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Early interaction between pseudomonas aeruginosa and polarized human bronchial epithelial cells Lo, Andy 2008

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EARLY INTERACTION BETWEEN PSEUDOMONAS AERUGINOSA AND POLARIZED HUMAN BRONCHIAL EPITHELIAL CELLS by ANDY LO B.Sc., University of British Columbia, 2002 A THESIS SUBMITED IN PARTIAL FUFFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology) UNIVERSITY OF BRITISH COLUMBIA January 2008 ©Andy Lo, 2008 Abstract Pseudomonas is the most common cause of chronic lung infections leading to death in cystic fibrosis patients. While chronic infection is extremely difficult to eradicate, the initial bacterial- host interactions prior to biofilm formation and establishment of chronic infections represents an attractive therapeutic target. It is clear that interaction between pathogens and the host is a very complex process and successful adaptation requires tight control of virulence factor expression. The aim of this project was to look for early changes in P. aeruginosa global gene expression in response to attachment to epithelial cells. P. aeruginosa PA01 was incubated with polarized HBE cells at a MOI of 100 for 4 hours and bacteria attached to epithelial cells (interacting) were collected separately from those in the supernatant (non-interacting). To minimize media effects observed by others, iron and phosphate were supplemented at appropriate levels to avoid expression changes due to limitation of these nutrients, as confirmed in our microarray experiments. Analysis of 3 independent experiments demonstrated that 766 genes were up or down regulated by more than 1.5 fold during attachment. Among these, 371 genes, including ion, oprC, as well as 3 genes in quorum-sensing systems and 9 genes involved in the pmrAB and phoPQ two-component regulatory systems were found to be induced in the interacting bacteria. On the other hand, 395 genes, including oprG outer membrane porin and pscP involved in type III secretion system were down regulated. To understand the roles of these differentially expressed genes, a cytotoxicity (LDH release) assay was performed and demonstrated that oprG and ion mutants were less capable than the wild type of killing HBE epithelial cells. These findings suggest that, under these interaction assay conditions, regulation of the expression of certain virulence factors provides a potential advantage for successful adaptation. In addition, a mutant lacking a filamentous hemagglutinin like protein was found to be less cytotoxic to HBE ii cells and also deficient in A549 epithelial cell binding, indicating that this probable non-pilin adhesin has multiple functions in P. aeruginosa. iii Table of Contents Abstract^ ii Table of Contents^ iv List of Tables vi List of Figures^ vii List of Abbreviations viii 1. Introduction^ 1 1.1 Pseudomonas aeruginosa^  1 1.2 P. aeruginosa in respiratory infection^  1 1.3 Initial bacterial-host interaction 2 1.3.1 Airway epithelium as the first line of defense ^ 3 1.3.2 P. aeruginosa in initial interaction: Attachment and motility^ 3 1.4 Additional virulence factors in P. aeruginosa 5 1.4.1 Type III secretion systems^ 5 1.4.2 Quorum-sensing systems 6 1.4.3 Iron acquisition^ 8 1.5 Differential expression of virulence factors for successful adaptation^ 8 1.6 Rationale and experimental goals^ 9 2. Materials and Methods^ 11 2.1 Bacterial strains and growth conditions^  11 2.2 Cell culture^  11 2.3 Interaction assay with polarized HBE cells^  12 2.4 RNA isolation and purification^  12 2.5 DNA Microarray^ 13 2.6 Real-time PCR 14 2.7 Cytotoxicity assay^ 15 2.8 Adhesion assay  16 3. Results^ 17 3.1 Early changes in global gene expression^  17 3.1.1^Introduction^ 17 3.1.2 Eukaryotic RNA contamination removal and total RNA amplification ^ 17 3.1.3 Total RNA microarray^ 20 3.1.4 Prokaryotic rRNA contamination removal and mRNA amplification 21 3.1.5 mRNA Microarray 22 3.1.6 Discovery of additional differentially expressed genes^ 24 3.2 Cytotoxicity studies^ 26 iv 3.3 Preliminary non-pilin adhesion screening^ 27 4. Discussion^ 30 4.1 Interaction assay: bacteria collection and RNA processing^ 30 4.2 Comparison between mRNA and total RNA microarray experiment^ 32 4.3 Discussion of microarray and Real-time PCR results^ 33 4.4 Genes differentially expressed: Functional correlation 35 4.4.1 Up regulation of two-component regulatory systems^ 35 4.4.2 Regulation of Type III secretion system, pilus and flagella 36 4.4.3 Upregulation of Lon Protease^ 38 4.4.4 Regulation of outer membrane proteins OprG and OprC^ 39 4.4.5 Regulation of quorum-sensing systems^ 40 4.5 Comparison to previous bacterial-host interaction studies 42 4.6 Regulation of genes involved in cytotoxicity^ 43 4.7 Non-pilin adhesins screening: fluctuations and inconsistency^ 44 4.8 Future areas for study^ 46 4.9 Conclusion^ 47 References^ 50 List of Tables Table 1. Primer sequences used in this study.^ 15 Table 2. Comparison of mRNA microarray result and total RNA microarray result^ 23 Table 3. Confirmation of differentially expressed genes using real-time PCR 24 Table 4. Discovery of two-component regulatory systems up regulation^ 25 Table 5. Mutants selected for prelimin. ary non-pilin adhesin screening 28 Supplementary Table S-1. Genes up-regulated in interacting bacteria^ 58 Supplementary Table S-2. Genes down-regulated in interacting bacteria 68 vi List of Figures Figure 1. Reduction of eukaryotic RNA contamination by RLT-pretreatment^ 18 Figure 2. Elimination of eukaryotic RNA contamination using MICROBEnrich kit. ^ 19 Figure 3. Removal of prokaryotic rRNA contamination using the MICROBExpress kit. 21 Figure 4. mRNA amplification using the MessageAmp II-Bacteria RNA amplification kit after MICROBExpress kit removal of prokaryotic rRNA contamination^ 22 Figure 5. Assessment of cytotoxicity induced by P. aeruginosa at MOI of 50 in HBE cells.^ 27 Figure 6. Preliminary screening of bacteria adhesion to the A549 human epithelial cell line. ^ 29 Figure 7. Schematic illustration of sample collection in the interaction assay^ 32 vii List of Abbreviations AIDS^acquired immune deficiency syndrome ATCC^American Type Culture Collection CF^cystic fibrosis CFTR^cystic fibrosis transmembrane conductance regulator DMEM^Dulbecco's Modified Eagle Medium DNA^deoxyribonucleic acid FBS^fetal bovine serum HBE^human bronchial epithelial IL-^interleukin LDH^lactate dehydrogenase LP S^lipopolysaccharide MIC^minimum inhibitory concentration MEM^minimum essential medium MOI^multiplicity of infection NF-KB^nuclear factor KB PBS^phosphate buffered saline RNA^ribonucleic acid RT-PCR^real-time polymerase chain reaction QS^Quorum-sensing T3SS^type III secretion system viii 1. Introduction 1.1 Pseudomonas aeruginosa P. aeruginosa is a ubiquitous Gram-negative bacterium. Since approximately 10% of genes encoded by its genome are involved in metabolism (107), P. aeruginosa can grow under both aerobic and anaerobic conditions, and can also utilize many different energy sources such as sugars, amino acids, alcohols, amines, small aromatic compounds, fatty acids, dicarboxylic acids and other organic acids (106). P. aeruginosa also demonstrates high intrinsic resistance to a range of antimicrobial agents (43). While the major element underlying this resistance is its low membrane permeability, which is significantly lower than that of E. coli (75), multidrug efflux systems further enhance the resistance by reversing uptake and thus preventing drug access to bacterial targets (100). In addition, P. aeruginosa is renowned for its possession of an impressive array of virulence factors including pilus, flagella, exotoxins, and type III secretion systems that allows the bacteria to compete to other organisms for survival (95). As a result, P. aeruginosa can colonize and successfully adapt to many different environments. 1.2 P. aeruginosa in respiratory infection As an opportunistic human pathogen, P. aeruginosa is capable of infecting immunocompromised individuals such as victims suffering from trauma or severe burns as well as patients with diseases including cancer, diabetes, AIDS, and cystic fibrosis (2, 8, 28, 105). P. aeruginosa can cause acute infections in patients with damaged airways by colonizing the injured respiratory tract, breaking down lung defenses and entering the bloodstream (95). Such infections can often lead to acute pneumonia and sepsis, resulting in rapid death of patients within hours or days (95). 1 P. aeruginosa is also the most common cause of chronic lung infections leading to death in cystic fibrosis patients (1). P. aeruginosa pathogenesis in chronic lung infection could be divided into four stages (88). In brief, (a) when P. aeruginosa first enter CF lungs, the bacteria often exhibit a non-mucoid phenotype and resemble motile environmental isolates. (b) Following the initial bacterial-host interaction and colonization, microcolonies are formed. (c) As the number of bacteria and cell density increases, quorum-sensing systems become activated and induce the expression of genes involved in biofilm formation (19, 49). (d) Over the course of infection and as the biofilm matures, gene mutations accumulate and bacteria exhibiting increased antibiotic resistance and reduced immunostimulatory potential are selected (102, 103). P. aeruginosa in mature biofilms is persistent and can live in the host for decades. In this chronic form of infection, although P. aeruginosa is usually found to be less cytotoxic than in acute infections, the bacteria are extremely difficult to eradicate in part due to their conversion to the biofilm state (39, 102). In addition, P. aeruginosa chronic infection in CF leads to not only degeneration in respiratory functions but often is life threatening (54, 86). 1.3 Initial bacterial-host interaction It is clear that acute infections in immunocompromised individuals and chronic infections in CF patients are both devastating, so a preventive measure avoiding infection is of great interest. Understanding the initial bacterial-host interaction prior to establishment of infection would therefore allow potential therapeutic development to clear P. aeruginosa at an early stage of pathogenesis. 2 1.3.1 Airway epithelium as the first line of defense The airway epithelium represents the first line of protection in respiratory infections and is the location where initial bacterial-host interaction takes place. Providing both mechanical and immunological defences (26, 48, 123), the surface of the airways is lined with a layer of highly differentiated polarized epithelial cells covered with airway surface liquid (ASL) and mucous sceretions. In healthy individuals, potential pathogens inhaled into the lung are trapped by this viscous layer and cleared from the lung by mucociliary movement. Tight junctions between cells enhance the mechanical defences by forming a physical barrier that prevents microbial invasion. In addition, airway epithelial cells also participate in the lung innate immunity. For instance, recognition of microbial antigens by Toll-like receptors on epithelial cells can induce production of pro-inflammatory molecules including adhesion molecules, enzyme, cytokines, and chemokines through the Nuclear factor KB (NF-x13) signalling pathways (16, 47). Airway epithelial cells can also produce lysozyme, lactoferrin, as well as host defence (antimicrobial) peptides such as human P-defensins and cathelicidin LL-37 as additional defence mechanisms. 1.3.2 P. aeruginosa in initial interaction: Attachment and motility As airway epithelium is the first barrier in host defences, bacterial attachment to airway epithelial cells is often considered the initial step in pathogenesis. In P. aeruginosa, the type IV pilus is an important adhesin in epithelial cell attachment (27, 119). As pili recognize and bind to asialo-GM1 on host epithelial cells that are abundant in the CF lung epithelium (96), type IV mediated attachment is considered especially important in CF pathogenesis. Other than pili, flagella have also been shown to be an important factor in epithelial attachment. The flagella cap protein FliD can bind to respiratory mucins as well as Muc 1 mucin on epithelial cells (9, 61). Mutants lacking these adhesins demonstrate reduced virulence in model lung infections (33, 3 110). Despite their prominent role in attachment, pili and flagella are not the sole adhesins in P. aeruginosa since mutants lacking both pili and flagella are still able to adhere to epithelial cells, albeit somewhat less effectively. Indeed, a variety of outer surface-associated structures involved in adherence have been described, including proteins of 75, 62, 89, 38, 28, 18 and 12 kDa (21), fibronectin-binding proteins of 70, 60, 48 and 36 kDa (89) or 50 kDa (92), and mucus binding proteins of 48, 46, 28 and 25 kDa (15). However, the identities of these adhesins remain uncertain. Identifying these non-pilin adhesins would therefore provide novel therapeutic targets to inhibit bacterial attachment to epithelial cells. Motility is likely another important virulence determinant during initial bacterial-host interaction as it is required for mobilization to the site of infection and for colonization on the host surface. P. aeruginosa has three different types of motilities including twitching on solid surface, swimming in aqueous environments and swarming on semisolid (viscous) media (44, 55, 68). Since twitching and swimming are mediated by type IV pili and flagella, respectively, these two motilities are probably involved in the initial bacterial-host interaction. However, the requirement of swarming to this early stage of pathogenesis is unclear. Expression of pili and flagella is , on one hand, essential as these appendages promote attachment, motility, and colonization; however, the presence of these virulence factors might also provoke immune responses and result in bacterial clearance. For example, attachment of type IV pilus to asialo-GM1 activates pro-inflammatory signalling through Toll-like receptor 2 (TLR2) pathway leading to activation of NF-KB and IL-8 production (104). Therefore, 4 expression of virulence factors acts as a double-edged sword in bacterial-host interaction. 1.4 Additional virulence factors in P. aeruginosa In this report, initial interaction is defined as the initial attachment and early colonization preceding the establishment of acute and chronic infections. While bacterial attachment to epithelial cells and motility are believed to be important factors for this early stage of pathogenesis, it is possible that additional virulence factors might also participate to allow, for example, the bacteria to circumvent the host immune response. Unfortunately, information about the factors involved in initial interactions with epithelial cells is limited. In the following sections, a number of virulence factors including cytotoxic effectors, quorum-sensing systems, and iron acquisition are reviewed as they may have an active role in the initial interaction. 1.4.1 Type III secretion systems It has been demonstrated that attachment of bacteria to epithelial cells can activate the type III secretion system of Yesinia and Shigella (50, 71, 93). Although such connections are not as well defined in P. aeruginosa (113), there is a possibility that the type III secretion system in P. aeruginosa might be induced upon epithelial cells contact and hence participate in these initial interactions. Conserved among a number of Gram-negative pathogens, the type III secretion system (T3SS) is a complex secretion machinery with a syringe-like apparatus that injects effector proteins, such as ExoS, ExoT, ExoU and ExoY (in P. aeruginosa), directly inside the cytosol of the target cells in a contact-dependent manner (74). It has been strongly implicated that expression of the type 5 III secretion system and the effector proteins is important for P. aeruginosa to cause severe disease and mortality in respiratory infections (45, 94). In addition, the T3SS also seems to have an important role in corneal infection since the regulator exsA, and effectors exoT and exoU were found necessary to cause disease (57). Expression of effector proteins varies among P. aeruginosa strains and clinical isolates. Once inside the target eukaryotic cells, effector proteins can often cause cytotoxicity and interfere with the host cell signal transduction system. For instance, ExoS and ExoT, possessing both Rho- dependent GTPase and ADP-ribosyltransferase activities, can alter the host cell physiology by causing disruption of actin filaments (36, 82), thus inhibiting tissue regeneration (11) and inducing apoptosis (53). In addition, these effector proteins were also found to be capable of reducing DNA synthesis, eliminating cell adhesion, and inducing microvillus effacement (78). ExoU, on the other hand, acts as a phospholipase and exerts its cytotoxic effect on host cells by destroying host cytoplasmic membranes (97). Although ExoU is expressed in only 10% of strains isolated from CF patients (22), the presence of ExoU is usually associated with increased virulence and a higher mortality rate in both corneal infection and acute respiratory tract infection (6, 34, 46, 101). Another T3SS effector in P. aeruginosa is ExoY that contributes to bacterial invasion by causing actin depolymerization of epithelial cells (20). 