Signal modulation of type III secretion system in pseudomonas aeruginosa

SIGNAL MODULATION OF TYPE III SECRETION SYSTEM IN PSEUDOMONAS AERUGINOSA ZHOU LIAN (B. Sc., Shanghai Jiaotong University) A THESIS SUBMITED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I wish to express my greatest gratitude and heartfelt appreciation to my supervisor, Prof. Zhang Lian-Hui, for his scientific guidance and thought-provoking advice. Sincere thanks are given to my PhD committee members, Prof. Wang Yue and Prof. Liu Ding-Xiang as well as my ex-PhD committee member A/Prof. Poh Chit Laa, for their valuable suggestions and critical evaluations. I would like to take this opportunity to thank all the past and present members in Lab of Microbial Quorum Sensing for their technical assistance and scientific discussions. In particular, I am very grateful to Dr. Wang Jing for her unreserved help in establishing the mouse pulmonary infection model and her suggestions on the mice infection experiments. Also thanks are given to Ms. Soh Pei Fen, the attachment student from Nanyang Polytechnic in 2005, for her extreme patience in screening for T3SS mutants. I would like to thank Ms. Linda Soo and Mr. Wen Chaoming from ex-Lab of Functional Genomics for their technical assistance in microarray analysis. Also thanks are given to IMCB DNA sequencing facility and A-star Biopolis Shared Facilities for their supports. Many thanks are given to Prof. Lin Zhi-Xin and Prof. Luo Jiu-Fu in Shanghai Jiaotong University, for their guidance during my undergraduate study. Thanks also extend to Prof. Xie Dao-Xin in Tsinghua University, A/Prof. Wen Zi-Long in the Hong Kong I University of Science and Technology, and Prof. Peng Jin-Rong in Zhe Jiang Univeristy, for introducing me to study in Singapore Last, but not least, I would like to thank my family members, my husband Ya-wen, my two kids Jeslyn and Eric, and my parents for their love and utmost support. II TABLE OF CONTENTS Page ACKNOWLEDGEMENTS . I TABLE OF CONTENTS III SUMMARY . VIII LIST OF TABLE . X LIST OF FIGURES . XI Chapter Literature Review 1.1 Pseudomonas aeruginosa and clinical importance 1.1.1 Taxonomy, description and distribution 1.1.2 Clinical importance of P. aeruginosa 1.1.2.1 P. aeruginosa infections in CF patients 1.1.2.2 P. aeruginosa infections in burn wounds 1.1.2.3 P. aeruginosa infections in cornea diseases 1.2 The major virulence factors of Pseudomonas aeruginosa 1.2.1 Bacterial cell membrane-associated virulence factors 1.2.1.1 Flagellum . 1.2.1.2 Type IV Pilus . 10 1.2.1.3 Lipopolysaccharide (LPS) 11 1.2.2 Extracellular virulence factors . 13 1.2.2.1 Exopolysaccharide (EPS) . 13 1.2.2.2 Extracellular enzymes 16 1.2.2.3 Extracellular chemical toxins 19 1.2.3 Type III Secretion System (T3SS) . 22 1.2.3.1 Structure of T3SS and the effectors 22 1.2.3.2 T3SS in P. aeruginosa 24 1.2.3.3 Clinical importance of T3SS in P. aeruginosa 30 1.3 Regulation of virulence factor production in P. aeruginosa . 31 1.3.1 Regulation of bacterial membrane associated virulence factors . 31 1.3.1.1 Regulation of flagellar biogenesis . 31 III 1.3.1.2 Regulation of type IV pili biogenesis 32 1.3.2 Regulation of extracellular virulence factors . 33 1.3.2.1 Regulation of EPS biosynthesis 33 1.3.2.2 Regulation of exotoxin and extracellular enzyme production 36 1.3.3 Regulation of T3SS . 37 1.3.3.1 Environmental cues and host signals 38 1.3.3.2 The exoenzyme S regulon 40 1.3.3.3 Regulation of T3SS through the cAMP-Vfr pathway 44 1.3.3.4 Reciprocal regulation of T3SS and biofilm . 45 1.3.3.5 Regulation of T3SS by stress 47 1.4 Treatment and prevention 48 1.5 Aims and scope of present study 51 Chapter Genetic screening of the novel genes involved in regulation of T3SS expression in P. aeruginosa . 53 2.1 Introduction . 53 2.2 Materials and methods . 55 2.2.1 Bacterial strains, plasmids and growth conditions 55 2.2.2 DNA manipulation 57 2.2.3 Construction of reporter strains . 