 2003), and the concentration used (2 µg/ml = one eighth MIC) was sub-inhibitory and caused no growth impairment towards wild-type or psrA mutant cells (data not shown). In addition, growth of cells in high Mg2+ with peptide was chosen to prevent low Mg2+-mediated induction of resistance, which is believed to involve different mechanisms than that of peptide-induced resistance (McPhee et al. 2003). As a positive control, in agreement with previous studies, arnB (first gene of the arnBCADTEF operon), was confirmed here by RT-qPCR to be 54 ± 8 up-regulated under these conditions, and microarray data confirmed that the indolicidin-regulated pmrA-pmrB operon was also upregulated in the presence of sub-inhibitory indolicidin, but the Mg2+- regulated oprH-phoP-phoQ operon was not. Contribution of psrA to intrinsic antimicrobial peptide and polymyxin B resistance. The influence of the psrA gene on intrinsic resistance to peptides was examined. Intrinsic resistance was assayed by growing cells under high (2 mM) Mg2+ conditions to suppress the possibility of induction by limiting Mg2+. The psrA mutant exhibited an intrinsic increased susceptibility to the polycationic lipopeptide polymyxin B as shown by killing curves (Fig. 2.1A). This super-susceptibility phenotype could be complemented to normal wild-type polymyxin B susceptibility by introducing a single wild-type psrA+ allele into the chromosome of the mutant by mini-Tn7 integration technology (Fig. 2.1A). Similarly, the psrA mutant demonstrated super-susceptibility to the cationic antimicrobial peptide indolicidin, which could be complemented back to wild-type susceptibility (Fig. 2.1B). Thus the psrA gene product appeared to be essential for normal expression of intrinsic peptide resistance.   26 The psrA mutation affected the permeabilization of the outer membrane. Polycationic molecules like polymyxin B and antimicrobial peptides pass across the outer membrane by self-promoted uptake. The first stage of self promoted uptake involves the interaction of the polycation with divalent cation binding sites on surface polyanionic LPS causing a disruption of the permeability barrier and subsequent uptake of the permeabilizing polycationic antibiotic. To address the possibility that altered outer membrane permeability was the basis for peptide super-susceptibility in the psrA mutant, NPN was used as probe for outer membrane permeabilization by indolicidin (Fig. 2.2). The hydrophobic fluorophore NPN is normally excluded from entering cells due to its inability to penetrate the outer membrane. Upon permeabilization of the outer membrane (as occurs during self promoted uptake) NPN is taken up and becomes strongly fluorescent in the nonpolar/hydrophobic environment of cell membranes. There was no obvious difference in the ability of psrA and wild type to exclude NPN. However, indolicidin at concentrations of 1.5 and 3.0 µg/ml was able to permeabilize the outer membranes of the psrA mutant to a greater extent than those of wild-type cells (Fig. 2.2A). Thus the super-susceptibility of the psrA mutant to indolicidin correlated with an outer membrane that was more easily permeabilized by this antimicrobial peptide. Similarly, polymyxin B also preferentially permeabilized the psrA mutant (Figure 2.2B). Contribution of psrA to biofilm formation and attachment. Other genes such as PhoQ, that regulate antimicrobial peptide resistance, also regulate biofilm formation and motility (Ramsey and Whiteley 2004). To assess the ability of the psrA mutant to form simple biofilms, static microtitre biofilm assays were employed. These experiments demonstrated that the psrA mutant displayed significant (P <0.001 by Student’s  27 t test) impairment in biofilm formation at 18 h, by more than 4-fold (Fig. 2.3A). Biofilm impairment could be successfully complemented by introducing the wild-type psrA allele into the mutant (Fig. 2.3A). No observable growth differences were observed when the OD600 of planktonic cells was measured as a function of time during the period of growth in the microtitre wells (Fig. 2.3B). Similarly, assessment of growth in defined medium in shaking flasks revealed no differences between mutant and wild type (data not shown), indicating no primary growth defect.  To determine whether this biofilm formation phenotype occurred during initial attachment stage or later during biofilm development, a rapid (30 min) attachment assay was performed. The psrA mutant displayed more than two-fold (P <0.001 by Student’s t test) impaired attachment, and this defect could be complemented with the psrA+ gene (Fig. 2.3C). Light microscopic observations of both psrA mutant and wild type cultures showed no obvious morphological differences under these conditions that might contribute to this phenotype. Requirement for PsrA for normal swarming. Mutant studies have revealed an intricate relationship between motility and biofilm formation in P. aeruginosa (Klausen et al. 2003; O’Toole and Kolter 1998). Therefore, the psrA mutant was assessed for its ability to undergo swimming, twitching, and swarming motility. Neither flagella-mediated swimming nor type IV-pilus-mediated twitching motility were significantly affected in the psrA mutant (data not shown). However, the psrA mutant demonstrated a severe impairment in swarming motility with a more than 2.5-fold (P <0.01 by Student’s t test) decrease in swarming zone size (Fig. 2.4). Introducing the wild-type  28 psrA allele restored wild-type swarming in the mutant, demonstrating that psrA is necessary for normal swarming motility (Fig. 2.4). Microarray analysis. The above-described complexity of phenotype indicated that PsrA might control the expression of a substantial regulon. To assess this and to identify candidate genes that might explain the observed psrA mutant phenotypes, microarray analysis was performed comparing the psrA mutant to wild type after growth to mid-logarithmic phase in BM2- glucose minimal medium containing 2 mM Mg2+. There were a total of 178 genes that were significantly (P ≤0.05) dysregulated ≥2-fold of which 70 were up-regulated and 108 down- regulated in the mutant relative to the wild-type. A selection of these genes (not including hypothetical and unclassified ORFs) is shown in Tables 2.2-2.4. Most previously identified genes with predicted PsrA binding sites in their promoters (Kojic et al. 2005) were identified by this microarray analysis (Table 2.2). In addition we observed dysregulation of the entire type III secretion apparatus and its effectors, certain adhesion and motility genes, 17 regulators (rpoS, pcrH, mdcR, toxR, arsA, PA0513, PA1399, PA1976, PA1978, PA2432, PA2469, PA2551, PA3077, PA3409, PA3630, PA4135, and PA4296), and a variety of metabolic and energy metabolism genes. Independent RT-qPCR analysis confirmed the regulation of 6 of these genes (indicated by * in Tables 2.2-2.4) and thus provided validation for our psrA microarray data. Additional mutant phenotypic analyses. The list of genes dysregulated in the psrA mutant provided a useful starting point towards understanding the basis for the observed psrA mutant phenotypes. To understand  29 the phenotypes associated with selected dysregulated genes, transposon mutants from the PA14 comprehensive non-redundant library were utilized (Table 2.1; Liberati et al. 2005). The microarray was examined to find genes that might influence peptide susceptibility (since the microarray and time kill experiments used similar growth conditions). The dysregulation of several genes of the wbp gene cluster (Table 2.4), which is involved in the biosynthesis of B-band (serotype O antigen) LPS (Burrows et al. 1996), suggested a possible role for B-band LPS in the observed supersuceptibility of the psrA mutant. Therefore, in order to look at the requirement of B-band LPS, we analyzed mutants in wbpI and wbpL, as well as a mutant in wbpM that had been previously shown to lack B-band LPS (Burrows et al. 1996; Burrows et al. 2000). In addition, a small panel of PA14 mutants including others related to energy metabolism was tested, since our preliminary unpublished observations have indicated a role for energy metabolism in resistance to antimicrobial peptides. It was hypothesized that one or more of these mutants would show peptide super-susceptibility, thereby indicating a putative contribution of the dysregulated genes in the intrinsic super- susceptibility of the psrA mutant. As shown in Fig. 2.5A, both wbpM and coxA (cytochrome C oxidase subunit 1) mutants showed modest super-susceptibility to indolicidin relative to wild-type at 25 min. No differences were seen for mutants in fhp, PA1883, mexC and wzz (data not shown). Unfortunately, an etfA (energy metabolism) mutant clumped during growth which made performing killing curves on this mutant difficult. No differences in O antigen chain length expression were seen when LPS was isolated from WT and psrA mutant and analyzed by SDS-PAGE and silver staining (data not shown). However, as small changes in LPS that influence functionality (such as substitution by  30 sugars, phosphates and fatty acids) could be affected by psrA mutation, we analyzed, mutants in wbpI, wbpL (both downregulated in the psrA mutant) and wbpM for possible outer membrane permeability phenotypes. Like the psrA mutant, all three of these mutants in LPS B-band biosynthetic genes showed increased outer membrane permeabilization by peptides relative to wild-type (Fig. 2.6A, B), although the effect observed with the wbpM mutant was more prominent (Fig. 2.6B). A panel of PA14 mutants were also analyzed for possible roles in swarming motility and biofilm formation. The wbpM mutant showed a significant swarming impairment phenotype, as did an etfA mutant, although in the latter mutant this might be related to the tendency of this mutant to clump (Fig. 2.5B and 2.5C). No swarming differences were seen for coxA and wzz (Fig. 2.5C) or mexC and PA1883 mutants (data not shown). The downregulation of certain genes of the type IVb pilus-encoding tad cluster (de Bentzmannn et al. 2006) led us to analyze mutants in these genes for possible biofilm formation phenotypes. In simple biofilm growth conditions, none of the tad mutants analyzed displayed biofilm impairment (Fig. 2.5D), confirming previously reported results (de Bentzmann et al. 2006). However, a pprB (PA4296) mutant, encoding a two-component response regulator located adjacent to the tad cluster and substantially down-regulated on the psrA arrays (Table 2.3) did demonstrate a significant (P <0.001 by Student’s t test) biofilm impairment phenotype (3-fold) at the time point analyzed (Fig. 2.5D). DISCUSSION The psrA gene of P. aeruginosa is an important regulator of both resistance to cationic antimicrobials and virulence features. It was up-regulated in response to the cationic antimicrobial peptide indolicidin and mediates intrinsic cationic peptide resistance and  31 certain virulence-related processes such as biofilm formation, rapid attachment, swarming motility. The involvement of PsrA in these phenotypes was supported by studies of a psrA mutant and single-copy complementation of the psrA defect. PsrA was previously characterized by the Venturi group as a positive regulator of transcription of the alternative sigma factor RpoS (Kojic and Venturi 2001; Kojic et al. 2002), as also confirmed here. In other Pseudomonas spp, PsrA is known to be involved in antifungal metabolite production (Chin et al. 2005) and the regulation of quorum sensing (Chatterjee et al. 2007). However, the direct signals that activate psrA were unknown, and the data here now demonstrates that the cationic antimicrobial peptide indolicidin is an activating signal for transcription, consistent with other studies demonstrating that peptides are key regulators of bacterial virulence and resistance processes (Bader et al. 2005). The demonstration that psrA contributes to cationic peptide resistance adds another regulator to the increasingly complex regulatory network influencing resistance, which already includes PhoP-PhoQ and PmrA-PmrB (Ernst et al. 1999; Macfarlane et al. 1999; McPhee et al. 2003; Moskowitz et al. 2004). However unlike these two-component regulators which mediate an increase in resistance to peptides in limiting Mg2+ growth conditions, PsrA mediates intrinsic resistance. Thus unlike psrA, pmrA and phoP mutants do not demonstrate supersusceptibility under non-inducing conditions and there appears to be no obvious regulatory hierarchy since psrA was not apparently transcriptionally regulated by PmrA or PhoP (or vice versa), nor was there any substantial overlap in dysregulated genes (McPhee et al. 2006). Polymyxin B and cationic antimicrobial peptides passage into cells via the self-promoted uptake pathway which involves interaction with divalent cation binding sites on LPS and subsequent distortion of the outer membrane permeability barrier (Falla et  32 al. 1996; Gilleland and Farley 1982; Jenssen et al. 2006). All three systems however appear to mediate resistance by influencing the ability of cationic agents to permeabilize outer membranes and we were able to demonstrate for PsrA that the increase in permeabilization by cationic agents (Fig. 2.2) correlated with the supersusceptibility of the psrA mutant to polymyxin B and indolicidin (Fig. 2.1). There were strong candidate genes that were dysregulated in the psrA mutant that probably contributed to the psrA peptide super-susceptibility phenotype through changes in outer membrane permeability. Three genes (wbpG, wbpI, wbpL; Table 2.4) involved in LPS B-band (serotype O antigen) biosynthesis were down-regulated by 2.0-to-4.4 fold suggesting that the entire wbpGHIJKLM locus would be transcriptionally repressed (consistent with the intrinsic limitations of microarray experiments; Table 2.4). Supporting this link between peptide super-susceptibility and outer membrane permeability of the psrA mutant, mutants in two genes (wbpI and wbpL) downregulated on the psrA array displayed increased outer membrane permeability (Fig. 2.6A). Further, a wbpM mutant, which has been previously demonstrated to be B-band deficient and necessary for LPS O antigen biosynthesis (Burrows et al. 1996; Burrows et al. 2000) was shown here to be peptide supersusceptible (Fig. 2.5A), and swarming deficient (Fig. 2.5B), and display substantial increased outer membrane permeability (Fig. 2.6B). This correlates with previous findings in Proteus mirabilis that LPS O antigen contributes to both antimicrobial peptide resistance and swarming motility (McCoy et al. 2001). Other possible candidates to explain peptide supersusceptibility would be gene products involved in energy generation and thus potentially in interaction of cationic peptides with the cytoplasmic membrane. One of the tested genes, coxA, encoding a subunit of cytochrome C oxidase, was 3.4-fold down-regulated in the arrays (Table 2.4) and a  33 mutant in this gene led to a modest supersusceptibilty phenotype relative to wild-type (Fig. 2.5A). The substantial swarming motility impairment displayed by the psrA mutant indicated that PsrA is involved in the complex regulatory mechanisms controlling this complex adaptation (Overhage et al. 2008). Although swarming requires both flagella and pili (Overhage et al. 2007), the psrA mutant did not exhibit a defect in either flagella-mediated swimming or type IV-pilus-mediated twitching motility, indicating that it did not control a primary motility organelle. PsrA regulation of swarming might reflect the down-regulation of both Lon protease (Table 2.3), which is required for normal swarming (Marr et al. 2007), and the LPS O antigen B-band biosynthetic gene cluster, since the wbpM mutant was also swarming deficient (Fig. 2.5B). Our observation that LPS O antigen biosynthetic genes (wbpG, wbpI, wbpL; Table 2.4) were down-regulated in the psrA mutant also may help explain the swarming impairment as LPS O antigen is required for swarming motility in other bacteria (Belas et al. 1995; McCoy et al. 2001; Toguchi et al. 2000). Biofilm formation in P. aeruginosa is initiated by initial attachment of cells to a surface, followed by complex steps leading to development of mature biofilms (Parsek and Greenberg 2005). The psrA biofilm impairment phenotype was likely related in part to early stages, as the psrA mutant displayed a significant impairment in rapid attachment to the polystyrene surface used for the simple biofilm experiments described here (Fig. 2.3C). Although psrA mutants were able to attach and form simple biofilms, they did this at significantly reduced levels comparable to wild-type. Two possible genes that might influence the regulation of biofilm formation by PsrA are lon (Marr et al. 2007) and the pprB response regulator gene (Fig. 2.5D; found directly adjacent to the tad gene cluster) in  34 the psrA mutant microarray since mutants in both displayed impaired biofilm formation. The finding that the psrA mutant displays both impaired biofilm formation and swarming motility suggests that PsrA is an integral component of the regulatory network that controls these two separate complex adaptations, and is consistent with observations that other regulators control both processes (Shrout et al. 2006; Caiazza et al. 2007; Overhage et al. 2007). Our results are consistent with previous observations that RpoS is a negative regulator of the type III secretion system, since rpoS is positively regulated by PsrA (Table 2.2; Kojic and Venturi 2002; Kojic et al. 2005). PsrA was previously shown to be a positive regulator of the type III secretion system in a mucoid strain of P. aeruginosa grown in complex medium (Shen et al. 2006). In contrast, the data presented here favor negative regulation of this secretion system by PsrA in the non-mucoid P. aeruginosa strain PAO1 grown in defined medium. We presume this is because of other underlying regulatory mutations that are known to occur in mucoid isolates of P. aeruginosa. Consistent with these observations, the psrA mutant presented here had no effect on cytotoxicity towards epithelial cells which is partially dependent on type III secretion (data not shown). PA0506, an acyl-CoA dehydrogenase, was highly up-regulated in the psrA mutant (43- fold according to qRT-PCR confirmation experiments). This gene was also a previously characterized target of PsrA (Kojic et al. 2005) and our microarray analysis confirmed PsrA as a negative regulator of this gene (Table 2.2). Noteworthy, PA0506 has previously been shown to be mutated in cystic fibrosis P. aeruginosa isolates, possibly indicating that mutation of this gene might favor chronic infection and that this gene might be involved in adaptation to the cystic fibrosis lung (Smith et al. 2006).  