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Type III Secretion System Associated Cytotoxicity of Pseudomonas aeruginosa Strain pa103

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Type III Secretion System Associated Cytotoxicity of Pseudomonas aeruginosa Strain pa103
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WANG, XIAOLING ( Author, Primary )
Copyright Date:
2008

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Bacteria ( jstor )
Cytotoxicity ( jstor )
Genes ( jstor )
Hela cells ( jstor )
Infections ( jstor )
Plasmids ( jstor )
Pseudomonas ( jstor )
Pseudomonas aeruginosa ( jstor )
Secretion ( jstor )
Transposons ( jstor )

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University of Florida
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University of Florida
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Copyright Xiaoling Wang. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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12/31/2008
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71324750 ( OCLC )

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Type III Secretion System Associated Cytotoxicity of Pseudomonas aeruginosa Strain pa103 By XIAOLING WANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Xiaoling Wang

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This thesis is dedicated to my family

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ACKNOWLEDGMENTS I am deeply indebted to my advisor, Dr. Shouguang Jin, for his support and encouragement throughout my two-year thesis research. He has made my graduate research experiences extremely interesting and rewarding. I am very grateful to my other committee members, Dr. Donna H. Duckworth and Dr. Paul A. Gulig, for their advice on my project. I also like to thank all the members in Dr. Jin’s group for their kind help and friendship. I would like to thank Dr. Jaewha Kim and Dr. Lin Zeng and Mr. Weihui Wu for their technical assistance. I would like to thank Dr. Mounia Alaoui El Azher for her valuable advice and also thank Mr. Jinhua Jia and Dr. Wei Lian for their helping hands on a daily basis. In addition, my deep appreciation goes to my parents, Zuoquan Wang and Fangqin Kou, for their love and support. My thanks also go to my dear sister and her fianc for their helpful comments on my thesis. Last but not least, my beloved husband Cheng-feng Tai and my newborn child Grace Tai deserve an award for their constant love and understanding; they are and will always be the sunshine, the rain, and the air in my life to nourish and encourage me to achieve further success in my life. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTER 1 BACKGROUND AND SIGNIFICANCE....................................................................1 Overview of Pseudomonas aeruginosa........................................................................1 Virulent Factors of Pseudomonas aeruginosa..............................................................2 Type III Secretion System in Pseudomonas aeruginosa..............................................2 Cytotoxicity of Pseudomonas aeruginosa....................................................................3 Invasive and Non-invasive Strains of Pseudomonas aeruginosa.................................4 Extra type III Secretion-Dependent Cytotoxicity in P. aeruginosa PA103.................4 Preliminary Data....................................................................................................4 Hypothesis.............................................................................................................5 2 MATERIALS AND METHODS.................................................................................8 Bacterial Strains and Plasmids......................................................................................8 Construction of exoY Deletion Mutant Strains.............................................................8 Cytotoxicity Assays....................................................................................................10 HeLa Cell Lifting Assay.............................................................................................11 Intracellular Survival Selection Assay........................................................................12 Construction of Transposon Tn5 Mutagenesis Library and Selection.......................12 Modification of a Cosmid Vector...............................................................................13 Other Methods............................................................................................................14 3 RESULTS...................................................................................................................18 Comparison of Cytotoxicity among PAKexoSexoT, PA103exoUexoT and PA103exsA.............................................................................................................18 Sequence Analysis of exoY and its Surrounding Genes in Different P. aeruginosa Strains....................................................................................................................19 v

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Cytotoxicity of Triple Mutants PAKexoSexoTexoYd and PA103exoUexoTexoYd....20 Screening Conditions for the Identification of Cytotoxin in PA103 .........................21 Transposon Tn5 Insertion Mutants of PA103exoUexoTexoY with Decreased Cytotoxicity............................................................................................................22 Sequence Analysis of the Genes Affecting Cytotoxicity in PA103exoUexoTexoYd.23 Effect of the Identified Genes on the Expression and Secretion of the Type III Effector Molecules.................................................................................................24 4 DISCUSSION.............................................................................................................39 Different Effect of ExoY in PAK Strain and PA103 Strain.......................................39 Analysis of the Identified Genes.................................................................................40 Group 1 Genes.....................................................................................................40 Group 2 Genes.....................................................................................................41 Group 3 Genes.....................................................................................................42 Future Directions........................................................................................................43 LIST OF REFERENCES...................................................................................................44 BIOGRAPHICAL SKETCH.............................................................................................49 vi

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LIST OF TABLES Table page 2-1 Bacteria strains.........................................................................................................15 2-2 Plasmids...................................................................................................................16 3-1 Sequences of the mutated genes in PA103...............................................................25 vii

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LIST OF FIGURES Figure page 1-1 Intracellular survival of different P. aeruginosa strains in HeLa cells......................6 1-2 Mutant strain PA103exoUexoT causes HeLa cell morphologic changes...................7 2-1 Physical map of the plasmid pXL0314....................................................................17 3-1 Comparison of HeLa cell lifting and death caused by PAK and PA103 derivatives................................................................................................................26 3-2 Gene sequences flanking exoY.................................................................................28 3-3 Comparison of cytotoxicity between mutants PAKexoSexoTexoY and PA103exoUexoTexoY...............................................................................................29 3-4 Kinetics of infection of HeLa cells by different Pseudomonas mutant strains........30 3-5 LDH release assay of HeLa cells infected by P. aeruginosa strains........................31 3-6 HeLa cell lifting assays at different MOIs and time points......................................32 3-7 Transposon mutagenesis delivery vector and insertion model.................................33 3-8 Cytotoxicity assays of the 7 Tn5 insertion mutants.................................................34 3-9 LDH release upon infection by the transposon insertion mutants............................35 3-10 ELISA assays of transposon mutant strains carrying ExoS-FLAG.........................36 3-11 Western blotting of the transposon-insertinal mutants carrying ExoS-FLAG plasmid.....................................................................................................................37 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science TYPE III SECRETION SYSTEM ASSOCIATED CYTOTOXICITY OF Pseudomonas aeruginosa STRAIN PA103 By Xiaoling Wang December 2004 Chair: Shouguang Jin Major Department: Molecular Genetics and Microbiology Pseudomonas aeruginosa is a gram-negative opportunistic pathogen. It inhabits soil, water, plants, animal tissues and various other environmental niches. P. aeruginosa is extremely difficult to eradicate once colonization has been established in immunocompromised patients because of its complicated virulence factors and resistance mechanisms. The most prominent virulence factor is the type III secretion system, through which P. aeruginosa injects effector proteins into the host cells causing various physiological changes. To date, four effector molecules, ExoS, ExoT, ExoU and ExoY, have been identified in P. aeruginosa. Previous experimental data suggested the presence of a yet unidentified type III secretion-dependent cytotoxin(s) in P. aeruginosa strain PA103. To identify the novel type III secretion-dependent bacterial toxins, I constructed a random transposon insertion mutant bank in PA103, established a selection system and identified seven genes that are associated with type III secretion mediated cytotoxicity. These genes encode cytochrome c551 peroxidase precursor (ccpR), ix

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dihydrolipoamide acetyltransferase (aceF), 23S ribosomal RNA (rrn), 50S ribosomal protein L19 (rplS), dihydrodipicolinate reductase (dapB), a hypothetical protein (PA3730) and transcriptional regulator PsrA (psrA). Mutation in ccpR, rplS or psrA had no effect on the synthesis or secretion of type III effector molecule ExoS, while mutation in the rrn or PA3730 only affected secretion but not expression, and mutation in the dapB gene resulted in a constitutive expression and secretion of the ExoS. Further characterization of those genes is needed to clarify their roles in type III dependent cytotoxicity. x

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CHAPTER 1 BACKGROUND AND SIGNIFICANCE Overview of Pseudomonas aeruginosa Pseudomonas aeruginosa is an opportunistic pathogen that inhabits soil, water, plants, animal tissues and various other environmental niches. It is a gram-negative rod-shaped bacterium. It can grow at 42oC, although the optimal growth temperature is 37oC (Ojeniyi, 1994). Pseudomonas aeruginosa cells have inner and outer membranes. The inner-cytoplasmic membrane covers the cytosol of the bacterium and consists of a phospholipid bilayer and proteins (Ojeniyi, 1994). The outer membrane is also a phospholipid bilayer with lipopolysaccharide (LPS) on the outside. Lipopolysaccharide is one of the cell-associated virulence factors in P. aeruginosa as well as in other gram-negative bacterial pathogens (Salyers et al,. 1994). The periplasmic space between the inner and outer membranes contains oligosaccharides, peptidoglycan and proteins (Ojeniyi, 1994). Patients with cystic fibrosis, burns or other wounds, and various immunocompromised conditions are at high risk of P. aeruginosa infections. In cystic fibrosis patients P. aeruginosa is rarely eradicated once colonization has been established (Banwart et al., 2002; Feltman et al., 2001; Garau & Gomez, 2003; Kerem et al., 1990; Moss et al., 2001; Roy-Burman et al., 2001). This is due to its complicated resistance mechanisms against both the host immune system and antibiotic chemotherapy and its ability to form biofilms in the host (Garau & Gomez, 2003; Hogardt et al., 2004; Lakkis & Fleiszig, 2001). Kerem et al. (1990) reported that P. aeruginosa can be cultured from up to 80% of cystic fibrosis patients’ sputum samples. Much of the morbidity and 1