1.4.2 Quorum-sensing systems During initial interaction, P. aeruginosa attaches to and colonizes epithelial cells. Increases in the number of bacteria and the cell density on the epithelial cell surface during this initial attachment may activate quorum-sensing systems. Therefore, it is possible that quorum-sensing may also participate in the early interaction and contribute to the adaptation process. 6 In P. aeruginosa, two quorum-sensing systems, LasRI and Rh1RI, allow bacteria to communicate with each other and coordinate expression of specific genes as a group (114). Normally, P. aeruginosa produces and exports a basal level of autoinducers N-(3-oxo-dodecanoy1)- homoserine lactone (3-oxo-C 12-HSL) and N-butanoyl-homoserine lactone (C4-HSL) to the surrounding medium (81). As cell density increases, these HSLs accumulate and are taken up by bacteria where they bind to their cognate transcriptional regulators LasR and Rh1R, not only inducing their own biogenesis but also regulating transcription of over 300 genes in the bacteria (99, 116). For instance, activation of quorum-sensing systems enhances the expression of various components in the Type II secretion system, including LasAB elastase, exotoxin A, as well as xcpP and xcpR encoding the secretory pathways (116), suggesting that the Type II secretion system might also participate in initial interactions. Unlike the contact-dependent cytotoxicity provided by the T3SS, Type II effector proteins are secreted to the extracellular space and can act remotely from the site of infection. An example of a virulence factor secreted by the Type II secretion system is protease IV, which is considered an important virulence factor in corneal infections (30). Acting as a serine protease to degrade immunoglobulins, complement components, fibrinogen, and plasminogen (29), protease IV also participates in respiratory infections by disrupting the host surfactant mediated defence mechanisms (65). Elastase, another major virulence factor in acute infections, is secreted by the Type II secretion system. This protease can rupture respiratory epithelium and disrupt the integrity of human lung tissue (10, 38). Lastly, Exotoxin A, a potent toxin capable of inhibiting protein synthesis and causing cell 7 death (117), has been shown to be an important virulence factor in corneal infections as mutants lacking this protein are more susceptible to host clearance (84). In addition to cell density dependent regulation, the activity of quorum-sensing systems is affected by factors such as sigma factor RpoS (98), global regulator GacA (90) and Vfr (5), LuxR-type regulator QscR (18) and VqsR (51), as well as quorum-sensing inhibitor RsaL (23). Furthermore, quorum-sensing systems are often considered important factors in bacterial-host interactions, as mutant lacking these systems show reduced virulence (58, 80, 120). 1.4.3 Iron acquisition Iron is an essential nutrient for both P. aeruginosa and the human host. Although iron is abundant in nature, the availability of assimilatable iron is limited under most physiological conditions (13). It has been shown that during bacterial-host interaction, the availability of iron is usually scarce as iron is actively sequestered and tightly bound by the host factors such as transferrin and lactoferrin (4). To adapt to the host environment, P. aeruginosa produce siderophores, including pyoverdine and pyochelin, to compete with transferrin for iron acquisition (121). Therefore, it is likely that iron acquisition genes are also important virulence factors for successful adaptation during initial bacterial-host interaction. 1.5 Differential expression of virulence factors for successful adaptation Although P. aeruginosa possesses a large number of virulence factors, they are not simply constitutively expressed but are tightly regulated in different situations. In fact, differential expression of these factors is critical as it allows the bacteria to exhibit optimized phenotypes to 8 adapt to different host environments. For example, while the T3SS is important for environmental survival and acute infection, its expression is significantly repressed in chronic infection (102, 103). Similarly, the highly immunogenic flagella, although essential for initial attachment, is suppressed after the colonization step (64, 118). The loss of these immunostimulatory factors in chronic infection reduces host defence responses and therefore contributes to the persistence of chronic infections. It is also interesting to note that, although Exotoxin A is highly expressed in CF lungs, it often contains mutations and shows reduced cytotoxicity in chronic infection (37), reiterating the importance of virulence regulation during bacterial-host interaction. 1.6 Rationale and experimental goals While we know that the bacterial factors involved in motility and attachment are essential in the early stages of P. aeruginosa-host interaction, information regarding the contributions of additional virulence factors remain limited. Two research groups have recently performed microarray experiments trying to examine genes differentially expressed in early bacterial-host interaction (17, 35); however, their findings are somewhat controversial as both studies suffered from certain technical limitations, especially regarding sample collection, RNA contamination, as well as possible medium effects (see Discussion 4.1). The aim of this study was to identify genes that are essential for initial bacterial-host interactions. To accurately assess the transcriptional profile of P. aeruginosa interacting with epithelial cells, an interaction assay that minimized bias in sample collection was performed. Differential lysis and a number of commercially available kits were employed to remove contamination from RNA samples, and sufficient amounts of bacterial mRNA for microarray experiment were 9 obtained by amplification. Real-time PCR was used to confirm differentially expressed genes identified by microarray studies. To understand the involvement of these genes in damage of epithelial cells, I conducted a time course cytotoxicity assay measuring LDH release induced by P. aeruginosa mutants. Since bacterial attachment is an important factor in initial bacterial-host interaction, a non-pilin adhesin screening was also performed. Mutants selected from our mutant library were screened for deficiency in A549 epithelial cell attachment. These experiments thus were designed to contribute to a better understanding in the P. aeruginosa early pathogenesis in respiratory tract. 10 2. Materials and Methods 2.1 Bacterial strains and growth conditions P. aeruginosa strains in the PAO1 mini-Tn5 lux (59) and PA14 MrT7 (60) mutant libraries were used in this study. Overnight cultures were inoculated (1:10) in BM2-succinate minimal medium and grown until mid-log phase at 37 °C for the experiments in this project. For bacterial selections, 100 pg/m1 tetracycline and 50 µg/ml gentamicin were used. 2.2 Cell culture Immortalized human bronchial epithelial cell line 16HBE4o-, a gift from Dr. D. Gruenert (University of California, San Francisco, CA), was cultured in Minimum Essential Medium (MEM) with Earle's salts (Technologies Invitrogen, ON), supplemented with 10% heat- inactivated fetal bovine serum (FBS) (Technologies Invitrogen, ON) and 2mM L-glutamine (Technologies Invitrogen, ON). HBE cells with a passage number between 5 to 12 were used in this project and the cells were to routinely cultured to 85-90% confluency in 100% humidity and 5% CO2 at 37 °C. When indicated, HBE cells were polarized in Corning 6-well Transwell plates (PET Membrane Clear Inserts, 0.4 ptM pore size; Corning, Acton, MA). Approximately 1x10 5 cells in a total of 1.5 ml of MEM supplemented with 10% FBS, 2mM L-glutamine and 1% penicillin/streptomycin (Technologies Invitrogen, ON) were seeded into each Transwell insert. Medium in the insert and the chamber were changed every other day until the HBE cells were polarized after 10-14 days. Cell polarization was monitored by measuring the transepithelial cell resistance with a Milicell electrical resistance system. (Millipore, MA). 11 Cells of the human tumorgenic alveolar epithelial cell line A549, obtained from the American Type Culture Collection (ATCC, VA), were routinely cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% heat-inactivated FBS and 2mM L-glutamine (Life Technologies Invitrogen, ON) to 85-90% confluence in 100% humidity and 5% CO2 at 37°C. A549 cells with a passage number between 9 to 12 were used in this project. 2.3 Interaction assay with polarized HBE cells P. aeruginosa PAO1 cells grown to the mid-logarithmic phase of growth in BM2-succinate minimal medium were added in triplicate to polarized HBE cells on the apical surface at a multiplicity of infections (MOI) of 100. Based on possible medium effects observed by Schurr and colleagues, 4 mM phosphate, 20 mM FeSO4, and 1 mM sodium citrate were supplemented into the assay medium (MEM containing 2 mM L-glutamine) to avoid iron and phosphate limitation. After 4 hours of incubation, non-interacting bacteria were collected in the supernatant and the polarized HBE monolayers were then washed three times with 2 ml of PBS to remove remaining non-associating bacteria. Bacteria attached to HBE cells were then collected by adding 0.5 mL of RLT lysing buffer. Both interacting bacteria and non-interacting bacteria collected were further processed and purified for microarray experiments and real-time PCR verification. 2.4 RNA isolation and purification Total RNA was isolated using the Qiagen RNeasy Mini kit (Qiagen, MD) and residual genomic DNA was removed using the DNA-free kit (Ambion Inc., TX). Total RNA was further purified 12 using the Ambion MICROBEnrich kit and MICROBExpress kit (Ambion Inc., TX) to remove mammalian RNA contamination and bacterial ribosomal RNA contamination, respectively. Purified prokaryotic mRNA was used as template for a) reverse transcription real-time PCR and b) amplification using Ambion MessageAmp-II Bacterial kit (Ambion Inc., TX) for microarray experiment. RNA samples were stored at -80 °C with 0.2 U/111 of SUPERase-In RNase inhibitor (Ambion Inc., TX). Integrity of RNA was monitored using the Agilent 2100 Bioanalyzer (Agilent Technologies) and quantity of RNA was measured using the Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies). 2.5 DNA Microarray To synthesize cDNA for the array experiment, 20 [ig of amplified mRNA was first annealed to 750 ng of random hexamers (NS) 5 (Alpha-DNA, Montreal) for 70 °C for 10 min and 25 °C for 10 min. Reaction mixture containing 1,500 U Superscript reverse transcriptase II (Invitrogen Co.), lx Superscript reverse transcriptase II buffer, 3mM amino-allyl dUTP (Ambion, Inc.), 100mM DTT, 10mM (each) dATP, dCTP, and dGTP (Invitrogen Co.), and 20 U SUPERase-In (Ambion, Inc.) was added and incubated for 37 °C for 1 hr, 42 °C for 3 hr and 72°C for 10min. Residual RNA was hydrolyzed by alkaline treatment and the cDNA product was then neutralized. P. aeruginosa PAO1 microarray slides were provided by The Institute for Genomic Research (TIGR) Pathogenic Functional Genomics Resource Center (http:/pfgrc.tigr.org/) . cDNA labeling, purification, and hybridization were performed according to TIGR "microbial RNA aminoallyl labeling" protocol (pfgrc.tigr.org/protocols/M007.pdf)  and "hybridization of labeled DNA probes" protocol (pfgrc.tigr.org/protocols/M008.pdf) . In brief, treated cDNA and control cDNA were labeled with cyanine-5 and cyanine-3 (GE Healthcare, Canada), respectively and hybridized to the array slides for 18 h in a solution containing 50% formamide, 5x SSC, 0.1% 13 sodium dodecyl sulphate, and 0.6 µg/µl salmon sperm DNA at 42 °C at 100% humidity. Slides were then washed, dried and scanned using a ScanArray Express scanner and software (Packard BioScience BioChip Technologies). The intensity of hybridization was quantified using ImaGene 6.0 Standard Edition software (BioDiscovery, Inc., EI Segundo, CA). Identity and fold change of differentially expressed genes were determined using the ArrayPipe FPMI version 1.398 protocol (http ://koch. path° genomic s. ca/cgi-bin/pub/arraypipe. pl ) . 2.6 Real-time PCR To synthesize cDNA template for real-time PCR experiments, 1µg of mRNA was mixed with 750 ng of random primers (Invitrogen), heated for 10 min at 70 °C and incubated for a further 10 min at 25 °C. The mixture was then added to 0.5 mM dNTPs, 500 U SuperRNase (Ambion), 10 mM DTT and 10,000 U SuperScriptll (Invitrogen). After incubating the reaction mixture at 37 °C for 1 h and 42 °C for 2 h, RNA was destroyed by alkaline treatment and the cDNA template was neutralized. Real-time PCR was carried out in 1 x SYBR Green PCR master Mix (Applied Biosystems) mixed with 1 pl and 200nM of forward and reverse primers using a ABI Prism 7000 system (Applied Biosystems). All reactions were normalized to the rpsL gene encoding the 30S ribosomal protein S12. RT-PCR primers used in this study were synthesized by A1phaDNA (Montreal, QC, Canada) and are listed in Table 1. 14 Table 1. Primer sequences used in this study. PA Gene Forward Sequence (5' — 3') Reverse Sequence (3' — 5') 0221 Aminotransferase GCTGGAACAACTGCCGTACT TAGATCGCGTGGCTGTAGTG 0921 phoPQ regulated (69) (69) 1079 flgD ATGAGCATCGATAACGTCAG AGCAGCTTGAGGAATTCGTC 1179 phoP GAATACCTCATGCGGCATCA CGGGATAGAGCTGTTCCATCA 1180 phoQ CGCGGCAACGAATTCC ACGTCGAACACGAAGAACTCCT 1343 phoPQ regulated (69) (69) 1559 pmrAB regulated (69) (69) 1695 pscP AGCTGAACGCCGTGGATAC TTCGAAGGCGATCTGCTG 1701 T3SS related CAGCAAACCTTTCTCCTCCA ACTCCAGGTCGAGTTTGTGG 1713 exsA ATGCAAGGAGCCAAATCTCT CATATACGCCCTCTTCCTTG 1721 pscH CGAATACCTGGCGCAACTG TGTCGAGCATGCCGTTGA 1724 pscK CATCCACGAGTCGCACCTAC GCAGTCCAATTCCAGTTGCT 1803 ion CGGCGACAAGCAAATCCT ATGCGGTACAGGCCATCTTC 2664 fhP ATTTCTACCGCACCATGCTC AGTTCCTGCAACTGGTCGAT 3620 mutS CGACCTCTCCCAGCACAC GCGTCCTCGTAGAACAGCTC 3790 oprC GCCGCTTTCAGCTTCGAC TCCTGGTGCTGTGAGTGTTC 3805 pilF GACGAGCGGAGATCAGAATC CGAGGCCAAGCTGGATATAG 4067 oprG AAGTCCTGGCTTACCGCTTC AGCTTGATGTCGGAACTGCT 4086 cupB1 CGCCGATCCCGAAGAAAT CGCTACCCAGTTTGACGTTGA 4552 pilW GCCTGTCCATGATCGAACTA GTAGTTGCGCTTGTTGTCCA 4776 pmrA AATACTGCTGGCCGAGGAC AGGTCGAACTCGTCGGTGAC 4777 pmrB CATCGACGGCTTCCAGATC CAGCGGGAACAGCGAGTAG 4781 pmrAB regulated (69) (69) 4782 pmrAB regulated (69) (69) 5117 typA GGAAAGCGAGCGGGTGAT GTGTTCTTCGCCAGGATGGT 2.7 Cytotoxicity assay Human bronchial epithelial cells 16HBE14o" were seeded in 24 well plates (Sarstedt, Newton, USA) at a density of 5x10 4 cells per well and grown to a confluent monolayer for approximately 48 hours at 37 °C in 5% CO2 in MEM supplemented with FBS and 2 mM L-glutamine. P. aeruginosa PAO1 grown to mid-log phase were added to the HBE cells at a MOI of 50 in triplicate and incubated for 4, 7, and 9 h. Supernatants collected at each time point were centrifuged at 10,000 x g for 2 min and were stored at 4 °C for up to 24 hours. The level of lactate dehydrogenase activity (LDH) in each sample was assayed in triplicate using the colorimetric Cytotoxicity Detection Kit (Roche, Mannheim, Germany). HBE cells treated with 15 1% Triton X-100 (Sigma, Canada) served as a positive control for maximum LDH release and HBE cells without treatment served as a negative control. 2.8^Adhesion assay A549 cells were seeded in 6 well plates at a density of 1.5x10 5 and grown in DMEM containing both 2mM of L-glutamine and 10% FBS to a confluent monolayer for approximately 48 hours at 37 °C in 5% CO2 . Immediately before the adhesion assay, confluent A549 cells were washed once with PBS and the medium was replaced with assay medium (DMEM containing L- glutamine). P. aeruginosa grown to mid-log phase was added to A549 cells at an MOI of 100 and incubated for 1 hour. Adhering bacteria were released from the monolayer by the addition of Triton X-100 and were enumerated by serial dilution and plate counting. 16 3. Results 3.1^Early changes in global gene expression 3.1.1 Introduction Although an array of virulence factors have been characterized in P. aeruginosa, their expression and contribution to host cell interaction and invasion during the initial stages of pathogenesis remains unclear. To assist in the evaluation of these factors, and specifically the gene responses accompanying bacterial interaction with epithelial cells, I utilized global gene expression analysis by microarray. An interaction assay was performed with modifications specifically designed to avoid responses to nutrient insufficiencies and interference by mammalian RNA contamination. I also developed an RNA isolation, purification and amplification workflow that permitted preparation of enriched prokaryotic mRNA for the microarray experiment and real- time PCR confirmation experiment to investigate the effect of epithelial cell attachment on P. aeruginosa PA01 gene expression. 