35 Our microarray gene lists uncovered many other interesting genes as part of the PsrA regulon. The downregulation of genes of the tad (tight adherence) cluster (Table 2.3), involved in the assembly of extracellular cell-surface Flp pili appendages (de Bentzmann et al. 2006; Tomich et al. 2007), was consistent with the attachment defect in psrA mutants in the face of normal piliation and twitching motility. However, no differences were seen in biofim formation by mutants in key components of the tad cluster (Fig. 2.5D). Probable type II secretion system genes (PA0683, PA2672, PA2673) also showed modest to strong repression (Table 2.2 and 2.3) and could encode adhesion-associated products (based on the similarity of pili to the components of the type II secretion system), and thus might contribute to the attachment and biofilm phenotype observed for the psrA mutant. Biofilm formation, attachment, and swarming motility appear to be very important in P. aeruginosa colonization and virulence, while it has been strongly suggested that Pseudomonas is exposed to cationic antimicrobial peptides during infections and occasionally polymyxin B during therapy. The involvement of PsrA in these processes, together with its inducibility by a cationic antimicrobial peptide, highlights the likely importance of this gene in adaptation to the lung environment through regulation of virulence and antimicrobial peptide resistance. The results presented here are consistent with the massive complexity of the regulatory network influencing these processes.  36 Table 2.1 P. aeruginosa strains and plasmids used in this study.  Strain or plasmid Genotype or characteristicsa  Source or reference WT Wild-type P. aeruginosa PAO1; H103 Lab collection UW WT UW wild-type P. aeruginosa PAO1 Jacobs et al. 2003 UW-psrA psrA::ISlacZ/hah-TcR; insertion at 46(702 bp) in psrA; derived from UW WT Jacobs et al. 2003 psrA mutant psrA::ISlacZ/hah-TcR, H103 background; TcR This study psrA (Tn7-psrA+) psrA mutant with Tn7-psrA+ integrated; TcR, GmR This study PA14 Wild-type P. aeruginosa PA14 coxA 05_2:A11;  derived from PA14 etfA 04_4:A12;  derived from PA14 fhp 09_1:F11;  derived from PA14 mexC 01_4:H2;  derived from PA14 pprB 08_3:C3;  derived from PA14 rhlG 05_3:A8;  derived from PA14 flp 14_1:F4;  derived from PA14 rcpA 01_2:B12;  derived from PA14 tadA 01_2:A7;  derived from PA14 tadB 04_2:H5;  derived from PA14 wbpM 03_4:E4;  derived from PA14 wzz 06_1:F2l;  derived from PA14 PA1883 (homolog) 12_1:A7; derived from PA14 Liberati et al. 2006 Liberati et al. 2006 Liberati et al. 2006 Liberati et al. 2006 Liberati et al. 2006 Liberati et al. 2006 Liberati et al. 2006 Libertai et al. 2006 Liberati et al. 2006 Liberati et al. 2006 Liberati et al. 2006 Liberati et al. 2006 Liberati et al. 2006 Liberati et al. 2006 wbpI wbpI:: ISlacZ/hah-TcR; insertion 807(1065 bp) Jacobs et al. 2003 wbpL wbpL:: ISlacZ/hah-TcR; insertion 302(1020 bp)  Jacobs et al. 2003  E .coli TOP10 F- mcrA (mrr-hsdRMS-mcrBC) 80lacZ M15 lacX74 recA1 ara 139 (ara-leu)7697 galU galK rpsL (StrR) endA1 nupG Invitrogen Inc. pCR-Blunt II-TOPO PCR cloning vector, KanR Invitrogen Inc. pCR-psrA+ pCR-BluntII-TOPO harboring 1.12 kb psrA amplicon This study pUC18-mini-Tn7T- Gm Suicide plasmid; GmR, AmpR Choi et al. 2005 pUC-Tn7-psrA+ pUC18-mini-Tn7T-Gm with 1.12 kb psrA fragment from pCR-psrA This study pTNS2 Transposition helper plasmid; AmpR Choi et al. 2005  a  Antibiotic resistance phenotypes: AmpR, ampicillin for E. coli and carbenicillin for P. aeruginosa; GmR, gentamicin; KanR, kanamycin; TcR, tetracycline.    37 Table 2.2 Known PsrA targets significantly dysregulated in psrA mutants as determined using microarray. Only genes showing ≥2-fold change in the psrA mutant are depicted. * indicates confirmation of gene regulation by qRT-PCR.  Gene IDa Name Fold changeb P value Descriptiona PA0506  16.2* <0.0001 probable acyl-CoA dehydrogenase PA2673 hplV −27.9 <0.0001 probable type II secretion system protein PA2951 etfA 3.3 <0.0001 electron transfer flavoprotein alpha-subunit PA2952 etfB 3.8 <0.0001 electron transfer flavoprotein beta-subunit PA2953  6.4* <0.0001 electron transfer flavoprotein-ubiquinone oxidoreductase PA3013 foaB 15.2* <0.0001 fatty-acid oxidation complex beta-subunit PA3014 faoA 11.6 <0.0001 fatty-acid oxidation complex alpha-subunit PA3622 rpoS −2.5* <0.0001 alternative sigma factor RpoS  a  Information according to the P. aeruginosa genome website (www.pseudomonas.com/) b  Fold regulation of genes differentially expressed in the psrA mutant relative to WT. A positive number indicates transcript up-regulation in the psrA mutant.   38 Table 2.3 Type III secretion, adhesion (tad), motility, and type II secretion genes significantly dysregulated in psrA mutants as determined using microarray. * indicates confirmation of gene regulation by qRT-PCR.  Gene IDa Name Fold changeb P value Descriptiona Type III secretion PA0044 exoT 4.6 0.0005 exoenzyme T; Type III secretion system effector PA1695 pscP 3.0 <0.0001 translocation protein in type III secretion PA1697 pscN 2.1 0.004 ATP synthase in type III secretion system PA1698 popN 2.6 0.004 outer membrane protein PopN PA1699  2.3 0.003 conserved protein in type III secretion PA1700  2.0 0.0009 conserved protein in type III secretion PA1701  4.5 0.0003 conserved protein in type III secretion PA1702  2.0 0.0002 conserved protein in type III secretion PA1703 pcrD 2.0 0.01 type III secretory apparatus protein PcrD PA1705 pcrG 3.8 0.002 regulator in type III secretion PA1706  2.1 0.001 type III secretion protein PcrV PA1707 pcrH 2.4 0.0002 regulatory protein PcrH PA1708 popB 3.1 0.005 translocator protein PopB PA1709 popD 2.7 0.002 translocator protein PopD PA1710 exsC 2.1 <0.0001 exoenzyme S synthesis protein C PA1712 exsB 2.0 0.001 exoenzyme S synthesis protein B PA1715 pscB 2.5 0.001 type III export apparatus protein PA1717 pscD 2.8 0.01 type III export protein PscD PA1721 pscH 2.0 0.01 type III export protein PscH PA1723 pscJ 2.1 0.01 type III export protein PscJ PA2191 exoY 2.1 0.0004 adenylate cyclase ExoY PA3841 exoS 2.6 0.001 exoenzyme S; Type III secretion effector PA3842 orf1 3.5 0.001 chaperone for ExoS secretion Adhesion and motility PA0176 aer2 −4.2 0.04 aerotaxis methyl-accepting chemotaxis protein PA1803 lon −2.0 0.05 ATP-dependent Lon protease PA4296 pprB −4.9* 0.05 PprB two-component response regulator PA4300 tadC −2.2 0.04 Flp pilus assembly protein, PilC-like PA4302 tadA −4.1* 0.01 TadA traffic ATPase in Flp pilus assembly PA4303 tadZ −2.7 0.02 Flp pilus assembly protein PA4305 rcpC −2.1 0.05 Flp pilus assembly protein Type II secretion PA0683 hxcY −4.4 0.008 Hxc type II secretion system membrane protein PA1871 lasA 10.5 0.002 LasA protease PA2672 hplW −2.5 0.003 type II secretion prepilin peptidase substrate  a  Information according to the P. aeruginosa genome website (www.pseudomonas.com/) b  Fold regulation of genes differentially expressed in the psrA mutant relative to WT. A positive number indicates transcript up-regulation in the psrA mutant.  39 Table 2.4 Other known genes significantly dysregulated in psrA mutants as determined using microarray. Dysregulated hypothetical or unclassified ORFs are not included. Only genes showing ≥2-fold change in the psrA mutant are depicted.  Gene IDa Name Fold changeb P value Descriptiona PA0106 coxA −3.4 0.01 cytochrome c oxidase, subunit I PA0217 mdcR −4.8 0.007 transcriptional regulator PA0459 clpC −3.5 0.05 ClpA/B protease ATP binding subunit PA0507  −3.2 0.001 probable acyl-CoA dehydrogenase PA0511 nirJ 4.8 0.007 heme d1 biosynthesis protein PA0512 nirH 2.3 0.04 conserved hypothetical protein PA0513 nirG 4.5 0.008 probable transcriptional regulator PA0517 nirC −2.6 0.001 c-type cytochrome PA0530  −4.3 0.04 pyridoxal phosphate-dependent aminotransferase PA0588 yeaG −3.3 0.001 conserved hypothetical protein PA0707 toxR −4.2 0.009 ToxR/RegA transcriptional regulator PA0719  −4.1 0.009 bacteriophage Pf1 protein PA0724 coaA 2.8 0.02 coat protein A of bacteriophage Pf1 PA0840  −8.9 0.006 probable oxidoreductase PA0852 cbpD 2.2 0.04 chitin-binding protein CbpD PA1041  −12.9 0.005 OmpA-family outer membrane protein PA1173 napB −2.6 0.02 cytochrome c-type protein NapB PA1187 lcaD −2.1 0.04 acyl-CoA dehydrogenase PA1399  −2.0 0.004 Probable LysR-family transcriptional regulator PA1648  −3.0 0.002 probable oxidoreductase PA1649  −6.3 0.0003 probable short-chain dehydrogenase PA1650  −2.0 0.008 probable transporter PA1828  −3.1 0.006 probable short-chain dehydrogenase PA1881  −2.2 0.03 probable oxidoreductase PA1883  −10.2 0.002 NADH-ubiquinone/plastoquinone oxidoreductase PA1927 metE 2.1 0.007 methionine synthase PA1976  −2.2 0.0009 two-component sensor kinase PA1978 agmR −2.9 0.003 two-component response regulator PA1982 exaA −2.7 0.03 quinoprotein ethanol dehydrogenase PA1983 exaB −2.7 0.02 cytochrome c550 PA1984 exaC −3.6 0.05 aldehyde dehydrogenase PA1985 pqqA −4.5 0.002 pyrroloquinoline quinone biosynthesis protein A PA2124  3.2 0.01 probable dehydrogenase PA2277 arsR  0.05 transcriptional regulator PA2278 arsB  0.02 ion transport membrane protein PA2339 mtlF −2.1 0.05 maltose/mannitol transport protein PA2350  3.1 0.02 probable ATP-binding component of ABC transporter PA2352  2.4 0.03 probable glycerophosphoryl diester phosphodiesterase PA2371 clpV3 −3.0 0.02 probable ClpA/B-type protease PA2396 pvdF 2.1 0.05 pyoverdine synthetase F  40 Gene IDa Name Fold changeb P value Descriptiona PA2398 fpvA 2.5 0.03 ferripyoverdine outer membrane receptor PA2432  5.8 0.03 probable transcriptional regulator PA2469  −4.1 0.01 probable transcriptional regulator PA2522 czcC −3.7 0.01 outer membrane efflux protein PA2535  −2.3 0.05 probable oxidoreductase PA2536 ynbB 2.2 0.03 phosphatidate cytidylyltransferase PA2550  −3.7 0.001 probable acyl-CoA dehydrogenase PA2551  −2.3 0.001 probable transcriptional regulator PA2573  −2.9 0.02 probable chemotaxis transducer PA2664 fhp −83.1 0.0004 flavohemoprotein PA2892  −2.2 0.04 probable short-chain dehydrogenase PA2893  −2.4 0.03 probable very-long-chain acyl-CoA synthetase PA2939 pepB −4.9 0.007 secreted aminopeptidase PA3077  2.5 0.03 two-component response regulator PA3145 wbpL −2.5 0.03 WbpL rhamnosyltransferase in LPS biosynthesis PA3148 wbpI −4.4 0.04 UDP-N-acetylglucosamine 2-epimerase WbpI PA3150 wbpG −2.0 0.05 LPS biosynthesis protein WbpG PA3152 hisH2 −2.3 0.03 glutamine amidotransferase PA3277  −4.2 0.0003 probable short-chain dehydrogenase PA3327  −2.7 0.02 probable non-ribosomal peptide synthetase PA3387 rhlG −2.7 0.0002 beta-ketoacyl reductase PA3409  −2.1 0.05 probable transmembrane sensor PA3418 ldh −3.0 <0.0001 leucine dehydrogenase PA3427  −2.6 0.002 probable short-chain dehydrogenases PA3454  −2.1 0.002 probable acyl-CoA thiolase PA3630  2.2 0.05 probable transcriptional regulator PA3723 yqiM −3.0 0.005 FMN oxidoreductase PA3877 narK1 −3.8 0.0008 nitrite extrusion protein 1 PA3957  3.7 0.01 probable short-chain dehydrogenase PA4135  −2.9 0.02 probable transcriptional regulator PA4497  −4.1 0.01 binding protein component of ABC transporter PA4599 mexC −2.0 0.008 RND multidrug efflux membrane fusion protein PA4654  −5.6 0.02 major facilitator superfamily (MFS) transporter PA4911  −6.6 0.004 probable permease of ABC amino acid transporter PA5020  −3.7 0.003 probable acyl-CoA dehydrogenase PA5097 hutT 2.7 0.02 amino acid permease PA5141 hisA 2.8 0.02 histidine biosynthesis protein PA5187  −3.2 0.0004 probable acyl-CoA dehydrogenase PA5188  −2.0 0.0004 probable 3-hydroxyacyl-CoA dehydrogenase PA5234  −2.2 0.005 probable oxidoreductase PA5302 dadX −3.3 0.02 catabolic alanine racemase a  Information according to the P. aeruginosa genome website (www.pseudomonas.com/) b  Fold regulation of genes differentially expressed in psrA mutant relative to WT. A positive number indicates transcript up-regulation in the psrA mutant.  41 Figure 2.1 0.0001 0.001 0.01 0.1 1 10 100 0 5 10 15 20 25 Time (min) Su rv iv a l (% ) WT psrA psrA +Tn7-psrA Su rv iv al  (% ) Ti  (% ( r +) Su rv iv a l (% ) Su rv iv al  (% )   0.001 0.01 0.1 1 10 100 0 10 20 30 Time (min) Su rv iv al  (% ) WT psrA psrA (Tn7- psrA+) Su rv iv al  (% )  Figure 2.1 Intrinsic polymyxin B and antimicrobial peptide super-susceptibility in psrA mutants. Intrinsic sensitivity was analyzed by first growing cells to mid-log in BM2- glucose with 2 mM Mg2+, then exposing them to 1 µg/ml polymyxin B (A) or 64 µg/ml indolicidin (B), and plating diluted aliquots for survivors. For each condition, one representative experiment is shown of 4 independent experiments that produced identical trends. A B  42 Figure 2.2 0 100 200 300 400 500 600 0 50 100 150 200 250 300 Time (s) Fl u o re sc en ce  (A U ) WT 3.0 psrA 3.0 WT 1.5 psrA 1.5 T 3.0 T 1.5 psr psr 3.0 Fl u o re sc en ce  (A U )  0 100 200 300 400 500 0 50 100 150 200 Time (s) Fl u o re sc en ce  (A U ) WT psrA  Figure 2.2 PsrA mutation effect on outer membrane permeabilization to peptides. Cells from mid-logarithmic phase cultures of wild-type and psrA mutant were exposed to 1.5 or 3 µg/ml of indolicidin (A) or 0.2 µg/ml of polymyxin B (B) and the increase in fluorescence due to peptide-stimulated partitioning of NPN into the outer membrane was measured. NPN experiments were repeated independently three times, each of which produced reproducible observed trends, and one representative experiment is shown. A B  43 Figure 2.3  0 0.05 0.1 0.15 0.2 0.25 0.3 WT psrA psrA+Tn7psrA R el at iv e bi o fil m  (A 60 0) psrA psrA (Tn7-psrA+) R el at iv e bi o fil m  (A 60 0)  0.01 0.1 1 0 100 200 300 400 500 Time (min) B ac te ria l c o n ce n tr at io n  (O D 60 0) WT psrA T srA B ac te ria l c o n ce n tr at io n  (O D 60 0)  A B  44 0 0.02 0.04 0.06 0.08 0.1 WT psrA psrA+Tn7psrA A tta c hm e n t (A  60 0) WT psrA psrA (Tn7-psrA+) A tta c hm e n t (A  60 0)   Figure 2.3 Defects in biofilm formation and attachment in psrA mutants. (A) Requirement for psrA in static biofilm formation. Cells were grown at 37°C for 18 h in polystyrene microtitre plates containing LB. Adherent biofilm cells were stained with crystal violet followed by ethanol solubilization of the crystal violet and quantification (A600) of stained wells. (B) Planktonic growth of psrA mutant under these biofilm conditions was unaffected. Planktonic cells were grown as in biofilm microtitre assays and turbidity was measured (OD600). (C) Requirement of psrA for rapid attachment. Rapid attachment was assayed using mid-log cells for 30 min. Attached cells were stained with crystal violet followed by ethanol extraction of the crystal violet for quantification at A600. Results are shown as averages ± standard deviation for three biological experiments, each with eight technical repeats.                     