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2 mortality of cystic fibrosis patients is initiated by acute and chronic P. aeruginosa infections because the P. aeruginosa can easily acquire resistance to antibiotics and produces complicated virulence factors. Many secreted toxins of the P. aeruginosa cause profound host tissue damage and functional interference. There is a great need for the development of new strategies for the control of this pathogen. Virulence Factors of P. aeruginosa A number of virulence factors contribute to P. aeruginosa pathogenesis, such as LPS, exopolysaccharide (alginate), pili, flagella, alkaline protease, elastase, phospholipase, pyocyanin, exotoxin A, exoenzymes and type III secretion system (TTSS) (Frank, 1997; Frank & Storey, 1994; Fraylick et al., 2002; Ha et al., 2003; Hakansson et al., 1996; Mecsas & Strauss, 1996; Salyers & Whitt, 1994; Wolfgang et al., 2003). These virulence factors are either secreted or cell-associated. The most prominent among these is the type III secretion system. Type III Secretion System in P. aeruginosa Type III secretion systems exist in many gram-negative pathogens, including Salmonella, Yersinia, Escherichia, Pseudomonas, Shigella, and the plant pathogen Pseudomonas syringae (Galan & Collmer, 1999). This protein secretion system is sec-independent, in which effectors are secreted without cleavage of a signal peptide, and only needs one step to cause proteins to cross both inner and outer membranes (Winstanley & Hart, 2001). The type III secretion system consists of over 20 genes, coding for secretion, translocation, and regulatory functions (Nanao et al., 2003). The secretion apparatus is encoded by pcr and psc operon genes. The translocation machinery of the system is encoded by the popB and popD genes. PopB and PopD have pore-forming activities on the eukaryotic cell membrane, and the effector proteins can be

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3 translocated into host cells through the pores (Dachex et al., 2001; Schoehn et al., 2003). ExsA and its interacting proteins play regulatory roles (Feltman et al., 2001). ExsA is a transcriptional activator which coordinately regulates the expression of exoenzymes under low calcium or upon contact with host cells (Ha & Jin, 2001). The bacterial effector proteins are delivered into host cells where they modulate host cellular functions. Four effector proteins have been identified, including ExoS, ExoT, ExoU, and ExoY, all of which have been shown to be cytotoxic to the eukaryotic cells (Feltman et al., 2001). Cytotoxicity of P. aeruginosa Unlike components of the type III secretion machinery, genes coding for effector proteins are distributed throughout the chromosome of P. aeruginosa strains (Feltman et al., 2001) ( http://www.pseudomonas.com , 2004). The prevalence of the type III secretion effectors varies among different P. aeruginosa strains: 72% of P. aeruginosa strains contained the exoS gene, while 28% contained the exoU gene. Interestingly, P. aeruginosa contained either exoS or exoU but not both; 89% of P. aeruginosa contained the exoY gene, and all of the P. aeruginosa isolates contained the exoT gene (Feltman et al., 2001). The effector proteins ExoS and ExoT, whose molecular masses are 49kDa and 53kDa respectively, both have ADP-ribosyltransferase activity and GTPase activating protein (GAP) activity (Kulich et al., 1993; McGuffie et al., 1998; Pederson et al., 1999). They share 75% amino acid identity; however ExoT has only 0.2% of the ADP-ribosyltransferase activity of the ExoS in vitro (Cowell et al., 2000; Yahr et al., 1996a). Besides the cytotoxic activities of the ExoS and ExoT, these two effectors can also confer anti-internalization function to the host cells, inhibiting the uptake of P. aeruginosa by epithelial cells. The 42-kDa ExoY protein has adenylate cyclase activity. Expression of exoY causes host cell rounding followed by increased cAMP level, which

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4 is stimulated by eukaryotic factors (Yahr et al., 1998). All three cytotoxins cause morphologic changes in eukaryotic cells, while ExoS can trigger effective host cell death by apoptosis (Vallis et al., 1999a). An acute cytotoxin ExoU, 74 kDa in size, has the most acute cytotoxicity and is able to break the host cell monolayer and cause epithelial and macrophage cell lysis during a short infection period (Finck-Barbancon et al., 1997; Phillips et al., 2003). Recently, ExoU was shown to have a lipase activity which is essential for its cytotoxicity (Philips et al., 2003). Invasive and Non-invasive Strains in P. aeruginosa There are two types of P. aeruginosa strains, invasive and non-invasive (cytotoxic strain), depending on the way they interact with non-phagocytic epithelial cells. These two types of strains contain different sets of type III secreted cytotoxins (Frithz-Lindsten et al., 1997; Yahr et al., 1996b). Invasive strains, such as PAK, encode ExoS, ExoT, and ExoY (Feltman et al., 2001; Ha & Jin, 2001), while cytotoxic strains, such as PA103 and PA14, encode ExoU, ExoT, and ExoY (Miyata et al., 2003; Yahr et al., 1998). Additional Type III Secretion-Dependent Cytotoxicity in P. aeruginosa Strain PA103 Preliminary Data Dr. Unhwan Ha of our laboratory discovered that although cytotoxic P. aeruginosa PA103 cannot invade epithelial cells, the exoU and exoT mutant of PA103 (named PA103exoUexoT::Tc) has the same invasion rate as the exoS and exoT mutant of invasive PAK (PAKexoS::exoT::Gm) (Ha & Jin, 2001), suggesting that it was the acute cytotoxic effect that interfered with the invasion assays. Interestingly, as shown in Figure 1-1, when the mutants were used to infect HeLa S3 epithelial cells, the intracellular survival rate of PA103exoUexoT was significantly lower than that of PAKexoSexoT (Ha

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5 & Jin, 2001). However, when a repressor of the type III system (PtrA) was overexpressed in the PA103exoUexoT background, the intracellular survival of the resulting mutant strain was increased (Ha and Jin, manuscript in preparation). Dr. Ha suggested that strain PA103exoUexoT harbors an additional type III dependent cytotoxin(s) (Ha and Jin, manuscript in preparation). During infection of tissue culture cells, strain PA103exoUexoT caused apparent epithelial cell morphological change by 8 hours post-infection while PAKexoSexoT failed to do so (Figure 1-2). In support of the hypothesis that this novel cytotoxicity is dependent upon the type III secretion system, a mutation in the type III master regulatory gene exsA completely eliminated the cytotoxicity of the strain PA103. Similar results have been reported in another non-invasive strain PA14 which was isolated from a human burn wound patient (Miyata et al., 2003). In this report, the moth caterpillar Galleria mellonella infection model was used to study the type III secretion system. Strain PA14 was used to infect G. mellonella larvae, and the LD50 was measured. It was reported that PA14exoT exoU and PA14exoT exoU exoY mutants had about 165-fold lower LD50 than that of PA14pscD which is completely defective of the type III secretion machinery, suggesting the presence of an additional type III dependent cytotoxin(s) in strain PA14 as well. Hypothesis Based on the above data, I hypothesized that PA103exoUexoT remains cytotoxic due to the presence of an additional type III associated toxin(s). In this project, my goal was to find the novel, unidentified virulence factor(s) associated with the type III secretion system in cytotoxic strain PA103.

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6 1.E+001.E+011.E+021.E+031.E+041.E+051.E+061.E+07261224364860Incubation time (hrs)Number of baterial cells (cfu) exoST exoUT Figure 1-1.Intracellular survival of different P. aeruginosa strains in HeLa cells. Bacteria were used to infect HeLa cells for 2 hours and washed away by PBS three times. HeLa cells were incubated at 37C in 5% CO2 with 400 g/ml amikacin to kill the extracellular bacteria for 2, 6, 12, 24, 36, 48 and 60 hours. After lysing the HeLa cells, the number of bacteria which survived in HeLa cells was measured by colony count. , PAKexoSexoT mutant strain; , PA103exoUexoT mutant strain. The error bars indicate standard deviation. (Ha & Jin, 2001)

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7 Figure 1-2.Mutant strain PA103exoUexoT causes HeLa cell morphologic changes. There are more HeLa cells killed by PA103exoUexoT infection than PAKexoSexoT after 8 hours post-infection. Uninfected cells were used as negative control, and PAKexoSexoT indicates double mutant of exoS and exoT in PAK background, while PA103exoUexoT indicates exoU and exoT double mutant in PA103 background. (Ha & Jin, manuscript in preparation).