3.1.2 Eukaryotic RNA contamination removal and total RNA amplification To reduce eukaryotic RNA contamination in the interacting bacteria, I adopted the RLT- pretreatment preferential lysis procedure, which has been shown to have negligible interference with E. coli microarray experiments (25). Lanes 1 and 4 in Figure 1 represent RNA isolated from HBE cells alone and P. aeruginosa alone, respectively. Lane 2 shows RNA isolated without RLT-pretreatment and indicates a significant amount of 28S and 18S eukaryotic RNA contamination in the RNA sample. In contrast, lane 3 shows a significant reduction in mammalian RNA contamination after RLT-pretreatment. 17 Since RLT alone effectively lysed HBE cells but not the gram-negative P. aeruginosa, the differential lysis plus the subsequent centrifugation step successfully retained intact bacteria in the sample while reducing the collection of eukaryotic RNA leaking out from the disrupted HBE cells in the final RNA sample. 1 ^ 2^3^4 Figure 1. Reduction of eukaryotic RNA contamination by RLT-pretreatment. RNA samples were analyzed using a 1.5% agarose gel run at 150 volts for 30 min. RNA were isolated from a mixture of P. aeruginosa and HBE cells before (lane 2) and after (lane 3) RLT- pretreatment. RNA isolated from HBE cells alone (lane 1) and P. aeruginosa alone (lane 4) were used as reference. To further purify the RNA sample, the MICROBEnrich kit was employed to capture human mRNA and rRNA by hybridization. The residual 28S eukaryotic rRNA found in the sample treated with the RLT differential lysis was completely eliminated in the RNA samples after the MICROBEnrich treatment (Figure 2). The purity of prokaryotic RNA was verified by the lack of eukaryotic rRNA bands. The quality of the prokaryotic RNA was subsequently confirmed by the rRNA band ratio > 1.7. 18 Figure 2. Elimination of eukaryotic RNA contamination using MICROBEnrich kit. RNA sample treated with MICROBEnrich kit was analyzed using Agilent Bioanalyzer 2100. Since each Transwell provided only 0.5 gg of RNA but approximately 12 to 15 pg of prokaryotic total RNA was required for a single microarray experiment, attempts were made to improve the yield. At first, six-well Transwell plates (insert membrane growth area size = 4.67 cm 2) were replaced with 100mm Transwell dishes (insert size of 44 cm2), so that more bacteria could be added to a larger surface area of polarized HBE cells without altering the multiplicity of infection (MOI). However the monolayers of polarized HBE on the Transwell dishes were more fragile and failed to tolerate the bacterial infections and extensive washes during sample collection. Another attempt to improve the yield was made by increasing the scale of the experiment and collecting RNA from 24 Transwells. However, the RNA isolated was found to be significantly degraded (rRNA ratio < 1.4; data not shown) probably due to the length of the experiment and bacterial RNA instability. In the end it was decided to maintain the original scale of the interaction assay and employ RNA amplification to prepare sufficient RNA for the microarray experiment. A bacterial RNA amplification protocol developed by The Institute of Genomic Research (TIGR) was tried. Unfortunately, the desired amplification could not be achieved since the TIGR protocol was not sufficiently optimized for efficient bacterial RNA amplification (data not shown). Therefore the 19 Ambion MessageAmp bacteria II amplification kit was chosen. This commercial amplification kit provided very consistent and successful RNA amplification (approximately 600 to 1000 fold). The average size of the amplified total RNA was found to be around 900nt (data not shown), matching the anticipated size as suggested by the kit manufacturer. 3.1.3 Total RNA microarray Total RNA microarray were performed to examine the transcriptome profile of P. aeruginosa during bacterial-host interaction compared to non-interacting bacteria, using samples collected from five biological repeats (independent experiments performed on separate days). Analyses of the microarray slides indicated that 88 genes were differentially expressed with a fold change above 1.5 (Data not shown). Among these, 45 genes were found to be down regulated and 43 genes were found to be up regulated. Ten genes (rpoD, tolQ, flgD, fleS, lasR, exsD, pilA, pilR, typA, and cycB) were selected for real-time PCR confirmation but their expression was not found to be significantly different under the selected conditions. One possible explanation for the failure of real-time PCR confirmation was the potential problem of interference by other RNA species. Since bacterial ribosomal RNA was the major species found in the total RNA sample prior to amplification, it was concluded that the concentration of rRNA was further enriched by amplification and become more dominant in the sample, interfering with subsequent microarray analysis. It seems possible that the amplified rRNA could significantly influence the hybridization of the minority yet critical mRNA molecules to the array slides. To tackle this problem, I included an additional step to remove the prokaryotic rRNA contamination. 20 3.1.4 Prokaryotic rRNA contamination removal and mRNA amplification A MICROBExpress kit with a Pseudomonas module, developed by Ambion, was employed to remove P. aeruginosa ribosomal RNA contamination (Figure 2). A significant amount of the rRNA in the sample was removed by the MICROBExpress treatment (Figure 3). 38 Figure 3. Removal of prokaryotic rRNA contamination using the MICROSExpress kit. RNA sample treated with MICROBExpress kit was analyzed using Agilent Bioanalyzer 2100. The purified mRNA was then amplified (1000x fold) using MessageAmp kit for microarray experiment. The shape of the size distribution curve of the amplified mRNA (Figure 4) matches the expected profile suggested by the amplification kit protocol. After rRNA removal amplification of the residual RNA sample gave a broad band that was more typical of mRNA. It is worth noting that both interacting and control non-interacting samples were subjected to the same treatments. 21 fl te e Figure 4. mRNA amplification using the MessageAmp II-Bacteria RNA amplification kit after MICROBExpress kit removal of prokaryotic rRNA contamination. mRNA amplified with MessageAmp II-Bacteria RNA amplification kit was analyzed using Agilent Bioanalyzer 2100. 3.1.5 mRNA Microarray RNA samples collected from three independent interaction assays were isolated and processed for mRNA microarray experiments as described in Materials and Methods. A total of 766 genes were found to be significantly differentially expressed (p<0.05 by Student's t-test) with a fold change greater than 1.5 upon epithelial cell interaction, when compared to non-interacting bacteria (Supplementary table 1 and 2). Among these genes, 215 genes exhibited fold changes greater than 2.0. Results obtained from the mRNA microarray and total RNA microarray are compared in Table 2. The number of genes identified by the microarray using enriched mRNA was substantially greater than that identified by the total RNA microarray suggesting a more efficient mRNA hybridization after rRNA contamination removal. 22 Table 2. Comparison of mRNA microarray result and total RNA microarray result mRNA microarray (3 slides) Total RNA microarray (5 slides) Fold change > 2.0 Up-regulation 102 25Down-regulation 113 30 Fold change > 1.5 Up-regulation 371 45Down-regulation 395 43 For confirmation, 14 genes identified only in the mRNA-enriched microarray and 1 gene (typA) identified in both microarray experiments were selected and tested using real-time PCR. Of the 14 genes identified only in the mRNA-enriched microarray, 10 were successfully confirmed (Table 3), verifying their differential expression during the initial interaction assay. On the other hand, the change in typA expression, that was found to be 1.7 fold down regulated in the mRNA- enriched and 1.6 fold down regulated in the total RNA array, was determined to be not significant (i.e. fold change below 1.5) using the more accurate method of Real-time PCR. Three additional type III secretion system genes that were not observed to be differentially regulated on the microarrays were also tested using Real-time PCR. However, none of these additional T3SS genes showed any significant fold change, confirming these negative microarray results (Table 3). 23 Table 3. Confirmation of differentially expressed genes using real-time PCR PA number GeneName Description mRNA array RT-PCR 4776 pmrA TCR response regulator 1.6 4.4 4552 pilW Pilus biogenesis 2.4 3.9 3805 pilF Pilus biogenesis -2.0 N/S 3790 oprC Outer membrane porin 4.3 51.4 4067 oprG Outer membrane porin -5.8 -8.7 1430 lasR QS transcriptional regulator 1.5 3.4 1431 rsaL QS inhibitor 3.1 3.3 3476 rhll QS autoinducer synthesis 1.8 4.2 2664 fhp Flavohemoglobin -3.2 -49.2 0221 - Aminotransferase 10.9 1.8 3006 psrA Transcriptional regulator -1.8 N/S 3620 mutS DNA repair system -1.9 N/S 5117 typA Regulatory protein(swarming motility) -1.7 N/S 1695 pscP T3SS biogenesis -2.2 -1.9 1701 - T3SS biogenesis -1.9 N/S 1713 exsA T3SS regulatory protein N/S N/S 1721 pscH T3SS biogenesis N/S N/S 1724 pscK T3SS biogenesis N/S N/S N/S indicates not significant as the fold change detected was below 1.5 fold change. 3.1.6 Discovery of additional differentially expressed genes Fifteen additional genes absent from the mRNA microarray results were also selected for study using Real-time PCR. Two of these genes (Ion and cupB1) were selected based on the interests of our laboratory and a third gene ( f IgD) was selected because it was identified in the total RNA microarray (8.0 fold up regulated) but not the mRNA microarray. In addition, due to the confirmation of pmrA and oprH as being up regulated in the initial real-time PCR experiment (Table 3) and our interest in the two-component regulatory systems in general, 10 additional genes related to the PhoPQ and PmrAB systems were selected and tested using real-time PCR. 24 Table 4. Discovery of two-component regulatory systems up regulation PA number Gene Name Description RNay Amrra RT-PCR 1079 flgD Flagella biogenesis N/S 1.7 1803 ion ATP-dependent protease (involved in motility, biofilm formation, antibiotic resistance) N/S 3.3 4086 cupB1 Potential adhesin N/S N/S 1178 oprH phoPQ regulated outer membraneprotein 1.5 4.7 1179 phoP TCR response regulator N/S 5.0 1180 phoQ TCR sensor kinase N/S 3.7 4776 pmrA TCR response regulator 1.6 4.4 4777 pmrB TCR sensor kinase N/S 6.3 0921 - pmrAB regulated gene(hypothetical protein) N/S 3.9 1343 - pmrAB regulated gene(hypothetical protein) N/S 5.1 1559 - pmrAB regulated gene(hypothetical protein) N/S 4.0 3552 pmrB pmrAB & phoPQ regulated(LPS modification) N/S 4.9 4359 feoA pmrAB regulated gene(ferrous iron uptake) N/S N/S 4781 - pmrAB regulated gene(uncharacterized response regulator) N/S 3.6 4782 - pmrAB regulated gene(hypothetical) N/S 2.0 N/S indicates not significant as the fold change detected was below 1 5 fold change Using real-time PCR, Ion was found up regulated in the interacting bacteria by 3.3 fold (Table 4). A moderate induction with a fold change of 1.7 fold was found for the flgD gene and no significant regulation could be measured for the cupBl gene. Although all the 10 additional two- component regulatory genes selected were not identified by the microarray experiments, almost all of them except one (feoA) were found to be differentially expressed in the interacting bacteria. The apparent discrepancy in microarray and RT-PCR is further discussed in section 4.3. 25 3.2 Cytotoxicity studies A cytotoxicity study was carried out to further examine epithelial cell killing and damage induced by P. aeruginosa. Selected genes identified in the initial interaction assay (flgD, typA, ion, oprG, fhp and oprC) as well as genes of interest to our laboratory (fha and psrA) were screened for deficiency in cytotoxicity. Although some of the genes listed have well characterized functions, for instance, the importance of pilus and flagella in attachment and motility (68, 111), the requirement of ion protease in motility and biofilm formation (67), as well as the involvement of two-component regulatory systems pmrAB and phoPQ in peptide resistance in low Mg2+ condition (69, 70), these genes were tested in the cytotoxicity assay to determine if they have an additional potential role in epithelial cell damage during early interaction. Cytotoxicity induced by P. aeruginosa was measured as a function of LDH release caused by epithelial cell lysis. All mutants showed similar cytotoxicity levels when compared to the wild type after 4 hours of incubation revealing that, under these assay conditions, the effect of the cytotoxicity deficiency is not observed until a later stage of the interaction. Mutants, including fha, flgD, ion and oprG, were all found less effective in HBE cells damaging after 7.0 hours (Figure 5). 26 Figure 5. Assessment of cytotoxicity induced by P. aeruginosa at MOI of 50 in JIBE cells. Mutants in the indicated genes were incubated with monolayer of HBE cells at a MOI of 50 for 4, 7 and 9 hours at 37°C. Supernatant was collected at each time point and cytotoxicity was assessed by measuring the release of cytosolic lactate dehydrogenase. Results are expressed as mean values of %LDH released compared to TX 100 treated control ± standard deviation of three independent experiments. Student's t-test analyses were used to compare %LDH release induced by the mutant to that induced by wild type bacteria. * denote p-value < 0.05. 3.3 Preliminary non-pilin adhesion screening Attachment to epithelial cells, usually considered the first step in pathogenesis, is one of the most important requirements in establishment of bacterial infections. In P. aeruginosa, while pilus is the major adhesion factor contributing to epithelial cell attachment, there is evidence suggesting the presence of a number of non-pilin adhesins, albeit their specific roles and/or identities remain unclear (15, 21, 89, 92). The objective of this study was to identify non-pilin adhesins by screening mini-Tn5 lux mutants for deficiency in attachment to A549 human epithelial cells. Twelve mutants were selected from our mini-Tn5 lux mutant library for the adhesion assay (Table 5) based on homology to known adhesins (PA0041, PA0222, PA0413, PA0690 , PA3707, PA4086, PA4541 and PA5498) and/ or possession of type I or II export signal (PA0041, 27 PA0046, PA0222, PA0690, PA3064, PA4086, PA5033, PA5191 and PA5498) as non-pilin adhesins could be surface proteins or exported proteins. A number of mutants with pilus deficiency were tested to serve as a positive control and pilYl was selected as it provided more consistent trend of attachment deficiency in the pilot experiments. PilY1 shares C-terminal homology to the Pi1C2 protein of Neisseria gonorrhoeae and is believed to be a fimbrial tip- associated adhesin (7). It has also been shown that pilYl mutant has significant deficiency in both twitching and swarming motility (79). In addition, mutant in flgL, which has been shown to be essential for both swarming and swimming motility (79), was also tested to determine the role of flagella in epithelial cells binding. Hypothetical protein PA5273 was chosen randomly from the genome to serve as a negative control of the experiment. Table 5. Mutants selected for preliminary non-pilin adhesin screening PA number Gene Note 0041 fha 43% similar to regions of filamentous hemagglutinin [B. pertussis];Type I export signal predicted 0046 Hypothetical Type II (lipoprotein) export signal predicted 0222 Hypothetical 50% similar to putative mannopine-binding periplasmic protein MotA[A. tumefaciens]; Type I export signal predicted 0413 chpA 99% similar to pilL [P. aeruginosa] 0690 Hypothetical 38% similar to high-molecular-weight surface-exposed protein[Haemophilus influenzae]; Type I export signal predicted 1087 flgL Flagella hook-associated protein 3064 pelA Type I export signal predicted 3707 wspB 50% similar to pill [P. aeruginosa] 4086 cupB1 Probable fimbrial subunit; Type I export signal predicted 4541 Hypothetical 38% similar to high-molecular-weight surface-exposed proteinHMW1 [H influenzae] 4554 pilY1 Type IV pilus biogenesis protein 5033 Hypothetical Type I export signal predicted 5191 Hypothetical Type I export signal predicted 5273 Hypothetical Unknown 5498 Hypothetical 59% similar to putative adhesin ZnuA [E. coli]; Type I export signalpredicted 28 Figure 6. Preliminary screening of bacteria adhesion to the A549 human epithelial cell line. Mutants in the indicated genes were incubated with monolayers of A549 at a MOI of 100 for 1 hour at 37°C. The monolayer was then washed 3 times with DMEM. Bacteria were recovered by addition of 0.1% Triton X100 and enumerated by serial dilution and plate counting. Representative experiments are shown. Results are expressed as mean values of number recovered per A549 cells ± standard deviation. Student's t-test was performed to compare the number of mutant cells recovered to the number of wild-type recovered. * denotes p-value < 0.05. A number of probable non-pilin adhesins were identified. Mutants lacking PA0690, PA0222, PA0041, PA0046 and PA4541 were all found to be less capable of binding to A549 epithelial cells (Figure 6). In addition, the participation of flagella in epithelial cell adhesion was also confirmed. However, these data were considered preliminary due to certain