C  45 Figure 2.4 0 10 20 30 40 50 60 70 WT psrA psrA + Tn7psrA Sw a rm  zo n e  (m m ) WT psrA psrA (Tn7-psrA+) Sw a rm  zo n e  (m m )        Figure 2.4 Swarming motility defect in psrA mutants. (A) Swarming motility was evaluated by spot inoculating cells onto BM2 swarm plates containing 0.5% agar at 37°C for 18 h. Diameters of the characteristic circular PAO1 swarm zones were measured and averages ± standard deviation are reported for three biological repeats each with three technical repeats. (B) Representative WT (top) and psrA mutant (bottom) swarming morphology. (C) Complemented psrA mutant swarming morphology: WT (top) and psrA (Tn7-psrA+; bottom). psrA (Tn7- psrA+) psrA WT WT A B C  46 Figure 2.5 0.1 1 10 PA14 wbpM coxA Su rv iv al  (% ) wbp Su rv iv al  (% )         wbpM WT rhlG etfA wzz coxA A B C  47 0 0.02 0.04 0.06 0.08 0.1 PA14 pprB tadB tadA rcpA flp B io fil m  fo rm at io n  (A  60 0) B io fil m  fo rm at io n  (A  60 0)   Figure 2.5 Peptide susceptibility, swarming and biofilm analysis of PA14 mutants in selected genes transcriptionally downregulated in psrA mutants. (A) Intrinsic indolicidin super-susceptibility time-course killing curve analysis of PA14 coxA and wbpM mutants compared to wild-type. Cells were grown to mid-logarithmic phase and exposed to 64 µg/ml indolicidin and survival was assessed after 25 min. Shown is the means ± standard deviation of three independent experiments are shown.  (B and C) PA14 mutants in wbpM and etfA cannot undergo normal swarming motility. Mid-logarithmic phase cultures were spot inoculated onto PA14-type swarm agar plates and incubated for 18 h at 37°C. Swarming assays were performed with three independent cultures of each strain and a representative swarm morphology was photographed.  (D) Biofilm impairment of the pprB mutant. Overnight cultures were diluted 1:500 and then grown for 18 h at 37°C in microtitre wells, followed by washing with dH20 and staining with crystal violet. Biofilm formation was repeated three times, each time with six technical replicates, and shown are the averages ± standard deviations of one experiment.   D  48 Figure 2.6 200 400 600 800 1000 0 100 200 300 Time (s) Fl u o re sc en ce  (A U ) WT wbpL wbpI  0 100 200 300 400 500 0 100 200 300 400 Time (s) Fl u o re sc en ce  (A U ) WT PA14 wbpM  Figure 2.6 Mutants in the B-band O antigen biosynthetic operon demonstrating altered outer membrane permeabilization by indolicidin antimicrobial peptides. (A) Cells from mid-logarithmic phase cultures of wild-type PAO1 and wbpI and wbpL mutants were exposed to 3.0 µg/ml indolicidin and the increase in fluorescence due to peptide-stimulated partitioning of NPN into the outer membrane was measured. (B) Cultures of wild-type PA14 and wbpM mutant were exposed to 1.5 µg/ml indolicidin. One representative experiment is shown from at least three independent trials, each of which produced the same trends  A B  49 REFERENCES Ausubel, F. M. 1987. Current protocols in molecular biology. Published by Greene Pub. Associates and Wiley-Interscience: J. Wiley, New York.  Bader, M. W., S. Sanowar, M. E. Daley, A. R. Schneider, U. Cho, W. Xu, R. E. Klevit, H. Le Moual, and S. I. Miller. 2005. Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 122:461-72.  Belas, R., M. Goldman, and K. Ashliman. 1995. Genetic analysis of Proteus mirabilis mutants defective in swarmer cell elongation. J Bacteriol 177:823-8.  de Bentzmann, S., M. Aurouze, G. Ball, and A. Filloux. 2006. FppA, a novel Pseudomonas aeruginosa prepilin peptidase involved in assembly of type IVb pili. J Bacteriol 188:4851-60.  Burrows, L. L., R. V. Urbanic, and J. S. Lam. 2000. Functional conservation of the polysaccharide biosynthetic protein WbpM and its homologues in Pseudomonas aeruginosa and other medically significant bacteria. Infect Immun 68:931-6.  Burrows, L. L., D.F., Charter, and J.S. Lam. 1996. Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B-band lipopolysaccharide gene cluster. Mol Microbiol 22: 481-495.  Caiazza, N. C., J. H. Merritt, K. M. Brothers, and G. A. O'Toole. 2007. Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14. J Bacteriol 189:3603-12.  Chatterjee, A., Y. Cui, H. Hasegawa, and A. K. Chatterjee. 2007. PsrA, the Pseudomonas sigma regulator, controls regulators of epiphytic fitness, quorum-sensing signals, and plant interactions in Pseudomonas syringae pv. tomato strain DC3000. Appl Environ Microbiol 73:3684-94.  Chin, A. W. T. F., D. van den Broek, B. J. Lugtenberg, and G. V. Bloemberg. 2005. The Pseudomonas chlororaphis PCL1391 sigma regulator psrA represses the production of the antifungal metabolite phenazine-1-carboxamide. Mol Plant Microbe Interact 18:244-53.  Choi, K. H., A. Kumar, and H. P. Schweizer. 2006. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64:391-7.  Choi, K. H., J. B. Gaynor, K. G. White, C. Lopez, C. M. Bosio, R. R. Karkhoff- Schweizer, and H. P. Schweizer. 2005. A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2:443-8.   50 Ernst, R. K., E. C. Yi, L. Guo, K. B. Lim, J. L. Burns, M. Hackett, and S. I. Miller. 1999. Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa. Science 286:1561-5.  Falagas, M. E., and I. A. Bliziotis. 2007. Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era? Int J Antimicrob Agents 29:630-6.  Falla, T. J., D. N. Karunaratne, and R. E. W. Hancock. 1996. Mode of action of the antimicrobial peptide indolicidin. J Biol Chem 271:19298-303.  Flick, M. R., and L. E. Cluff. 1976. Pseudomonas bacteremia. Review of 108 cases. Am J Med 60:501-8.  Gibson, R. L., J. L. Burns, and B. W. Ramsey. 2003. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am J Respir Crit Care Med 168:918-51.  Gilleland, H. E., Jr., and L. B. Farley. 1982. Adaptive resistance to polymyxin in Pseudomonas aeruginosa due to an outer membrane impermeability mechanism. Can J Microbiol 28:830-40.  Hancock, R. E. W., and H. G. Sahl. 2006. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24:1551-7.  Hancock, R. E. W., and D. P. Speert. 2000. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resist Updat 3:247-255.  Jacobs, M. A., A. Alwood, I. Thaipisuttikul, D. Spencer, E. Haugen, S. Ernst, O. Will, R. Kaul, C. Raymond, R. Levy, L. Chun-Rong, D. Guenthner, D. Bovee, M. V. Olson, and C. Manoil. 2003. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 100:14339-44.  Jenssen, H., P. Hamill, and R. E. W. Hancock. 2006. Peptide antimicrobial agents. Clin Microbiol Rev 19:491-511  Karlowsky, J. A., S. A. Zelenitsky, and G. G. Zhanel. 1997. Aminoglycoside adaptive resistance. Pharmacotherapy 17:549-55.  Kielhofner, M., R. L. Atmar, R. J. Hamill, and D. M. Musher. 1992. Life-threatening Pseudomonas aeruginosa infections in patients with human immunodeficiency virus infection. Clin Infect Dis 14:403-11.  Klausen, M., A. Aaes-Jorgensen, S. Molin, and T. Tolker-Nielsen. 2003. Involvement of bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms. Mol Microbiol 50:61-8.   51 Kojic, M., B. Jovcic, A. Vindigni, F. Odreman, and V. Venturi. 2005. Novel target genes of PsrA transcriptional regulator of Pseudomonas aeruginosa. FEMS Microbiol Lett 246:175-81.  Kojic, M., C. Aguilar, and V. Venturi. 2002. TetR family member psrA directly binds the Pseudomonas rpoS and psrA promoters. J Bacteriol 184:2324-30.  Kojic, M., and V. Venturi. 2001. Regulation of rpoS gene expression in Pseudomonas: involvement of a TetR family regulator. J Bacteriol 183:3712-20.  Li, J., R. L. Nation, J. D. Turnidge, R. W. Milne, K. Coulthard, C. R. Rayner, and D. L. Paterson. 2006. Colistin: the re-emerging antibiotic for multidrug-resistant Gram- negative bacterial infections. Lancet Infect Dis 6:589-601.  Liberati, N.T., J.M. Urbach, S. Miyata, D.G. Lee, E. Drenkard, G. Wu, J. Villanueva, T. Wei and F.M. Ausubel. 2006. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc Natl Acad Sci U S A 103: 2833- 2838.  Loh, B., C. Grant, and R.E.W. Hancock. 1984. Use of the fluorescent probe 1-N- phenylnapthamine to study the interactions of the aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa. Antimicrob. Agents Chemother 26:546-551.  Lyczak, J. B., C. L. Cannon, and G. B. Pier. 2000. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2:1051-60.  Ma, L., K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak. 2006. Analysis of Pseudomonas aeruginosa conditional psl variants reveals roles for the psl polysaccharide in adhesion and maintaining biofilm structure postattachment. J Bacteriol 188:8213-21.  Macfarlane, E. L., A. Kwasnicka, M. M. Ochs, and R. E. W. Hancock. 1999. PhoP- PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer-membrane protein OprH and polymyxin B resistance. Mol Microbiol 34:305-16.  Marr, A. K., J. Overhage, M. Bains, and R. E. W. Hancock. 2007. The Lon protease of Pseudomonas aeruginosa is induced by aminoglycosides and is involved in biofilm formation and motility. Microbiology 153:474-82.  Marr, A. K., W. J. Gooderham, and R. E. W. Hancock. 2006. Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Curr Opin Pharmacol 6:468-72.  McCoy, A. J., H. Liu, T. J. Falla, and J. S. Gunn. 2001. Identification of Proteus mirabilis mutants with increased sensitivity to antimicrobial peptides. Antimicrob Agents Chemother 45:2030-7.   52 McPhee, J. B., M. Bains, G. Winsor, S. Lewenza, A. Kwasnicka, M. D. Brazas, F. S. Brinkman, and R. E. W. Hancock. 2006. Contribution of the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to Mg2+-induced gene regulation in Pseudomonas aeruginosa. J Bacteriol 188:3995-4006.  McPhee, J. B., S. Lewenza, and R. E. W. Hancock. 2003. Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol Microbiol 50:205-17.  Mesaros, N., P. Nordmann, P. Plesiat, M. Roussel-Delvallez, J. Van Eldere, Y. Glupczynski, Y. Van Laethem, F. Jacobs, P. Lebecque, A. Malfroot, P. M. Tulkens, and F. Van Bambeke. 2007. Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium. Clin Microbiol Infect 13:560-78.  Moskowitz, S. M., R. K. Ernst, and S. I. Miller. 2004. PmrAB, a two-component regulatory system of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid A. J Bacteriol 186:575-9.  O'Toole, G. A., and R. Kolter. 1998. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30:295-304.  O'Toole, G. A., and R. Kolter. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 28:449-61.  Overhage, J., M. Bains, M.D. Brazas, and R.E.W. Hancock. 2008. Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance. J Bacteriol 190: 2671-2679.  Overhage, J., S. Lewenza, A. K. Marr, and R. E. W. Hancock. 2007. Identification of genes involved in swarming motility using a Pseudomonas aeruginosa PAO1 mini-Tn5-lux mutant library. J Bacteriol 189:2164-9.  Parsek, M. R., and E. P. Greenberg. 2005. Sociomicrobiology: the connections between quorum sensing and biofilms. Trends Microbiol 13:27-33.  Parsek, M. R., and P. K. Singh. 2003. Bacterial biofilms: an emerging link to disease pathogenesis. Annu Rev Microbiol 57:677-701.  Ramsey, M. M., and M. Whiteley. 2004. Pseudomonas aeruginosa attachment and biofilm development in dynamic environments. Mol Microbiol 53:1075-87.  Rowe, S. M., S. Miller, and E. J. Sorscher. 2005. Cystic fibrosis. N Engl J Med 352:1992- 2001.   53 Rozen, S., and H. Skaletsky. 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365-86.  Selsted, M. E., M. J. Novotny, W. L. Morris, Y. Q. Tang, W. Smith, and J. S. Cullor. 1992. Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J Biol Chem 267:4292-5.  Shen, D. K., D. Filopon, L. Kuhn, B. Polack, and B. Toussaint. 2006. PsrA is a positive transcriptional regulator of the type III secretion system in Pseudomonas aeruginosa. Infect Immun 74:1121-9.  Shrout, J. D., D. L. Chopp, C. L. Just, M. Hentzer, M. Givskov, and M. R. Parsek. 2006. The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional. Mol Microbiol 62:1264-77.  Smith, E. E., D. G. Buckley, Z. Wu, C. Saenphimmachak, L. R. Hoffman, D. A. D'Argenio, S. I. Miller, B. W. Ramsey, D. P. Speert, S. M. Moskowitz, J. L. Burns, R. Kaul, and M. V. Olson. 2006. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A 103:8487-92.  Toguchi, A., M. Siano, M. Burkart, and R. M. Harshey. 2000. Genetics of swarming motility in Salmonella enterica serovar typhimurium: critical role for lipopolysaccharide. J Bacteriol 182:6308-21.  Tomich, M., P. J. Planet, and D. H. Figurski. 2007. The tad locus: postcards from the widespread colonization island. Nat Rev Microbiol 5:363-75.  Yahr, T. L., and M. C. Wolfgang. 2006. Transcriptional regulation of the Pseudomonas aeruginosa type III secretion system. Mol Microbiol 62:631-40.  Zavascki, A. P., L. Z. Goldani, J. Li, and R. L. Nation. 2007. Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review. J Antimicrob Chemother., in press.  54  CHAPTER 3 – PhoQ of Pseudomonas aeruginosa*  INTRODUCTION   Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium and is also an opportunistic pathogen capable of causing both acute and chronic infections (Stover et al. 2000). P. aeruginosa acute infections are often associated with the immunocompromised, especially burn victims and patients requiring continuous mechanical ventilation (El Solh et al. 2008), and it is the third most predominant nosocomial pathogen in North America (Fridkin et al. 1999). P. aeruginosa also is well known as a dominant pathogen in chronic lung infections in individuals with cystic fibrosis, the most prevalent inherited disorder of Caucasian populations (Lyczak et al. 2002; Gibson et al. 2003). Both P. aeruginosa infection types are noted for being intrinsically refractory to antimicrobial treatment, which contributes to the high degree of morbidity and mortality associated with P. aeruginosa infections (Hancock and Speert 2000; Mesaros et al. 2007).  A number of genetic differences have been observed between P. aeruginosa strains causing chronic or acute infections (Furukawa et al. 2006). Acute P. aeruginosa infections have been fairly well-characterized and are broad range, affecting humans, animals, plants, nematodes and amoebae (Rahme et al. 2000). In contrast to acute infections, chronic infections differ most markedly because of their narrow host range, being observed primarily in the lungs of humans with cystic fibrosis and also on catheters and other indwelling medical devices.  *  A version of this chaper will be submitted for publication. Gooderham, W. J., J. B. McPhee, M. Bains, S. Gellatly, F. Sanschagrin, C. Cosseau, R. C. Levesque, and R. E.W. Hancock. Regulation of virulence by the PhoQ sensor kinase in Pseudomonas aeruginosa.   55  The PhoP-PhoQ two-component regulatory system has been well studied in a number of organisms, including Salmonella (Miller et al. 1989; Bader et al. 2005) and P. aeruginosa (Macfarlane et al. 1999; Macfarlane et al. 2000). This classical-type two- component system comprises a membrane-bound sensor histidine kinase PhoQ which recognizes a stimulus (limiting Mg2+ and in Salmonella but not Pseudomonas cationic antimicrobial peptides) and then acts to auto-phosphorylate and phosphorylate and/or dephosphorylate its cognate response regulator PhoP, thus influencing this regulator’s ability to bind DNA and influence gene expression (Groisman 2001). In S. Typhimurium, both PhoP and PhoQ are required for full virulence (Miller et al. 1989). In contrast, in P. aeruginosa a mutant in phoQ, but not phoP, was attenuated for virulence in the neutropenic mouse model of acute infection (Macfarlane et al. 1999). In response to limiting concentrations of extracellular Mg2+, P. aeruginosa PhoPQ is involved in resistance to polymyxin B and antimicrobial peptides (Macfarlane et al. 1999). Polymyxins have recently re-emerged as systemic therapeutic agents due to their good activity against multi-drug resistant Gram-negatives including P. aeruginosa (Li et al. 2006; Zavascki et al. 2007). Here, we demonstrate that a P. aeruginosa PAO1 strain containing a mutation in phoQ was highly attenuated for persistence in a chronic lung infection model. In addition, the phoQ mutant displayed reduced twitching motility and was less cytotoxic towards human bronchial epithelial cells, suggesting that the in vivo virulence defect observed may be partly due to these phenotypes. Through the use of microarrays it was shown that the loss of PhoQ led to up-regulation of PhoP- and PmrA-regulated genes and that the PhoQ regulon additionally includes non-Mg2+ regulated genes. The potential of all of these regulatory alterations to contribute to virulence are discussed.  56 MATERIALS AND METHODS Tissue culture, bacterial strains, primers, and growth conditions. The bacterial strains and plasmids used in this study are described in Table 3.1. All primers were synthesized by AlphaDNA (Montreal, QC, Canada). Cultures were routinely grown in Luria-Bertani (LB) broth or BM2-glucose minimal medium containing low (20 µM) or high (2 mM) MgSO4 concentrations. Antibiotics for selection were used at the following concentrations: tetracycline, 50 µg/ml, carbenicillin, 300 µg/ml, and gentamicin, 30 µg/ml. Routine genetic manipulations were carried out according to standard molecular biology procedures (Ausubel 1987).  