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CHAPTER 2 MATERIALS AND METHODS Bacterial Strains and Plasmids The bacterial strains and plasmids used in this study are listed in Table 2-1 and Table 2-2. Escherichia coli strains were cultured in Luria (L)-agar or L-both at 37C with appropriate antibiotics. P. aeruginosa strains were grown in L-agar or L-broth at 37C in most cases and at 42C when used for transposon mutagenesis. Because the restriction system of P. aeruginosa is shut down at 42C, the transposon-containing plasmid conjugated into P. aeruginosa will not be digested, thus higher transposon insertion frequency can be obtained. Construction of a transposon mutagenesis library of P. aeruginosa strains has been described later in this chapter. Antibiotics were used at the final concentrations of: ampicillin (Ap) 100 g/ml, gentamicin (Gm) 50 g/ml, kanamycin (Ka) 20 g/ml, spectinomycin (Sp) 50 g/ml, streptomycin (Sm) 25 g/ml, tetracycline (Tc) 10 g/ml for E. coli strains; and carbenicillin (Cb) 150 g/ml, gentamicin 200 g/ml, spectinomycin 200 g/ml, streptomycin 200 g/ml, tetracycline 100 g/ml, neomycin (Neo) 300 g/ml, Amikacin (Ak) 400 g/ml for P. aeruginosa. Construction of exoY Deletion Mutant Strains To amplify exoY genes from PAK and PA103 strains, polymerase chain reaction (PCR) primers were designed based on the P. aeruginosa PA01 genome sequence (www.pseudomonas.com). The oligonucleotides used to clone the exoY gene were 5’-exoY ’-CGGATGGCGGAATATGCGATGAGCCTCGACT-3’” and 3’-exoY ’-CGGCATTGAGCACGTTGAGCACGGTCTCAC-3’”. DNA fragments containing the 8

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9 exoY gene from PAK and PA103 were cloned after PCR amplification, using a TA-cloning vector, pCR2.1-TOPO (Invitrogen, Carlsbad, CA); the resulting plasmid was named pXL0307. To make an exoY deletion construct, a 5.4-kb SacII-SalI fragment of pXL0307 was isolated and ligated with a 1.6-kb SmaI fragment of pTZ18Tc-FRT to insert Flp recombinase target cassettes (FRT), resulting in plasmid pXL0314 (tetracycline resistance), as shown in Figure 2-1. FRT cassettes contained antibiotic markers (i.e., gentamicin or tetracycline resistance), making it easier to select for a deletion mutant (Hoang et al., 1998). The plasmid pXL0323 (Gentamicin resistance) was made from pTZ18Gm-FRT ligated with pXL0307, and used for exoY deletion mutant in PA103exoUexoT background. The suicide delivery plasmid pXL0315 with tetracycline resistance marker and pXL0324 with gentamicin resistance marker were used to make exoY deletion in strains PAKexoS:: exoT::Gm, and PA103exoUexoT::Tc, respectively, obtaining triple mutants PAKexoS:: exoT::GmexoY (PAKexoSexoTexoY) and PA103exoUexoT::TcexoY (PA103exoUexoTexoY). However, tests on these new mutants indicated that both PAKexoSexoTexoY and PA103exoUexoTexoY had lost their twitching motility, which makes them unsuitable for our purpose, since type IV pili as well as twitching motility might be required for the full function of type III secretion system in P. aeruginosa. This phenomenon has been observed in the study of other P. aeruginosa mutants constructed similarly by using the Flp recombinase-expressing plasmid to excise the FRT cassette. It is possible that certain twitching related genes harbor Flp recombinase recognition sequences and got disrupted. However, the insertional mutants with FRT cassettes intact, intermediate strains obtained prior to the excision of the FRT cassette, still maintained normal level of twitching motility.

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10 Therefore, mutants PAKexoS::exoT::GmexoY::FRT(Tc) (PAKexoSexoTexoYd) and PA103exoUexoT::TcexoY::FRT(Gm) (PA103exoUexoTexoYd), which were PAKexoSexoTexoY and PA103exoUexoTexoY mutants containing FRT cassette, were used in this study. Cytotoxicity Assays Two cytotoxicity assays were used to assess the cytotoxic effect exerted in the host cells by bacteria. The crystal violet staining can easily assess the degree of host cell lifting caused by P. aeruginosa infection, while LDH cytotoxicity assay measures the amount of LDH (Lactate dehydrogenase) released from lysed host cells. Crystal violet can stain the eukaryotic cells and is soluble in several solvents (e.g., ethanol) (Merritt et al., 1998). This assay can be used to measure HeLa cell lifting by measuring the amount of HeLa cells that are attached to the bottom of the culture plates at the end of the infection. Briefly, at several hours post infection with P. aeruginosa, HeLa cells in 24-well cell culture plate were washed twice with pre-warmed phosphate buffered saline (PBS), followed by staining with 0.1% crystal violet. After a brief wash with water, 200l per well of 95% ethanol was used to dissolve the crystal violet stain, and the absorption at 590nm was measured to quantify the amount of crystal violet dye which correlates with the number of HeLa cells attached to the well. Lactate dehydrogenase (LDH) release assay can quantitatively measure the cytotoxicity. LDH is a stable cytosolic enzyme of eukaryotic cells that is released during cell lysis. The activity of released LDH was measured at 6, 18, 24, 30, and 40 hours post-infection and calculated using the following formula according to the instruction provided with the CytoTox96 Non-radioactive Cytotoxicity Assay Kit (Promega, Madison, WI):

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11 % Cytotoxicity = (Experimental LDH – Spontaneous LDH from HeLa and bacteria) / (Maximum LDH – Spontaneous LDH from HeLa) 100 The crystal violet assay is quicker and easier to perform compared to the LDH cytotoxicity assay and is very sensitive in assessing the effect of bacterial type III dependent cytotoxicity. However, it is not the ideal tool to measure the absolute cytotoxicity, as both the lifted yet viable host cells and dead host cells will be washed away during the process. Therefore, we used both crystal violet staining and the LDH cytotoxicity assays to accurately assess the cytotoxicity caused by the P. aeruginosa infections. HeLa Cell Lifting Assay A total of 5 104 HeLa S3 epithelial cells were seeded on a 24 well culture plate (Falcon), while 2.5 104 HeLa cells were used in a 48-well cell culture plate, 0.5 ml of Dulbecco’s Modified Eagle Media (DMEM) containing 5% fetal bovine serum (FBS) was added into each well and incubated at 37C in 5% CO2 for 24 hours. After washing twice with warm PBS, 0.5 ml of DMEM containing 5% FBS was added to the HeLa cells, followed by the addition of 0.3 ml for the 24-well plate or 0.2 ml for the 48-well plate of bacterial suspension in DMEM. This resulted in an MOI of 10 for PAK and its derivatives or MOI of 100 for PA103 and its derivatives, because PA103 had 10-fold less binding due to its motility defect (Ha & Jin, 2001). The infected HeLa cells were incubated at 37C in 5% CO2 for 18 hours. After three washes with PBS, HeLa cells were stained by 0.1% Crystal Violet solution to estimate the number of attached HeLa cells on the bottom of culture plate. LDH cytotoxicity assays were also used to determine the cytotoxicity of different bacterial strains.

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12 Intracellular Survival Selection Assay A total of 5 104 HeLa S3 epithelial cells in 0.5 ml of DMEM containing 5% FBS were seeded into each well of the 24-well plates and incubated at 37C in 5% of CO2 for 24 hours. After two washes with warmed PBS, 0.25 ml of DMEM containing 5% FBS was added to the HeLa cells, followed by the addition of 0.25 ml of bacterial suspension in DMEM. This yielded a MOI of 100 for PA103 mutant strains. The infected HeLa cells were incubated at 37C in 5% CO2 for 2 hours. After three washes with PBS, 0.5 ml of DMEM containing 5% FBS and 400 g/ml of amikacin was added and incubated at 37C in 5% CO2 for 36 hours post-infection. After washing the HeLa cells three times with warm PBS, trypsin was used to detach the infected HeLa cells from the culture plates, then 1 ml of DMEM with 5% FBS was added to inhibit the trypsin reaction. The HeLa cells were spun down (1 min, 13000 rpm), re suspended in a 0.25% (w/v) Triton-X100 and plated on L-agar plates containing appropriate antibiotics. Construction of a Tn5 Transposon Mutant Library Recipient P. aeruginosa strain, PA103exoUexoTexoYd, which is PA103exoUexoTexoY mutant containing FRT cassette and with normal twitching motility, and PA103exoUexoT were cultured over-night at 42C in LB with appropriate antibiotics. The restriction system of P. aeruginosa strain was turned off by the increased temperature, so that the transposon-containing plasmids conjugated into P. aeruginosa become much more stable, thus the transposon insertion frequency is higher. When the OD600 value of P. aeruginosa reached around 2.0 to 3.6 and the E. coli strain containing the Tn5 plasmid (Larsen et al., 2002) reached 1.0, the bacterial cultures were centrifuged for 1 minute at the maximum speed, and resuspended in 3 ml LB medium to wash away the antibiotics. About 108 109 E. coli and 108 P. aeruginosa cells were mixed in a sterile