technical difficulties (see Discussion section 4.7) 29 4. Discussion 4.1^Interaction assay: bacteria collection and RNA processing As mentioned in the introduction, two groups have previously investigated the transcriptome profile of P. aeruginosa interacting with epithelial cells, but both studies had certain limitations created by experimental design. When Frisk et al. and colleagues (35) studied the interaction between P. aeruginosa and primary normal human airway epithelial (PNHAE) cells, they extracted a mixture of mammalian and bacterial RNA in their sample for use in their microarray experiments. Thus instead of eliminating mammalian RNA contamination to avoid binding competition, they hybridized a sample containing only PNHAE RNA to the Pseudomonas array chip to permit baseline subtraction. This procedure would likely be problematic because of the intrinsic limitations of accuracy of microarrays, and the fact that the majority of RNA in the interacting bacterial sample was in fact mammalian RNA. The presence of a large amount of mammalian RNA contamination could interfere with the bacterial RNA hybridization by competing with probes on the Pseudomonas array slide and also by potentially binding to the bacteria RNA. In addition, the results of this study were considered controversial because eight genes involved in iron acquisition were found to be repressed 4 hours post-infection. To explain their observations, the authors suggested that P. aeruginosa was able to acquire an ample amount of iron from the epithelial cells during infection. This finding and explanation contradicted a substantial amount of previous data demonstrating that iron is limiting during bacterial-host interaction (41, 72, 109). Although it was not possible to pinpoint the exact source of any potential error, it was believed that the repression of iron acquisition genes observed was not a direct result of epithelial interactions but potentially an artifact of their experimental procedure possibly due to medium effects. To provide a more representative analysis of epithelial interactions, I decided to (a) remove all mammalian RNA contamination in my array experiment 30 to eliminate interference in the microarray hybridization step and (b) to supplement the assay medium to eliminate possible nutrient depletion, such that my experiments would highlight the regulation of gene expression that resulted directly from bacterial-host interaction. The second published investigation of the effect of epithelial cells on the P. aeruginosa transcriptome expression was by the Greenberg group (17). While the main objective of their project was to examine the differences in gene expression profile upon epithelial cell interaction when comparing a wildtype P. aeruginosa and a Type 3 secretion system mutant, they also compared the gene expression profile of the wild type strain interacting with epithelial cells to the same strain grown overnight in nutrient rich medium. These researchers minimized mammalian RNA contamination by washing the interacting bacteria off the epithelial surface. However the bacterial populations compared were far from ideal as the growth conditions and medium of the two populations compared were quite different. Therefore, in order to accurately investigate the influence of bacterial-host interaction on bacterial gene expression, I set up my experiments in such a way that both interacting and non- interacting bacteria were collected and compared from the same Transwell in which the bottom of the well was covered with a monolayer of polarized HBE cells (Figure 7). After 4 hours of infection at a MOI of 100, the supernatant layer was collected as the control non-interacting bacteria and, after washing off any bacteria weakly associating with the HBE cells, the strongly attached bacteria remaining at the bottom, together with the mammalian cells, were collected as the interacting bacteria. By doing this, I collected more comparable interacting and non- interacting bacteria that had been exposed to the same culture conditions. 31 Since a mixture of bacteria and mammalian cells were collected together in the interacting bacteria sample, attempts were made to remove the mammalian RNA contamination to avoid interference in the microarray hybridization. To enrich bacterial RNA from the mixture of RNA sample, an RLT-pretreatment differential lysis step was adopted to remove part of the mammalian RNA contamination (25). The remaining mammalian RNA contamination was further eliminated using the Ambion MICROBEnrich kit. Next, prokaryotic ribosomal RNA was removed using the Ambion MICROBExpress kit. Enhanced mRNA were then used as template for a) reverse transcription and real-time PCR analysis, and b) amplification using the Ambion MessageAmp II-bacteria kit for microarray experiment. HI 03 Non-interactirig Bacteria Figure 7. Schematic illustration of sample collection in the interaction assay. 4.2 Comparison between mRNA and total RNA microarray experiment Although the exact cause was not known, we believed that the limited success of the total RNA microarray is contributed by the presence of a large amount of amplified rRNA in the sample. In fact, removing rRNA from the total RNA before amplification allowed a more accurate and sensitive mRNA microarray experiment. While only 88 genes were identified in the total RNA to have fold change larger than 1.5 (Table 2), 766 genes were found in the mRNA microarray. A 32 closer inspection of the two results showed that 25 out of 88 genes (i.e. 28%) identified in the total RNA were also present in the mRNA microarray analysis. All except for one (PA4545) of these 25 genes were found to demonstrate regulation in the same direction (up- or down regulation) in the array experiments. The common gene list included mostly hypothetical proteins, as well as typA, rpoD, trpG, efp, rpsP, rpmJ, rplV, comL and rpoZ. Only one of the twenty-five genes common to both lists was tested (typA) using the more sensitive and accurate Real-time PCR, and the differential expression could not be confirmed. There is no evidence indicating any major difference between the two microarray experiments, except that the mRNA microarray showed greater sensitivity. 4.3 Discussion of microarray and Real-time PCR results In the interaction assay, 15 genes were selected from the mRNA microarray result for real-time PCR confirmation (Table 3). Among these genes, 8 of them (pilW, pilF, oprC, oprG, rsaL, fhp, pscP and aminotransferase) showed regulation of greater than two-fold differences in microarray and 7 of these were successfully confirmed using Real-time PCR (87.5% confirmation rate). Most of the genes that failed Real-time PCR confirmation were the ones with a fold change of less than 2.0 in the microarray. Since a fold change of 2.0 is usually considered the cut off point for array analysis, the testing of these genes was pushing the limit of microarray technology. Nonetheless, it should be noted that although pmrA was also found to have a fold change of less than 2.0 by microarray, it was successfully confirmed using real-time PCR. As a result, at least for the 15 genes tested in this project, while a cut off of 2.0 fold change is effective in eliminating many false positive results, this value might fail to report differentially expressed genes such as pmrA in this case. 33 The confirmation of pmrA and oprH induction ultimately led to the discovery of 8 additional differentially expressed genes in the PhoPQ and PmrAB two-component regulatory systems. Since all of these 8 genes were absent in the microarray experiment, the rate of false negatives, at the first glance, is almost alarming. One possible explanation for this high rate of the false negatives is that insufficient repeats of the mRNA microarray were performed. As a result, genes showing no variation in expression level in any one of the triplicate experiments might have a dominated the statistical analysis and such a gene would be more likely to be discarded from the resulting gene list. Therefore, repeating the experiment two more times might have allowed the microarray to identify more genes, especially those showing stronger variations among the original three biological repeats. In addition, basal gene expression levels could also affect the sensitivity of microarray. Genes with low expression levels would exhibit lower mRNA copy numbers in the sample and therefore might not be as readily detected by the microarray due to reduced hybridization to the array slides. In contrast, real-time PCR has a much higher sensitivity and specificity for these same genes, since custom designed primers are used to amplify the mRNA for more accurate and responsive detection. Therefore, another possible explanation for the high rate of false negatives observed could be due to low gene expression levels. Quantification of these genes is required to verify this speculation. Finally, the discrepancy between the microarray result and the RT-PCR confirmation might be accounted for by the potential bias introduced through RNA amplification. However, since most of the genes exhibiting strong fold change in the array (amplified mRNA) could be confirmed by the real- time PCR (original non-amplified mRNA), the amplification step was justified as a sufficient amount of mRNA to perform microarrays would not be available without the magnification. 34 Since three independent biological repeats were tested and all of them showed very similar expression profiles, and because ten related genes belonging to the same two-component regulatory systems were all found to be up-regulated, I have confidence in the results obtained in these initial bacterial-host interaction studies. 4.4 Genes differentially expressed: Functional correlation A number of genes, including components of the PhoPQ and PmrAB two-component regulatory systems, lon protease, pilW and flgD involved in pilus and flagella biogenesis, pscP involved in the Type III secretion system, and outer membrane proteins oprC and oprG as well as rh1I, lasR and rsaL from the quorum-sensing system were all found differentially expression during bacterial-host interaction. Some of these genes were also found to be involved in HBE cytotoxicity. The potential functional correlations of these genes are discussed in this section. 4.4.1 Up regulation of two-component regulatory systems The PhoPQ and PmrAB two-component regulatory systems in P. aeruginosa have previously been shown to be involved in cationic antimicrobial peptide and polymyxin B resistance induced by Mg2+ limitation (40, 62, 63, 70). In addition, the expression of pmrAB, but not phoPQ, is activated by a range of cationic peptides, such as polymyxins B and E, cattle indolicidin and LL- 37 (70). Although only a modest subset of genes differentially expressed in Mg 2+ limitation are regulated by PhoP or PmrA, these two systems have regulatory effects on an array of genes, including regulation of oprH-phoP-phoQ, pmrHFIJKLM-ugd operon, PA0921 and PA 1343 by the PhoPQ system, as well as regulation of PA1559-1560, PA4782-4781,feoAB, PA4773-4775- pmrAB and pmrHFIJKLM-ugd operons by the PmrAB system (69). In particular, pmrH, which is 35 regulated by both PmrAB and PhoPQ two-component regulatory systems, was found up regulated during epithelial cell interaction. Since the pmrH-ugd operon encodes genes responsible for adding aminoarabinose to Lipid A on the LPS (73), a modification that occurs in P. aeruginosa isolated from chronic infection in CF lungs (32), the induction of this operon during interaction with epithelial cells indicates that these LPS modification may occur at an early stage of pathogenesis and consequently protect bacteria from peptides in the host environment prior to biofilm formation. On the other hand, feoA was the only tested gene that is regulated by these two-component regulatory systems that showed no significant differential expression during initial bacterial-host interaction. It is possible that the expression of feoA, which is involved in ferrous iron uptake (66), was suppressed as a sufficient amount of iron was made available through iron supplementation of the interaction assay medium. In this study, we observed the up-regulation of both the PmrAB and PhoPQ two-component regulatory systems during initial bacterial-host interaction. Induction of these two systems might allow the bacteria to become more resistance to antimicrobial peptides including LL-37 and (3- defensin produced by the innate immune system. In addition, induction of the pmrH gene would lead to LPS modifications that might have an impact during initial bacterial-host interaction. For example Lipid A modification impacts on recognition by Toll-like receptor 4 (31). Together, the bacteria would be more prepared to evade the host immune response. 4.4.2 Regulation of Type III secretion system, pilus and flagella In this study, differential expression of pilus, flagella and the T3SS system were indicated. Genes involved in pilus (pilW) and flagella (figD) biogenesis were found upregulated by 2.4 and 1.7 fold, respectively (Table 3). PilW is an ancillary protein with pre-pilin like leader sequences 36 (other examples are PilE, PilV, Pi1X, FimT, and FimU) and is required to construct a functional pilus as mutants lacking the pilW gene are phage resistant and twitching deficient (52, 68). On the other hand, flgD is also essential for flagella functions since P. aeruginosa mutants lacking flgD showed a reduction in swimming and swarming motility (79). In fact, it has been shown that in Salmonella, a flgD mutant fails to form functional flagella, since the hook of the flagella cannot be assembled correctly (77). The increased induction of pilus and flagella described here provide supporting evidence that these two systems are important factors in initial interaction possibly by mediating motility and/or attachment. It is interesting to note that out of 37 genes previously found to be required for swarming motility (79), 4 genes were examined in this study for their differential expression in the interacting bacteria. Among them, three (Ion, flgD and rhll) were found upregulated and one gene (typA) was not differentially expressed. Although the data presented in this study may hint a possible role of swarming motility in initial bacterial-host interaction, the association is far from conclusive and further investigations are required. On the other hand, although it has been proposed that attachment to eukaryotic cells may provide a sufficient signal to activate T3SS (113), my microarray experiment and real-time PCR confirmation demonstrated that pscP was repressed and hence T3SS might not be important during the initial bacterial-host interaction. A region of pscP shares 38% sequence identity with yscP, an important T3SS regulatory factor in Yesinia. YscP acts as a molecular ruler and monitors the length of the T3SS needle (3). When a certain length is achieved, YscP interacts with YscU and switches the substrate specificity from needle subunits (YscF) to Yesinia outer membrane proteins (Yop). Mutants in yscP cannot control the length of the T3SS needles and fail to secret Yops. Therefore, reduced expression of pscP during initial bacterial-host interaction might render the bacteria less cytotoxic as less effector proteins could be exported. However, 37 since exsA and other genes involved in T3SS were not repressed, there was no apparent global regulation of T3SS and it is not possible to judge the impact of such a subtle change in pscP expression on production of the Type III secretion apparatus and Type III effectors during this initial stage of pathogenesis. It should be noted that, since only isolated genes, instead of complete operons, involved in pilus and flagella biogenesis and the T3SS have been tested for differential expression, further investigations are required to confirm the regulation of these three systems. 4.4.3 Upregulation of Lon Protease Lon is an ATP-dependent protease that is important for cellular functioning by maintaining protein quality control (112). Our laboratory has recently demonstrated that Lon in P. aeruginosa is involved antibiotic resistance as a ion mutant was found more susceptible to ciprofloxacin and expression of ion was induced in the presence of sub-inhibitory ciprofloxacin concentrations (14). In another microarray experiment, Ion was found to be upregulated when bacteria were exposed to sub MIC aminoglycosides (67). Aside from the involvement of protein quality control and antibiotic resistance, ion is also involved in motility and biofilm formation, as P. aeruginosa mutants lacking ion were found to be deficient in swimming, swarming and twitching motility as well as having a reduced capacity to develop biofilms (67). In this project, Lon protease was found to be upregulated in the bacteria interacting with epithetical cells (Table 4). It is possible that, during initial bacterial-host interaction, the induction of expression of ion reflects the importance of motility in attachment and localization. The contribution of the lon induction to biofilm formation and antibiotic resistance at this early 38 stage of bacterial-host interaction is unclear as biofilms are usually developed after initial colonization and neither ciprofloxacin nor aminoglycosides are present naturally in the assay system. On the other hand, since a tighter regulation of virulence factor expression is critical for successful adaptation to epithelial cell interactions, and Lon is responsible for controlling certain short-lived regulatory proteins that are involved in virulence genes expression (91, 108), another possible benefit of Lon protease induction is therefore modulation of virulence gene expression. It is clear that Lon has multiple roles in pathogenesis and represents a novel therapeutic target in the early bacterial-host interactions. 