The SV40-transformed, immortalized 16HBE4o- cell line (human bronchial epithelial cells) was a gift from Dr. D. Gruenert (University of California, San Francisco, CA). It was grown in cell culture flasks (Costar, Cambridge, MA) at 37°C in a 5% CO2 atmosphere in MEM with Earle’s salts (Invitrogen Inc., Canada) containing 10% FBS and 2 mM L-glutamine. Cells were passaged by treating the monolayer with Trypsin-EDTA (Invitrogen Inc., Canada) at 37°C for 5 min to dissociate the cells from the flask. The detached cells were transferred to a 50 ml centrifuge tube containing 20 ml complete MEM medium and then centrifuged for 5 min at 1000 × g. The supernatant was discarded and the cells were resuspended in MEM complete medium. Competitive index (CI) determination. For in vivo assays, bacteria were enmeshed into agar beads and the rat model of chronic lung infection was used (Cash et al. 1979). Bacterial cells embedded in agarose beads were prepared as described (van Heeckeren and Schluchter 2002). Briefly, cultures were grown in tryptic soy broth (TSB). A culture from the wild-type strain PAO1 containing  57 the pUCP19 plasmid and a culture from each mutant strain were grown overnight at 37°C. A 200 µl aliquot of an overnight culture was diluted 1:5 into fresh TSB to give a final concentration of approximately 1 × 1010 CFU/ml. 500 µl of a 1:1 mixture of wild-type and mutant bacteria was added to 4.5 ml of TSB. This culture was mixed with 50 ml of 2% sterile agarose (Nusieve GTG, FMC, Rockland, Maine) in phosphate-buffered saline (PBS), pH 7.2 at 48°C. The agarose-broth mixture was added to a 250 ml Erlenmeyer flask containing 200 ml of heavy mineral oil at 48°C and rapidly stirred on a magnetic stirrer in a water bath (setting 500-600 rpm on a Hotplate Stirrer, Model M13, Staufen, Germany). The mixture was cooled gradually with ice chips to 0°C over a period of 5 min. The agarose beads were transferred to a sterile 500 ml Squibb separator funnel and washed once with 200 ml of 0.5% deoxycholic acid sodium salt (SDC) in PBS, once with 200 ml of 0.25% SDC in PBS, and 3 times with 200 ml of PBS. Agarose beads were incubated on ice and the remaining PBS was removed. Prepared agarose beads were stored at 4°C; bacterial counts were stable for up to one month. To determine the competitive index (CI), 1 ml of bead slurry was diluted in 9 ml PBS, homogenized and serial dilutions were plated on Pseudomonas isolation agar (PIA) and on Mueller Hinton agar (MHA, Difco) supplemented with appropriate antibiotics. CFUs were determined after 18h at 37°C and were used to calculate the input ratio of mutant to wild-type bacterial cells. Male Sprague-Dawley rats of 500 g in weight were used according to the recommendations of the ethics committee for animal treatment. The animals were anaesthetized using Isofluorane; inoculation into the lungs was done by intubation with ~120 µl of an agarose bead suspension containing a total of 105 bacterial cells. At 7 days post-infection, animals were sacrificed, lungs were removed and homogenized tissues were  58 plated on Mueller Hinton agar supplemented with appropriate antibiotics. After the in vivo passage, CFUs on plates represented the total number of bacteria present in the rat lungs. The CI is defined as the CFU output ratio of mutant when compared to wild-type strain, divided by the CFU input ratio of mutant to wild-type strain (Lehoux et al. 2000). The final CI was calculated as the geometric mean for animals in the same group and experiments were done at least in triplicate (Hava and Camilli 2002). Each in vivo competition was tested for statistical significance by Student’s two-tailed t test (Hava and Camilli 2002). RNA extraction, cDNA synthesis and hybridization to DNA microarrays. P. aeruginosa PAO1 microarray slides were provided by The Institute of Genomic Research-Pathogenic Functional Genomics Resource Center (http://pfgrc.tigr.org/). Microarrays were performed using five biologically independent experiments (for each strain, five independent cultures). Cultures were grown for 18 hr in BM2-glucose medium supplemented with 2 mM MgSO4. Cultures were then diluted 1/100 into the fresh media and cells were harvested at mid-logarithmic phase (high Mg2+ OD600 0.5) after growth at 37°C with shaking (250 rpm). RNA was isolated using the Qiagen RNeasy Midi RNA isolation kit according to the manufacturer’s protocols (Qiagen Inc., Canada). Contaminating genomic DNA was removed by treatment with the DNA-free kit (Ambion Inc., Austin, TX, USA). RNA was stored at -80°C with 0.2 units/µl of SUPERase-In RNase Inhibitor (Ambion Inc., Austin, TX, USA). RNA quality was assessed spectrophotometrically and by agarose gel electrophoresis. RNA was converted to cDNA and hybridized as previously described (McPhee et al. 2006).    59 Analysis of DNA Microarrays. Data analysis of DNA microarrays was carried out as previously described (McPhee et al. 2006). Slide images from the 5 biologically independent experiments were quantified using ImaGene 6.0 Standard Edition software (BioDiscovery, Inc., El Segundo, CA, USA). Assessment of slide quality, normalization, detection of differential gene expression and statistical analysis was carried out with ArrayPipe (version 1.7) a web-based, semi- automated software specifically designed for processing of microarray data (http://koch.pathogenomics.ca/cgi-bin/pub/arraypipe.pl; Hokamp et al. 2004) using genome annotation from the Pseudomonas genome database (www.pseudomonas.com; Winsor et al. 2005). The following processing steps were applied: (1) flagging of markers and control spots, (2) subgrid-wise background correction, using the median of the lower 10% foreground intensity as foreground intensity as an estimate for the background noise, (3) data-shifting, to rescue most of the negative spots, (4) printTip LOESS normalization, (5) merging of replicate spots, (6) two-sided, one-sample Student’s t-test on the log2-ratios within each experiment, and (7) averaging of biological replicates to yield overall fold- changes for each treatment group. Two-sided one-sample Student’s t test for the log2 ratios within each experiment and averaging of the five biological replicates to obtain overall fold changes for psrA mutant relative to wild-type. Greater than or equal to 2-fold changes and Student’s t test P value of ≤0.05 were used as the cut-offs for reporting expression changes. Quantitative Real-Time PCR (RT-qPCR). Total RNA was isolated, using RNeasy mini columns (Qiagen Inc., Mississauga, ON), from mid-logarithmic phase P. aeruginosa grown in BM2-glucose minimal media with 2 mM Mg2+. RNA samples were treated with DNase I (Invitrogen Inc., Carlsbad, CA) to  60 remove contaminating genomic DNA. Four µg of total RNA was combined with 0.5 µM dNTPs, 500 U/ml SuperaseIN (Ambion, Austin, TX), 10 µM DTT, in 1 × reaction buffer and reverse transcribed for 1 hour at 37°C and 2 hr at 42°C with 10,000 U/ml Superscript II reverse transcriptase (Invitrogen Inc., Carlsbad, CA). The RNA was subsequently destroyed by the addition of 170 mM NaOH and incubation at 65°C for 10 min. The reaction was then neutralized by the addition of HCl, and the cDNA was used as a template for Q-PCR. Analysis was carried out in the ABI Prism 7000 sequence detection system (Applied Biosystems) using the two step qRT-PCR kit with SYBR Green detection (Invitrogen). Melting curve analysis was performed to ensure specificity. Fold-change was determined using the comparative Ct method by comparison to the rpsL gene, encoding the 30S ribosomal protein S12. Experiments were repeated with three independent cultures, each assayed in duplicate, with the average ± standard deviation reported. Cytotoxicity assays. For the interaction assay, 16HBE4o- cells were seeded in 96-well tissue culture plates (Corning Life science, New York, USA) at 1 × 104 cells/well in complete MEM containing 10% FBS and 2 mM L-glutamine. The cells were incubated at 37°C and 5% CO2 for approximately 2 days to achieve >90% confluency. Bacteria were grown in LB media to logarithmic phase, washed with PBS, and resuspended and diluted in serum-free MEM. The interaction assay was performed at a multiplicity of infection (MOI) of 50 bacteria per epithelial cell in serum-free MEM with 2 mM L-glutamine at 37°C and 5% CO2. At the post-infection time point, cell supernatants were removed, placed in microfuge tubes and spun for five minutes at maximum speed to pellet the bacteria. The level of LDH in the supernatant was then assayed in triplicate using a colorimetric Cytotoxicity Detection Kit  61 (Roche Inc., Mannheim, Germany). As a positive control for maximum LDH release, cells were treated with 2% Triton X-100 (Sigma Inc., Oakville, Canada), resulting in complete cell lysis, while untreated cells were used to assess background (0%) LDH release. Minimal inhibitory concentration determination (MICs). MICs were assessed using standard broth microdilution procedures in BM2-glucose minimal medium containing 2 mM Mg2+ (Macfarlane et al. 1999). Growth was scored following 24 hr incubation at 37°C. For measuring MICs against cationic antimicrobial peptides, a modified assay was used to prevent artificially high MICs due to aggregation of peptides and binding to polystyrene (Wu and Hancock 1999). Twitching motility analysis. Twitch motility plates were made with 1.5 % (wt/vol) LB and 1 % (wt/vol) agar. Bacteria were grown overnight in LB media, diluted 1:100 into fresh LB media and grown to mid-logarithmic phase, where 1 µl was used to inoculate twitch motility plates by stabbing down to the agar-plastic interface. The diameter of the twitch zone, visible at the interface between the agar and plastic bottom, was measured after 24 h incubation at 37°C. Lettuce leaf model of infection. We followed a protocol previously described (Rahme et al. 1997; Filiatrault et al. 2006). Briefly, lettuce leaves from Romaine hearts were washed with dH20 and 0.1% bleach. Mid-logarithmic phase cultures of P. aeruginosa were washed twice with 10 mM MgSO4. Lettuce leaf midribs were then inoculated with 10 µL of P. aeruginosa at a density of 1 × 108 cells/ml (~1 × 106 cells). Leaves were then placed in humid plastic containers at 37°C and symptoms were monitored for several days. Experiments were repeated three times on different days.  62 RESULTS PhoQ mutants were highly attenuated for virulence in a model of chronic lung infection. In a previous study, we examined the contribution of the PhoPQ and PmrAB two- component systems to Mg2+ regulation in P. aeruginosa (McPhee et al. 2006). Given the importance of the PhoPQ and PmrAB systems to virulence in S. Typhimurium, we examined P. aeruginosa phoPQ and pmrAB mutants in a rat model of chronic lung infection. These experiments indicated that the phoQ mutant was highly attenuated for maintenance in this model system (Table 3.2). Somewhat surprisingly, mutants in the cognate response regulator PhoP and in genes encoding the PmrA response regulator and the PmrB sensor kinase were not attenuated for virulence in this model of infection and were as capable of surviving as the wild-type parental strain (Table 3.2). It therefore appeared that the PhoQ sensor kinase was required for full virulence in this model of chronic lung infection. Mutants in PA4773 (first gene in pmrAB operon) and pmrE (last gene in arnBCADTEF-pmrE aminoarabinose LPS modification operon) genes, which are part of operons important for resistance to polymyxin B and cationic antimicrobial peptides (McPhee et al. 2003; Lewenza et al. 2005), were similarly un-attenuated for maintenance in this model of infection. Also, mutants in the feoAB (PA4358/9) ferrous iron transport system and the putP (PA0783) sodium/proline symporter were similarly un-attenuated for virulence in this model (Table 3.2).    63 PhoQ mutants demonstrated reduced cytotoxicity toward human bronchial epithelial cells. Human airway epithelial cells (16HBE4o-) were used to monitor the in vitro cytotoxicity of the phoQ mutant compared to its isogenic wild-type parent. This cell line maintains many of the properties of primary airway epithelial cells including the ability to form tight junctions and differentiation to produce microvilli and cilia (Cozens et al. 1994). In control experiments, wild-type and phoQ mutant cells cultured on the epithelial cells were shown to display the same growth properties (data not shown). The cytotoxic effects of both strains were monitored via lactate dehydrogenase (LDH) activity released from the dying cells. Both phoQ and wild-type strains induced LDH release on 16HBE4o- cells showing that they both displayed some cytotoxicity toward this cell line. However, wild-type P. aeruginosa displayed a 2.2-fold (P <0.05) greater cytotoxicity than the phoQ mutant after 8.5 hours of interaction (Fig. 3.1). This difference remained at 16 hours with the wild-type strain always showing greater cytotoxicity than the phoQ mutant (Fig. 3.1). Introducing the wild-type phoQ+ allele into the phoQ mutant in trans restored cytotoxicity to wild-type levels at both time points (Fig. 3.1). These results show that PhoQ is necessary for wild-type levels of cytotoxicity towards human bronchial epithelial cells in vitro. PhoQ mutants were impaired in twitching motility.  Twitching of P. aeruginosa is an important form of motility dependent on type IV pili. These motility appendages have been associated with colonization, attachment and biofilm formation (O’Toole and Kolter 1998). As the phoQ mutant has been previously shown to have reduced swarming motility (Brinkman et al. 2001) and also a defect in biofilm formation (Ramsey and Whiteley 2004), we chose to analyze twitching motility in  64 this mutant due to the interrelatedness of these three processes. The phoQ mutant displayed a significant (P <0.001) impairment in, though not complete abolishment of, twitching motility (Fig. 3.2). This impaired twitching phenotype could be successfully restored back to wild-type twitching levels by providing a wild-type phoQ+ allele in trans on a plasmid (Fig. 3.2). This demonstrates that PhoQ is necessary for normal wild-type twitching motility. Reduced lettuce virulence of phoQ mutants. Like other phytopathogenic Pseudomonas spp, P. aeruginosa can infect an assortment of plants. The lettuce leaf model, originally developed using whole Arabidopsis plants, is a simple model geared towards analyzing large differences in virulence phenotypes between different P. aeruginosa strains (Rahme et al. 1997). Here, P. aeruginosa cells are inoculated into the Romaine lettuce leaf stem midrib and macroscopic symptoms elicited by infection are followed over several days. Relative to wild-type PAO1, the phoQ mutant was consistently found to have an impairment in its ability to cause spreading soft-rot destruction inside the leaf midrib outwards from the point of inoculation (Fig. 3.3). Thus it appears PhoQ was necessary for full virulence expression in this model of plant infection. Analysis of the altered transcriptome in the phoQ mutant. To investigate how phoQ mutation contributes to gene expression in Pseudomonas and possibly to the virulence phenotypes observed, microarray studies were performed to compare the phoQ mutant to wild-type. Of note, we used a freshly constructed phoQ mutant based on the cloning protocol originally described, since our original mutant underwent phenotypic dilution over time. For the microarray, cells were grown in BM2-glucose containing 2 mM Mg2+, a condition under which PhoP is phosphorylated and constitutively active (Macfarlane et al. 1999; Groisman 2000; McPhee et al. 2003). Using these conditions  65 we observed 474 genes that were differentially regulated (P ≤0.05) by more than 1.5-fold under high Mg2+ conditions, with 296 transcriptionally up-regulated and 178 down- regulated. A selection of these genes is presented in Table 3.3. The phoQ mutation affected the regulation of many genes that were previously shown to be regulated by PhoP and/or PmrA in an Mg2+ dependent manner (McPhee et al. 2006). Many previously-identified genes positively dependent on PhoP and Mg2+ limitation appeared dysregulated on our phoQ array and included PA0921, oprH-phoP, the LPS- modification operon arnBCADTEF, PA3885, PA4010-11, PA4453-5 and PA1343. PmrA- dependent genes shown to be up-regulated included feoAB, PA1559-60, PA4357-9, PA4773-8 (includes pmrAB), arnBCADTEF, and PA4286 (Table 3.3). The up-regulation of pmrA was confirmed using qRT-PCR, demonstrating that this gene was induced 4.2 ± 1.7 fold in the phoQ mutant (Table 3.4). Similarly the up-regulation of arnB was confirmed by qRT-PCR and shown to be 505 ± 21 fold up-regulated (Table 3.4). The massive up- regulation of arnB together with the rest of the genes of this LPS modification operon directly correlated with the high-level resistance to antimicrobial peptides such as polymyxin B in the phoQ mutant. Indeed, the freshly made phoQ mutant was >64-fold more resistant to polymyxin B than wild-type cells as assayed by broth dilution MIC in high Mg2+ BM2-glucose medium. The switch to the mucoid (alginate producing) phenotype in most cystic fibrosis isolates is caused by mutations in the mucA gene (encoding an anti-sigma factor) and the resulting block in sequestration and activation of AlgU, an alternative sigma factor. The phoQ microarray revealed transcriptional dysregulation of the algU-mucABCD alginate regulatory gene cluster, namely up-regulation of algU and the corresponding AlgU-  66 dependent (mucoidy) genes (Firoved and Deretic 2003) including slyB, encoding a putative porin, the osmC and osmE osmotically induced lipoproteins, and the pfpI protease (Table 