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13 tube, and the mixture was filtered through a filter membrane. The ratio of E. coli: P. aeruginosa was about 2:1. The filter membrane was placed on a nutrient agar plate and incubated at 37C for 7 hours. It was then placed into a sterile tube, mixed with one milliliter LB and vortexed to wash off the bacteria from the membrane. Serial dilutions were made, and the frequency of transposition was measured by plate counting Tn5-containing P. aeruginosa on LB plates containing proper antibiotics. LB containing antibiotics was subsequently added to the remaining bacteria, cultured for 4 hours for amplification and the bacteria were then collected for future use. In order to select mutants that can survive within HeLa cells, the Tn5 insertion mutants were used to infect the HeLa cells which had been grown in a 24-well plate for 24 hours. These were then incubated at 37oC in 5% CO2 and incubated for 2 hours. After washing twice with warm PBS, 400g/ml amikacin with DMEM containing 5% FBS was added into the wells with HeLa cells containing intracellular bacteria. The mixture was incubated for 36 hours post-infection. PBS was used to wash the plate twice, trypsin was then added to lift the HeLa cells and subsequently DMEM containing 5% FBS was added to inhibit the trypsin. The mixture was then spun down at top speed for 1 minute. LB containing 0.25% Triton-X100 was then added to lyse the HeLa cells. The lysates were serially diluted and spread on the LB agar plates containing the proper antibiotics. Bacterial colonies are pooled together and were used to repeat the above process to isolate less cytotoxic mutants. Modification of Cosmid Vector There are many ways to look for the cytotoxin genes based on the whole bacterial genome, two among which are easier and faster. One is by screening a transposon

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14 insertion library and the other by complementation with a cosmid library. Both have been carried out in parallel. The commercial cosmid vector SuperCos1 was available in our laboratory (Stratagene), but it can not replicate within P. aeruginosa strains because of its replication origin. Therefore, the RK2 origin which can promote plasmid replication in P. aeruginosa was isolated from a broad-host-range plasmid pTR102 by EcoR1 and Nru1 enzyme digestion and introduced into the SuperCos1 vector, resulting in pSCoriV which is now able to replicate in Pseudomonas (Weinstein et al., 1992). Other Methods Standard methods were used for preparing plasmid DNA, restriction enzyme digestion and molecular cloning (Sambrook et al., 1989). DNA restriction enzyme sites and DNA clone maps were generated using Clone Manager for Windows (version 4.1). Primer design was done with Primer Designer for Windows (version 3.0). DNA sequence analysis was by PCR-mediated Taq DiDeoxy Terminator Cycle sequence using Applied Biosystems model 373A DNA sequencer. Enzyme-linked Immunosorbent Assays (ELISA) followed standard protocol. Western blot used a monoclonal antibody against FLAG peptide from Sigma and an anti-mouse immunoglobulin conjugated with horseradish peroxidase together with the ECL plus labeling and detection kit from Amersham.

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15 Table 2-1. Bacterial strains Strains Relevant genotype or phenotypea Reference or source E. coli strains E. coli DH5 F-80 lacZ M15 endA1 recA1 hsdR17 supE44 thi-1 relA1 (lacZYA-argF) gyrA96 deoR BRL E. coli DH5/pir 80dlacZM15(lacZYA-argF)U169 recA1 hsdR17 deoR thi-1 supE44 gyrA96 relA1/pir (Larsen et al., 2002) E. coli BW20767 RP4-2-Tc::Mu-1 Kan::Tn7 integrant leu-63::IS10 recA1 zbf-5 creB510 hsdR17 endA1 thi uidA (Mlu1::pir+) (Larsen et al., 2002) P. aeruginosa strains PAK Clinical isolate of wild-type invasive strain (Davis & Mingioli, 1950) PA103 Clinical isolate of wild-type cytotoxic strain (Liu, 1966) PAKexsA PAK with chromosomal disruption of the exsA locus; Spr Smr (Frank et al., 1994) PA103exsA PA103 with chromosomal disruption of the exsA locus; Spr Smr S. Jin PAKexoSexoT PAK with chromosomal disruption of the exoS and exoT loci; Spr Smr Gmr (Kaufman et al., 2000) PAKexoSexoTexoYd PAK with chromosomal disruption of the exoS and exoT loci and exoY; Spr Smr Gmr Tcr This study PA103exoUexoT PA103 with chromosomal disruption of exoU and exoT locus; Tcr (Vallis et al., 1999b) PA103exoUexoTexoYd PA103 with chromosomal disruption of the exoU, exoT and exoY; Tcr Gmr This study a Phenotypes: Apr, ampicillin resistance marker; Tcr, tetracycline resistance marker; Kmr, kanamycin resistance marker; Gmr, gentamicin resistance marker; Spr, spectinomycin resistance marker; Smr, streptomycin resistance marker; Neor, neomycin resistance marker

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16 Table 2-2. Plasmids Plasmids Relevant genotype or phenotypea Reference or source pCR2.1-TOPO PCR cloning vector; Apr Kmr Invitrogen pTR102 the broad-host-range plasmid has origin RK2, Tcr (Weinstein et al., 1992) pEX18Tc Vector for a broad host range recombination by site specific excision; Sucs, Tcr (Hoang et al., 1998) pEX18Ap Vector for a broad host range recombination by site specific excision; Sucs, Apr (Hoang et al., 1998) SuperCos1 Cosmid vector; Apr Neor Stratagene SCoriV Modified Cosmid vector containing RK2 replication origin; Apr This study pXL0307 exoY and its flanking genes of PAK in pCR2.1; Apr This study pXL0311 exoY and its flanking genes of PA01 in pCR2.1; Apr This study pXL0312 Deletion of exoY from pXL0307, Apr This study pXL0314 Insertion FRT (Tcr) into exoY from pXL0312, Apr Tcr This study pXL0315 pEX18AP with a fragment containing deletion of exoY from pXL0314 This study pXL0323 Insertion FRT (Gmr) into exoY from pXL0312, Apr Gmr This study pXL0324 pEX18AP with a fragment containing deletion of exoY from pXL0323 This study pPS880 FRT cassette vector contained a -lactamase (bla) gene and pMB-derived origin of replication, Tcr (Hoang et al., 1998) pTZ18Tc-FRT pTZ18R vector contain FRT cassette and tetracycline resistance markers (Tcr), multiple cloning sites : BamH1-Sma1-Kpn1-Sac1 S. Jin pFLP2 Flp recombinase-expression plasmid, Apr (Hoang et al., 1998) pHW0029 PAK ExoS-FLAG in pucp18 vector, Apr (Ha & Jin, 2001) a Phenotypes: Apr, ampicillin resistance marker; Kmr, kanamycin resistance marker; Gmr, gentamicin resistance marker; Spr, spectinomycin resistance marker; Smr, streptomycin resistance marker; Tcr, tetracycline resistance marker. Neor, neomycin resistance marker; Sucs, Sucrose sensitive encoded in sacB.

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17 TcFRTFRTSalISacIIIIATPexoYPA2189pUC oriampknfloriPA2193pXL0314 7177bps Figure 2-1. Physical map of the plasmid pXL0314. Only selected restriction enzyme sites are shown. To make deletion of gene exoY, the SacII-SalI fragment of exoY in pXL0307 was replaced with a 1.6-kb SmaI fragment of pTZ18Tc-FRT, containing a Tc resistance marker as well as Flp recombinase target cassettes (FRT) (Hoang et al., 1998). The resultant plasmid, named pXL0314, was used to further construct the suicide-delivery plasmid.