4.4.4 Regulation of outer membrane proteins OprG and OprC Previously, since OprG was induced when P. aeruginosa is grown in low iron conditions, it has been suggested to be involved in low-affinity iron uptake (15). In fact, expression of OprG could be affected by a large array of conditions including growth phase, growth temperature, Mg 2+ deficiency, presence of certain carbon sources as well as certain lipopolysaccharide alterations (76) . In addition, OprG has also been proposed to be involved in quinolone uptake and susceptibility (85, 115). The current project has provided new insights in characterizing the functions of the OprG porin. Firstly, as a modest deficiency in damaging HBE cells was found for the oprG mutant (Figure 5), OprG seems to be somehow associated with enhancing cytotoxicity, although the mechanism of action remains unknown. In addition, as well as the modest role in enhancing cytotoxicity, it is clear that the presence of epithelial cells also has a regulatory effect on the expression of OprG. OprG is repressed in bacteria interacting with epithelial cells (Table 3), which may be required to suppress its negative effect on bacterial-host interaction. Thus like the T3SS, OprG is an example of down regulation of a gene involved in cytotoxicity, supporting the idea that cytotoxicity might not be desirable the early stages of 39 bacterial-host interaction. Further investigation is required to explore the function of OprG and the mechanism of OprG related cytotoxicity. OprC, on the other hand, has been described as a channel-forming protein that shows zinc- binding specificity. Expression of OprC is repressed under anaerobic condition and regulated by exogenous Cu2+ concentration (42, 122). Therefore, the substantial upregulation of OprC (51- fold; Table 3) observed in this study reflects a possible adaptation to changes in metabolism during epithelial interaction. 4.4.5 Regulation of quorum-sensing systems As mentioned in the introduction, quorum-sensing systems are important for cell-to-cell communication when cell density is high. Activation of QS systems can regulate expression of a number of virulence factors. In this study, rhlI from the Rh1RI system and the lasR from the LasRI system were found to be upregulated during epithelial cell interaction. This activation is unlikely to have occurred just because of increased cell density of the interacting bacteria as it did not occur in non-interacting bacteria. However in the interaction assay, although only a smaller portion of bacteria was interacting with the epithelial cells, these were confined to the bottom of the Transwell; and it is possible that these interacting bacteria had a higher cell density compared to the non-interacting bacteria. This change in cell density could enrich the local concentration of HSL and contribute to the quorum-sensing system activation. Conversely there are many global regulators that control quorum sensing which has been observed even in logarithmic phase bacteria and upregulatioon of these systems might have been in response to other signals. 40 Another gene rsaL involved in quorum sensing was also confirmed to be upregulated in the interaction assay. RsaL is believed to be a negative inhibitor of the LasRI system as overexpression of RsaL leads to dramatic reduction of 3-oxo-C 12-HSL (23) and mutants lacking RsaL show significant induction in HSL production (87). It has been shown that RsaL inhibits the LasRI system by competing to LasR for binding to the las]. promoter (87). Therefore, the induction of rsaL in the interaction assay might actually antagonize the activity of the LasRI system. In particular, elevated level of RsaL ensures individual bacterium only produces a basal level of Iasi so that autoinducer threshold could only be met when a large number of bacteria each producing a small amount of 3-oxo-C 12-HSL gather together. This mechanism can ensure cell-to-cell communication and avoid false LasRI activation due to hyper-expression of 3-oxo- C12-HSL in a small number of bacteria. The situation might be different for the Rh1RI systems. LasR bound to HSL can induce expression of Rh1R thus placing the LasRI system hierarchically above the Rh1RI system (83). Although the activity of LasRI might be partially suppressed, this antagonistic effect would not extend to the Rh1RI system as Rh1I gene was found to be upregulated in the interaction assay. It has been suggested that LasRI is more important for cell-to-cell communication during the initial phase of biofilm development when there are a larger number of cells, while the activity of Rh1RI system is more important when the cell density is relatively lower (24). During the initial interaction and under the assay conditions utilized here, it is reasonable to propose that the Rh1RI system may have a more prominent role as the number of bacteria in this situation would be lower than found in microcolonies immediately before biofilm formation. Induction of the rhll and rsaL genes provide supporting evidence for the potential importance of rhll during initial interaction and are consistent with a lesser importance of the LasRI system. Nonetheless, a closer 41 inspection of the microarray results revealed no significant induction of genes known to be controlled by the Rh1RI system suggesting that either the microarray is not sensitive enough to detect these genes or there might be additional factors delaying or impacting on Rh1RI activity. Further experimentation is required to understand the role and functions of the Rh1RI system during the initial bacterial-host interaction. 4.5 Comparison to previous bacterial-host interaction studies When I first began working on this bacterial-host interaction studies, one clear objective was to eliminate the possible medium effect observed by Schurr and colleagues. Although the exact cause is unknown, there are a few possible reasons to account for their observations, including potential medium effects and/or technical errors such as the interference with hybridization caused by mammalian RNA contamination in the microarray experiment. My goal was to set up an interaction assay and microarray experiment that would highlight the effects of epithelial cell interaction. To do so, I specifically 1) supplemented the assay medium with iron to avoid possible nutrient depletion, 2) used non-interacting bacteria, grown in the same medium, as the control population and 3) developed a protocol that would allow us to prepare enriched Pseudomonas mRNA from a mixture of bacteria and mammalian cells. While Frisk et al. (35) indicated that there was a significant repression of iron acquisition when they compared RNA isolated from a mixture of bacteria and epithelial cells to RNA isolated from bacteria alone, Chugani and Greenberg (17) obtained the opposite results as they found that iron acquisition genes were induced in the bacteria interacting with epithelial cells when compared to bacteria grown in overnight culture. In contrast, my study suggests that iron acquisition is not significantly regulated by interaction with epithelial cells per se (or affected to 42 the same extent in interacting and non-interacting cells). Comparisons between these findings clearly indicate that the gene expression pattern is sensitive to assay conditions. It should be noted that, although the current study successfully avoided the probable medium effect as observed by Frisk et al. (35), as iron is an essential nutrient and its availability can significant impact the bacterial physiology, the addition of iron to the assay medium could itself introduce changes in the bacteria (12, 56, 109). 4.6 Regulation of genes involved in cytotoxicity. A number of genes identified in the mRNA microarray were further characterized for their involvement in cytotoxicity (Figure 5). Among them, 3 genes (oprG, flgD and ion) were found to have a positive influence in damage of HBE cells based on the suppressive effects of mutants in these genes. It is clear that genes involved in cytotoxicity could be either repressed (oprG) or induced (flgD and ion) in the interacting bacteria suggesting that there is no simple theme in the regulation of these genes during initial bacterial-host interaction. In other words, genes involved in HBE cell damage might not be only regulated for their ability to promote cytotoxicity but might also be affected by other factors. P. aeruginosa Fha (PA0041), which shares 43% similarity to region of filamentous hemagglutinin in B. pertussis, was involved in both A549 epithelial cell attachment (Figure 6) and HBE cells damage (Figure 5). The reduced cytotoxicity might be a direct result of the reduced numbers of mutant bacteria attaching to the epithelial cells. The reduction of cytotoxicity induced by the flagella mutant (flgD) and the Lon mutant might also be explained similarly as a flagella mutant (figL) was also found to be less capable of epithelial cell attachment. One interesting observation in the cytotoxicity assay was the identification of a 43 modest deficiency in the oprG outer membrane protein mutant, as its involvement in cytotoxicity has not been suggested. An elevated level of cytotoxicity might not be desirable during the very initial stage of bacterial- host interaction as the resultant triggering of a host immune response would impact on the success of colonization and thus impact on early adaptation. Therefore, based on this speculation, it is possible that, even though the induction of flgD and ion expression would inevitably increase epithelial cell damage and hence incite host immune response, these two genes were upregulated regardless because of their significance in bacterial-host interaction. Thus the induction of flgD would promote more efficient motility and/or attachment while ion induction would promote enhanced motility and subsequently biofilm formation as well as improved protein expression accuracy. All these functions are important factors for successful adaptation as they allow the bacteria to colonize lung epithelial cell surfaces and develop into infections. Further experiments are required to confirm these speculations. 4.7 Non-pilin adhesins screening: fluctuations and inconsistency It should be noted that the non-pilin adhesin screening reported here suffered from certain technical difficulties and inconsistencies. First, the adhesion assay protocol contained many variables especially in steps such as washing, bacteria re-suspension and serial dilution. Since only a small portion of bacteria, even for the wild type strain, remained attached to the A549 monolayer at the end of the experiment, the differences observed in attachment between bacterial strains tended to be relatively modest compared to the experimental error. Therefore although many experiments were performed, only representative experiments that provided consistent trends are reported in this thesis, and a more reproducible procedure is required to verify these 44 results. Thus these non-pilin adhesion studies are included in this thesis as a record of preliminary results. A number of probable non-pilin adhesins were identified in this study and one of the important discoveries is the identification of the fha gene. Filamentous hemagglutinin is a major adhesin in B. pertussis. It is required for the bacteria to colonize lower respiratory tract and is important for biofilm formation. In the current study, fha in P. aeruginosa was found to be important for both cytotoxicity in HBE cells and attachment to A549 epithelial cells. Due to the well known role of pilus in adherence (mediating about 50% of attachment to host cells), and to highlight the impact of PA0041 fha on attachment to A549 cells, a PA0041 pilus double mutant was made using PO4 phages. Unfortunately, the assessment of this double mutant in binding deficiency suffered from stronger than usual variation among the 5 experiments performed and a conclusion could not be made until more screening assays are performed. Aside from the filamentous hemagglutinin, another larger outer membrane protein PA0690 (hypothetical protein 38% similar to high molecular weight surface exposed protein in H. influenzae) was also found less capable of binding to A549 epithelial cells. In this non-pilin adhesin screening, twelve mutants were selected: five of them (PA0041, PA0222, PA0690, PA4086 and PA5498) have both adhesion-like export signal and sequence homology to known adhesins (category 1), four of them (PA0046, PA3064, PA5033 and PA5191) possess adhesion-like export signal alone (category 2), and three of them (PA0413, PA3707 and PA4541) have sequence homology to known adhesins alone (category 3). In category 1, all mutants except PA4086 showed various amount of binding deficiency; this high confirmation rate (80%) justifies the selection criteria. On the other hand, only PA0046 and 45 PA4541 were found deficient in A549 attachment in the other two groups of mutants, making the confirmation rate 25% in category 2 and 33% in category 3. Since a number of possible adhesins were identified in this project, using more sensitive measurement techniques and constructing a library of non-pilin mutants with defective pilus using PO4 phage selection would allow a more accurate assessment of the role of these factors in epithelial cells attachment. In addition, performing multiple knockouts on a pilus negative strain to create a library of mutants that express single non-pilin adhesins would highlight the binding capacity of each non-pilin adhesin and allow more accurate assessment. 4.8 Future areas for study Although the effect of epithelial cells on the expression profile of P. aeruginosa has been examined in this project and a number of genes were identified as important for initial bacterial- host interaction, the function and benefit of these differential expressions remain largely unclear. Adhesion and cytotoxicity assays might provide additional information about these genes. In addition, although the current study suggests that the expression of pilus (pilW), flagella (figD) and the Type III secretion system (pscP) may be regulated in the interacting bacteria, electron microscopy should be employed to examine the effect of these mutations on the biogenesis and expression of the corresponding systems. Bacteria could also be recovered immediately after the interaction assay to test for changes in phenotypes such as resistance to antimicrobial peptide (mediated by PhoPQ & PmrAB induction) and to ciprofloxacin (mediated by Lon) to confirm the induction of these systems; although it should be noted that recovering the bacteria without introducing changes might be a challenge as detergent is required to release the bacteria from epithelial attachment. Provided a lux transcriptional fusion mutant is present and the lux 46 expression is strong, mutants in the identified genes could be used in an interaction assay and the expression of the gene could be monitored in real time by measurement of luminescence, providing additional supporting evidence for the differential expression. Furthermore, since a working protocol has been successfully developed in this project to study initial bacterial-host interaction between P. aeruginosa PAO1 wild type strain and polarized HBE cell line, interactions between clinical isolates and physiological cells (for instance CF primary epithelial cells) could be studied using this procedure to assess genes important for the early stages of pathogenesis in a more clinically relevant setting. Finally, for genes upregulated in the interacting bacteria, especially ion which seems to have multiple roles in P. aeruginosa pathogenesis, animal model studies of survival rate and infection establishment could be designed to test for their significance in successful adaptation to the host environment. 4.9 Conclusion Differential expression of virulence factors contributes significantly to successful P. aeruginosa adaptation. In the case of acute and chronic lung infections, a subset of genes responsible for cytotoxicity and persistence are tightly regulated so that the bacteria can adapt to the unique challenges in each environment. In this thesis project, the interaction between P. aeruginosa and human bronchial epithelial cells was studied using a variety of techniques. In particular, the effect of epithelial cells on the P. aeruginosa transcriptome profile was studied using microarray experiments, and the involvement of differentially-expressed genes in epithelial cell damage was further characterized using a cytotoxicity assay. In addition, a non-pilin adhesin screening was also carried out to identify factors important for epithelial cell attachment during this early stage of pathogenesis. 