3.3). These three genes are among the strongest AlgU-dependent genes (Firoved and Deretic 2003) and were the only members of the AlgU stimulon that appeared on our microarray. This is not entirely unsurprising, as the phoQ mutant possessed a wild-type copy of mucA (which was similarly induced) and so the anti-AlgU sigma factor activity of MucA would likely therefore limit the degree to which less robust AlgU targets are induced. Of note, the alginate biosynthetic cluster (algABCD) did not appear to be significantly dysregulated in phoQ mutants, and phoQ mutants did not possess a mucoid phenotype. The algU-mucAB genes are also positively regulated by low Mg2+ (McPhee et al. 2006) and this is relevant as cells grown in low Mg2+ media have LPS analogous to that from cells isolated from CF lungs which become colonized with mucoid P. aeruginosa (Ernst et al. 1999). Microarray analysis also indicated many interesting genes that while Mg2+ regulated, were strongly affected by mutation of the phoQ gene. These included norC (PA0523; nitric- oxide reductase subunit C) which was strongly down-regulated and two type II secretion genes, hxcQ (PA0685; probable type II secretion system protein) and xqhA (PA1868; type II secretion protein) that were down-regulated. Since down-regulation of two type II secretion genes (hxcQ and xqhA) was observed, we chose to analyze the regulation of the type II secretion-dependent lipase major virulence factor, lipA, which was demonstrated to be 3.2 ± 0.6-fold down-regulated in the phoQ mutant (Table 3.4). Interestingly, the Pseudomonas quinolone signal (PQS) biosynthetic genes pqsBCD were up-regulated. Also induced were four iron scavenging-related genes: pyoverdine genes pvcA (PA2254; pyoverdine biosynthesis protein), pvdA (PA2386; ornithine oxygenase in pyoverdine biosynthesis) and  67 fpvA (PA2398; ferripyoverdine receptor) and another siderophore gene pchC (PA4229; pyochelin biosynthesis thioesterase). The up-regulation of these green pigmented siderophore genes was supported by the observation that phoQ mutants were a darker green colour on plates and in broth culture (Fig. 3.4). Additional regulation of genes associated with virulence was observed in phoQ mutants. The AlgR response regulator regulates a diverse assortment of virulence determinants including twitching and swarming motility and biofilm formation (Lizewski et al. 2002; Belete and Wozniak 2008). The transcriptional up-regulation of algR was confirmed by qPCR and showed that this gene was actually more strongly induced at 4.6 ± 2.6-fold in the phoQ mutant (Table 3.4). Of note, the major transcriptional regulator of the type III secretion system, exsA, was very modestly down-regulated on our phoQ microarray (−1.6, P <0.01; Appendix II). We also analyzed the level of regulation of the GacS/GacA/rsmZ pathway which operates at the level of post-translational regulation, since previous work indicated some overlap in phenotypes between GacA and PhoPQ. RsmZ is a small non-coding RNA that post-translationally sequesters the RNA-binding protein RsmA and thus indirectly regulates a broad range of virulence properties including liapse production (Heurlier et al. 2004). However, we were unable to demonstrate a significant level of regulation of gacA or rsmZ, thus indicating that this pathway may not play a role in the phoQ virulence defects. DISCUSSION  In this study it is demonstrated that the PhoQ sensor kinase of P. aeruginosa was capable of regulating virulence in a rat model of chronic lung infection. Consistent with this, the phoQ mutant displayed reduced cytotoxicity towards polarized human bronchial  68 epithelial cells, reduced twitching motility, and impaired virulence in lettuce leaves relative to wild-type. Microarray analysis of the phoQ mutant produced a large list of dysregulated genes and uncovered new genes that are likely to form part of the PhoQ regulon. The PhoP-PhoQ and PmrA-PmrB two-component systems of P. aeruginosa are responsible for regulating the adaptive response of P. aeruginosa to limiting concentrations of cations (e.g. Mg2+; McPhee et al. 2006). They are also of critical importance in controlling resistance to cationic antimicrobial peptides in response to low Mg2+ conditions (Ernst et al. 1999; Macfarlane et al. 1999; McPhee et al. 2003; Moskowitz et al. 2004). Somewhat surprisingly, mutants in the PhoP and PmrA response regulators, and the PmrB sensor kinase were not attenuated for virulence in this model of infection and were fully capable of competing with the wild-type parental strain in vivo (Table 3.2). Furthermore, a mutant in pmrE (PA3559), a component of the aminoarabinaose lipid A LPS modification operon (arnBCADTEF-pmrE) was not impaired in virulence in this model (Table 3.2). As these operons are major mediators of intrinsic and mutational resistance to cationic antimicrobial peptides (McPhee et al. 2003; Moskowitz et al. 2004; Lewenza et al. 2005), this would seem to indicate that an increased susceptibility to such peptides did not impact on fitness of P. aeruginosa. Similarly, the increased antimicrobial peptide resistance of the PhoQ mutant was associated with decreased fitness in this model rather than increased resistance to host defence mechanisms, suggesting that altered virulence and polymyxin B/antimicrobial peptide resistance are independent phenotypes of the PhoQ mutant. The PhoP-PhoQ system of Salmonella is a master regulator of virulence (Miller et al. 1989). We have previously compared the extensive differences in the architecture of these systems between P. aeruginosa and S. Typhimurium (McPhee et al. 2003), a theme that has  69 been mirrored in several other studies on Yersinia (Marceau et al. 2004; Winfield and Groisman, 2004; Winfield et al. 2004). The Yersinia PhoP-PhoQ system is important for regulating virulence gene expression since phoP mutants were less virulent during Yersinia pseudotuberculosis and Y. pestis intra-macrophage survival (Grabenstein et al. 2004). However, a Salmonella strain constitutively expressing PhoP was attenuated for virulence in mice and this was linked to a requirement to initiate an initial large induction of positive autoregulation (Shin et al. 2006). It is possible that this constitutive PhoP phenotype in Salmonella is similar to that which is occurring in the attenuated virulence phenotype displayed by our P. aeruginosa phoQ mutant which also has a constitutive PhoP phenotype. One of the most fundamental differences between the Salmonella and Pseudomonas systems is that Salmonella PhoQ appears able to both phosphorylate and dephosphorylate PhoP whereas in Pseudomonas the primary role of PhoQ appears to be in the dephosphorylation of PhoP and we suspect that another kinase may activate Pseudomonas PhoP (Macfarlane et al. 1999; Macfarlane et al. 2000). In contrast to the situation in enteric S. Typhimurium and insect-pathogenic Photorhabdus luminescens (Bennett and Clarke 2005; Gunn et al. 2000), mutation of the pmrE (PA3559) gene of the arnBCADTEF-pmrE LPS modification operon in P. aeruginosa did not result in decreased survival in the chronic infection mouse model (Table 3.2). PmrE, a UDP-glucose dehydrogenase that has been referred to as ugd (McPhee et al. 2006), is required for P. aeruginosa resistance to antimicrobial peptides (Lewenza et al. 2005). However, it is possible that because upstream genes are not affected by the pmrE transposon mutation, the other arn genes in this operon could still have been required for virulence in this chronic model. Mice inoculated with S. enterica serovar typhimurium pmrA and  70 pmrHFIJKLM (arnBCADTEF) mutant strains demonstrated virulence attenuation when administered orally but not when administered intraperitoneally, indicating that aminoarabinose addition to LPS may be important for resistance to host innate defences within certain specific tissues (Gunn et al. 2000). This observation is similar to that which has been made for Y. pseudotuberculosis (a soil and waterborne enteropathogen), in which the pmrF-containing operon was dispensable for full virulence in mice, even though it was required for resistance to cationic antimicrobial peptides and polymyxin B (Marceau et al. 2004). It would be interesting to analyze P. aeruginosa mutants in the first genes of the arnB operon to see if these genes had virulence attenuation phenotypes. In this study we have determined the dysregulated in vitro transcriptome of a phoQ mutant and used this gene list to attempt to explain our findings regarding the in vivo chronic rat lung infection attenuation phenotype. We are aware of a number of problems with this sort of comparison. The growth condition (BM2-glucose with high Mg2+) used for our in vitro microarray experiment is expected to be quite different from the growth conditions found in the rat lung and as a result, the gene expression patterns might be quite different. Of note, a defined synthetic artificial cystic fibrosis sputum medium (based on analysis of cystic fibrosis sputum) for growth of P. aeruginosa contained a variety of carbon sources, with glucose having the largest concentration of any carbon source (Palmer et al. 2007). Nevertheless, a phoQ mutation results in a PhoP response regulator that would be relatively insensitive to additional stimuli (i.e. constitutively active) and might therefore be expected to have some of the same direct effects on gene expression regardless of growth conditions. Also, these microarrays are directly comparable to those utilized for previous  71 studies of the adaptive response to Mg2+ limitation, and the effects observed with phoP and pmrA mutants in vitro. A P. aeruginosa phoQ mutant (in strain PA14) was shown to be impaired in the formation of both microtitre plate and mature flow-cell mushroom-shaped biofilms (Ramsey and Whiteley 2004). It was suggested in this study that the biofilm defect was multi-factorial and potentially involved changes in LPS, as LPS was required for biofilm formation in P. aeruginosa and P. fluorescens (de Lima Pimenta et al. 2003; Sabra et al. 2003). While in agreement with a potential role for LPS due to the strong upregulation of the arnBCADTEF operon, our data is consistent with an influential role for the observed impaired twitching motility of the phoQ mutant (Fig. 3.2), since twitching motility has been implicitly linked to biofilm formation (O’Toole and Kolter 1998). How the transcriptional dysregulation of this type IV-pili-dependent form of motility occurs in the phoQ mutant is not immediately apparent, given that the array and twitching motility experiments were done under different conditions. However, the phoQ microarray list did show down-regulation of some type IV- pili genes (data not shown as fold changes were significant but less than two-fold; Appendix II). It is also possible that the biofilm defect in this strain was indeed multi-factorial given the large number of genes differentially regulated in the phoQ mutant. Mutation of phoQ affects a large number of genes outside of the PhoP regulon (McPhee et al. 2006), as indicated by comparison of microarray analyses. At present it is not known how the membrane-bound sensor kinase PhoQ accomplishes this. As PhoQ itself is not directly regulating these genes, it presumably is capable of modifying the phosphorylation state of another as yet unknown regulatory protein. If so, in the phoQ  72 mutant this activity would be lost and consequently gene expression would be modified. Experiments attempting to uncover such a biochemical target of PhoQ are ongoing.  Despite the phoQ mutant being highly antimicrobial peptide resistant in vitro, data are presented here that demonstrate that this mutant is attenuated for survival in a chronic lung infection model. In reflection, two considerations are relevant to this phenotype. First, other phoQ phenotypes such as reduced twitching motility and cytotoxicity may mask or contribute more than the hyper-resistance phenotype to the final in vivo rat lung phenotype. Second, these seemingly contradictory findings are perhaps not surprising given the growing body of knowledge regarding the indirect roles of host defense (antimicrobial) peptides as important regulators of the immune system, rather than direct antimicrobial compounds (Jenssen et al. 2006) and the complete lack of α-defensins in mouse phagocytes, in contrast to human neutrophils where they are a major mediator of direct microbicidal killing of pathogens.  73 Table 3.1  P. aeruginosa strains and plasmids used in this study Strain Genotype or characteristicsa Source or reference  WT Wild-type P. aeruginosa PAO1; strain H103 Lab collection phoQ phoQ::xylE-GmR; PAO1 background Macfarlane et al. 1999 phoQ (pUC-phoQ+) phoQ mutant with pUCP22-phoQ+; GmR, CbR Macfarlane et al. 1999 phoP phoP::xylE:aacC1; GmR derivative of WT Macfarlane et al. 1999 pmrA pmrA::xylE:aacC1; GmR derivative of WT McPhee et al. 2003 pmrB pmrB::xylE:aacC1; GmR derivative of WT McPhee et al. 2003 putP PA0783::luxCDABE-TcR; derivative of WT Lewenza et al. 2005 PA4773 PA4773::luxCDABE-TcR; inserted between PA4773 and PA4774; derivative of WT Lewenza et al. 2005 pmrE PA3559::luxCDABE-TcR; derivative of WT Lewenza et al. 2005 feoA PA4359::luxCDABE-TcR; derivative of WT Lewenza et al. 2005  Plasmid pUCP19, 22 Escherichia-Pseudomonas shuttle vectors West et al. 1994  a  Antibiotic resistance: AmpR, ampicillin for E. coli, carbenicillin for P. aeruginosa; GmR, gentamicin; TcR, tetracycline.    Table 3.2 Competitive index (CI) analysis of P. aeruginosa mutant strains grown with the wild type PAO1 strain after 7 days of in vivo passage in the rat lung. The ability of wild-type PAO1 and mutant strains to compete with each other was determined in the rat lung model of chronic infection.  Gene disrupted  Geometric mean of CIa No. of animals phoP 0.52 3 phoQ <0.00019 (P <0.02) 6 pmrA 1.27 3 pmrB 0.70 2 putP 0.54 2 PA4773 1.06 2 pmrE 1.51 2 feoA 0.80 3  a  Competitive index (CI) analysis of mutant relative versus wild-type. The CI is defined as the CFU output ratio of mutant when compared to wild-type strain, divided by the CFU input ratio of mutant to wild-type strain. A CI <1 theoretically repesents a competitive disadvantage of the mutant strain compared to the wild-type parental strain.  74 Table 3.3 Microarray analysis of genes significantly dysregulated in the phoQ mutant relative to wild-type. Dysregulated hypothetical and/or unclassified ORFs are not included and only genes showing ≥2 fold change in the phoQ mutant are depicted.  