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CHAPTER 3 RESULTS Comparison of Cytotoxicity among PAKexoSexoT, PA103exoUexoT and PA103exsA To compare the cytotoxicity between mutant strains PA103exoUexoT and PAKexoSexoT, the HeLa cell lifting assay measuring cytotoxicity of the mutant bacteria was applied. Overnight cultures of the bacteria were used to infect HeLa cells which were grown 24 hours in a 24-well cell culture plate, reaching 50-60% confluency. After 2 hours of infection, the cells were washed twice with pre-warmed PBS, added DMEM containing 5% FBS plus 400 g/ml amikacin and incubated at 37C under 5% CO2. The cells were then washed and stained after 3, 24, 48, 60 hours post-infection. HeLa cells infected by PA103exoUexoT were killed and detached from the culture plate significantly more than those infected by PAKexoSexoT mutant strain. Figure 3-1 (C) shows the difference in virulence between PAKexoSexoT and PA103exoUexoT by 16 hours post-infection either with or without amikacin. As shown in Figure 3-1 (A, B), PA103exoUexoT causes higher cell lifting than PAKexoSexoT, while the PAK wild type caused almost complete cell lifting (positive control). On the other hand, when the exsA gene was knocked out in the wild type PA103, most of the cytotoxicity disappeared like PAKexsA mutants, suggesting the presence of a novel ExsA controlled cytotoxin in the PA103 background. 18

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19 Sequence Analysis of exoY and its Surrounding Genes in Different P. aeruginosa Strains The ExsA protein is a transcriptional activator of the type III secretion system, thus the exsA mutant is defective in the secretion of the type III secretion system effectors. It is therefore conceivable that the exsA mutant of P. aeruginosa is missing of all type III secretion dependent cytotoxicity. Indeed, as shown in Figure 3-1 (C), when PAKexoSexoT was compared to PAKexsA or PA103exoUexoT compared to PA103exsA, there was about 2-fold more HeLa cell lifting caused by the double mutant strains of the PAK and PA103 than the corresponding exsA mutants. This suggested that there are other ExsA-dependent effector(s) in PAK and PA103 besides the ExoS, ExoT and ExoU. The fourth effector, ExoY, is known to be present in both PAK and PA103 strains (Miyata et al., 2003; Vallis et al., 1999a; Yahr et al., 1998). In order to test the role of ExoY in the cytotoxicity of the above double mutants, we further generated exoY deletion mutants in the backgrounds of PAKexoSexoT and PA103exoUexoT. Based on the PAO1 genome ( http://www.pseudomonas.com , 2004), a pair of oligonucleotides, 5’-exoY ’-CGG ATG GCG GAA TAT GCG ATG AGC CTC GAC T-3’” and 3’-exoY ’-CGG CAT TGA GCA CGT TGA GCA CGG TCT CAC-3’”, were designed to amplify from PA2189 to PA2193 where exoY is encoded by PA2191. Interestingly, as shown in Figure 3-2, the PCR products from PAK and PA103 were shorter than that of PA01. After cloning the PCR products from PAK and PA103 into pCR2.1-TOPO vector, the resulting plasmids pXL0307 and pXL0311, respectively, were subjected to sequence analysis. The sequence analysis showed that the PA01 contained two genes flanking the exoY gene, PA2190 and PA2192 ( http://www.pseudomonas.com , 2004), while PAK and PA103 did not (Figure 3-2B). The sequences of the exoY genes were the same in PAK,

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20 PA103, and PA01. Gene exoY is encoded on a 1.1kb nucleotide stretch containing an ATP binding site with and phosphates of the bound nucleotide contacting the site (conserved region II) (Yahr et al., 1998). To generate exoY deletions in PAK and PA103, 960bp nucleotides from the ATP binding site to the conserved region II were deleted from the corresponding exoY clones by SalI SacII enzyme digestion (Figure 2-1). Cytotoxicity of Triple Mutants PAKexoSexoTexoY and PA103exoUexoTexoY To test whether ExoY is the “unknown” cytotoxic effector, cytotoxicity assays were run to compare the cytotoxicity of the triple mutants PAKexoSexoTexoY and PA103exoUexoTexoY. As seen in Figure 3-3, PA103exoUexoT caused more HeLa cell lifting than PAKexoSexoTexoY and PA103exoUexoTexoY whose cytotoxicities were similar to those of PAKexsA and PA103exsA, respectively, regardless of the addition of amikacin. However, further analyses have found that the newly generated exoY mutants had twitching motility defects. The loss of twitching motility was also seen in other P. aeruginosa mutant strains when using the FRT cassette in our laboratory. It seems the Flp recombinase-expressing plasmid, which excises the FRT cassette from the mutant gene in the last step of the mutagenesis, causes the twitching motility defect. Since the twitching motility of the mutant PAKexoSexoTexoY containing FRT cassette (before the introduction of Flp recombinase-expressing plasmid), named PAKexoSexoTexoYd (PAKexoS::exoT::GmexoY::FRT(Tc)), as well as the mutant strain PA103exoUexoTexoYd (PA103exoUexoT::TcexoY::FRT(Gm)) displayed normal twitching motility, the cytotoxicities of these two strains were then measured. As shown in Figure 3-4, PAKexoSexoTexoYd did lose the cytotoxicity, while PA103exoUexoTexoYd still had a similar level of cytotoxicity as PA103exoUexoT. Both PA103exoUexoTexoYd and PA103exoUexoT caused more HeLa cell damage than

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21 PAKexoSexoT. To accurately quantify the cytotoxicity of these strains, the LDH cytotoxicity assay was used. As shown in Figure 3-5, PAKexoSexoTexoYd had a reduced cytotoxicity compared to PAKexoSexoT. However, PA103exoUexoTexoYd was as cytotoxic as PA103exoUexoT, and both were over 6-fold more cytotoxic than PA103exsA, suggesting the presence of additional ExsA-dependent, thus type III secretion dependent cytotoxin(s). Screening Conditions for the Identification of Cytotoxin in PA103 To find the genes responsible for the type III-dependent cytotoxicity in strain PA103exoUexoTexoY, it was necessary to have a fast and easy screening method. As mentioned before, the lifting assay has the advantage of being easy and fast to perform, and also cheaper compared to other methods. In order to choose an optimal condition for the lifting assay, infection with P. aeruginosa strains at various MOI and time points were tested. As shown in Figure 3-6, four different MOI were used: 2, 5, 10, 15 for PAK and its derivatives while 20, 50, 100, and 150 for PA103 and its derivatives, respectively. The reason for using different MOI for PAK and PA103 strains was that PA103 has about 10-fold less binding capacity than PAK, due to the lack of flagella (Ha & Jin, 2001). After bacterial infection of the HeLa cells, different time points were chosen to measure the attached HeLa cells as described in the Materials and Methods (Chapter 2). From the experimental results as shown in Fig. 3-6, the order of severity of HeLa cell lifting and death caused by P. aeruginosa strains was: PA103exoUexoTexoYd = PA103exoUexoT > PA103exsA > PAKexoSexoTexoYd. At the MOI of 10 for PAK and MOI of 100 for PA103, the differences in cytotoxicity were more obvious and stable than that of any other MOIs. Also, the infection time from 18 to 24 hours showed the most obvious difference in cytotoxicity among different strains. Therefore, an infection time of 18

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22 hours to 24 hours at MOI 10 for PAK derivatives and 100 for PA103 derivatives were chosen for further screening analysis. Transposon Tn5 Insertion Mutants of PA103exoUexoTexoYd with Decreased cytotoxicity Recipient strain PA103exoUexoTexoYd was mixed at a ratio of 2:1 with donor strain E. coli BW20767 containing transposon delivery vector pRL27 which encodes a Tn5 transposase and a mini-Tn5 element encoding the kanamycin and neomycin resistance gene (aph) (Larsen et al., 2002). Figure 3-7 shows the plasmid with transposon and the model of transposon inserted into the chromosomal DNA. In my hand, around 109 recipient bacteria produce 104 – 105 transposon insertion mutants after 7 hours conjugation with donor BW20767. Therefore, the transposon insertion frequency was approximately 1-4 to 1-5 per recipient cell. This frequency is enough to cover the whole P. aeruginosa genome which is about 6.3 Mbp. Theoretically, about 34 random insertion mutants would be a near-saturation library for P. aeruginosa (Jacobs et al., 2003). In our experiment, about 16 insertion mutants were obtained by combining transposon mutants derived from four mating experiments, and this provided about 30-fold coverage of predicted genes. To identify mutants with less cytotoxicity, an intracellular survival assay was used to screen the transposon insertion mutants, as described in the Materials and Methods (Chapter 2). Basically, after bacteria contact and invade the HeLa cells, cytotoxic bacteria will kill and lyse the HeLa cells and be released into the culture medium. After 400g amikacin per ml was added into the culture medium, the extracellular bacteria would be killed and only bacteria which survive inside the HeLa cell (i.e., do not cause cytotoxicity) will survive. An MOI of 100 was chosen in this selection assay, so that each HeLa cell can maximally be invaded by a single PA103

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23 mutant bacterium. The whole transposon insertion mutant library of PA103exoUexoTexoYd was used to infect the HeLa cells. After the first round of screening, 323 transposon mutants survived within the HeLa cells. These transposon mutants were picked and used to infect HeLa cells individually to measure the cytotoxicity by lifting assay followed by LDH cytotoxicity assay. Eventually, 48 transposon insertion mutants showed lower cytotoxicity than the PA103exoUexoTexoYd strain. Sequence Analysis of the Genes Affecting Cytotoxicity in PA103exoUexoTexoYd Forty-eight individual mutants were picked for sequencing. Chromosomal DNA was digested by BamHI and self-ligated. Since the transposon does not contain BamHI restrictions site (Larsen et al., 2002), this resulted in circular chromosomal fragments containing the transposon as well as replication origin oriR6K. These were transformed into E. coli DH5/pir and selected for the kanamycin resistant marker encoded on the transposon (Larsen et al., 2002). The rescued plasmids contain part of the PA103 chromosomal DNA flanking transposon insertion sites, thus could be directly subjected to sequencing analysis. The transposon-specific primers were used for sequencing. Tn-seq1: 5'-TGTGGACAACAAGCCAGGGATGTAAC-3' and Tn-seq2: 5'-GCAACACCTTCTTCACGAGGCAGACC-3'. The 48 mutants involved transposon insertion at 7 different genes, including cytochrome c551 peroxidase precursor (ccpR), dihydrolipoamide acetyltransferase (aceF), 23S ribosomal RNA (rrn), 50S ribosomal protein L19 (rplS), dihydrodipicolinate reductase (dapB), transcriptional regulator PsrA and a hypothetical membrane protein. Interestingly, 30 out of 48 transposon insertion mutants were in 23S ribosomal RNA and 8 out of 48 mutants were in rplS gene. Table 3-1 summarizes these mutated genes and their known functions.