57 2.2.4 Transposon mutagenesis . 58 2.2.5 Quantitative -galactosidase assay 59 2.3 Results . 59 2.3.1 The transcriptional fusion reporters pC-lacZ and pT-lacZ monitor the expression of exsCEBA and exoT respectively . 59 2.3.2 Screening the transposon mutants with decreased T3SS expression . 63 2.3.3 DNA sequence analysis of the transposon mutants 63 2.4 Discussion . 72 2.4.1 The roles of five previously characterized T3SS regulators in modulation of T3SS . 72 2.4.2 Mutants with Tn insertion at the genes functionally or physically related to T3SS regulators . 73 IV 2.4.3 Mutants associated with bacterial motility and EPS production . 75 2.4.4 Mutants associated with metabolic imbalance . 76 2.4.5 Transposon insertion mutants with defects in genes involved in transport of small molecules . 77 2.4.6 Other mutants . 78 Chapter Modulation of bacterial type III secretion system by a spermidine transporterdependent signaling pathway . 80 3.1 Introduction . 80 3.2 Materials and methods . 81 3.2.1 Bacterial strains, plasmids and growth conditions 81 3.2.2 Gene cloning and deletion . 81 3.2.3 RNA Extraction and microarray analysis . 87 3.2.4 Protein isolation and western blotting analysis 88 3.2.5 Quantitative -galactosidase assay 89 3.2.6 Culture of P. aeruginosa with mouse liver extract . 89 3.2.7 HeLa cell culture and cytotoxicity assay 90 3.3 Results . 91 3.3.1 Induction of exsCEBA required the major spermidine uptake transporter encoded by spuDEFGH . 91 3.3.2 Null mutation of the spermidine transporter down-regulated the transcription of T3SS genes 95 3.3.3 Deletion of spuE impaired the production and secretion of the T3SS effector ExoS 96 3.3.4 Exogenous addition of spermidine induced the expression of T3SS and secretion of effector ExoS 101 3.3.5 Spermidine was the most effective polyamine signal for T3SS induction 104 3.3.6 Mutation of spermidine transporter decreased the host cell extract-induced expression of T3SS genes and attenuates the T3SS-mediated cytotoxicity . 104 3.4 Discussion . 107 Chapter A novel spermidine-responsive transcriptional regulator TsrA modulates the expression of T3SS genes in P. aeruginosa . 112 V 4.1 Introduction . 112 4.2 Materials and methods . 115 4.2.1 Strains and culture conditions 115 4.2.2 Gene cloning and deletion . 115 4.2.3 RNA Extraction and microarray analysis . 120 4.2.4 Extracellular protein isolation and western blotting analysis 120 4.2.5 Quantitative -galactosidase assay 121 4.2.6 HeLa cell culture and cytotoxicity assay 121 4.2.7 Purification of recombinant GST-TsrA . 121 4.2.8 Electrophoretic Mobility Shift Assay (EMSA) 122 4.2.9 Isothermal titration calorimetry (ITC) analysis 122 4.3 Results . 123 4.3.1 Deletion of tsrA (pa2432) encoding a putative transcription regulator in the mutant spuE partially restores the expression level of exsCEBA 123 4.3.2 In trans expression of tsrA down-regulated the expression of T3SS Genes 128 4.3.3 Enhanced expression of tsrA reduced the production of T3SS effector ExoS and attenuated T3SS-mediated cytotoxicity 134 4.3.4 Vfr is involved in the TsrA regulatory pathway . 135 4.3.5 Spermidine modulates the interaction between TsrA and the promoter of vfr (Pvfr) . 141 4.3.6 Self repression of tsrA expression through direct binding between TsrA and tsrA promoter (PtsrA) 148 4.3.7 Deletion of tsrA partially attenuates the response of P. aeruginosa to exogenous addition of spermidine 150 4.4 Discussion . 151 Chapter Protection against mice pulmonary pseudomonal infection by active immunization with polyamine transport protein SpuD of P. aeruginosa 161 5.1 Introduction . 161 5.2 Material and methods . 163 5.2.1 Bacterial strains and growth conditions . 163 5.2.2 Intra-tracheal challenge with P. aeruginosa . 164 VI 5.2.3 Quantification of P. aeruginosa load in mice lungs . 164 5.2.4 Cytokine levels following infection with P. aeruginosa 165 5.2.5 Purification of recombinant SpuD . 165 5.2.6 Endotoxin removal from the SpuD protein solution . 