Gene IDa Namea Fold changeb P value Descriptiona PA0048   −19.1 0.002 probable transcriptional regulator PA0059 osmC 2.1 0.003 osmotically inducible protein OsmC PA0062  3.0 0.0001 hypothetical protein PA0082  2.0 0.04 hypothetical protein PA0135  −5.5 0.008 hypothetical protein PA0161  −3.7 0.001 hypothetical protein PA0165  −2.5 0.01 hypothetical protein PA0198 exbB1 −2.1 0.01 transport protein ExbB PA0199 exbD1 −2.0 0.02 transport protein ExbD PA0224  4.0 0.01 probable aldolase PA0307  −2.1 0.005 hypothetical protein PA0327  2.3 <0.0001 hypothetical protein PA0329  2.6 <0.0001 conserved hypothetical protein PA0355 pfpI 2.4 0.05 intracellular protease PfpI PA0459 clpC −2.1 0.02 probable ClpA/B protease ATP binding subunit PA0460  5.0 <0.0001 hypothetical protein PA0490  3.0 <0.0001 hypothetical protein PA0521 nirO −2.2 0.02 probable cytochrome c oxidase subunit PA0523 norC −13.2 0.002 nitric-oxide reductase subunit C PA0529  5.4 0.02 conserved hypothetical protein PA0537   2.2 <0.0001 conserved hypothetical protein PA0567 yqaE  2.0 0.01 conserved hypothetical protein PA0569   −2.8 0.0004 hypothetical protein PA0623   2.1 0.0008 probable bacteriophage protein PA0631   2.0 0.003 hypothetical protein PA0637   2.3 0.0004 conserved hypothetical protein PA0685  hxcQ −3.0 0.03 probable type II secretion system protein PA0715   −2.5 0.04 hypothetical protein PA0718   2.1 0.0004 hypothetical protein of bacteriophage Pf1 PA0737   2.2 0.01 hypothetical protein PA0739   3.1 0.02 probable transcriptional regulator PA0762 algU 6.4 <0.0001 sigma factor AlgU PA0763 mucA 6.5 <0.0001 anti-sigma factor MucA PA0764 mucB 2.7 <0.0001 negative regulator for alginate biosynthesis PA0765 mucC 2.2 0.001 positive regulator for alginate biosynthesis PA0766 mucD 2.7 0.005 serine protease MucD precursor PA0801   2.2 0.004 hypothetical protein PA0802   2.0 0.004 hypothetical protein PA0806   −4.5 0.01 hypothetical protein PA0814   2.8 0.03 conserved hypothetical protein  75 Gene IDa Namea Fold changeb P value Descriptiona PA0833   8.2 <0.0001 hypothetical protein PA0853   3.4 <0.0001 probable oxidoreductase PA0854 fumC2 3.1 0.0001 fumarate hydratase PA0874   −2.1 0.02 hypothetical protein PA0879   −3.7 0.02 probable acyl-CoA dehydrogenase PA0885  dctQ −2.4 0.05 probable C4-dicarboxylate transporter PA0910   3.3 0.02 hypothetical protein PA0911   2.4 <0.0001 hypothetical protein PA0914   −4.5 0.01 hypothetical protein PA0919   2.3 <0.0001 hypothetical protein PA0920   3.2 0.0001 hypothetical protein PA0921   14.5 <0.0001 hypothetical protein PA0929  pirR 2.3 0.0004 PirR two-component response regulator PA0939   −5.8 0.008 hypothetical protein PA0949 wrbA 4.5 0.003 Trp repressor binding protein WrbA PA0984   −2.1 0.05 colicin immunity protein PA0997 pqsB 2.5 0.0001 quinolone signal (PQS) biosynthesis PA0998 pqsC 2.4 0.0007 quinolone signal (PQS) biosynthesis PA0999 pqsD 2.3 0.0007 quinolone signal (PQS) biosynthesis PA1001 phnA 2.6 0.0002 pyocyanin biosynthesis PA1041   2.5 0.03 probable outer membrane protein PA1053  slyB 4.1 <0.0001 outer membrane lipoprotein PA1149   −2.6 0.04 hypothetical protein PA1178 oprH 33.1 <0.0001 outer membrane protein OprH PA1179 phoP 82.6 <0.0001 two-component response regulator PhoP PA1317 cyoA −2.8 0.005 cytochrome o ubiquinol oxidase subunit II PA1318 cyoB −2.7 0.002 cytochrome o ubiquinol oxidase subunit I PA1319 cyoC −2.4 0.01 cytochrome o ubiquinol oxidase subunit III PA1321 cyoE −3.5 0.007 cytochrome o ubiquinol oxidase protein CyoE PA1323   6.6 <0.0001 hypothetical protein PA1324   3.4 0.0008 hypothetical protein PA1325  yybH −2.6 0.0007 conserved hypothetical protein PA1326 ilvA2 −2.9 0.0003 threonine dehydratase PA1343   16.6 <0.0001 hypothetical protein PA1344  yvaG 9.1 <0.0001 probable short-chain dehydrogenase PA1346   −2.7 0.04 hypothetical protein PA1385   −3.4 0.02 probable glycosyl transferase PA1403   −2.6 0.03 probable transcriptional regulator PA1471   7.1 <0.0001 hypothetical protein PA1494   3.3 <0.0001 conserved hypothetical protein PA1498 pykF 3.7 0.02 pyruvate kinase I PA1506   −2.5 0.008 hypothetical protein PA1547   −2.4 0.0007 hypothetical protein PA1549  fixI −2.4 0.006 probable cation-transporting P-type ATPase  76 Gene IDa Namea Fold changeb P value Descriptiona PA1559   5.7 0.002 hypothetical protein PA1560   6.0 0.001 hypothetical protein PA1562 acnA 2.0 0.0007 aconitate hydratase 1 PA1579   2.4 <0.0001 hypothetical protein PA1592   14.6 <0.0001 hypothetical protein PA1596 htpG −3.2 0.03 heat shock protein HtpG PA1631   6.3 0.007 probable acyl-CoA dehydrogenase PA1656   −2.0 0.001 hypothetical protein PA1660   10.7 0.003 hypothetical protein PA1680   2.0 0.05 hypothetical protein PA1688   2.6 <0.0001 hypothetical protein PA1689   3.3 <0.0001 conserved hypothetical protein PA1715 pscB −2.4 0.0004 type III export apparatus protein PA1797  4.0 0.0002 hypothetical protein PA1824  −3.8 0.02 conserved hypothetical protein PA1868 xqhA −3.9 0.02 type II secretion protein XqhA PA1917  2.4 0.05 hypothetical protein PA1920 nrdD 2.7 0.03 ribonucleotide reductase PA1975  −2.5 0.04 hypothetical protein PA1977  −2.4 0.005 hypothetical protein PA1978 agmR −2.4 0.05 transcriptional regulator AgmR PA1979 exaD −3.3 0.02 two-component sensor kinase ExaD PA1981  −8.6 0.004 hypothetical protein PA1982 exaA −10.9 0.003 quinoprotein alcohol dehydrogenase PA1983 exaB −9.6 0.004 cytochrome c550 PA1984 exaC −9.6 0.02 aldehyde dehydrogenase PA2011 gnyL 2.6 0.04 hydroxymethylglutaryl-CoA lyase PA2019 amrA 2.7 0.02 RND multidrug efflux membrane fusion protein PA2021  2.5 0.007 hypothetical protein PA2023 galU 2.2 0.001 UTP--glucose-1-phosphate uridylyltransferase PA2078  −2.9 0.03 hypothetical protein PA2106  2.2 0.009 hypothetical protein PA2121  2.0 0.02 probable transcriptional regulator PA2147 katE −3.4 0.02 catalase HPII PA2186  −3.4 0.02 hypothetical protein PA2194 hcnB 2.1 0.02 hydrogen cyanide synthase HcnB PA2252  2.1 0.002 AGCS sodium/alanine/glycine symporter PA2254 pvcA 5.1 0.009 pyoverdine biosynthesis protein PvcA PA2258 ptxR 2.6 0.02 transcriptional regulator PtxR PA2280 arsH −4.6 0.01 conserved protein in arsenic resistance PA2298  −2.9 0.005 probable oxidoreductase PA2353  2.5 0.04 conserved hypothetical protein PA2358  7.9 <0.0001 hypothetical protein PA2373  3.1 0.02 conserved hypothetical protein  77 Gene IDa Namea Fold changeb P value Descriptiona PA2384  2.0 0.01 hypothetical protein PA2386 pvdA 4.3 0.01 Pyoverdine biosyntheis, ornithine oxygenase PA2398 fpvA 4.0 0.0005 ferripyoverdine receptor PA2403   3.1 0.04 hypothetical protein PA2404   4.0 0.003 hypothetical protein PA2405   5.2 0.0002 hypothetical protein PA2406   2.8 0.0009 hypothetical protein PA2407   2.2 0.003 probable adhesion protein PA2409   2.7 0.0004 probable permease of ABC transporter PA2433   2.3 0.001 hypothetical protein PA2434   3.2 0.05 hypothetical protein PA2435   4.2 0.02 probable cation-transporting P-type ATPase PA2436   2.5 0.04 hypothetical protein PA2460   −2.0 0.002 hypothetical protein PA2467   2.5 0.04 probable transmembrane sensor PA2470 gtdA 2.4 0.05 gentisate 1,2-dioxygenase PA2485  5.5 <0.0001 hypothetical protein PA2486   3.4 <0.0001 hypothetical protein PA2506   −2.9 0.03 hypothetical protein PA2548   −3.1 0.02 hypothetical protein PA2562   3.2 <0.0001 hypothetical protein PA2569   2.3 0.0008 hypothetical protein PA2650  ybaJ −2.5 0.04 conserved methylase protein PA2653  yuiF −4.8 0.01 probable transporter PA2658   2.0 0.0002 hypothetical protein PA2659   2.3 0.0002 hypothetical protein PA2662   −10.1 <0.0001 conserved hypothetical protein PA2663   −14.9 0.008 hypothetical protein PA2664 fhp −30.5 0.02 flavohemoprotein PA2779   2.9 0.0004 hypothetical protein PA2787 cpg2 2.7 0.03 carboxypeptidase G2 precursor PA2815 yafH 2.0 0.004 probable acyl-CoA dehydrogenase PA2823  2.4 0.05 conserved hypothetical protein PA2929  −2.8 0.03 hypothetical protein PA2987 ycfV 2.1 0.01 ATP-binding component of ABC transporter PA3001  −6.1 0.007 glyceraldehyde-3-phosphate dehydrogenase PA3031  2.6 0.0001 hypothetical protein PA3069  3.6 <0.0001 hypothetical protein PA3189 gltF 2.4 0.02 probable permease of ABC sugar transporter PA3239  2.0 0.003 conserved hypothetical protein PA3277  −2.1 0.03 probable short-chain dehydrogenase PA3391 nosR −3.0 0.004 regulatory protein NosR PA3404 opmM 13.0 0.003 outer membrane protein PA3405 hasE −4.6 0.01 metalloprotease secretion protein  78 Gene IDa Namea Fold changeb P value Descriptiona PA3425  −2.6 0.04 hypothetical protein PA3436  −2.7 0.003 hypothetical protein PA3474 yigM −2.1 0.02 conserved membrane protein PA3475 pheC −2.4 0.003 cyclohexadienyl dehy. (phenylalanine synthesis) PA3506   3.4 0.02 probable decarboxylase PA3515   2.9 0.02 hypothetical protein PA3516   3.8 0.02 probable lyase PA3517   3.7 0.04 probable lyase PA3530 bfd 3.4 0.02 bacterioferritin-associated ferredoxin PA3537 argF 2.5 0.04 ornithine carbamoyltransferase, Arg biosynthesis PA3552 arnB 44.9 <0.0001 Aminotransferase in L-Ara4N biosynthesis PA3553 arnC 75.4 <0.0001 Glycosyltransferase in L-Ara4N biosynthesis PA3554 arnA 77.8 <0.0001 Dual function enzyme in L-Ara4N biosynthesis PA3556 arnT 45.4 <0.0001 Transferase in L-Ara4N biosynthesis PA3557 arnE 45.6 <0.0001 Transport system for L-Ara4N PA3558 arnF 69.0 <0.0001 Transport system for L-Ara4N PA3559 pmrE 3.4 0.004 UDP-glucose dehydrogenase PA3560 fruA 2.0 0.04 fructose IIBC phosphotransferase system PA3598 ypqQ 4.6 0.03 conserved hypothetical protein PA3607 potA −2.5 0.04 polyamine transport protein PotA PA3610 potD −3.2 0.02 polyamine transport protein PotD PA3691  6.5 <0.0001 hypothetical protein PA3692  6.2 <0.0001 probable outer membrane protein PA3713  −2.8 0.02 hypothetical protein PA3788  2.0 0.005 hypothetical protein PA3819 ycfJ 12.0 <0.0001 putative porin PA3840 ybiN −2.3 0.0002 conserved protein (methylase) PA3867  −2.5 0.04 probable DNA invertase PA3885   3.2 <0.0001 hypothetical protein PA3889   2.2 0.04 binding protein component of ABC transporter PA3890   2.4 0.0002 probable permease of ABC transporter PA3891   2.5 0.04 ATP-binding component of ABC transporter PA3922   2.6 0.002 conserved hypothetical protein PA3940   3.1 0.005 probable DNA binding protein PA4010   5.0 <0.0001 hypothetical protein PA4011   9.2 <0.0001 hypothetical protein PA4040   −2.5 0.04 hypothetical protein PA4092 hpaC −6.2 0.007 4-hydroxyphenylacetate 3-monooxygenase PA4129   −3.5 0.008 hypothetical protein PA4154  ygiM 2.6 0.001 conserved hypothetical protein PA4204   3.2 <0.0001 conserved hypothetical protein PA4229 pchC 10.7 0.003 thioesterase PchC, pyochelin biosynthesis PA4338   4.4 0.01 hypothetical protein PA4357  yhgG 3.2 0.009 conserved hypothetical protein  79 Gene IDa Namea Fold changeb P value Descriptiona PA4358  feoB 3.0 0.003 ferrous iron transport protein PA4359  feoA 3.7 <0.0001 ferrous iron transport protein PA4370 icmP 2.2 0.003 outer-membrane insulin-cleaving protease PA4452   5.2 <0.0001 conserved hypothetical protein PA4453   5.9 <0.0001 conserved hypothetical protein PA4454  yrbD 6.3 <0.0001 conserved hypothetical protein PA4455  yrbE 5.3 <0.0001 probable permease of ABC transporter PA4456 yrbF  4.1 <0.0001 ATP-binding component of ABC transporter PA4469   3.6 0.02 hypothetical protein PA4479 mreD 3.2 0.05 rod shape-determining protein MreD PA4495   3.7 <0.0001 hypothetical protein PA4514  piuA 2.9 0.03 outer membrane receptor for iron transport PA4517   6.8 <0.0001 conserved hypothetical protein PA4621   −2.0 0.002 probable oxidoreductase PA4635  mgtC 3.1 <0.0001 conserved membrane protein MgtC PA4644   −4.0 0.01 hypothetical protein PA4675  iutA 2.0 0.007 probable TonB-dependent receptor PA4713   2.0 0.02 hypothetical protein PA4762 grpE −2.7 0.03 heat shock protein GrpE PA4773   7.2 0.0003 hypothetical protein PA4774   8.0 0.0002 hypothetical protein PA4776 pmrA 4.0 0.0003 two-component response regulator PA4777 pmrB 3.2 0.005 two-component sensor PA4800   −2.5 0.04 hypothetical protein PA4817   −2.0 0.01 hypothetical protein PA4826   2.6 <0.0001 hypothetical protein PA4873   −3.0 0.03 probable heat-shock protein PA4876 osmE 4.5 <0.0001 osmotically inducible lipoprotein OsmE PA4880   6.6 <0.0001 probable bacterioferritin PA4892 ureF −2.0 0.002 urease accessory protein UreF PA5020   −2.3 0.05 probable acyl-CoA dehydrogenase PA5030  ynfM −2.5 0.0006 probable MFS transporter PA5053 hslV −2.0 0.002 heat shock protein HslV PA5061 phaI 4.4 <0.0001 polyhydroxyalkanoic acid biosynthesis PA5107 blc 2.6 0.005 outer membrane lipoprotein Blc PA5144  −2.5 0.04 hypothetical protein PA5157 marR −2.0 0.003 transcriptional regulator MarR PA5158 opmG −2.8 0.001 outer membrane protein PA5169  2.5 0.04 probable C4-dicarboxylate transporter PA5172 arcB −2.7 0.0005 ornithine carbamoyltransferase, catabolic PA5178  3.6 0.0004 conserved hypothetical protein PA5182  4.6 <0.0001 hypothetical protein PA5183  2.4 0.02 hypothetical protein PA5188  -2.2 0.01 probable 3-hydroxyacyl-CoA dehydrogenase  80 Gene IDa Namea Fold changeb P value Descriptiona PA5212  8.3 <0.0001 hypothetical protein PA5231 yhiH −3.4 0.03 ATP-binding/permease fusion ABC transporter PA5302 dadX −2.9 0.004 catabolic alanine racemase PA5390  −2.6 0.04 probable peptidic bond hydrolase PA5424 yeaQ 3.4 0.0002 conserved hypothetical protein PA5473 yjbB 2.6 0.001 conserved hypothetical protein PA5493 polA 3.4 <0.0001 DNA polymerase I PA5517  −2.2 0.004 conserved hypothetical protein PA5526  7.0 <0.0001 hypothetical protein PA5531 tonB 2.5 0.009 TonB protein PA5537  2.0 0.008 hypothetical protein  a  Information according to the P. aeruginosa genome website (www.pseudomonas.com/) b  Fold regulation of genes differentially expressed in the phoQ mutant relative to WT. A positive number indicates transcript up-regulation in the phoQ mutant.         Table 3.4 qPCR gene expression analysis of select genes in phoQ mutants relative to wild-type. Experiments were performed using three independent biological samples each with two technical replicates and the average ± standard deviation is reported.  Gene IDa Namea  Fold changeb Descriptiona  PA0762 algU 5.1 ± 0.9 Sigma factor AlgU PA2862 lipA 3.2 ± 0.6 LipA lactonizing lipase precursor PA3552 arnB 505 ± 21 Aminotransferase in L-Ara4N biosynthesis PA4776 pmrA 4.1 ± 1.7 PmrA response regulator PA5261 algR 4.6 ± 2.6 AlgR response regulator  a  Information according to the P. aeruginosa genome website (www.pseudomonas.com/) b  Fold regulation of genes differentially expressed in the phoQ mutant relative to WT. A positive number indicates transcript up-regulation in the phoQ mutant.   81 Figure 3.1  0 10 20 30 40 50 Wild type PhoQ- PhoQ complement C yt o to x ici ty  (% ) 8.5 hours 16 hours pho phoQ (pUC- phoQ+) WT C yt o to x ici ty  (% )  Figure 3.1 PhoQ mutants displayed reduced in vitro cytotoxicity towards human bronchial epithelial cells. The ability of the wild-type PAO1 and phoQ mutant strains to induce cell damage was determined by monitoring the release of intracellular lactate dehydrogenase (LDH) into the supernatant from human bronchial epithelial cells. Bacteria were co-cultured with the cells and LDH release was monitored at the time point indicated. Data represent the mean of 3 biological repeats, each assayed in triplicate, with the data reported as averages ± standard deviation.  Figure 3.2 0 5 10 15 20 25 WT phoQ phoQ + pUC-phoQ Tw itc h zo n e  (m m ) phoQ (pUC- phoQ+) Tw itc h zo n e  (m m )  Figure 3.2 PhoQ mutants displayed reduced twitching motility. Twitching motility was assessed by inoculating cells from mid-logarithmic phase cultures into thin LB agar (1% wt/vol) plates, down to the agar-plastic interface, and measuring colony diameter after 24 hr incubation at 37°C. Results shown are averages of several independent biological replicates for each strain. *** represents a statistically significant difference (P <0.001) between phoQ mutant and wild-type as determined by Student’s t test. ***  82 Figure 3.3      Figure 3.3 PhoQ mutants were attenuated for virulence in lettuce leaves. Day 4 symptoms of Romaine leaf infections after midribs were inoculated with 1 × 106 CFU of P. aeruginosa. Leaves were incubated at 37°C in small containers with moistened paper towels. Shown is one representative leaf of several that produced similar symptoms.        Figure 3.4      Figure 3.4 Pigmentation differences of mid-logarithmic phase phoQ mutant and wild- type liquid cultures. Photograph of cultures grown in BM2-glucose minimal medium containing 2 mM MgSO4. WT WT phoQ phoQ  83 REFERENCES  Ausubel, F. M. 1987. Current protocols in molecular biology. Published by Greene Pub. Associates and Wiley-Interscience: J. Wiley, New York.  Bader, M. W., S. Sanowar, M. E. Daley, A. R. Schneider, U. Cho, W. Xu, R. E. Klevit, H. Le Moual, and S. I. Miller. 2005. Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 122:461-72.  Belete, B., H. Lu, and D. J. Wozniak. 2008. Pseudomonas aeruginosa AlgR regulates type IV pilus biosynthesis by activating transcription of the fimU-pilVWXY1Y2E operon. J Bacteriol 190:2023-30.  Bennett, H. P., and D. J. Clarke. 2005. The pbgPE operon in Photorhabdus luminescens is required for pathogenicity and symbiosis. J Bacteriol 187:77-84.  Bonomo, R. A., and D. Szabo. 2006. Mechanisms of multidrug resistance in Acinetobacter species and Pseudomonas aeruginosa. Clin Infect Dis 43 Suppl 2:S49-56.  Brinkman, F. S., E. L. Macfarlane, P. Warrener, and R. E. Hancock. 2001. Evolutionary relationships among virulence-associated histidine kinases. Infect Immun 69:5207-11.  Cash, H. A., D. E. Woods, B. McCullough, W. G. Johanson, Jr., and J. A. Bass. 1979. A rat model of chronic respiratory infection with Pseudomonas aeruginosa. Am Rev Respir Dis 119:453-9.  Cozens, A. L., M. J. Yezzi, K. Kunzelmann, T. Ohrui, L. Chin, K. Eng, W. E. Finkbeiner, J. H. Widdicombe, and D. C. Gruenert. 1994. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 10:38-47.  de Lima Pimenta, A., P. Di Martino, E. Le Bouder, C. Hulen, and M. A. Blight. 2003. In vitro identification of two adherence factors required for in vivo virulence of Pseudomonas fluorescens. Microbes Infect 5:1177-87.  El Solh, A. A., M. E. Akinnusi, J. P. Wiener-Kronish, S. V. Lynch, L. A. Pineda, and K. Szarpa. 2008. Persistent Infection with Pseudomonas Aeruginosa in Ventilator Associated Pneumonia. Am J Respir Crit Care Med. (In Press)  Ernst, R. K., E. C. Yi, L. Guo, K. B. Lim, J. L. Burns, M. Hackett, and S. I. Miller. 1999. Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa. Science 286:1561-5.   84 Filiatrault, M. J., K. F. Picardo, H. Ngai, L. Passador, and B. H. Iglewski. 2006. Identification of Pseudomonas aeruginosa genes involved in virulence and anaerobic growth. Infect Immun 74:4237-45.  Firoved, A. M., and V. Deretic. 2003. Microarray analysis of global gene expression in mucoid Pseudomonas aeruginosa. J Bacteriol 185:1071-81.  Fridkin, S. K., C. D. Steward, J. R. Edwards, E. R. Pryor, J. E. McGowan, Jr., L. K. Archibald, R. P. Gaynes, and F. C. Tenover. 1999. Surveillance of antimicrobial use and antimicrobial resistance in United States hospitals: project ICARE phase 2. Project Intensive Care Antimicrobial Resistance Epidemiology (ICARE) hospitals. Clin Infect Dis 29:245-52.  Furukawa, S., S. L. Kuchma, and G. A. O'Toole. 2006. Keeping their options open: acute versus persistent infections. J Bacteriol 188:1211-7.  Gibson, R. L., J. L. Burns, and B. W. Ramsey. 2003. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am J Respir Crit Care Med 168:918-51.  Grabenstein, J. P., M. Marceau, C. Pujol, M. Simonet, and J. B. Bliska. 