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24 Figure 3-8 and 3-9 showed the cytotoxicity of the representative mutations in the 7 genes. As expected, all of them had lower cytotoxicity than PA103exoUexoTexoYd. A mutation in aceF gene resulting in inactive type III secretion has previously been reported (Dacheux et al., 2002), further validating our selection results. Effect of the Identified Genes on the Expression and Secretion of the Type III Effector Molecules In order to confirm whether those mutated genes are related to the type III gene expression or secretion, we transformed plasmid encoding exoS-FLAG, named pHW0029 (Ha & Jin, 2001), into the 7 representative transposon insertion mutants. ELISA assay (Fig. 3-10) and Western blot (Fig. 3-11) were used to measure the ExoS-FLAG expression (intracellular level of ExoS-Flag) and secretion (ExoS-Flag in supernatant), and compared with PA103exoUexoTexoYd, PA103exsA and PA103exoUexoT containing the pHW0029. Interestingly, mutation in the dapB gene secreted the same amount of ExoS in the presence or absence of EGTA (about 5 g/50uL supernatant), while normally the type III secretion system of P. aeruginosa can not be induced without EGTA or without contacting host cells. Mutations in the 23S Ribosomal RNA, rplS, transcriptional regulator PsrA, or the hypothetical gene resulted in reduced ExoS secretion. Western blot analysis was consistent with the ELISA results (Fig. 3-11).

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Table 3-1. Sequences of the mutated genes in PA103 Name PA namea Proteina Functionsa I-36 PA4587 (ccpR) cytochrome c551 peroxidase precursor Energy metabolism I-40 PA5016 (aceF) dihydrolipoamide acetyltransferase Carbon compound catabolism;Energy metabolism III-14 PA0668.4 (rrn) 23S ribosomal RNA Non-coding RNA gene XI-3 PA3742 (rplS) 50S ribosomal protein L19 Translation, post-translational modification, degradation XII-2 PA4759 (dapB) dihydrodipicolinate reductase Amino acid biosynthesis and metabolism XII-7 PA3730 hypothetical protein Unknown Membrane proteins (3 predicted transmembrane helices) XII-38 PA3006 (psrA) transcriptional regulator PsrA Transcriptional regulators a. PA number, protein name and their functions are blast search compared with pseudomonas PA01 genome ( http://www.pseudomonas.com , 2004).

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26 (A) Amikacin00.050.10.150.20.250.30.353 h24 h48 h60 hTime (h)OD 590 nm Control PAK wt PAK st PA103 ut (B) Non-amikacin00.511.522.53616222938Time (h)OD 590 nm PAKst PAKexsA PA103ut PA103exsA Figure 3-1.Comparison of HeLa cell lifting and death caused by PAK and PA103 derivatives. (A) Intracellular survival selection assay was used to measure HeLa cells lifting and death after adding bacteria into medium containing HeLa cells for 2 hours and then using 400g/ml amikacin to kill extracellular bacteria. Finally, 0.1% crystal violet was used to stain the HeLa cells attached to the cell culture plate after 3, 24, 48 and 60 hours post-infection. (B) HeLa cells were infected by directly adding bacteria without adding amikacin and measuring the OD 590nm value by time course. (C) Comparison of HeLa cell lifting and death caused by PAK strains and PA103 strains at 16 hours infection with or without amikacin. The error bars indicate standard deviations. Control is HeLa cells without bacterial infection as negative control. PAKst is PAKexoSexoT, PA103ut is PA103exoUexoT. NK is the non amikacin infection and AK means adding amikacin after 2 hours bacteria infection. Standard deviations were shown. P value * indicated P<0.05, ** indicated P<0.01, *** indicated P<0.001.

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27 (C) 00.511.522.533.54PAKstPAKexsAPA103utPA103exsAOD 590 nm NA AK********* 00.511.522.533.54PAKstPAKexsAPA103utPA103exsAOD 590 nm NA AK********* 00.511.522.533.54PAKstPAKexsAPA103utPA103exsAOD 590 nm NA AK********* Figure 3-1. Continued

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28 (A) (B) PA2192 PA2189 PA2190 hcnA exoY PCR primer PCR primer Figure 3-2. Gene sequences flanking exoY. (A) The PCR products produced by using the same primers in PA01, PAK and PA103 strains. The procedure was indicated in Materials and Methods (Chapter 2). (B) The model showed exoY gene and its flanking genes. PA01 contained gene PA2190 and PA2192, while PAK and PA103 did not. Transcription direction, The missing sequences in PAK and PA103 strain. ( http://www.pseudomonas.com , 2000)

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29 (A) (B) 00.511.522.533.54PAKstPAKstyPAKexsAPA103utPA103utyPA103exsAOD 590 nm NA AK********* PAK strainsPA103 strains Figure 3-3. Comparison of cytotoxicity between mutants PAKexoSexoTexoY and PA103exoUexoTexoY. (A) The crystal violet staining assay shows HeLa cells that were infected by different P. aeruginosa strains at 16 hours post-infection with amikacin and without amikacin (400g/ml). (B) Measurement of OD 590 nm for crystal violet staining assay plate as shown in figure 3-3 (A). NA, no amikacin added after infection; AK, 400g/ml amikacin added after 2 hours infection. The error bars show standard deviations.To indicate the significant difference from the control samples, P value (student t test) was performed for comparison between exoSexoT and exoSexoTexoY with exsA mutant of PAK, exoUexoT and exoUexoTexoY with exsA mutants of PA103. * , P<0.05; **, P<0.01; ***, P<0.001.

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30 11.522.533.544.512182436Time (h)OD 590 nm PAKst PAKstyD PAKexsA PA103ut PA103utyD PA103exsA contr. Figure 3-4. Kinetics of infection of HeLa cells by different Pseudomonas mutant strains. The mutants were added into the plate where HeLa cells were incubate for 24 hours in 370C under 5% CO2. After 2 hours infection, 400mg/ml amikacin were added into the infection assay and the mixture was continually incubated for 12, 18, 24 and 36 hours. Crystal violet staining assay was used to measure lifting and dead HeLa cells. PAKst is PAKexoSexoT; PAKstyD is PAKexoSexoTexoYd; PA103ut is PA103exoUexoT; PA103utyD is PA103exoUexoTexoYd; contr is HeLa cells without bacterial infection as a control. Assays were done in triplicate and means are shown. The error bars show standard deviations.

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31 010203040506070PAKstPAKstyDPAKexsAPA103utPA103utyDPA103exsACytotoxicity (%)****** 010203040506070PAKstPAKstyDPAKexsAPA103utPA103utyDPA103exsACytotoxicity (%)****** Figure 3-5. LDH release assay of HeLa cells infected by P. aeruginosa strains. The incubation was for 18 hours after adding 400g amikacin per ml. PAKst is PAKexoSexoT; PAKstyD is PAKexoSexoTexoYd; PA103ut is PA103exoUexoT; PA103utyD is PA103exoUexoTexoYd. The error bars indicate standard deviations. Samples were assayed in triplicates. Student t test was used to measure P value. P values compared the exoSexoT and exoSexoTexoY mutants with exsA mutant of PAK, exoUexoT and exoUexoTexoY mutants with exsA mutant of PA103 as following. * indicated P<0.05, ** indicated P<0.01, *** indicated P<0.001.

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32 (B) (A) MOI = 2/2000.511.522.561218243648Time (h)OD 590 nm UTYd UT STYd 103exsA MOI = 5/5000.511.5261218243648Time (h)OD 590 nm UTYd UT STYd 103exsA (C) (D) MOI = 10/10000.511.522.561218243648Time (h)OD 590 nm UTYd UT STYd 103exsA MOI = 15/15000.511.522.561218243648Time (h)OD 590 nm UTYd UT STYd 103exsA Figure 3-6.HeLa cell lifting assays at different MOIs and time points. UTYd is PA103exoUexoTexoYd, UT is PA103exoUexoT, STYd is PAKexoSexoTexoYd, 103exsA is PA103exsA. Error bars indicate standard deviations.