166 5.2.7 Immunogenicity assay of mice by ELISA analysis 166 5.3 Results . 167 5.3.1 Null mutation of spermidine uptake transporter in P. aeruginosa reduces the host mortality in a mouse pulmonary infection model 167 5.3.2 The spuE mutant shows a slow lung clearance post infection 168 5.3.3 Mice infected with spuE have reduced cytokine production 169 5.3.4 SpuD Immunization increased the chance for mice to survive in the Intratracheal challenge model 174 5.4 Discussion . 180 Chapter General Conclusions and Future Directions . 185 6.1 General conclusions . 185 6.2 Future directions 190 Reference List . 193 VII SUMMARY Pseudomonas aeruginosa is an opportunistic pathogen that causes infection mainly in immuno-compromised patients, such as AIDS patients, cystic fibrosis patients and severe burn victims. Various virulence factors have been identified and experimentally evaluated, among which a conserved secretion system, the Type III Secretion System (T3SS), holds particular interests to the scientists studying hostpathogen interactions. By utilizing this system, the pathogen is able to directly inject its protein effectors into host cells, where they manipulate host cellular functions. Expression of T3SS genes is under the tight control of its master regulator ExsA and other regulatory proteins encoded by the exsCEBA operon and induced by calcium depletion and contact with host cells. However, the molecular mechanism by which host cells induce T3SS expression remains elusive. By generating a chromosomal integrated reporter strain PAO1pClacZ, transposon mutagenesis was conducted, which led to identification of a range of mutants defective in T3SS expression under calcium-depletion conditions, including the mutants of spuE, spuF and spuG, As the spuDEFGH genes encode a major uptake system for spermidine, which is abundant in host cells, this transporter system has thus become the focus of this study. Genomic and biochemical analysis showed that mutation of the transporter substantially reduced the expression of most T3SS genes, including the exsCEBA operon, and impaired the secretion of effector ExoS, under calcium-depletion conditions. The mutation also decreased the host cell extract-dependent induction of T3SS expression and attenuated bacterial cytotoxicity towards HeLa cells. Consistently, exogenous addition of spermidine to the wild type strain PAO1 enhanced the expression of exsCEBA and VIII induced the secretion of the effector ExoS, demonstrating that a spermidine transporterdependent signaling pathway is involved in T3SS regulation. By deletion analysis of the genes up-regulated in spu transporter mutants, a LysR type transcriptional regulator TsrA that controls T3SS expression was identified. Further analysis showed that TsrA negatively regulates T3SS expression through downregulating the expression of vfr, which encodes a known positive regulator of T3SS, by binding to its promoter Pvfr. Addition of spermidine activates T3SS expression by interfering with the interaction of the suppressor TsrA with Pvfr. Cumulatively, these data have depicted a novel spermidine transporter-dependent regulation cascade, which plays an essential role in signal modulation of T3SS expression in P. aeruginosa. 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P aeruginosa is not dependent on a single TLR is likely due to the redundancy of TLRs in the host innate immune system In addition, MyD88 adaptor protein has been shown to be important in modulating host inflammatory responses through recruitment of neutrophils (Power et al., 2004) 1.1.2.2 P aeruginosa infections in burn wounds In healthy individuals, the intact skin forms a barrier to protect against... (Ostroff and Vasil, 1987), ferripyochelin-binding protein (Sokol, 1987), LPS and the effector proteins secreted by the type III secretion system (Nicas and Iglewski, 1985; Sokol, 1987; Goldberg et al., 1995), which promote its own dissemination