2004. The response regulator PhoP of Yersinia pseudotuberculosis is important for replication in macrophages and for virulence. Infect Immun 72:4973-84.  Groisman, E. A. 2001. The pleiotropic two-component regulatory system PhoP-PhoQ. J Bacteriol 183:1835-42.  Gunn, J. S., S. S. Ryan, J. C. Van Velkinburgh, R. K. Ernst, and S. I. Miller. 2000. Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium. Infect Immun 68:6139-46.  Hancock, R. E., and D. P. Speert. 2000. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resist Updat 3:247-255.  Hava, D. L., and A. Camilli. 2002. Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol Microbiol 45:1389-406.  van Heeckeren, A. M., and M. D. Schluchter. 2002. Murine models of chronic Pseudomonas aeruginosa lung infection. Lab Anim 36:291-312.  Heurlier, K., F. Williams, S. Heeb, C. Dormond, G. Pessi, D. Singer, M. Camara, P. Williams, and D. Haas. 2004. Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1. J Bacteriol 186:2  Hokamp, K., F. M. Roche, M. Acab, M. E. Rousseau, B. Kuo, D. Goode, D. Aeschliman, J. Bryan, L. A. Babiuk, R. E. W. Hancock, and F. S. Brinkman. 2004.  85 ArrayPipe: a flexible processing pipeline for microarray data. Nucleic Acids Res 32:W457- 9.  Jenssen, H., P. Hamill, and R. E. W. Hancock. 2006. Peptide antimicrobial agents. Clin Microbiol Rev 19:491-511.  Lehoux, D. E., F. Sanschagrin, and R. C. Levesque. 2000. Genomics of the 35-kb pvd locus and analysis of novel pvdIJK genes implicated in pyoverdine biosynthesis in Pseudomonas aeruginosa. FEMS Microbiol Lett 190:141-6.  Lewenza, S., R. K. Falsafi, G. Winsor, W. J. Gooderham, J. B. McPhee, F. S. Brinkman, and R. E. W. Hancock. 2005. Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes. Genome Res 15:583-9.  Li, J., R. L. Nation, J. D. Turnidge, R. W. Milne, K. Coulthard, C. R. Rayner, and D. L. Paterson. 2006. Colistin: the re-emerging antibiotic for multidrug-resistant Gram- negative bacterial infections. Lancet Infect Dis 6:589-601.  Lizewski, S. E., D. S. Lundberg, and M. J. Schurr. 2002. The transcriptional regulator AlgR is essential for Pseudomonas aeruginosa pathogenesis. Infect Immun 70:6083-93.  Macfarlane, E. L., A. Kwasnicka, and R. E. W. Hancock. 2000. Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides. Microbiology 146:2543-54.  Macfarlane, E. L., A. Kwasnicka, M. M. Ochs, and R. E. W. Hancock. 1999. PhoP- PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer-membrane protein OprH and polymyxin B resistance. Mol Microbiol 34:305-16.  Marceau, M., F. Sebbane, F. Ewann, F. Collyn, B. Lindner, M. A. Campos, J. A. Bengoechea, and M. Simonet. 2004. The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence. Microbiology 150:3947-57.  Marr, A. K., W. J. Gooderham, and R. E. W. Hancock. 2006. Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Curr Opin Pharmacol 6:468-72.  McPhee, J. B., M. Bains, G. Winsor, S. Lewenza, A. Kwasnicka, M. D. Brazas, F. S. Brinkman, and R. E. W. Hancock. 2006. Contribution of the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to Mg2+-induced gene regulation in Pseudomonas aeruginosa. J Bacteriol 188:3995-4006.  McPhee, J. B., S. Lewenza, and R. E. W. Hancock. 2003. Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to  86 polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol Microbiol 50:205-17.  Mesaros, N., P. Nordmann, P. Plesiat, M. Roussel-Delvallez, J. Van Eldere, Y. Glupczynski, Y. Van Laethem, F. Jacobs, P. Lebecque, A. Malfroot, P. M. Tulkens, and F. Van Bambeke. 2007. Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium. Clin Microbiol Infect 13:560-78.  Miller, S. I., A. M. Kukral, and J. J. Mekalanos. 1989. A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence. Proc Natl Acad Sci U S A 86:5054-8.  Moskowitz, S. M., R. K. Ernst, and S. I. Miller. 2004. PmrAB, a two-component regulatory system of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid A. J Bacteriol 186:575-9.  O'Toole, G. A., and R. Kolter. 1998. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30:295-304.  Overhage, J., M. Bains, M. D. Brazas, and R. E. W. Hancock. 2008. Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance. J Bacteriol 190:2671-9.  Palmer, K. L., L. M. Aye, and M. Whiteley. 2007. Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum. J Bacteriol 189:8079-87.  Rahme, L. G., F. M. Ausubel, H. Cao, E. Drenkard, B. C. Goumnerov, G. W. Lau, S. Mahajan-Miklos, J. Plotnikova, M. W. Tan, J. Tsongalis, C. L. Walendziewicz, and R. G. Tompkins. 2000. Plants and animals share functionally common bacterial virulence factors. Proc Natl Acad Sci U S A 97:8815-21.  Rahme, L. G., M. W. Tan, L. Le, S. M. Wong, R. G. Tompkins, S. B. Calderwood, and F. M. Ausubel. 1997. Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors. Proc Natl Acad Sci U S A 94:13245-50.  Ramsey, M. M., and M. Whiteley. 2004. Pseudomonas aeruginosa attachment and biofilm development in dynamic environments. Mol Microbiol 53:1075-87.  Sabra, W., H. Lunsdorf, and A. P. Zeng. 2003. Alterations in the formation of lipopolysaccharide and membrane vesicles on the surface of Pseudomonas aeruginosa PAO1 under oxygen stress conditions. Microbiology 149:2789-95.  Shin, D., E. J. Lee, H. Huang, and E. A. Groisman. 2006. A positive feedback loop promotes transcription surge that jump-starts Salmonella virulence circuit. Science 314:1607-9.   87 Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. W. Hancock, S. Lory, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959-64.  West, S. E., H. P. Schweizer, C. Dall, A. K. Sample, and L. J. Runyen-Janecky. 1994. Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148:81-6.  Winsor, G. L., R. Lo, S. J. Sui, K. S. Ung, S. Huang, D. Cheng, W. K. Ching, R. E. W. Hancock, and F. S. Brinkman. 2005. Pseudomonas aeruginosa Genome Database and PseudoCAP: facilitating community-based, continually updated, genome annotation. Nucleic Acids Res 33:D338-43.  Wu, M., and R. E. W. Hancock. 1999. Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane. J Biol Chem 274:29-35.  Zavascki, A. P., L. Z. Goldani, J. Li, and R. L. Nation. 2007. Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review. J Antimicrob Chemother 60:1206-15.   88 CHAPTER 4 – Concluding remarks  INTRODUCTION  The opportunistic pathogen P. aeruginosa causes serious infections including those associated with burn wounds, ventillator-associated pneumonia, and inside the lungs of cystic fibrosis patients. Without the development of new drugs active against these and other hospital-acquired Gram-negative bacterial infections, a foundation of modern medicine faces an ominous future (Falagas and Bliziotis 2007). The studies on PsrA and PhoQ presented in this thesis highlight the complex nature of P. aeruginosa regulation of virulence and antimicrobial peptide resistance. Mutation of either gene is associated with several phenotypic differences relative to wild-type and accompanied by the transcriptional dysregulation of a relatively large number of genes. The ability of PsrA and PhoQ to affect a wide-range of important overlapping processes in virulence is noteworthy. Further, the inclusion of antimicrobial peptide resistance with these virulence phenotypes highlights the significance of these findings. PsrA As described in Chapter 2, the PsrA transcriptional regulator appears capable of regulating intrinsic resistance to antimicrobial peptides and polymyxin B, swarming motility and biofilm formation. The finding that psrA was one of the few regulatory protein-encoding genes induced by antimicrobial peptides is significant given that peptides are known to regulate an assortment of resistance and virulence genes in other bacteria. The downregulation of the pprB gene on the PsrA microarray was possibly partly responsible for the psrA mutant’s biofilm formation phenotype as a pprB mutant was defective in biofilm formation. PprB is a response regulator and forms a two-component  89 regulatory system with its cognate sensor kinase PprA. Interestingly, this system was found to have a role in membrane permeability, antibiotic resistance, virulence factor production, and modulation of quorum sensing (Dong et al. 2005; Wang et al. 2003), similar phenotypes to those of PsrA. Determining whether PsrA could have a clinically significant role is an interesting subject. PsrA does regulate processes that are necessary for virulence, such as biofilm formation and motility. Further, P. aeruginosa would be exposed to antimicrobial peptides during both therapeutic administration (e.g inhaled polymyxins) and during meetings on epithelial or mucosal surfaces (e.g. host defensins). For this reason, it would be of interest to examine whether sub-inhibitory concentrations of peptides modulate the observed psrA phenotypes in wild-type P. aeruginosa (e.g. swarming or biofilms) and then see if this modulation is lost in the psrA mutant. Swarming motility itself is a complex motility adaptation where cells become more antibiotic resistant and increase production of virulence factors (Overhage et al. 2008). Further, biofilm formation is widely recognized now to be a leading determinant in the antibiotic resistance of some chronic infections. As such, the finding that PsrA can regulate swarming and biofilms adds more insight into the regulation of these virulence determinants in P. aeruginosa. Indeed, the inverse regulation of type III secretion and biofilm formation of the psrA mutant is reminiscent of the two main phenotypes of the ladS mutant in P. aeruginosa (Ventre et al. 2006). LadS is a hybrid-type sensor kinase and a master regulator capable of inversely controlling chronic (e.g. biofilm) and acute (e.g. type III secretion) virulence determinants (Ventre et al. 2006).    90 PhoQ The phoQ work documents an interesting dilemma between antimicrobial peptides and virulence (Chapter 3). That is, although the phoQ mutant possesses the most antimicrobial peptide super-resistant phenotype known in P. aeruginosa, this mutant was nevertheless highly attenuated for virulence. Further, from our laboratory’s recent screens of P. aeruginosa transposon mutant libraries for increased peptide resistance, the phoQ mutation represents one of the few single gene mutations which increases antimicrobial peptide resistance. Conversely, screens for mutants resistant to conventional antibiotics such as the aminoglycoside tobramycin or the fluoroquinolone ciprofloxacin have uncovered large numbers of genes conferring resistance upon knockout mutation. This inherent difficulty in developing mutational antimicrobial peptide resistance is significant and lends added support for the continued development of antimicrobial peptides as therapeutics. FUTURE RESEARCH DIRECTIONS Although cationic antimicrobial peptides are being pursued as novel antimicrobials against these infections, some antimicrobial resistant determinants have already been described. As mentioned, sub-inhibitory concentrations of these peptides have been shown to have a variety of effects on bacteria independent of their lethal effects at higher concentrations. A central goal has been to find the P. aeruginosa regulator of adaptive peptide resistance. That is, finding the regulatory system that senses sub-inhibitory concentrations of peptides and subsequently promotes resistance to normally lethal concentrations of peptides. In Salmonella, it is PhoQ that directly senses peptides and activates its cognate regulator PhoP to promote resistance, but P. aeruginosa PhoQ does not perform this task as the periplasmic sensing domains of these proteins are quite dissimilar  91 (Bader et al. 2005). PsrA is also not the regulator that responds to peptides (even though psrA was transcriptionally induced by peptides), as growing P. aeruginosa with sub-lethal levels of peptides (to promote resistance) and then killing with high concentrations showed that the psrA mutant was not super-sensitive relative to wild-type. It is possible that one of the many uncharacterized two-component regulatory systems in P. aeruginosa functions as this elusive peptide sensor/regulator. Further, this regulator is likely a homolog of PmrA due to the fact that the mystery regulator must recognize similar PmrA target promoters (PA4773-pmrAB and arnBCADTEF) to promote resistance through LPS modification. Although no differences were seen in the lettuce model for the psrA mutant, it is interesting nevertheless to speculate on the nature of the virulence phenotype for the psrA mutant in an in vivo model (e.g. the rat model of chronic lung infections). The capacity to form biofilms is generally associated with a chronic infection state, so this mutant might be predicted to be attenuated for virulence. Indeed, the other psrA mutant phenotypes such as impaired swarming and antimicrobial peptide susceptibility would lend support to this prediction. However, a mutant possessing multiple phenotypes makes predicting such an outcome difficult (as was described for PhoQ). It is interesting to speculate how antimicrobial peptides are capable of inducing psrA transcription. The first step would be to determine whether this peptide-mediated regulation is direct (peptides interacting with PsrA) or indirect (peptides affecting another protein which then effects psrA transcription). Recently, it was shown that both sub-inhibitory concentrations of both indolicidin and human LL-37 cationic peptides are able to inhibit P. aeruginosa biofilm formation while not killing the bacteria (Overhage and Hancock, unpublished). Due to the similar overlap of these peptide and biofilm associated phenotypes  92 in the psrA mutant, it would be interesting to test this peptide-influenced biofilm formation in the psrA mutant to see if PsrA modulates this effect. Mutation of phoQ was shown to affect a large number of genes outside of the PhoP regulon. However, at present it is not known how the membrane-bound sensor kinase PhoQ accomplishes this. As membrane-bound PhoQ cannot be directly regulating these genes, presumably it is capable of modifying the phosphorylation state of an unknown intermediate regulatory protein. In the phoQ mutant this activity is lost and consequently gene expression is modified. Finding such a protein would greatly add an important mechanistic aspect to a presently quite descriptive PhoQ story. The future study on P. aeruginosa regulatory genes (known and uncharacterized) involved in virulence and antibiotic resistance is anticipated to shed more light on these important processes and may ultimately help establish connections and hierarchy between these regulatory systems in this important opportunistic human pathogen.   93 REFERENCES  Bader, M. W., S. Sanowar, M. E. Daley, A. R. Schneider, U. Cho, W. Xu, R. E. Klevit, H. Le Moual, and S. I. Miller. 2005. Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 122:461-72.  Dong, Y. H., X. F. Zhang, H. M. Soo, E. P. Greenberg, and L. H. Zhang. 2005. The two- component response regulator PprB modulates quorum-sensing signal production and global gene expression in Pseudomonas aeruginosa. Mol Microbiol 56:1287-301.  Falagas, M. E., and I. A. Bliziotis. 2007. Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era? Int J Antimicrob Agents 29:630-6.  Overhage, J., M. Bains, M. D. Brazas, and R. E. W. Hancock. 2008. Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance. J Bacteriol 190:2671-9.  Ventre, I., A. L. Goodman, I. Vallet-Gely, P. Vasseur, C. Soscia, S. Molin, S. Bleves, A. Lazdunski, S. Lory, and A. Filloux. 2006. Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc Natl Acad Sci U S A 103:171-6.  Wang, Y., U. Ha, L. Zeng, and S. Jin. 2003. Regulation of membrane permeability by a two-component regulatory system in Pseudomonas aeruginosa. Antimicrob Agents Chemother 47:95-101.   94 APPENDIX I.  Other genes significantly dysregulated in psrA mutants and displaying ≥ 1.5-fold change as determined using microarray. This list includes hypothetical genes and other genes not included in Table 2.4.  