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33 (A) aphoriTtetAptnporiR6K pRL274050 bps (B) Primer 2 Primer 1 Chrom DNA Chrom DN A oriR6K aph BamH1 BamH1 Figure 3-7.Transposon mutagenesis delivery vector and insertion model. (A) Transposon delivery vector pRL27 carries a Tn5 transposase gene (tnp) under the control of the tetA promoter (tetAp) from plasmid RP4. It also contains the Tn5 element encoding kanamycin resistance (KmR) (Larsen et al., 2002). (B) Cartoon shows the Tn 5 transposon inserted into chromosomal DNA carrying the oriRBK origin and Neomycin resistance marker. Two outward-directed primers on the transposon were used for sequencing. The BamH1 restriction enzyme cleavage site which does not cut within the transposon could be used for digestion of chromosomal DNA from a transposon-insertion mutant.

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34 (A) 00.511.522.533.54I-36I-40III-14XI-3XII-2XII-7XII-38103exsAUTYdUTSTYdHeLaP. aeruginosaOD 590nm (B) 05101520253035404550HeLaSTYdUTUTYd103exsAXII-38XII-7XII-2XI-3III-14I-40I-36P. aeruginosa%Cytotoxicity Figure 3-8.Cytotoxicity assays of the 7 Tn5 insertion mutants. (A) HeLa cell lifting assay for the strains carrying the 7 mutated genes infecting HeLa cells for 24 hours. XII-2 and XII-6 are strains containing the same mutated gene, XII-38 and XII-42 also contained the same mutated gene. STYd is PAKexoSexoTexoYd, UT is PA103exoUexoT and UTYd is PA103exoUexoTexoYd strain, 103exsA is PA103exsA mutant strain. (B) The LDH cytotoxicity assay was performed by the same mutants as (A) shown to infect HeLa cells for 24 hours. Samples were assayed in triplicates. Error bars indicate standard deviations.

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35 051015202530354045504.5h8h16h24hTime (h)%C y totoxicit y HeLa UTYd 103exsA XII-38 XII-7 XII-2 XI-3 III-14 I-40 I-36 Figure 3-9. LDH release upon infection by the transposon insertion mutants. The strains are the same as in Figure 3-8. LDH cytotoxicity was measured at 4.5, 8, 16, and 24 hours after mutant strains infecting HeLa cells. PA103exoUexoTexoYd and PA103exsA are the positive controls and HeLa cell without infection is the negative control. The mean of three independent replicate tests are shown.

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36 (A) 15 5 1.7 0.6 (ug/50ul) 1 1 0 (B) 15 5 1.7 0.6 (ug/50ul) 1 1 0 Figure 3-10.ELISA assays of transposon mutant strains carrying ExoS-FLAG. (A) Sandwich ELISA assays were performed in the EGTA induced condition (EGTA 5mM). The right panel is the ExoS standard concentration. (B) Sandwich ELISA assays were performed in the EGTA non-induced condition. Error bars indicate standard deviations.

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37 Figure 3-11.Western blotting of the transposon-insertinal mutants carrying ExoS-FLAG plasmid. (A)Mutant strains pellet and supernatant western blotting in the EGTA induced condition. (B) Second group mutant strains pellet and supernatant western blotting in the EGTA induced condition. (C) The third group mutant strains western blotting in EGTA induced and non-induced conditions. The bacterial mutants were cultured overnight in 5 mM EGTA induced condition and the pellet and culture supernatant were collected separately. Anti-Rabbit-lgG to ExoS was used as detecting antibody.

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38 Figure 3-11. Continued.

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CHAPTER 4 DISCUSSION Pseudomonas aeruginosa intoxicates epithelial cells through the activity of the type III secretion system. To shed light on the mechanism of epithelial cell intoxication by P. aeruginosa strain PA103, I have constructed a transposon mutant library in cytotoxic strain PA103, developed assay conditions and screened for mutants with reduced cytotoxicity. This allowed us to search for the genes that affect the type III secretion system-dependent cytotoxicity on epithelial cells. Different Effect of ExoY in PAK and PA103 Strains In this study, we found that mutant PA103exoUexoTexoYd was more cytotoxic than mutant PA103exsA which is defective in the expression of all type III related genes, while the cytotoxicity of PA103exoUexoTexoYd was the same as that of mutant PA103exoUexoT. These are shown in Figure 3-4. Based on these data, we may assume that ExoY plays no role in the cytotoxicity of P. aeruginosa strain PA103, and there is additional cytotoxic factor(s) associated with the Type III secretion system. As shown in the same figure, it was found that PA103exoUexoTexoYd was more cytotoxic than the PAKexoSexoTexoYd mutant. However, PAKexoSexoTexoYd lost almost all its cytotoxicity compared to mutant PAKexsA. These data suggested that cytotoxin ExoY was not responsible for the difference in cytotoxicity between PAKexoSexoT and PA103exoUexoT. There must be other cytotoxic factor (s) existing in P. aeruginosa strain PA103. 39

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40 Analysis of the Identified Genes By screening transposon insertion mutants, seven genes were found to affect the cytotoxicity of strain PA103, and each gene mutant was characterized based on their effect on the synthesis and secretion of type III effector proteins ExoS. One of the genes named aceF (also named aceB), which belongs to the aceAB operon encoding pyruvate dehydrogenase, has previously been reported to be essential for the functional type III secretion system of P. aeruginosa (Dacheux et al., 2002). Isolation of the aceF mutant in our selection system further validated our assay system. The rest of the mutated genes could be divided into three groups based on their effect on the expression and secretion of effector molecule ExoS. The Group one mutants has normal synthesis and secretion level of effector ExoS. Group two has normal synthesis but less secretion of ExoS, and group three can constitutively secrete ExoS into the culture medium without the type III inducing signal (EGTA). Group 1 Genes Mutants I-36 (the ccpR gene was mutated), XI-3 (the rplS gene was mutated) and XII-38 (the psrA gene was mutated) were grouped into the first group which were shown to have the same synthesis and secretion level of ExoS as parent strain PA103exoUexoTexoYd (Figure 3-10 and 3-11). The gene ccpR encodes cytochrome c peroxidase precursor ( www.pseudomonas.com , 2004). This peroxidase is a periplasmic enzyme reducing H2O2 and prevents bacterial cell lysis, thus allowing increased growth yield (Fulop et al. 1995). Although the mechanism of action is not clear, it is interesting that the ccpR gene product contains a calcium binding site (Hu et al. 1997), since type III secretion system requires a low calcium signal. The rplS gene codes for 50s ribosomal protein L19 and belongs to the trmD operon. The trmD operon contains four genes

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41 encoding ribosomal protein S16, 21KDa protein, tRNA methyltransferase, and ribosomal protein L19 (rplS). These proteins are essential for the assembly of 50S ribosomal subunit (Persson et al. 1995). Surprisingly, disruption of the L19 did not result in decreased bacterial growth, but decreased the bacterial cytotoxicity. Therefore, a mutant lacking L19 resulted in a specific deficiency in cytotoxin(s) and/or type III secretion associated proteins. The mechanism for such specific effect of general translational component on type III secretion system is not clear, however, this not the first such examples. Previously, a mutation in an rRNA modifying enzyme, tRNA pseudouridine synthase, has been reported to affect type III secretion system specifically, despite its predicted effect on general translation (Ahn, et al. 2004). PsrA is pseudomonas sigma regulator involved in rpoS transcription. It was reported that PsrA has the ability to induce RpoS expression at stationary phase (Kojic & Venturi, 2001). RpoS is a stationary phase sigma factor which represses the exoS transcription (Hogardt et al. 2004). Base on these information, the effector ExoS expression would be reduced when the psrA gene were interrupted. However, in our study when we mutated the psrA gene in PA103, the expression of ExoS was the same as control PA103exoUexoTexoYd. The reason for the discrepancy between the published work and our own is not clear at this moment, further confirmation of our result is needed as outlined in the “Future studies” (see below) Group 2 Genes Mutant III-14 and XII-7 had the characteristics of normal exoS expression level but lower secretion of ExoS under type III secretion system induced condition. III-14 carried mutation in a gene coding for 23S rRNA and XII-7 in a gene coding for a putative membrane protein whose function is unknown ( www.pseudomonas.com , 2004). These data indicated that these two mutants have type III secretion defect. 23SrRNA genes are

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42 conserved in various bacterial species. It is a component of ribosome which is necessary for protein synthesis (Maeda et al., 1998). In our study, I found that thirty of forty-eight mutants isolated had transposon-insertional disruption of the 23SrRNA gene. It has been report that the 23SrRNA gene associated with clarithromycin resistance (Maeda et al., 1998). However, we used amikacin to select for the transposon-insertional mutant with less cytotoxicity in our study. It is possible that mutated 23SrRNA might make P. aeruginosa PA103 resistant to amikacin which belongs to aminoglycoside antibiotics. However, it is still hard to explain why 23SrRNA mutant of PA103 has less cytotoxicity. Similar to the case of tRNA pseudouridine synthase (Ahn et al., 2004), the 23SrRNA may also affect the translation of key component in the type III secretion while the putative membrane protein might affect the translocation of the type III effector molecules. Group 3 Genes Group three was grouped with high secretion of effector ExoS without the type III inducing signal (presence of 5mM EGTA in culture medium). The most interesting gene found in this study was dapB, which is L-2, 3-dihydrodipicolinate reductase. The gene dapB is required for both lysine and diaminopimelate biosynthesis (Liu & Shaw, 1997). In our experiments, mutation in the dapB gene caused a certain amount of effector protein ExoS to be secreted into the culture medium even without EGTA, while its effector synthesis level was the same as wild type. In this mutant, majority of its effectors might be secreted into the culture medium constitutively, reducing the amount that can be effectively injected into the host cell, thus explains why its cytotoxicity is reduced when infecting HeLa cells. The ionic form of the amino acid diaminopimelic acid (DAP) is found in the murein peptidoglycans of bacterial cell walls, thus the dapB