47 A number of genes, including ion protease, oprC outer membrane protein, lasI, rsaL and rhlI in quorum-sensing system, as well as a subset of genes in the pmrAB and phoPQ two-component regulatory systems were found to be significantly induced in the bacteria interacting with epithelial cells, revealing the potential importance of these genes in successful adaptation to the host environment. In contrast, oprG outer membrane protein was found to be down-regulated indicating that its expression might be less critical during this stage of pathogenesis. While the function of OprG still remains unclear, the cytotoxicity study performed in this project has provided new insight as a mutant in OprG was found less capable of mediating HBE cell damage. Genes in the pilus, flagella, and the Type III secretion systems were also found regulated; however, since only a single gene was confirmed in each system, further experiments are required to conclude the differential expression of these systems in the interacting bacteria. In addition quorum-sensing systems were found to be upregulated in the interacting bacteria; however, the regulatory effects of these systems on QS controlled factors might not be significant at this stage due to the lack of induction in Rh1RI-regulated genes in the microarray as well as the fact that LasRI system was potentially antagonized by the induction of inhibitory protein RsaL. Another significant observation in this project was the involvement of Lon protease in cytotoxicity and its induction in P. aeruginosa interacting with epithelial cells. Since Ion has previously been documented to be essential for motility and biofilm formation, it is now clear that this gene has a very important role in pathogenesis and has an impact on most of the life cycle of P. aeruginosa in the human lung. Therefore, this protein could represent be a novel target for therapeutic development. 48 Initial bacterial-host interaction is a complex process. In this study, it was clear that a large number of genes in the bacteria must be tightly controlled in order to regulate attachment, motility, and cytotoxicity as well as resistance to the host immune response, all of which are important factors for the bacteria to colonize epithelial cells while maintaining a low immunostimulatory profile. 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Zhang, P., W. R. Summer, G. J. Bagby, and S. Nelson. 2000. Innate immunity and pulmonary host defense. Immunol Rev 173:39-51. 57 Supplementary Table S-1. Genes up-regulated in interacting bacteria. PA number Gene Name Description Fold change P-value PA0022 ; Hypothetical, unclassified, unknown 1.5 0.017 PA0026 plcB ; Hypothetical, unclassified, unknown 2.1 7.79E-05 PA0033 ; Hypothetical, unclassified, unknown 2.7 0.0002 PA0034 ; Two-component regulatory systems ; Transcriptional regulators 2.8 1.53E-05 PA0035 trpA ; Amino acid biosynthesis and metabolism 4.8 1.50E-06 PA0036 trp8 ; Amino acid biosynthesis and metabolism 1.7 0.0016 PA0040 ; Hypothetical, unclassified, unknown 1.5 0.0014 PA0053 ; Hypothetical, unclassified, unknown 1.8 0.0001 PA0054 ; Hypothetical, unclassified, unknown 1.9 0.0002 PA0055 ; Hypothetical, unclassified, unknown 1.6 0.0002 PA0080 ; Hypothetical, unclassified, unknown 1.7 0.0007 PA0107 ; Energy metabolism 1.6 0.0009 PA0115 ; Hypothetical, unclassified, unknown 4.4 1.08E-05 PA0122 ; Hypothetical, unclassified, unknown 2.3 0.0117 PA0142 ; Putative enzymes 1.6 0.0075 PA0154 pcaG ; Carbon compound catabolism 1.6 0.0002 PA0208 mdcA ; Carbon compound catabolism 2.5 1.29E-05 PA0221 ; Putative enzymes 10.9 1.32E-08 PA0249 ; Putative enzymes 1.6 0.0023 PA0259 ; Hypothetical, unclassified, unknown 1.9 0.0036 PA0260 ;^Membrane proteins ; Hypothetical, unclassified, unknown 2.1 0.0003 PA0264 ; Hypothetical, unclassified, unknown 3.8 5.91E-06 PA0266 gabT ;^Amino acid biosynthesis and metabolism ; Carbon compound catabolism ; Central intermediary metabolism 1.5 0.0032 PA0296 ; Putative enzymes 2.4 0.001 PA0297 spuA ; Amino acid biosynthesis and metabolism 2.5 0.0004 PA0298 spuB ; Putative enzymes 2.7 7.86E-06 PA0299 spuC ; Putative enzymes 1.9 3.58E-05 PA0301 spuE ; Transport of small molecules 1.7 0.0004 PA0307 ; Hypothetical, unclassified, unknown 1.7 0.0001 PA0311 ; Hypothetical, unclassified, unknown 2.3 7.82E-06 PA0313 ; Transport of small molecules ; Membrane proteins 1.6 0.0114 PA0422 ; Hypothetical, unclassified, unknown 2.0 0.0013 PA0433 ; Hypothetical, unclassified, unknown 2.2 2.17E-05 PA0435 ; Hypothetical, unclassified, unknown 2.2 1.23E-05 PA0446 ; Hypothetical, unclassified, unknown 1.9 3.95E-05 PA0450 ; Transport of small molecules ; Membraneproteins 1.7 0.003 PA0482 glcB ;^Carbon compound catabolism ; Central intermediary metabolism 1.9 0.0004 PA0495 ; Hypothetical, unclassified, unknown 1.5 0.003 PA0497 ; Hypothetical, unclassified, unknown 1.6 0.0203 PA0498 ; Hypothetical, unclassified, unknown 1.6 0.0007 58 PA number Gene Name Description Fold change P-value PA0499 ;^Chaperones & heat shock proteins ; Motility & Attachment 2.1 0.0059 PA0558 ; Hypothetical, unclassified, unknown 1.7 0.0019 PA0560 ; Hypothetical, unclassified, unknown 1.7 0.0235 PA0561 ; Hypothetical, unclassified, unknown 2.3 0.0003 PA0566 ; Hypothetical, unclassified, unknown 1.7 0.001 PA0580 gcp ; Translation, post-translational modification,degradation 1.5 0.0082 PA0620 ^ ; Related to phage, transposon, or plasmid ; Related to phage, transposon, or plasmid 1.8 1.5 0.0001 0.0107PA0643 PA0644 ; Related to phage, transposon, or plasmid 2.3 0.0001 PA0645 ; Related to phage, transposon, or plasmid 2.7 6.41E-06 PA0649 trpG ; Amino acid biosynthesis and metabolism ; Biosynthesis of cofactors, prosthetic groups and carriers ; Energy metabolism 1.7 0.014 PA0651 trpC ; Amino acid biosynthesis and metabolism 1.8 0.0002 PA0661 ; Membrane proteins 1.5 0.0272 PA0689 ;^Transport of small molecules ; Hypothetical, unclassified, unknown 1.5 0.0018 PA0712 ; Hypothetical, unclassified, unknown 1.6 0.0331 PA0719 ;^Related to phage, transposon, or plasmid ; Hypothetical, unclassified, unknown 2.0 0.0076 PA0729 ; Hypothetical, unclassified, unknown 1.5 0.0012 PA0730 ; Putative enzymes 1.5 0.0034 PA0740 ; Putative enzymes 1.6 0.0093 PA0744 ; Putative enzymes 1.9 7.27E-05 PA0745 ; Putative enzymes 1.5 0.0051 PA0755 ; Transport of small molecules ; Membrane proteins 1.6 0.0011 PA0761 nad8 ;^Biosynthesis of cofactors, prosthetic groups and carriers ; Amino acid biosynthesis and metabolism 1.6 0.0035 PA0791 ; Transcriptional regulators 1.5 0.0128 PA0792 prpD ; Carbon compound catabolism 1.9 0.0003 PA0794 ; Energy metabolism 1.8 0.0007 PA0795 prpC ; Carbon compound catabolism ; Central intermediary metabolism 2.0 0.0034 PA0796 prpB ; Carbon compound catabolism ; Central intermediary metabolism ; Fatty acid and phospholipid metabolism 1.8 0.0161 PA0804 ; Putative enzymes 2.0 3.36E-05 PA0821 ; Hypothetical, unclassified, unknown 3.6 1.89E-05 PA0823 ; Hypothetical, unclassified, unknown 1.5 0.0032 PA0824 ; Hypothetical, unclassified, unknown 2.6 0.0008 PA0863 ; Putative enzymes 2.4 1.20E-05 PA0865 hpd ; Amino acid biosynthesis and metabolism 3.5 3.91E-05 PA0870 phhC ; Amino acid biosynthesis and metabolism 1.9 0.0003 PA0871 phhB ; Amino acid biosynthesis and metabolism 2.0 8.59E-05 PA0874 ; Hypothetical, unclassified, unknown 1.9 0.0184 PA0887 acsA •^Central intermediary metabolism ; Carbon'compound catabolism 2.9 4.01E-06 59 PA number Gene Name Description Fold change P-value PA0911 ; Hypothetical, unclassified, unknown 1.6 0.0005 PA0936 1pxO2 ;^Cell wall / LPS / capsule ; Putative enzymes 1.6 0.0188 PA0959 ; Hypothetical, unclassified, unknown 2.0 9.85E-05 PA0975 ; Putative enzymes 2.9 1.04E-05 PA0976 ; Hypothetical, unclassified, unknown 4.4 1.02E-05 PA0977 ; Hypothetical, unclassified, unknown 2.0 8.43E-05 PA0979 ; Related to phage, transposon, or plasmid 1.7 0.0028 PA0982 ; Hypothetical, unclassified, unknown 1.6 0.001 PA0988 ; Hypothetical, unclassified, unknown 1.9 0.0148 PA0990 ; Hypothetical, unclassified, unknown 1.6 0.0013 PA0995 ogt ; DNA replication, recombination, modification and repair 1.5 0.007 PA1034 ; Hypothetical, unclassified, unknown 1.5 0.0208 PA1054 ;^Putative enzymes ; Membrane proteins 1.6 0.0039 PA1123 ; Hypothetical, unclassified, unknown 1.6 0.0054 PA1150 pys2 ;^Secreted Factors (toxins, enzymes, alginate); Adaptation, Protection 1.7 0.0023 PA1178 oprH ;^Adaptation, Protection ; Transport of small molecules ; Membrane proteins 1.5 0.0015 PA1187 ; Putative enzymes 2.4 0.002 PA1309 ; Transcriptional regulators 1.7 0.0015 PA1313 ; Transport of small molecules ; Membrane proteins 1.5 0.0277 PA1371 ; Hypothetical, unclassified, unknown 1.7 0.0235 PA1377 ; Hypothetical, unclassified, unknown 2.2 0.0004 PA1387 ; Hypothetical, unclassified, unknown 1.7 0.0225 PA1430 lasR • Transcriptional regulators ; Adaptation, 'Protection 1.5 0.0032 PA1431 rsaL ;^Adaptation, Protection ;^Transcriptional regulators ; Secreted Factors (toxins, enzymes, alginate) 3.1 1.28E-06 PA1439 ; Hypothetical, unclassified, unknown 1.7 0.001 PA1503 ; Hypothetical, unclassified, unknown 1.5 0.0028 PA1504 ; Transcriptional regulators 1.6 0.001 PA1505 moaA2 • Biosynthesis of cofactors, prosthetic groups ' and carriers 2.0 0.0001 PA1507 ; Transport of small molecules ; Membrane proteins 1.6 0.0012 PA1514 ; Hypothetical, unclassified, unknown 1.6 0.0041 PA1560 ; Hypothetical, unclassified, unknown 1.6 0.0019 PA1562 acnA ; Energy metabolism 1.7 0.02 PA1565 ; Putative enzymes 1.5 0.0011 PA1571 ; Hypothetical, unclassified, unknown 1.9 0.0016 PA1574 ; Hypothetical, unclassified, unknown 1.9 0.0006 PA1577 ;^Hypothetical, unclassified, unknown ;Membrane proteins 2.0 2.59E-05 PA1580 gltA ; Energy metabolism 1.9 8.38E-05 PA1581 sdhC ; Energy metabolism 1.8 0.0019 PA1601 ; Putative enzymes 1.6 0.0002 PA1602 ; Carbon compound catabolism 2.4 8.97E-06 60 PA number Gene Name Description Fold change P-value PA1606 ; Hypothetical, unclassified, unknown 1.6 0.0006 PA1623 ; Hypothetical, unclassified, unknown 2.0 0.0018 PA1624 ; Hypothetical, unclassified, unknown 1.6 0.0022 PA1639 ; Hypothetical, unclassified, unknown 2.1 0.0031 PA1652 ;^Hypothetical, unclassified, unknown ;Membrane proteins 1.6 0.0007 PA1654 ; Putative enzymes 2.4 4.81E-05 PA1655 ; Putative enzymes 1.9 5.21E-05 PA1661 ; Hypothetical, unclassified, unknown 4.3 2.72E-06 PA1733 ; Hypothetical, unclassified, unknown 1.8 0.0131 PA1743 ; Hypothetical, unclassified, unknown 2.2 1.65E-05 PA1753 ; Hypothetical, unclassified, unknown 1.8 0.0015 PA1811 ; Transport of small molecules 1.6 0.0008 PA1852 ; Hypothetical, unclassified, unknown 3.0 1.27E-06 PA1882 ; Transport of small molecules ; Membrane proteins 2.0 0.0007 PA1935 ; Hypothetical, unclassified, unknown 1.5 0.0027 PA1940 ; Hypothetical, unclassified, unknown 1.8 0.0005 PA1942 ; Hypothetical, unclassified, unknown 2.4 0.0004 PA1951 ; Hypothetical, unclassified, unknown 1.5 0.001 PA1978 ; Transcriptional regulators 1.6 0.0076 PA1999 ;^Carbon compound catabolism ; Amino acidbiosynthesis and metabolism 2.8 1.00E-05 PA2000 ; Carbon compound catabolism ; Amino acid biosynthesis and metabolism 2.3 3.26E-05 PA2001 atoB • Fatty acid and phospholipid metabolism ;; Central intermediary metabolism 2.6 7.23E-06 PA2003 bdhA ; Carbon compound catabolism 1.8 0.0001 PA2008 fahA ; Carbon compound catabolism 1.8 0.0019 PA2009 hmgA ; Carbon compound catabolism 1.8 0.0006 PA2014 gnyB ; Carbon compound catabolism 1.8 0.0035 PA2015 gnyD ; Carbon compound catabolism 2.2 2.99E-05 PA2016 gnyR ; Transcriptional regulators 1.9 0.0009 PA2039 ;^Hypothetical, unclassified, unknown ;Membrane proteins 1.9 0.0016 PA2102 ; Hypothetical, unclassified, unknown 1.9 0.0039 PA2103 ; Biosynthesis of cofactors, prosthetic groups and carriers 1.7 0.0013 PA2118 ada • DNA replication, recombination, modification 'and repair ; Transcriptional regulators 3.9 3.22E-07 PA2128 cupAl ; Motility & Attachment 1.8 0.0004 PA2247 bkdAl ; Amino acid biosynthesis and metabolism 1.9 4.14E-05 PA2271 ; Putative enzymes 1.7 0.0054 PA2287 ; Hypothetical, unclassified, unknown 1.9 0.0068 PA2291 ; Transport of small molecules 1.7 0.0146 PA2318 ; Hypothetical, unclassified, unknown 2.4 0.0003 PA2359 ; Transcriptional regulators 1.5 0.002 PA2372 ; Hypothetical, unclassified, unknown 1.8 0.0001 PA2422 ; Hypothetical, unclassified, unknown 1.8 0.0002 PA2455 ; Hypothetical, unclassified, unknown 2.5 1.62E-05 61 PA number Gene Name Description Fold change P-value PA2456 ; Hypothetical, unclassified, unknown 3.8 2.48E-06 PA2486 ; Hypothetical, unclassified, unknown 1.5 0.0121 PA2491 ; Putative enzymes 2.0 0.0046 PA2493 mexE • Antibiotic resistance and susceptibility ;'Transport of small molecules 1.8 0.0018 PA2541 ; Putative enzymes 1.8 0.0004 PA2551 ; Transcriptional regulators 2.1 0.0004 PA2552 ; Putative enzymes 1.6 0.002 PA2553 ; Putative enzymes 2.2 3.91E-05 PA2554 ; Putative enzymes 1.8 0.0003 PA2555 ; Putative enzymes 5.0 1.64E-05 PA2586 gacA ; Transcriptional regulators 1.7 0.0004 PA2592 ; Transport of small molecules 1.6 0.0056 PA2595 ; Hypothetical, unclassified, unknown 2.1 0.0004 PA2616 trx131 ; Nucleotide biosynthesis and metabolism 1.6 0.0013 PA2623 icd ; Amino acid biosynthesis and metabolism ; Energy metabolism ; Carbon compound catabolism 1.5 0.0078 PA2666 ; Biosynthesis of cofactors, prosthetic groups and carriers 1.5 0.0201 PA2668 ; Hypothetical, unclassified, unknown 1.9 9.90E-05 PA2691 ; Hypothetical, unclassified, unknown 1.8 0.0003 PA2712 ;^Hypothetical, unclassified, unknown ; Membrane proteins 1.9 0.0119 PA2746 ;^Hypothetical, unclassified, unknown ;Membrane proteins 1.9 0.0003 PA2755 eco •Translation, post-translational modification, 'degradation 2.2 2.58E-05 PA2761 ;^Hypothetical, unclassified, unknown ;Membrane proteins 1.5 0.0005 PA2776 ; Hypothetical, unclassified, unknown 1.6 0.0045 PA2777 ;^Hypothetical, unclassified, unknown ; Membrane proteins 1.7 0.0003 PA2791 ; Hypothetical, unclassified, unknown 2.1 0.0002 PA2795 ; Hypothetical, unclassified, unknown 1.5 0.0168 PA2819 ; Hypothetical, unclassified, unknown 1.7 0.0003 PA2822 ; Hypothetical, unclassified, unknown 1.6 0.0005 PA2824 ; Two-component regulatory systems 1.7 0.0381 PA2855 ; Hypothetical, unclassified, unknown 1.6 0.0005 PA2878 ; Hypothetical, unclassified, unknown 2.1 0.0016 PA2897 ; Transcriptional regulators 1.6 0.002 PA2919 ;^Hypothetical, unclassified, unknown ;Membrane proteins 1.6 0.0112 PA2943 ; Amino acid biosynthesis and metabolism 1.9 0.002 PA2946 ;^Hypothetical, unclassified, unknown ; Membrane proteins 1.6 0.0013 PA3016 ; Hypothetical, unclassified, unknown 1.8 0.0005 PA3038 ; Transport of small molecules 3.2 2.68E-05 PA3104 xcpP ; Protein secretion/export apparatus 1.7 0.0036 PA3129 ; Hypothetical, unclassified, unknown 2.0 8.93E-05 PA3140 ; Hypothetical, unclassified, unknown 1.5 0.0008 62 PA number Gene Name Description Fold change P-value PA3143 ; Related to phage, transposon, or plasmid 1.8 0.0002 PA3159 wbpA ;^Cell wall / LPS / capsule ; Putative enzymes 1.6 0.0021 PA3180 ; Hypothetical, unclassified, unknown 1.7 0.0062 PA3182 pg/ ; Central intermediary metabolism 1.5 0.0166 PA3190 ; Transport of small molecules 1.9 2.71E-05 PA3192 gltR ; Transcriptional regulators ; Two-component regulatory systems ; Carbon compound catabolism 1.6 0.0033 PA3193 glk ; Carbon compound catabolism ; Energy metabolism 1.9 0.0026 PA3194 edd • Carbon compound catabolism ; Energy'metabolism 3.2 4.03E-06 PA3196 ; Hypothetical, unclassified, unknown 3.1 2.56E-06 PA3224 ; Hypothetical, unclassified, unknown 1.7 0.0065 PA3234 ; Transport of small molecules ; Membrane proteins 2.2 2.14E-05 PA3235 ;^Hypothetical, unclassified, unknown ;Membrane proteins 2.3 1.50E-05 PA3256 ; Putative enzymes 1.5 0.0015 PA3262 ; Chaperones & heat shock proteins ; Translation, post-translational modification, degradation 1.5 0.0018 PA3319 pIcAl ; Secreted Factors (toxins, enzymes, alginate) 1.7 0.0005 PA3325 ; Hypothetical, unclassified, unknown 2.2 0.0002 PA3326 ; Translation, post-translational modification, degradation 2.5 1.44E-05 PA3348 ;^Chemotaxis ; Adaptation, Protection 1.5 0.0014 PA3350 ; Hypothetical, unclassified, unknown 2.3 1.34E-05 PA3356 ; Hypothetical, unclassified, unknown 1.6 0.0013 PA3367 ; Hypothetical, unclassified, unknown 1.9 7.20E-05 PA3422 ; Hypothetical, unclassified, unknown 2.4 0.0345 PA3436 ; Hypothetical, unclassified, unknown 2.1 3.11E-05 PA3437 ; Putative enzymes 1.5 0.0071 PA3438 folE1 • Biosynthesis of cofactors, prosthetic groups ' and carriers 1.8 0.0017 PA3439 foIX • Biosynthesis of cofactors, prosthetic groups ' and carriers 2.0 0.0019 PA3442 ; Transport of small molecules 1.7 0.0015 PA3476 rh/I ; Adaptation, Protection 1.8 0.001 PA3486 ; Hypothetical, unclassified, unknown 1.5 0.0027 PA3487 pldA ; Fatty acid and phospholipid metabolism 1.7 0.0032 PA3536 ; Hypothetical, unclassified, unknown 1.6 0.0033 PA3564 ; Hypothetical, unclassified, unknown 2.3 1.01E-05 PA3577 ; Hypothetical, unclassified, unknown 2.0 5.37E-05 PA3581 g/pF ; Transport of small molecules 1.6 0.0007 PA3602 ;^Hypothetical, unclassified, unknown ; Membrane proteins 1.7 0.0003 PA3619 ; Hypothetical, unclassified, unknown 1.5 0.0021 PA3622 rpoS ; Transcriptional regulators 1.9 0.0002 PA3659 ; Putative enzymes 2.2 0.0002 PA3663 ; Hypothetical, unclassified, unknown 1.6 0.0003 63 PA number Gene Name Description Fold change P-value PA3688 ; Hypothetical, unclassified, unknown 1.7 0.0003 PA3689 ; Transcriptional regulators 2.0 0.0007 PA3695 ; Hypothetical, unclassified, unknown 1.6 0.0143 PA3751 purT ;^Nucleotide biosynthesis and metabolism ; Amino acid biosynthesis and metabolism 1.8 0.0012 PA3752 ; Hypothetical, unclassified, unknown 1.9 0.0006 PA3754 ; Hypothetical, unclassified, unknown 1.9 0.0009 PA3755 ; Hypothetical, unclassified, unknown 1.6 0.0304 PA3756 ; Hypothetical, unclassified, unknown 1.5 0.0033 PA3783 ; Hypothetical, unclassified, unknown 2.3 6.27E-05 PA3787 ; Hypothetical, unclassified, unknown 3.5 0.0003 PA3788 ;^Hypothetical, unclassified, unknown ;Membrane proteins 3.1 1.07E-05 PA3790 oprC ; Transport of small molecules 4.3 1.01E-05 PA3791 ;^Membrane proteins ; Hypothetical, unclassified, unknown 1.7 0.0171 PA3843 ; Hypothetical, unclassified, unknown 2.6 5.85E-06 PA3845 ; Transcriptional regulators 1.6 0.0113 PA3864 ; Hypothetical, unclassified, unknown 2.3 0.0001 PA3885 ; Hypothetical, unclassified, unknown 1.6 0.0014 PA3911 ; Hypothetical, unclassified, unknown 1.6 0.0388 PA3922 ; Hypothetical, unclassified, unknown 3.7 2.18E-06 PA3923 ; Hypothetical, unclassified, unknown 2.1 0.0003 PA3928 ; Hypothetical, unclassified, unknown 2.1 4.31E-05 PA3929 cioB ; Energy metabolism 2.2 0.0002 PA3991 ; Hypothetical, unclassified, unknown 1.5 0.0231 PA3992 ; Hypothetical, unclassified, unknown 1.8 0.0022 PA4017 ; Hypothetical, unclassified, unknown 1.5 0.0114 PA4022 ; Putative enzymes 4.2 6.07E-07 PA4023 ; Transport of small molecules 2.6 3.97E-05 PA4035 ; Hypothetical, unclassified, unknown 1.8 0.0004 PA4041 ; Putative enzymes 6.9 5.48E-08 PA4062 ; Hypothetical, unclassified, unknown 2.1 5.20E-05 PA4074 ; Transcriptional regulators 1.7 0.0003 PA4079 ; Putative enzymes 1.7 0.0068 PA4080 ;^Transcriptional regulators ; Transport of small molecules 1.6 0.0015 PA4090 ; Hypothetical, unclassified, unknown 1.8 0.0071 PA4092 hpaC ; Carbon compound catabolism 1.5 0.0089 PA4108 ; Hypothetical, unclassified, unknown 1.7 0.0382 PA4111 ; Hypothetical, unclassified, unknown 1.5 0.0099 PA4114 ; Putative enzymes 2.0 4.98E-05 PA4115 ; Hypothetical, unclassified, unknown 1.7 0.0037 PA4116 ; Hypothetical, unclassified, unknown 1.6 0.0039 PA4149 ; Hypothetical, unclassified, unknown 1.5 0.004 PA4171 ; Putative enzymes 1.7 0.0122 PA4182 ; Hypothetical, unclassified, unknown 2.1 0.0002 PA4183 ; Hypothetical, unclassified, unknown 1.5 0.0018 PA4200 ; Hypothetical, unclassified, unknown 2.7 2.08E-05 64 PA number Gene Name Description Fold change P-value PA4289 ; Transport of small molecules ; Membrane proteins 1.8 0.0002 PA4297 ;^Hypothetical, unclassified, unknown ; Membrane proteins 1.5 0.0021 PA4316 sbcB . DNA replication, recombination, modification'and repair 1.8 0.0002 PA4330 ; Putative enzymes 2.0 0.0082 PA4336 ; Hypothetical, unclassified, unknown 1.7 0.0003 PA4340 ; Hypothetical, unclassified, unknown 1.8 0.0135 PA4366 sod8 ; Adaptation, Protection 1.6 0.0125 PA4385 groEL ; Chaperones & heat shock proteins 1.8 0.0005 PA4386 groES ; Chaperones & heat shock proteins 1.6 0.0001 PA4440 ; Hypothetical, unclassified, unknown 1.7 0.0004 PA4488 ; Hypothetical, unclassified, unknown 1.5 0.0149 PA4496 ; Transport of small molecules 1.5 0.0234 PA4498 ; Translation, post-translational modification, degradation 1.6 0.0018 PA4520 ;^Chemotaxis ; Adaptation, Protection 1.5 0.0044 PA4526 pi/8 ; Motility & Attachment 2.2 6.70E-05 PA4527 pi1C ; Motility & Attachment 1.6 0.0012 PA4545 comL ; Cell wall / LPS / capsule 1.7 0.0063 PA4552 pilW ; Motility & Attachment 2.4 5.26E-05 PA4555 pi/Y2 ; Motility & Attachment 1.8 0.0175 PA4556 pilE ; Motility & Attachment 2.0 0.0214 PA4570 ; Hypothetical, unclassified, unknown 2.0 0.0023 PA4590 pra ;^Carbon compound catabolism ; Transport of small molecules 1.8 0.0001 PA4606 ; Adaptation, Protection 2.0 0.0002 PA4607 ; Hypothetical, unclassified, unknown 1.9 0.0001 PA4634 ; Hypothetical, unclassified, unknown 2.0 7.20E-05 PA4635 ; Hypothetical, unclassified, unknown 1.9 8.54E-05 PA4652 ; Hypothetical, unclassified, unknown 1.7 0.0006 PA4703 ; Hypothetical, unclassified, unknown 1.6 0.0046 PA4770 IldP ; Transport of small molecules 1.9 0.0002 PA4776 pmrA ; Two-component regulatory systems 1.6 0.0047 PA4787 ; Transcriptional regulators 1.6 0.0041 PA4827 ;^Putative enzymes ; Adaptation, Protection 1.5 0.0102 PA4834 ;^Hypothetical, unclassified, unknown ; Membrane proteins 1.8 0.0002 PA4871 ; Hypothetical, unclassified, unknown 1.8 0.002 PA4895 ; Transcriptional regulators ; Membrane proteins 1.6 0.0121 PA4910 ; Transport of small molecules 1.7 0.0223 PA4937 mr ; Transcription, RNA processing and degradation 1.8 8.32E-05 PA4968 ; Hypothetical, unclassified, unknown 1.5 0.0008 PA4971 aspP ; Energy metabolism 1.8 0.0011 PA4983 ; Two-component regulatory systems ; Transcriptional regulators 1.6 0.003 PA4994 ; Putative enzymes 1.6 0.0016 PA5031 ; Putative enzymes 1.6 0.0009 65 PA number Gene Name Description Fold change P-value PA5058 phaC2 ; Central intermediary metabolism 1.9 0.0001 PA5066 hisl ; Amino acid biosynthesis and metabolism 1.5 0.003 PA5068 tatA ; Protein secretion/export apparatus 1.6 0.0049 PA5081 ; Hypothetical, unclassified, unknown 1.6 0.0021 PA5087 ; Hypothetical, unclassified, unknown 1.7 0.0007 PA5089 ; Hypothetical, unclassified, unknown 1.7 0.0006 PA5094 ; Transport of small molecules 4.9 6.02E-05 PA5100 hutU ; Amino acid biosynthesis and metabolism 3.3 2.33E-06 PA5123 ; Hypothetical, unclassified, unknown 1.7 0.0018 PA5154 ; Transport of small molecules ; Membrane proteins 1.8 0.0005 PA5180 ; Hypothetical, unclassified, unknown 1.7 0.001 PA5185 ; Hypothetical, unclassified, unknown 1.7 0.0038 PA5186 ; Putative enzymes 1.6 0.002 PA5204 argA ; Amino acid biosynthesis and metabolism 1.8 0.0008 PA5205 ;^Hypothetical, unclassified, unknown ; Membrane proteins 4.2 1.50E-07 PA5206 argE ; Amino acid biosynthesis and metabolism 1.7 9.41E-05 PA5239 rho ; Transcription, RNA processing and degradation 1.8 0.0018 PA5244 ;^Hypothetical, unclassified, unknown ; Membrane proteins 1.5 0.0009 PA5246 ; Hypothetical, unclassified, unknown 2.0 5.09E-05 PA5268 corA • Transport of small molecules ; Membrane 'proteins 1.7 0.0487 PA5273 ; Hypothetical, unclassified, unknown 1.6 0.001 PA5282 ; Transport of small molecules ; Membrane proteins 1.8 0.0005 PA5287 amtB • Transport of small molecules ; Membrane; proteins 1.7 0.0003 PA5290 ; Hypothetical, unclassified, unknown 2.3 1.18E-05 PA5301 ; Transcriptional regulators 2.0 0.0033 PA5312 ; Putative enzymes 3.7 0.0004 PA5319 radC • DNA replication, recombination, modification; and repair 1.7 0.0008 PA5347 ; Hypothetical, unclassified, unknown 1.6 0.0007 PA5356 glcC ; Transcriptional regulators 1.8 0.0003 PA5362 ; Hypothetical, unclassified, unknown 1.8 0.0031 PA5363 ; Hypothetical, unclassified, unknown 1.5 0.0011 PA5372 betA • Adaptation, Protection ; Amino acid; biosynthesis and metabolism 1.6 0.0207 PA5380 ; Transcriptional regulators 3.1 2.84E-06 PA5381 ; Hypothetical, unclassified, unknown 2.5 1.72E-05 PA5396 ; Hypothetical, unclassified, unknown 1.7 0.0006 PA5397 ; Hypothetical, unclassified, unknown 1.6 0.0334 PA5452 wbpW ; Cell wall / LPS / capsule 1.6 0.0007 PA5474 ; Translation, post-translational modification, degradation 1.5 0.0099 PA5509 ; Hypothetical, unclassified, unknown 1.5 0.0079 PA5517 ; Hypothetical, unclassified, unknown 1.6 0.0015 PA5519 ; Hypothetical, unclassified, unknown 1.5 0.011 66 PA number Gene Name Description Fold change P-value PA5523 ; Putative enzymes 1.5 0.0028 PA5529 ; Transport of small molecules ; Membrane proteins 1.5 0.0015 PA5544 ;^Hypothetical, unclassified, unknown ; Membrane proteins 1.6 0.0088 PA5566 ; Hypothetical, unclassified, unknown 1.7 0.0133 67 Supplementary Table S-2. Genes down-regulated in interacting bacteria. PA number Gene Name Description Fold Change P-value PA0008 glyS ;^Translation, post-translational modification, degradation ; Amino acid biosynthesis and metabolism -1.7 0.0005 PA0010 tag ; DNA replication, recombination, modification and repair -1.5 0.0099 PA0046 ; Hypothetical, unclassified, unknown -1.7 0.0003 PA0047 ; Hypothetical, unclassified, unknown -2 5.57E-05 PA0048 ; Transcriptional regulators -1.7 0.0003 PA0051 phzH ; Putative enzymes -1.7 0.0221 PA0098 ; Hypothetical, unclassified, unknown -1.7 0.0201 PA0128 ; Hypothetical, unclassified, unknown -2.7 0.0021 PA0141 ; Hypothetical, unclassified, unknown -1.6 0.0129 PA0163 ; Transcriptional regulators -1.6 0.0017 PA0165 ; Hypothetical, unclassified, unknown -1.6 0.0368 PA0185 ; Transport of small molecules ; Membraneproteins -1.5 0.0011 PA0198 exbB1 ; Transport of small molecules -1.5 0.0027 PA0199 exbD1 ; Transport of small molecules -1.6 0.0036 PA0200 ; Hypothetical, unclassified, unknown -1.6 0.0003 PA0201 ; Hypothetical, unclassified, unknown -3.4 1.22E-06 PA0202 ; Putative enzymes -2.3 4.02E-05 PA0224 ; Putative enzymes -1.8 0.0017 PA0277 ; Hypothetical, unclassified, unknown -1.5 0.0007 PA0278 ; Hypothetical, unclassified, unknown -1.9 0.008 PA0347 glpQ ; Fatty acid and phospholipid metabolism -2 2.96E-05 PA0359 ; Hypothetical, unclassified, unknown -1.6 0.0009 PA0364 ; Putative enzymes -1.5 0.0122 PA0382 micA • DNA replication, recombination, modification' and repair -1.7 0.0002 PA0391 ; Hypothetical, unclassified, unknown -1.5 0.0458 PA0402 pyrB ;^Nucleotide biosynthesis and metabolism ; Amino acid biosynthesis and metabolism -1.5 0.0056 PA0403 pyrR ;^Transcriptional regulators ; Nucleotidebiosynthesis and metabolism -1.5 0.0024 PA0408 pilG ;^Chemotaxis ;^Motility & Attachment ; Two- component regulatory systems -1.5 0.002 PA0463 creB • Two-component regulatory systems ; 'Transcriptional regulators -1.5 0.0101 PA0518 nirM • Energy metabolism ; Biosynthesis of 'cofactors, prosthetic groups and carriers -1.7 0.0034 PA0519 nirS ; Energy metabolism -3 2.43E-06 PA0525 ; Energy metabolism -2.4 9.18E-06 PA0526 ; Hypothetical, unclassified, unknown -4.6 0.0002 PA0545 ; Putative enzymes -1.7 0.0019 PA0546 metK • Central intermediary metabolism ; Amino acid 'biosynthesis and metabolism -1.9 4.49E-05 PA0549 ; Hypothetical, unclassified, unknown -1.9 0.0005 PA0553 ; Hypothetical, unclassified, unknown -2.9 0.0003 68 PA number Gene Name Description Fold Change P-value PA0554 ; Hypothetical, unclassified, unknown -1.5 0.0022 PA0557 ; Hypothetical, unclassified, unknown -1.5 0.0009 PA0576 rpoD ; Transcriptional regulators -1.5 0.0012 PA0581 ; Hypothetical, unclassified, unknown -2 3.25E-05 PA0591 ; Hypothetical, unclassified, unknown -1.6 0.0039 PA0594 surA ;^Adaptation, Protection ;^Chaperones & heat shock proteins ; Translation, post-translational modification, degradation -1.6 0.008 PA0728 ;^Related to phage, transposon, or plasmid ; Putative enzymes -1.6 0.0293 PA0767 lepA ;^Protein secretion/export apparatus ; Translation, post-translational modification, degradation -2 6.27E-05 PA0768 /epB ;^Protein secretion/export apparatus ; Translation, post-translational modification, degradation -1.8 0.0004 PA0772 recO • DNA replication, recombination, modification 'and repair -1.5 0.0339 PA0773 pdxJ ; Biosynthesis of cofactors, prosthetic groups and carriers -1.5 0.0013 PA0798 pmtA ; Fatty acid and phospholipid metabolism -1.8 0.0118 PA0807 ; Hypothetical, unclassified, unknown -1.7 0.0208 PA0836 ; Putative enzymes -3.8 2.08E-07 PA0840 ; Putative enzymes -2.1 5.66E-05 PA0842 ; Putative enzymes -2.4 0.0001 PA0845 ; Hypothetical, unclassified, unknown -2 2.33E-05 PA0851 ; Hypothetical, unclassified, unknown -1.6 0.0132 PA0890 aotM ; Tra nsport of small molecules ; Membrane proteins -1.7 8.65E-05 PA0891 ; Hypothetical, unclassified, unknown -1.8 0.0003 PA0903 alaS ;^Translation, post-translational modification, degradation ; Transcription, RNA processing and degradation -1.9 4.45E-05 PA0915 ; Hypothetical, unclassified, unknown -1.6 0.0109 PA0916 ; Hypothetical, unclassified, unknown -3 9.73E-07 PA0917 kup ; Transport of small molecules -1.6 0.0014 PA0944 purN ; Nucleotide biosynthesis and metabolism -1.9 9.57E-05 PA0945 purM ; Nucleotide biosynthesis and metabolism -1.6 0.0006 PA0964 ; Hypothetical, unclassified, unknown -1.6 0.0011 PA0965 ruvC • DNA replication, recombination, modification; and repair -1.5 0.0019 PA1011 ; Hypothetical, unclassified, unknown -1.6 0.001 PA1015 ; Transcriptional regulators -1.7 0.0007 PA1044 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.5 0.0083 PA1050 ; Hypothetical, unclassified, unknown -1.8 0.0128 PA1069 ; Hypothetical, unclassified, unknown -1.7 0.0012 PA1070 braG ; Transport of small molecules -1.8 0.0002 PA1076 ; Hypothetical, unclassified, unknown -1.7 0.0005 PA1103 ;^Motility & Attachment ; Cell wall / LPS / capsule -1.5 0.0346 69 PA number Gene Name Description Fold Change P-value PA1156 nrdA ; Nucleotide biosynthesis and metabolism -1.6 0.0019 PA1193 ; Hypothetical, unclassified, unknown -1.8 0.0001 PAl227 ; Hypothetical, unclassified, unknown -2.3 1.47E-05 PAl228 ; Hypothetical, unclassified, unknown -1.6 0.0079 PAl235 ; Transcriptional regulators -1.8 0.0153 PAl272 cobO • Biosynthesis of cofactors, prosthetic groups 'and carriers -1.5 0.048 PA1303 ; Putative enzymes -1.6 0.0378 PA1321 cyoE ; Energy metabolism -1.7 0.0176 PA1340 ; Transport of small molecules -1.6 0.0066 PA1414 ; Hypothetical, unclassified, unknown -8 4.18E-08 PA1415 ; Hypothetical, unclassified, unknown -2.7 9.21E-05 PA1487 ; Putative enzymes -1.6 0.0248 PA1545 ; Hypothetical, unclassified, unknown -3.4 1.53E-06 PA1546 hemN • Biosynthesis of cofactors, prosthetic groups; and carriers -5.1 3.37E-07 PA1548 ; Hypothetical, unclassified, unknown -1.6 0.0148 PA1553 ; Energy metabolism -1.8 0.0001 PA1557 ; Energy metabolism -2.3 2.03E-05 PA1561 aer ;^Chemotaxis ; Adaptation, Protection -1.9 8.60E-05 PA1583 sdhA ; Energy metabolism -1.5 0.006 PA1634 kdpB ; Transport of small molecules -2.1 0.0008 PA1636 kdpD ; Two-component regulatory systems -1.8 0.0127 PA1642 selD • Translation, post-translational modification, ' degradation -1.6 0.0047 PA1649 ; Putative enzymes -2.2 2.84E-05 PA1668 ; Hypothetical, unclassified, unknown -1.6 0.0291 PA1673 ; Hypothetical, unclassified, unknown -1.9 0.0002 PA1674 folE2 • Biosynthesis of cofactors, prosthetic groups 'and carriers -2.2 1.41E-05 PA1680 ; Hypothetical, unclassified, unknown -1.5 0.0011 PA1687 speE ; Amino acid biosynthesis and metabolism -2.1 0.0008 PA1689 ; Hypothetical, unclassified, unknown -1.6 0.0009 PA1695 pscP ; Protein secretion/export apparatus -2.1 0.0038 PA1699 ;^Hypothetical, unclassified, unknown ; Proteinsecretion/export apparatus -1.7 0.0059 PA1701 ;^Hypothetical, unclassified, unknown ; Proteinsecretion/export apparatus -1.9 0.0002 PA1715 pscB ; Protein secretion/export apparatus -1.6 0.0056 PA1716 pscC ; Protein secretion/export apparatus -1.5 0.0019 PA1718 pscE ; Protein secretion/export apparatus -1.5 0.0075 PA1726 bglX ; Carbon compound catabolism -1.6 0.0006 PA1747 ; Hypothetical, unclassified, unknown -2.4 1.79E-05 PA1786 ; Hypothetical, unclassified, unknown -1.5 0.0499 PA1789 ; Hypothetical, unclassified, unknown -1.6 0.0003 PA1790 ; Hypothetical, unclassified, unknown -1.9 0.0002 PA1799 ; Two-component regulatory systems ; Transcriptional regulators -2 0.0063 PA1804 hupB • DNA replication, recombination, modification; and repair -1.7 0.0434 70 PA number Gene Name Description Fold Change P-value PA1828 ; Putative enzymes -2.3 1.19E-05 PA1890 ; Putative enzymes -1.7 0.0013 PA1891 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.7 0.0014 PA1892 ; Hypothetical, unclassified, unknown -1.6 0.0022 PA1958 ; Transport of small molecules ; Membrane proteins -2.2 0.0031 PA1960 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.5 0.0047 PA1971 braZ ; Transport of small molecules -1.7 0.0008 PA2023 ga/U ; Central intermediary metabolism -1.6 0.0019 PA2024 ; Putative enzymes -1.6 0.0003 PA2036 ; Hypothetical, unclassified, unknown -1.7 0.021 PA2044 ; Hypothetical, unclassified, unknown -1.8 8.96E-05 PA2064 pcoB ; Adaptation, Protection -2.3 8.23E-06 PA2066 ; Hypothetical, unclassified, unknown -1.7 0.0223 PA2070 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.9 0.0074 PA2110 ; Hypothetical, unclassified, unknown -1.6 0.0021 PA2127 ; Hypothetical, unclassified, unknown -1.7 0.0003 PA2136 ; Hypothetical, unclassified, unknown -4 6.07E-05 PA2162 ; Putative enzymes -1.6 0.039 PA2235 psIE ; Hypothetical, unclassified, unknown -2 9.37E-05 PA2280 ; Hypothetical, unclassified, unknown -1.5 0.0018 PA2353 ; Hypothetical, unclassified, unknown -1.5 0.0032 PA2376 ; Transcriptional regulators -1.7 0.0006 PA2409 ; Transport of small molecules ; Membrane proteins -2.2 0.003 PA2418 ; Hypothetical, unclassified, unknown -1.6 0.016 PA2446 gcvH2 ; Amino acid biosynthesis and metabolism -2.3 1.14E-05 PA2449 ; Transcriptional regulators -1.5 0.0092 PA2453 ; Hypothetical, unclassified, unknown -1.6 0.0007 PA2500 ; Transport of small molecules ; Membrane proteins -3.2 6.33E-05 PA2501 ;^Hypothetical, unclassified, unknown ; Membrane proteins -3.8 2.16E-07 PA2504 ; Hypothetical, unclassified, unknown -1.6 0.0067 PA2505 ; Transport of small molecules -2.8 0.0008 PA2549 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.5 0.0011 PA2578 ; Putative enzymes -1.8 0.0002 PA2603 ; Putative enzymes -1.5 0.0147 PA2630 ; Hypothetical, unclassified, unknown -1.9 4.77E-05 PA2631 ; Putative enzymes -1.5 0.0009 PA2639 nuoD ; Energy metabolism -1.7 0.0003 PA2641 nuoF ; Energy metabolism -1.5 0.0009 PA2653 ; Transport of small molecules ; Membrane proteins -1.8 0.0022 PA2663 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.5 0.044 71 PA number Gene Name Description Fold Change P-value PA2664 fhp ; Energy metabolism -3.1 1.49E-05 PA2718 ; Transcriptional regulators -2 0.0255 PA2753 ; Hypothetical, unclassified, unknown -1.8 0.0048 PA2759 ; Hypothetical, unclassified, unknown -6.9 6.00E-08 PA2768 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.7 0.0037 PA2799 ; Hypothetical, unclassified, unknown -1.7 0.0002 PA2807 ; Hypothetical, unclassified, unknown -2.5 1.33E-05 PA2817 ; Hypothetical, unclassified, unknown -1.7 0.0006 PA2840 ; Transcription, RNA processing anddegradation -2.1 2.48E-05 PA2841 ; Putative enzymes -1.5 0.0026 PA2847 ; Hypothetical, unclassified, unknown -2.7 0.001 PA2851 efp ; Translation, post-translational modification,degradation -2.2 0.001 PA2867 ;^Chemotaxis ; Adaptation, Protection -3.3 4.84E-07 PA2904 cob' • Biosynthesis of cofactors, prosthetic groups'and carriers -1.9 0.0002 PA2911 ; Transport of small molecules ; Membrane proteins -1.7 0.0017 PA2964 pabC ; Biosynthesis of cofactors, prosthetic groupsand carriers -2.4 5.19E-05 PA2966 acpP ; Fatty acid and phospholipid metabolism -1.5 0.0489 PA2970 rpmF ; Translation, post-translational modification, degradation -2 1.96E-05 PA2971 ; Hypothetical, unclassified, unknown -1.9 4.31E-05 PA2982 ; Hypothetical, unclassified, unknown -1.8 0.0018 PA2986 ; Hypothetical, unclassified, unknown -1.6 0.002 PA2996 nqrD ; Energy metabolism -1.5 0.0019 PA2997 nqrC ; Energy metabolism -2.2 0.0002 PA2998 nqrB ; Energy metabolism -1.8 0.0001 PA3006 psrA ; Transcriptional regulators -1.7 0.0004 PA3011 topA •DNA replication, recombination, modification 'and repair -1.6 0.0082 PA3014 faoA • Fatty acid and phospholipid metabolism ; 'Amino acid biosynthesis and metabolism -1.9 0.0069 PA3017 ; Hypothetical, unclassified, unknown -3.1 1.00E-05 PA3084 ; Hypothetical, unclassified, unknown -2 0.007 PA3087 ; Hypothetical, unclassified, unknown -1.8 0.0117 PA3107 metZ ; Amino acid biosynthesis and metabolism -1.6 0.0004 PA3108 purF ;^Nucleotide biosynthesis and metabolism ;Amino acid biosynthesis and metabolism -1.7 0.001 PA3116 ; Amino acid biosynthesis and metabolism -1.6 0.0352 PA3134 gltX ; Translation, post-translational modification, degradation -1.5 0.0018 PA3162 rpsA ; Translation, post-translational modification,degradation -1.9 9.11E-05 PA3167 serC ;^Biosynthesis of cofactors, prosthetic groups and carriers ; Amino acid biosynthesis and metabolism -1.5 0.0045 72 PA number Gene Name Description Fold Change P-value PA3169 ; Translation, post-translational modification, degradation -1.5 0.0013 PA3201 ; Membrane proteins -1.7 0.0009 PA3206 ; Two-component regulatory systems -3 0.0005 PA3266 capB ;^Adaptation, Protection ; Transcriptionalregulators -1.8 0.0114 PA3274 ; Hypothetical, unclassified, unknown -1.5 0.0202 PA3278 ;^Hypothetical, unclassified, unknown ; Membrane proteins -5.8 1.72E-08 PA3285 ; Transcriptional regulators -1.6 0.0057 PA3308 hepA ; Transcription, RNA processing and degradation -2.7 2.02E-05 PA3309 ; Hypothetical, unclassified, unknown -4.9 3.99E-07 PA3310 ;^Hypothetical, unclassified, unknown ; Membrane proteins -2.1 0.0002 PA3336 ; Transport of small molecules ; Membrane proteins -4.2 2.69E-07 PA3337 rfaD ; Cell wall / LPS / capsule -2.7 3.27E-05 PA3340 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.5 0.0054 PA3343 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.5 0.0086 PA3377 ;^Hypothetical, unclassified, unknown ; Transport of small molecules -3.8 0.0295 PA3385 ; Hypothetical, unclassified, unknown -1.8 8.15E-05 PA3453 ; Hypothetical, unclassified, unknown -1.8 0.0018 PA3497 ; Hypothetical, unclassified, unknown -1.8 0.0002 PA3519 ; Hypothetical, unclassified, unknown -2.1 0.0039 PA3528 mt ; Transcription, RNA processing and degradation ; DNA replication, recombination, modification and repair -1.5 0.0009 PA3572 ; Hypothetical, unclassified, unknown -1.7 0.0006 PA3573 ; Transport of small molecules ; Adaptation, Protection -2 0.0002 PA3574 ; Transcriptional regulators -4.6 1.76E-06 PA3609 potC • Transport of small molecules ; Membrane 'proteins -1.6 0.0072 PA3612 ; Hypothetical, unclassified, unknown -1.7 0.0045 PA3620 mutS • DNA replication, recombination, modification 'and repair -1.9 4.50E-05 PA3628 ; Putative enzymes -3.4 3.60E-06 PA3629 adhC ; Central intermediary metabolism -2.6 2.59E-06 PA3633 ygbP ; Biosynthesis of cofactors, prosthetic groups and carriers -1.5 0.0376 PA3635 eno ; Carbon compound catabolism ; Energy metabolism ; Translation, post-translational modification, degradation -1.8 5.55E-05 PA3636 kdsA • Cell wall / LPS / capsule ; Carbon compound ' catabolism -1.6 0.0007 PA3643 Ipx8 ; Cell wall / LPS / capsule -1.5 0.0025 PA3644 IpxA ; Cell wall / LPS / capsule -1.6 0.0119 73 PA number Gene Name Description Fold Change P-value PA3646 IpxD ; Cell wall / LPS / capsule -1.7 0.0023 PA3648 ; Transport of small molecules ; Membrane proteins -1.6 0.0011 PA3651 cdsA ; Fatty acid and phospholipid metabolism -1.8 0.0007 PA3653 fir •Translation, post-translational modification,'degradation -1.7 0.0013 PA3654 pyrH ; Nucleotide biosynthesis and metabolism -2.2 0.0012 PA3685 ; Hypothetical, unclassified, unknown -1.5 0.0029 PA3692 ; Transport of small molecules ; Membrane proteins -1.8 0.0478 PA3700 lysS ;^Translation, post-translational modification, degradation ; Amino acid biosynthesis and metabolism -2.7 0.0002 PA3715 ; Hypothetical, unclassified, unknown -2.2 2.93E-05 PA3718 ; Transport of small molecules ; Membrane proteins -1.6 0.0332 PA3725 red • DNA replication, recombination, modification' and repair -1.6 0.0019 PA3742 rpIS ; Translation, post-translational modification,degradation -2 0.0001 PA3743 trmD •Transcription, RNA processing and'degradation -2.2 0.0006 PA3744 rimM •Transcription, RNA processing and'degradation -1.6 0.0003 PA3745 rpsP ;^Translation, post-translational modification, degradation ; DNA replication, recombination, modification and repair -1.9 0.0001 PA3763 purL ; Nucleotide biosynthesis and metabolism -1.6 0.0054 PA3769 guaA • Nucleotide biosynthesis and metabolism ;'Amino acid biosynthesis and metabolism -1.6 0.0017 PA3803 gcpE ; Putative enzymes -1.5 0.0028 PA3804 ; Hypothetical, unclassified, unknown -1.6 0.0015 PA3805 pilF ;^Motility & Attachment ; Protein secretion/export apparatus -1.9 0.0078 PA3806 ; Hypothetical, unclassified, unknown -1.8 8.97E-05 PA3823 tgt ;^Translation, post-translational modification, degradation ; Transcription, RNA processing and degradation -1.8 0.0002 PA3826 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.8 0.0004 PA3827 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.7 0.0004 PA3838 ; Transport of small molecules -1.6 0.002 PA3861 rhl •Transcription, RNA processing and 'degradation -2 6.49E-05 PA3872 nail ; Energy metabolism -3 0.0005 PA3880 ; Hypothetical, unclassified, unknown -2.1 0.0003 PA3901 fecA • Transport of small molecules ; Membrane'proteins -1.5 0.0058 PA3907 ; Hypothetical, unclassified, unknown -2.2 0.0039 PA3908 ; Hypothetical, unclassified, unknown -1.6 0.0008 PA3913 ; Putative enzymes -1.6 0.0383 74 PA number Gene Name Description Fold Change P-value PA3915 moaBl • Biosynthesis of cofactors, prosthetic groups 'and carriers -1.8 0.0138 PA3934 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.5 0.0019 PA4007 proA ;^Biosynthesis of cofactors, prosthetic groups and carriers ; Amino acid biosynthesis and metabolism -1.8 0.0006 PA4049 ; Hypothetical, unclassified, unknown -1.6 0.0171 PA4051 thiL • Biosynthesis of cofactors, prosthetic groups'and carriers -1.6 0.0005 PA4052 nusB • Transcription, RNA processing and ' degradation -1.7 0.0003 PA4067 oprG ; Membrane proteins -5.7 2.47E-08 PA4127 hpcG ; Carbon compound catabolism -3 1.25E-06 PA4128 ; Putative enzymes -1.6 0.0004 PA4129 ; Hypothetical, unclassified, unknown -3.2 4.26E-06 PA4130 ; Central intermediary metabolism -2.1 3.22E-05 PA4134 ; Hypothetical, unclassified, unknown -1.5 0.0013 PA4135 ; Transcriptional regulators -4 1.93E-06 PA4143 ;^Membrane proteins ; Transport of small molecules ; Protein secretion/export apparatus -3 0.0006 PA4153 ; Carbon compound catabolism -2.2 0.0004 PA4155 ; Hypothetical, unclassified, unknown -1.6 0.0301 PA4234 uvrA • DNA replication, recombination, modification 'and repair -1.7 0.0014 PA4235 bfrA • Adaptation, Protection ; Transport of small 'molecules -2.3 0.0002 PA4236 katA ; Adaptation, Protection -1.9 0.0054 PA4242 rpmJ • Translation, post-translational modification, ' degradation -1.6 0.0074 PA4245 rpmD ; Translation, post-translational modification, degradation -2.1 0.0004 PA4247 rpIR ; Translation, post-translational modification, degradation -1.7 0.0005 PA4258 rpIV ; Translation, post-translational modification, degradation -1.5 0.0052 PA4259 rpsS ; Translation, post-translational modification, degradation -1.5 0.0023 PA4260 rpIB ; Translation, post-translational modification, degradation -1.7 0.0034 PA4261 rpIW •Translation, post-translational modification, 'degradation -1.8 0.0209 PA4262 rpID ;^Translation, post-translational modification, degradation ; Transcription, RNA processing and degradation -1.7 0.0004 PA4264 rpsJ ;^Translation, post-translational modification, degradation ; Transcription, RNA processing and degradation -1.6 0.0016 PA4279 ; Hypothetical, unclassified, unknown -1.6 0.0014 PA4333 ; Energy metabolism -1.6 0.0002 PA4334 ; Transport of small molecules ; Membrane proteins -1.6 0.0032 75 PA number Gene Name Description Fold Change P-value PA4345 ; Hypothetical, unclassified, unknown -1.9 0.0102 PA4348 ; Hypothetical, unclassified, unknown -3.4 1.33E-06 PA4350 ; Hypothetical, unclassified, unknown -2.1 0.0006 PA4351 ; Fatty acid and phospholipid metabolism -2.3 1.78E-05 PA4352 ; Hypothetical, unclassified, unknown -2.8 7.44E-06 PA4429 ; Energy metabolism -1.9 0.0012 PA4430 ; Energy metabolism -2 0.0001 PA4431 ; Putative enzymes -1.5 0.0005 PA4432 rpsI ; Translation, post-translational modification, degradation -1.5 0.002 PA4449 hisG ; Amino acid biosynthesis and metabolism -1.5 0.0023 PA4450 murA ; Cell wall / LPS / capsule -1.5 0.0029 PA4479 mreD ;^Cell division ; Cell wall / LPS / capsule -2.9 1.72E-06 PA4480 mreC ;^Cell division ; Cell wall / LPS / capsule -2.8 0.001 PA4481 mreB ;^Cell division ; Cell wall / LPS / capsule -1.5 0.0176 PA4484 gatB ; Translation, post-translational modification, degradation -1.6 0.002 PA4490 ; Hypothetical, unclassified, unknown -1.5 0.01 PA4519 ; Amino acid biosynthesis and metabolism -1.5 0.002 PA4530 ; Hypothetical, unclassified, unknown -1.6 0.005 PA4543 ; Hypothetical, unclassified, unknown -1.8 8.64E-05 PA4563 rpsT • Translation, post-translational modification, 'degradation ; Central intermediary metabolism -2.1 0.0026 PA4571 ; Energy metabolism -1.6 0.0006 PA4577 ; Hypothetical, unclassified, unknown -5.1 3.98E-08 PA4588 gdhA ; Amino acid biosynthesis and metabolism -1.8 0.0002 PA4602 g/yA3 ; Amino acid biosynthesis and metabolism -1.6 0.0005 PA4603 ; Hypothetical, unclassified, unknown -1.9 0.0082 PA4610 ; Hypothetical, unclassified, unknown -2.4 2.34E-05 PA4611 ; Hypothetical, unclassified, unknown -3 1.27E-05 PA4623 ; Hypothetical, unclassified, unknown -1.5 0.002 PA4630 ; Hypothetical, unclassified, unknown -1.7 0.0002 PA4637 ; Hypothetical, unclassified, unknown -1.5 0.0012 PA4644 ; Hypothetical, unclassified, unknown -2.5 5.80E-06 PA4645 ; Nucleotide biosynthesis and metabolism -4.2 2.61E-05 PA4646 upp ; Nucleotide biosynthesis and metabolism -1.7 0.0002 PA4647 uraA • Transport of small molecules ; Membrane ' proteins -1.9 0.0087 PA4648 ; Hypothetical, unclassified, unknown -2.5 0.0001 PA4664 hemK • Biosynthesis of cofactors, prosthetic groups ' and carriers -2.6 0.0001 PA4665 prfA ; Translation, post-translational modification, degradation -1.8 0.0011 PA4672 ; Translation, post-translational modification, degradation -1.7 0.0028 PA4678 riml •Translation, post-translational modification, 'degradation -1.6 0.0028 PA4686 ; Hypothetical, unclassified, unknown -1.5 0.0017 PA4728 folK • Biosynthesis of cofactors, prosthetic groups 'and carriers -1.7 0.0002 76 PA number Gene Name Description Fold Change P-value PA4740 pnp ; Transcription, RNA processing and degradation -1.6 0.0259 PA4769 ; Transcriptional regulators -1.8 7.23E-05 PA4839 speA ; Amino acid biosynthesis and metabolism -1.7 0.0015 PA4840 ; Hypothetical, unclassified, unknown -1.9 5.30E-05 PA4843 ; Two-component regulatory systems ;Transcriptional regulators -1.8 0.0003 PA4851 ; Hypothetical, unclassified, unknown -1.6 0.0209 PA4852 ; Hypothetical, unclassified, unknown -1.6 0.0021 PA4853 fig ;^DNA replication, recombination, modification and repair ;^Transcription, RNA processing and degradation ; Transcriptional regulators -2.7 0.0005 PA4877 ; Hypothetical, unclassified, unknown -1.7 0.0009 PA4897 ; Hypothetical, unclassified, unknown -1.6 0.0258 PA4918 ; Hypothetical, unclassified, unknown -1.8 0.0038 PA4924 ; Membrane proteins -1.8 0.0001 PA4928 ; Hypothetical, unclassified, unknown -1.5 0.0008 PA4931 dnaB • DNA replication, recombination, modification; and repair -1.5 0.0179 PA4935 rpsF ; Translation, post-translational modification,degradation -1.9 0.0005 PA4960 ; Amino acid biosynthesis and metabolism -1.8 0.0121 PA5004 ; Putative enzymes -1.6 0.0005 PA5005 ; Putative enzymes -1.5 0.003 PA5020 ; Putative enzymes -1.6 0.0008 PA5034 hemE • Biosynthesis of cofactors, prosthetic groups ' and carriers -1.5 0.0406 PA5046 ; Central intermediary metabolism -1.5 0.0009 PA5048 ; Putative enzymes -1.6 0.001 PA5051 argS ; Translation, post-translational modification, degradation -1.8 0.0002 PA5110 fbp ; Carbon compound catabolism ; Central intermediary metabolism -2.1 5.53E-05 PA5113 ;^Hypothetical, unclassified, unknown ; Membrane proteins -1.6 0.0211 PA5117 typA ; Adaptation, Protection -1.6 0.0015 PA5118 thiI • Biosynthesis of cofactors, prosthetic groups 'and carriers -1.7 0.0014 PA5131 pgm ; Carbon compound catabolism -1.6 0.0004 PA5139 ; Hypothetical, unclassified, unknown -1.7 0.0007 PA5146 ; Hypothetical, unclassified, unknown -1.5 0.0051 PA5156 ; Hypothetical, unclassified, unknown -1.5 0.0323 PA5170 arcD ; Amino acid biosynthesis and metabolism ; Transport of small molecules ; Membrane proteins -4 1.27E-07 PA5171 arcA ; Amino acid biosynthesis and metabolism -5.9 2.39E-07 PA5172 arcB ; Amino acid biosynthesis and metabolism -2.3 0.0002 PA5192 pckA ; Energy metabolism ; Carbon compound catabolism -1.5 0.0008 PA5207 ; Transport of small molecules ; Membrane proteins -2.3 0.0001 77 PA number Gene Name Description Fold Change P-value PA5208 ; Hypothetical, unclassified, unknown -3.6 2.17E-06 PA5215 gcvT1 ; Amino acid biosynthesis and metabolism ; Central intermediary metabolism -1.6 0.0006 PA5232 ; Hypothetical, unclassified, unknown -2.6 0.0004 PA5286 ; Hypothetical, unclassified, unknown -2.2 0.0034 PA5337 rpoZ ; Transcription, RNA processing and degradation -1.7 0.0005 PA5338 spoT ;^Adaptation, Protection ; Nucleotide biosynthesis and metabolism -1.6 0.0073 PA5408 ; Hypothetical, unclassified, unknown -1.7 0.0002 PA5425 purK ; Nucleotide biosynthesis and metabolism -1.7 0.0003 PA5427 adhA • Carbon compound catabolism ; Energy 'metabolism -1.7 0.0005 PA5429 aspA ; Amino acid biosynthesis and metabolism -1.5 0.001 PA5440 ;^Putative enzymes ; Translation, post-translational modification, degradation -1.7 0.0016 PA5468 ; Transport of small molecules -1.5 0.0045 PA5469 ;^Hypothetical, unclassified, unknown ; Membrane proteins -2 9.34E-05 PA5470 ; Translation, post-translational modification, degradation -2.5 4.93E-06 PA5472 ; Hypothetical, unclassified, unknown -2.1 6.83E-05 PA5475 ; Hypothetical, unclassified, unknown -1.8 0.0003 PA5479 gltP ; Transport of small molecules ; Membraneproteins -1.6 0.0001 PA5480 ; Hypothetical, unclassified, unknown -4.4 7.38E-08 PA5484 ; Two-component regulatory systems -1.5 0.0009 PA5494 ; Hypothetical, unclassified, unknown -1.6 0.0003 PA5504 ; Transport of small molecules ; Membrane proteins -1.7 0.0015 PA5510 ; Transport of small molecules ; Membrane proteins -1.8 0.0133 PA5513 ; Hypothetical, unclassified, unknown -1.5 0.0087 PA5556 atpA ; Energy metabolism -2 0.0064 PA5558 atpF ; Energy metabolism -1.7 0.0002 PA5559 atpE ; Energy metabolism -1.5 0.0012 PA5560 atpB ; Energy metabolism -1.8 6.78E-05 PA5564 gidB ; Cell division -1.7 0.0001 PA5568 ;^Hypothetical, unclassified, unknown ; Membrane proteins -2 0.0002 PA5569 mpA •Translation, post-translational modification, 'degradation -1.9 7.33E-05 PA5570 rpmH • Translation, post-translational modification, 'degradation ; Central intermediary metabolism -1.6 0.0003 78

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