Gene IDa Gene namea Fold changeb P value  Descriptiona  PA0050  −1.7 0.02 hypothetical protein PA0095 vgr −1.7 0.004 conserved hypothetical protein PA0109  −1.5 0.04 hypothetical protein PA0172  1.5 0.008 hypothetical protein PA0177  −1.5 0.03 purine-binding chemotaxis protein PA0178  −1.8 0.04 probable two-component sensor PA0179  −1.8 0.008 two-component response regulator PA0321  −1.6 0.01 probable acetylpolyamine aminohydrolase PA0460  −1.8 0.03 hypothetical protein PA0484  −1.6 0.02 conserved hypothetical protein PA0524 norB −1.9 0.0008 nitric-oxide reductase subunit B PA0586 ycgB −1.6 0.01 conserved hypothetical protein PA0587 yeaH −1.9 0.02 conserved hypothetical protein PA0617  −1.5 0.02 probable bacteriophage protein PA0645  −1.7 0.008 hypothetical protein PA0803  1.8 0.01 hypothetical protein PA0805  −1.8 0.05 hypothetical protein PA0808  −2.0 0.06 hypothetical protein PA0816  1.6 0.01 probable transcriptional regulator PA0841  −1.6 0.01 hypothetical protein PA0861  −1.5 0.007 hypothetical protein PA0873 phhR −1.5 0.02 transcriptional regulator PhhR PA0912  −1.6 0.06 hypothetical protein PA0997 pqsB −1.7 0.02 hypothetical protein PA1065  −2.1 0.01 conserved hypothetical protein PA1090  −1.5 0.01 hypothetical protein PA1107  −1.7 0.004 conserved hypothetical protein PA1124 dgt −1.6 0.01 triphosphohydrolase PA1133  −1.6 0.04 hypothetical protein PA1150 pys2 −1.5 0.02 pyocin S2 PA1175 napD −1.9 0.003 NapD of periplasmic nitrate reductase PA1311 phnX −1.6 0.006 2-phosphonoacetaldehyde hydrolase PA1366  −1.6 0.01 hypothetical protein PA1370  −1.7 0.02 hypothetical protein PA1373 fabF2 1.7 0.02 3-oxoacyl-acyl carrier protein synthase II PA1436  −1.6 0.02 probable RND efflux transporter PA1437  −1.7 0.05 two-component response regulator PA1450  −2.0 0.02 conserved hypothetical protein  95 Gene IDa Gene namea Fold changeb P value  Descriptiona  PA1469  1.7 0.005 hypothetical protein PA1577  −1.6 0.06 hypothetical protein PA1692 pscS 1.6 0.002 translocation protein in type III secretion PA1694 pscQ 1.6 0.004 translocation protein in type III secretion PA1696 pscO 1.6 0.0003 translocation protein in type III secretion PA1704 pcrR 1.7 0.02 transcriptional regulator protein PcrR PA1711  1.7 0.001 hypothetical protein PA1714 exsD 1.8 0.02 hypothetical protein PA1718 pscE 1.8 0.03 type III export protein PscE PA1720 pscG 1.7 0.01 type III export protein PscG PA1829  −1.6 0.005 hypothetical protein PA1875  −1.5 0.03 hypothetical protein PA1876  −1.6 0.03 ATP-binding fusion ABC transporter PA1891  1.7 0.03 hypothetical protein PA1903 phzE2 −1.7 0.02 phenazine biosynthesis protein PhzE PA1988 pqqD −1.9 0.009 pyrroloquinoline quinone biosynthesis PA2231 pslA −1.6 0.05 probable glycosyl transferase PA2313  −2.0 0.06 hypothetical protein PA2318  2.2 0.04 hypothetical protein PA2324  −2.2 0.04 hypothetical protein PA2358  1.8 0.01 hypothetical protein PA2363  −1.9 0.02 hypothetical protein PA2365  −4.1 0.01 conserved hypothetical protein PA2367  −5.4 0.006 hypothetical protein PA2369  −4.6 0.008 hypothetical protein PA2373  −2.5 0.03 conserved hypothetical protein PA2412  −2.7 0.02 conserved hypothetical protein PA2428  3.6 0.002 hypothetical protein PA2430  2.6 0.03 conserved hypothetical protein PA2439  2.30 0.04 hypothetical protein PA2462  −2.2 0.005 hypothetical protein PA2463  1.5 0.009 hypothetical protein PA2504  2.0 0.06 hypothetical protein PA2539 ynbD 1.6 0.02 conserved hypothetical protein PA2546  −1.6 0.01 probable ring-cleaving dioxygenase PA2562  −2.0 0.004 hypothetical protein PA2565  −2.3 0.04 hypothetical protein PA2597  1.7 0.03 hypothetical protein PA2622 cspD −1.5 0.002 cold-shock protein CspD PA2655  −1.5 0.003 hypothetical protein PA2662  −7.5 0.004 conserved hypothetical protein PA2668  −1.51 0.008 hypothetical protein PA2706  −1.6 0.004 hypothetical protein PA2729  −1.6 0.01 hypothetical protein  96 Gene IDa Gene namea Fold changeb P value  Descriptiona  PA2731  −2.1 0.02 hypothetical protein PA2732  −1.7 0.002 hypothetical protein PA2750  1.5 0.04 hypothetical protein PA2790  −1.6 0.03 hypothetical protein PA2833  −2.0 0.0005 conserved hypothetical protein PA3012  3.5 <0.001 hypothetical protein PA3097 xcpX −1.5 0.006 general secretion pathway protein K PA3125  3.9 0.01 hypothetical protein PA3136  −1.6 0.02 probable secretion protein PA3261  −1.5 0.004 hypothetical protein PA3300 fadD2 −1.8 0.02 long-chain-fatty-acid--CoA ligase PA3314  1.5 0.03 ATP-binding cmpt of ABC transporter PA3320  −2.1 0.006 hypothetical protein PA3327  −2.7 0.02 probable non-ribosomal peptide synthetase PA3335  −3.5 0.01 hypothetical protein PA3360  2.0 0.009 probable secretion protein PA3394 nosF 1.9 0.03 NosF protein PA3424  1.5 0.04 hypothetical protein PA3426  −1.6 0.009 probable enoyl CoA-hydratase/isomerase PA3448 ygaM −1.7 0.01 probable permease of ABC transporter PA3451  −2.1 0.05 hypothetical protein PA3452 mqoA −1.6 0.004 malate:quinone oxidoreductase PA3533 ydhD −1.50 0.02 conserved hypothetical protein PA3534  −1.9 0.0007 probable oxidoreductase PA3575  1.7 0.01 hypothetical protein PA3592 baiF −2.1 0.05 conserved hypothetical protein PA3667  1.52 0.02 pyridoxal-phosphate dependent enzyme PA3720  10.4 0.002 hypothetical protein PA3773  −6.4 0.004 hypothetical protein PA3789  18.4 0.001 hypothetical protein PA3843  2.7 0.0007 hypothetical protein PA3852  −1.8 0.009 hypothetical protein PA3855  −1.9 0.01 hypothetical protein PA3858 aapJ −1.6 0.007 probable amino acid-binding protein PA3945  −1.7 0.002 conserved hypothetical protein PA3972 aidB −1.8 0.02 probable acyl-CoA dehydrogenase PA3973  −1.8 0.03 probable transcriptional regulator PA3986  −2.5 0.01 hypothetical protein PA4015  −2.0 0.003 conserved hypothetical protein PA4096 phlE 2.0 0.05 probable MFS transporter PA4242 rpmJ −1.9 0.01 50S ribosomal protein L36 PA4245 rpmD −1.7 0.02 50S ribosomal protein L30 PA4296  −2.9 0.06 two-component response regulator PA4298  −3.7 0.01 hypothetical protein  97 Gene IDa Gene namea Fold changeb P value  Descriptiona  PA4300  −2.2 0.04 hypothetical protein PA4303  −2.7 0.02 hypothetical protein PA4305  −2.1 0.05 hypothetical protein PA4364  −1.8 0.01 hypothetical protein PA4479 mreD 1.7 0.04 rod shape-determining protein MreD PA4487  1.5 0.03 conserved hypothetical protein PA4507  5.7 0.005 hypothetical protein PA4531  −4.5 0.02 hypothetical protein PA4586  1.5 0.01 hypothetical protein PA4591  −2.0 0.007 hypothetical protein PA4624  −1.6 0.03 hypothetical protein PA4625  −3.7 0.006 hypothetical protein PA4632  1.5 0.04 hypothetical protein PA4658  1.6 0.02 hypothetical protein PA4691  −1.7 0.03 hypothetical protein PA4738 yjbJ −1.8 0.0007 conserved hypothetical protein PA4739  −1.6 0.02 conserved hypothetical protein PA4783 yedA 1.7 0.02 conserved hypothetical protein PA4822  −2.4 0.03 hypothetical protein PA4889  1.5 0.03 probable oxidoreductase PA4908  −6.3 0.005 hypothetical protein PA5178  −2.7 0.02 conserved hypothetical protein PA5185  −1.5 0.04 conserved hypothetical protein PA5255 algQ −1.52 0.02 Alginate regulatory protein AlgQ PA5348  1.7 0.03 probable DNA-binding protein PA5479 gltP 1.5 0.04 proton-glutamate symporter  a  Information according to the P. aeruginosa genome website (www.pseudomonas.com/) b  Fold regulation of genes differentially expressed in psrA mutant relative to WT. A positive number indicates transcript up-regulation in the psrA mutant.   98 APPENDIX II.  Significantly dysregulated genes showing ≥ 1.5-fold change in phoQ mutants as determined using microarray. Genes represent those not included in Table 3.3.  Gene IDa Name  Fold changeb P value  Descriptiona  PA0034   1.8 0.05 probable two-component response regulator PA0038   1.7 0.003 hypothetical protein PA0060   1.6 0.01 conserved hypothetical protein PA0069   −1.7 0.05 conserved hypothetical protein PA0084   1.6 0.004 conserved hypothetical protein PA0120   −1.8 0.01 probable transcriptional regulator PA0125   −1.8 0.03 hypothetical protein PA0266 gabT 1.5 0.008 4-aminobutyrate aminotransferase PA0360   −1.5 0.09 hypothetical protein PA0371   1.7 0.005 hypothetical protein PA0372   1.5 0.01 probable zinc protease PA0423  yceI 1.7 0.02 conserved hypothetical protein PA0426 mexB 1.6 0.001 RND multidrug efflux transporter MexB PA0432 sahH −1.5 0.01 S-adenosyl-L-homocysteine hydrolase PA0454  yccS −1.6 0.05 conserved hypothetical protein PA0471  fiuR 1.7 0.02 probable transmembrane sensor PA0481   −1.5 0.023 hypothetical protein PA0500 bioB −1.6 0.004 biotin synthase PA0536   1.6 0.0007 hypothetical protein PA0552 pgk 1.5 0.02 phosphoglycerate kinase PA0565   −1.6 0.01 conserved hypothetical protein PA0568   −1.6 0.007 hypothetical protein PA0570   −1.8 0.004 hypothetical protein PA0571   −1.7 0.001 hypothetical protein PA0604   1.7 0.03 binding protein component of ABC transporter PA0605   1.9 0.01 probable permease of ABC transporter PA0613   1.6 0.0006 hypothetical protein PA0616   1.5 0.008 hypothetical protein PA0617   1.7 0.001 probable bacteriophage protein PA0618   1.7 0.0008 probable bacteriophage protein PA0624   1.5 0.002 hypothetical protein PA0625   1.6 0.001 hypothetical protein PA0626   1.6 0.01 hypothetical protein PA0627   1.6 0.002 conserved hypothetical protein PA0628   1.6 0.001 conserved hypothetical protein PA0632   1.9 0.007 hypothetical protein PA0634   1.9 0.0002 hypothetical protein PA0635   1.9 0.0002 hypothetical protein PA0638   1.9 0.03 probable bacteriophage protein  99 Gene IDa Name  Fold changeb P value  Descriptiona  PA0639   1.8 0.002 conserved hypothetical protein PA0643   1.7 0.004 hypothetical protein PA0667  yebA 1.5 0.02 conserved hypothetical protein PA0792 prpD −1.6 0.008 propionate catabolic protein PrpD PA0797   −1.5 0.002 probable transcriptional regulator PA0834   −1.7 0.03 conserved hypothetical protein PA0840   −1.6 0.02 probable oxidoreductase PA0852 cbpD 1.7 0.03 chitin-binding protein CbpD precursor PA0876   −1.7 0.02 probable transcriptional regulator PA0887 acsA 1.6 0.01 acetyl-coenzyme A synthetase PA0924   1.9 0.03 hypothetical protein PA0943   1.5 0.01 hypothetical protein PA0958 oprD −1.5 0.01 outer membrane porin protein OprD precursor PA0968  ybgC 1.5 0.005 conserved hypothetical protein PA1002 phnB 1.6 0.002 anthranilate synthase component II PA1048   1.8 0.002 probable outer membrane protein PA1106   1.5 0.02 hypothetical protein PA1159   1.7 0.008 probable cold-shock protein PA1175 napD 1.5 0.01 NapD protein of periplasmic nitrate reductase PA1249 aprA 1.9 0.001 alkaline metalloproteinase precursor PA1250 aprI 1.6 0.001 alkaline proteinase inhibitor AprI PA1282   −1.6 0.04 probable MFS transporter PA1296   1.9 0.0008 probable 2-hydroxyacid dehydrogenase PA1320 cyoD −1.8 0.01 cytochrome o ubiquinol oxidase subunit IV PA1327   −1.9 0.04 probable protease PA1389   1.7 0.02 probable glycosyl transferase PA1402   −1.8 0.03 hypothetical protein PA1437   1.6 0.01 probable two-component response regulator PA1535   −1.6 0.01 probable acyl-CoA dehydrogenase PA1542   −1.8 0.05 hypothetical protein PA1696 pscO −1.7 0.03 translocation protein in type III secretion PA1707 pcrH −1.6 0.05 regulatory protein PcrH PA1713 exsA −1.6 0.005 transcriptional regulator ExsA PA1726 bglX 1.5 0.03 periplasmic beta-glucosidase PA1800 tig 1.8 0.05 trigger factor PA1804 hupB 1.5 0.05 DNA-binding protein HU PA1882   1.8 0.02 probable transporter PA1912   −1.8 0.03 probable sigma-70 factor, ECF subfamily PA1930   1.5 0.005 probable chemotaxis transducer PA2071 fusA2 1.9 0.004 elongation factor G PA2177   1.8 0.002 probable sensor/response regulator hybrid PA2186   −3.4 0.02 hypothetical protein PA2193 hcnA 1.7 0.0007 hydrogen cyanide synthase HcnA PA2232 pslB −1.8 0.002 probable phosphomannose isomerase/GDP-  100 Gene IDa Name  Fold changeb P value  Descriptiona  mannose pyrophosphorylase PA2234 pslD −1.5 0.005 probable exopolysaccharide transporter PA2236 pslF −1.7 0.005 hypothetical protein PA2237 pslG −1.7 0.0003 probable glycosyl hydrolase PA2238 pslH −1.7 0.005 hypothetical protein PA2299   −1.9 0.04 probable transcriptional regulator PA2302   1.8 0.002 probable non-ribosomal peptide synthetase PA2303   1.8 0.0002 hypothetical protein PA2307   −1.9 0.02 probable permease of ABC transporter PA2344 mtlZ −1.6 0.04 fructokinase PA2364   −1.8 0.02 hypothetical protein PA2365   −1.5 0.04 conserved hypothetical protein PA2366   −1.6 0.04 conserved hypothetical protein PA2367   −1.7 0.03 hypothetical protein PA2396 pvdF 1.6 0.05 hypothetical protein PA2444 glyA2 −1.8 0.003 serine hydroxymethyltransferase PA2461   −1.6 0.02 hypothetical protein PA2482   −1.6 0.02 probable cytochrome c PA2527  yegN 1.6 0.007 RND efflux transporter PA2528  yegM 1.7 0.0005 RND efflux membrane fusion protein precursor PA2529   1.6 0.01 hypothetical protein PA2539  −1.8 0.01 conserved hypothetical protein PA2630  −1.5 0.02 conserved hypothetical protein PA2650  −2.5 0.04 conserved hypothetical protein PA2656   1.9 0.0006 probable two-component sensor PA2657   1.9 0.001 probable two-component response regulator PA2728   1.7 0.002 hypothetical protein PA2771   1.5 0.02 conserved hypothetical protein PA2777   1.6 0.04 conserved hypothetical protein PA2778   1.6 0.03 hypothetical protein PA2847   −1.9 0.002 conserved hypothetical protein PA2850 ohr −1.5 0.02 organic hydroperoxide resistance protein PA2895   1.9 0.0002 hypothetical protein PA2896   1.5 0.01 probable sigma-70 factor, ECF subfamily PA2958   −1.7 0.02 hypothetical protein PA2972  yceF −1.7 0.05 conserved hypothetical protein PA3038  opdQ 1.7 0.003 porin PA3040  yqjD 1.8 0.004 conserved hypothetical protein PA3041  yqjE 1.6 0.0007 hypothetical protein PA3042   1.7 0.008 hypothetical protein PA3119  yafE −1.5 0.004 conserved protein PA3136   −1.7 0.002 probable secretion protein PA3160 wzz 1.9 0.0002 O-antigen chain length regulator PA3180   1.8 0.05 hypothetical protein  101 Gene IDa Name  Fold changeb P value  Descriptiona  PA3188  gltG 1.8 0.009 permease of ABC sugar transporter PA3197   −1.6 0.03 hypothetical protein PA3234  yjcG 1.8 0.001 sodium:solute symporter PA3235  yjcH 1.8 0.007 conserved protein PA3243 minC 1.8 0.003 cell division inhibitor MinC PA3268   1.7 0.04 probable TonB-dependent receptor PA3304   −1.5 0.003 conserved hypothetical protein PA3310   1.9 0.004 conserved hypothetical protein PA3385   1.8 0.0005 hypothetical protein PA3441  ssuF −1.6 0.009 molybdopterin-binding protein PA3459  asnB 1.7 0.005 probable amidotransferase PA3461  yhfE 1.5 0.006 conserved protein PA3532   −1.9 0.02 hypothetical protein PA3581 glpF −1.8 0.0005 glycerol uptake facilitator protein PA3584 glpD −1.9 0.007 glycerol-3-phosphate dehydrogenase PA3603 dgkA 1.5 0.02 diacylglycerol kinase PA3677   1.5 0.05 RND efflux membrane fusion protein precursor PA3760  nagF 1.9 0.02 phosphotransferase protein PA3762   1.6 0.01 hypothetical protein PA3770 guaB −1.6 0.03 inosine-5-monophosphate dehydrogenase PA3795   1.6 0.002 probable oxidoreductase PA3815   −1.8 0.002 conserved hypothetical protein PA3817   −1.5 0.02 probable methyltransferase PA3857 pcs 1.5 0.005 conserved hypothetical protein PA3902   1.9 0.0008 hypothetical protein PA3962   1.6 0.001 hypothetical protein PA3979   −1.5 0.04 hypothetical protein PA3990   −1.5 0.05 conserved hypothetical protein PA4190 pqsL 1.5 0.009 probable FAD-dependent monooxygenase PA4297   −1.7 0.03 hypothetical protein PA4312   1.9 0.03 conserved hypothetical protein PA4345   1.6 0.03 hypothetical protein PA4366 sodB 1.6 0.02 superoxide dismutase PA4372   1.7 0.003 hypothetical protein PA4374   1.5 0.02 RND efflux membrane fusion protein precursor PA4377   1.8 0.0003 hypothetical protein PA4378 inaA 1.8 0.008 InaA protein PA4379   1.9 0.001 conserved hypothetical protein PA4391   −1.5 0.03 hypothetical protein PA4448 hisD −1.6 0.02 histidinol dehydrogenase PA4496   1.8 0.003 binding protein component of ABC transporter PA4550 fimU 1.8 0.001 type 4 fimbrial biogenesis protein FimU PA4552 pilW 1.8 0.0003 type 4 fimbrial biogenesis protein PilW PA4582   −1.6 0.007 conserved hypothetical protein  102 Gene IDa Name  Fold changeb P value  Descriptiona  PA4606  cstA 1.6 0.002 conserved protein PA4620   −1.9 0.002 hypothetical protein PA4638   1.6 0.013 hypothetical protein PA4717   1.7 0.009 conserved hypothetical protein PA4735   1.6 0.004 hypothetical protein PA4781   1.9 0.004 probable two-component response regulator PA4782   1.6 0.005 hypothetical protein PA4785   1.7 0.004 probable acyl-CoA thiolase PA4786   1.7 0.01 probable short-chain dehydrogenase PA4874   1.6 0.02 conserved hypothetical protein PA4885 irlR -1.7 0.0004 two-component response regulator PA4891 ureE -1.6 0.01 urease accessory protein UreE PA4917   -1.9 0.004 hypothetical protein PA4919 pncB1 -1.8 0.03 nicotinate phosphoribosyltransferase PA4983  dmsR -1.6 0.01 two-component response regulator PA4995   -1.6 0.004 probable acyl-CoA dehydrogenase PA5026   -1.9 0.01 hypothetical protein PA5041 pilP -1.5 0.007 type 4 fimbrial biogenesis protein PilP PA5042 pilO -1.7 0.02 type 4 fimbrial biogenesis protein PilO PA5043 pilN -1.7 0.004 type 4 fimbrial biogenesis protein PilN PA5054 hslU -1.8 0.02 heat shock protein HslU PA5108   1.9 0.0001 hypothetical protein PA5116   1.8 0.008 probable transcriptional regulator PA5139   -1.7 0.04 hypothetical protein PA5173 arcC -1.7 0.05 carbamate kinase PA5179   -1.5 0.04 probable transcriptional regulator PA5217   1.9 0.01 binding protein of ABC iron transporter PA5235 glpT -1.7 0.008 glycerol-3-phosphate transporter PA5248   1.5 0.04 hypothetical protein PA5261 algR 1.8 0.007 alginate biosynthesis regulatory protein AlgR PA5367 pstA -1.8 0.01 membrane protein of phosphate transporter PA5369   -1.7 0.001 hypothetical protein PA5450 wzt 1.8 0.002 ABC subunit of A-band LPS efflux transporter PA5451 wzm 1.5 0.01 subunit of A-band LPS efflux transporter PA5452 wbpW 1.6 0.003 phosphomannose isomerase/ WbpW PA5483 algB 1.8 0.005 two-component response regulator AlgB PA5542   1.5 0.02 hypothetical protein  a  Information according to the P. aeruginosa genome website (www.pseudomonas.com/) b  Fold regulation of genes differentially expressed in phoQ mutant relative to WT. A positive number indicates transcript up-regulation in the phoQ mutant. 

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