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43 mutant might have an abnormnal membrane structure which allows type III effectors to be leaked out of the cells. (Broms et al., 2003; Schoehn et al., 2003). Future Directions This project is still in its primary stage. The data presented and methods used would be the corner-stone for future studies. The selection system for detecting type III secreted cytotoxicity factor (s) was established. Some interesting genes associated with type III secretion cytotoxicity were identified, although their real functions in the type III secretion system are not clear yet. Complementation tests of the mutated genes should be done first to confirm that the observed phenotypes are indeed due to the transposon insertional mediated gene disruption. The genes can be further mutagenized in the background of PAKexoSexoTexoYd and assess the cytotoxicity of the resulting mutants. Then, individual genes can be cloned into a eukaryotic expression vector and transfection assays conducted to see if any of those genes have cytotoxic effects. Since none of the genes found in this study has typical cytotoxin structure, we might need to screen more transposon insertion mutants. Alternatively, we could generate a cosmid library of PA103exoUexoTexoYd chromosomal DNA, and then transform the cosmid clones into null cytotoxicity mutant PAKexoSexoTexoYd background and look for those becoming cytotoxic to HeLa cells.

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46 Jacobs, M. A., Alwood, A., Thaipisuttikul, I., Spencer, D., Haugen, E., Ernst, S., Will, O., Kaul, R., Raymond, C., Levy, R., Chun-Rong, L., Guenthner, D., Bovee, D., Olson, M. V. & Manoil, C. (2003). Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U S A 100, 14339-14344. Kaufman, M. R., Jia, J., Zeng, L., Ha, U., Chow, M. & Jin, S. (2000). Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of exoS. Microbiology 146 ( Pt 10), 2531-2541. Kerem, E., Corey, M., Gold, R. & Levison, H. (1990). Pulmonary function and clinical course in patients with cystic fibrosis after pulmonary colonization with Pseudomonas aeruginosa. J. Pediatr. 116, 714-719. Kojic, M. & Venturi, V. (2001). Regulation of rpoS gene expression in Pseudomonas: involvement of a TetR family regulator. J. Bacteriol. 183, 3712-3720. Kulich, S. M., Frank, D. W. & Barbieri, J. T. (1993). Purification and characterization of exoenzyme S from Pseudomonas aeruginosa 388. Infect. Immun. 61, 307-313. Lakkis, C. & Fleiszig, S. M. (2001). Resistance of Pseudomonas aeruginosa isolates to hydrogel contact lens disinfection correlates with cytotoxic activity. J. Clin. Microbiol. 39, 1477-1486. Larsen, R. A., Wilson, M. M., Guss, A. M. & Metcalf, W. W. (2002). Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch. Microbiol. 178, 193-201. Liu, L. & Shaw, P. D. (1997). Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis. J. Bacteriol. 179, 507-513. Liu, P. V. (1966). The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. 3. Identity of the lethal toxins produced in vitro and in vivo. J. Infect. Dis. 116, 481-489. Maeda, S., Yoshida, H., Ogura, K., Kanai, F., Shiratori, Y. & Omata, M. (1998). Helicobacter pylori specific nested PCR assay for the detection of 23S rRNA mutation associated with clarithromycin resistance. Gut 43, 317-321. McGuffie, E. M., Frank, D. W., Vincent, T. S. & Olson, J. C. (1998). Modification of Ras in eukaryotic cells by Pseudomonas aeruginosa exoenzyme S. Infect. Immun. 66, 2607-2613. Mecsas, J. J. & Strauss, E. J. (1996). Molecular mechanisms of bacterial virulence: type III secretion and pathogenicity islands. Emerg. Infect. Dis. 2, 270-288.

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47 Merritt, K., Gaind, A. & Anderson, J. M. (1998). Detection of bacterial adherence on biomedical polymers. J. Biomed. Mater. Res. 39, 415-422. Miyata, S., Casey, M., Frank, D. W., Ausubel, F. M. & Drenkard, E. (2003). Use of the Galleria mellonella Caterpillar as a Model Host To Study the Role of the Type III Secretion System in Pseudomonas aeruginosa Pathogenesis. Infect. Immun. 71, 2404-2413. Moss, J., Ehrmantraut, M. E., Banwart, B. D., Frank, D. W. & Barbieri, J. T. (2001). Sera from adult patients with cystic fibrosis contain antibodies to Pseudomonas aeruginosa type III apparatus. Infect. Immun. 69, 1185-1188. Nanao, M., Ricard-Blum, S., Di Guilmi, A. M., Lemaire, D., Lascoux, D., Chabert, J., Attree, I. & Dessen, A. (2003). Type III secretion proteins PcrV and PcrG from Pseudomonas aeruginosa form a 1:1 complex through high affinity interactions. BMC Microbiol. 3, 21. Ojeniyi, B. (1994). Polyagglutinable Pseudomonas aeruginosa from cystic fibrosis patients. A survey. APMIS Suppl. 46, 1-44. Pederson, K. J., Vallis, A. J., Aktories, K., Frank, D. W. & Barbieri, J. T. (1999). The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins. Mol. Microbiol. 32, 393-401. Phillips, R. M., Six, D. A., Dennis, E. A. & Ghosh, P. (2003). In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors. J. Biol. Chem. 278, 41326-41332. Roy-Burman, A., Savel, R. H., Racine, S., Swanson, B. L., Revadigar, N. S., Fujimoto, J., Sawa, T., Frank, D. W. & Wiener-Kronish, J. P. (2001). Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J. Infect. Dis. 183, 1767-1774. Salyers, A. A. & Whitt, D. D. (1994). Bacterial Pathogenesis: a Molecular Approach. American Society for Microbiology. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, N. Y.: Cold Spring Harbor Laboratory Press. Schoehn, G., Di Guilmi, A. M., Lemaire, D., Attree, I., Weissenhorn, W. & Dessen, A. (2003). Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas. Embo. J 22, 4957-4967. Vallis, A. J., Finck-Barbancon, V., Yahr, T. L. & Frank, D. W. (1999a). Biological effects of Pseudomonas aeruginosa type III-secreted proteins on CHO cells. Infect. Immun. 67, 2040-2044.

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48 Vallis, A. J., Yahr, T. L., Barbieri, J. T. & Frank, D. W. (1999b). Regulation of ExoS production and secretion by Pseudomonas aeruginosa in response to tissue culture conditions. Infect. Immun. 67, 914-920. Weinstein, M., Roberts, R. C. & Helinski, D. R. (1992). A region of the broad-host-range plasmid RK2 causes stable in planta inheritance of plasmids in Rhizobium meliloti cells isolated from alfalfa root nodules. J. Bacteriol. 174, 7486-7489. Winstanley, C. & Hart, C. A. (2001). Type III secretion systems and pathogenicity islands. J. Med. Microbiol. 50, 116-126. Wolfgang, M. C., Kulasekara, B. R., Liang, X., Boyd, D., Wu, K., Yang, Q., Miyada, C. G. & Lory, S. (2003). Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa. PNAS 100, 8484-8489. Yahr, T. L., Barbieri, J. T. & Frank, D. W. (1996a). Genetic relationship between the 53and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa. J. Bacteriol. 178, 1412-1419. Yahr, T. L., Goranson, J. & Frank, D. W. (1996b). Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway. Mol. Microbiol. 22, 991-1003. Yahr, T. L., Vallis, A. J., Hancock, M. K., Barbieri, J. T. & Frank, D. W. (1998). ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system. Proc. Natl. Acad. Sci. USA 95, 13899-13904.

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BIOGRAPHICAL SKETCH Xiaoling Wang worked as a physician in Xi’an province hospital for several years before pursuing graduate research at the University of Florida. Despite all the excitement and accomplishments in saving patients from all kinds of malicious diseases in clinical work, she found that she was particularly interested in enriching herself with basic medical research. She believed that this might help her to further understand the diseases and treatments rather than simply following the medical instructions. Therefore, she joined Dr. Jin’s research group in 2002, and spent her time in basic research in genetics and microbiological sciences. She met many great people there and enjoyed discussing with them all kinds of questions. At the same time, she met her beloved husband, Cheng-feng Tai, and started a happy new life. 49