throughout the host 1.1.2.3 P aeruginosa infections in cornea diseases P aeruginosa causes severe eye infections even in healthy individuals Previously, eye infections... Structures of common polyamines 159 Fig 4-15 Proposed spermidine signaling pathway required for T3SS gene expression in P aeruginosa 160 Fig 5-1 The growth patterns of PAO1 and spuE 170 Fig 5-2 Survival plots of FVB/N mice infected by P aeruginosa 171 Fig 5-3 P aeruginosa clearance in mouse lung after intra-tracheal infection 172 Fig 5-4 Expression of recombinant SpuD... (TLR5); the event leads to activation of the MyD88-dependent signaling pathway and is subsequently provoking the innate inflammatory response (Feuillet et al., 2006) 1.2.1.2 Type IV Pilus Type IV pili are strong and flexible filaments that mediate diverse cellular functions in P aeruginosa First of all, type IV pili are the major adhesins in P aeruginosa acting by binding to the glycolipids asialo-GM1... 2008) In addition, P aeruginosa is associated with urinary tract infections, nosocomial pneumonia and severe eye disease due to the use of contact lenses in healthy individuals This pathogen is also easily found in clinics and hospitals, leading to infections in patients through colonization of respiratory tubes and intravascular catheters (Pollack et al., 1995) 1.1.2.1 P aeruginosa infections in CF... Genetic organization of the P aeruginosa exoenzyme S regulon 26 Fig 1-2 Occurrence of the protein homologues of T3SS genes in Pseudomonas and other bacterial families 28 Fig 1-3 Schematic model of the Type III Secretion System (T3SS) in P aeruginosa 29 Fig 1-4 The environmental signals and regulatory systems which regulate the expression of the P aeruginosa T3SS 39 Fig 1-5... advantages for P aeruginosa in the CF airways P aeruginosa alginate is an acetylated polymer composed of non-repetitive monomers of -1,4 linked L-guluronic and D-mannuronic acids (Ryder et al., 2007) Overproduction of alginate causes the mucoid phenotype for P aeruginosa The CF patients carrying the mucoid P aeruginosa strains have a worse prognosis than those colonized with non-mucoid strains (Govan and... liquid in CF decreases mucociliary clearance of P aeruginosa from the airway and allows the retention of organisms within the airway lumen (Worlitzsch et al., 2002) In addition to be a chloride channel, CFTR has also been identified as an important epithelial cell receptor for clearance of P aeruginosa from lungs In healthy individuals, CFTR recognizes P aeruginosa through specific binding of the amino... corneal injury happens In this case, instead of clearing the bacterial cells, the CFTR- 8 expressing epithelial cells function as a reservoir for P aeruginosa since the bacterialaden cell layer is buried beneath several epithelial cell layers (Fleiszig et al., 1995; Zaidi et al., 1999) 1.2 The major virulence factors of Pseudomonas aeruginosa P aeruginosa employs a range of virulence determinants as... nucleotide binding domain (NBD) (Lyczak et al., 2002) The membrane-spanning 3 domain of CFTR determines the diameter of the pore for the chloride ion (Sheppard et al., 1996) The NBD is responsible for the binding and hydrolysis of ATP to provide energy as well as for the regulation of the opening and closing of the ion channel pore (Anderson et al., 1991; Gadsby and Nairn, 1999) In addition, CFTR contains . 1 1.1.2 Clinical importance of P. aeruginosa 3 1.1.2.1 P. aeruginosa infections in CF patients 3 1.1.2.2 P. aeruginosa infections in burn wounds 6 1.1.2.3 P. aeruginosa infections in cornea diseases. chemical toxins 19 1.2.3 Type III Secretion System (T3SS) 22 1.2.3.1 Structure of T3SS and the effectors 22 1.2.3.2 T3SS in P. aeruginosa 24 1.2.3.3 Clinical importance of T3SS in P. aeruginosa 30 1.3. SIGNAL MODULATION OF TYPE III SECRETION SYSTEM IN PSEUDOMONAS AERUGINOSA ZHOU LIAN (B. Sc., Shanghai Jiaotong University) A THESIS SUBMITED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT