PROTEOMICS APPROACH ON THE IDENTIFICATION OF VIRULENCE FACTORS OF ENTEROPATHOGENIC AND ENTEROHEMORRHAGIC ESCHERICHIA COLI (EPEC AND EHEC) AND FURTHER CHARACTERIZATION OF TWO EFFECTORS: ESPB AND NLEI BY LI MO (M. SC.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS I wish to express my heartfelt gratitude to my main supervisor, Professor Hew Choy Leong for his care and guidance. His clement character and esteemed research passion inspirit me through my study. I wish to express my heartful thanks to my co-supervisor, Associate Professor Leung Ka Yin for his supervision and advice. He had planned properly and provided many opportunities for my research. I would sincerely thank Professor Ilan Rosenshine from the Hebrew University, Israel, for his sound instruction and generous sharing of ideas and experiences. My Special thanks will also go to my PhD. Committee members. Thank you for spending so much time in reviewing my thesis and audit my presentation. I would like to take this opportunity to thank Dr. Lin Qingsong, Dr. John Foo, Ms Wang Xianhui and Ms Kho Say Tin from the Protein and Proteomics Center for their kind assistance in mass spectrometry, protein sequencing and data analysis. Further thank goes to Mr. Shashikant Joshi for his reviewing and suggestions on my thesis. My appreciation also goes to my previous and present lab members: Dr Yu Hongbing, Dr Seng Eng Khuan, Dr Srinivasa Rao, Dr Tan Yuan Peng, Dr Yamada, Dr. Li Zhengjun, Dr. Xie Haixia , Ms Tung Siew Lai, Mr. Zheng Jun, Mr. Peng Bo, Mr. Zhou Wenguang, Ms Tang Xuhua, Mr. Liu Yang, Mr. Wang Fan, Mr. Chen Liming, for their encouragement, help, assistance and company. I would also like to thank my good friends Wang Xiaoxing, Hu Yi, Qian Zuolei, Sheng Donglai, Luo Min, Tu Haitao, Sun Deying, Alan John Lowton. I appreciate their friendship and their valuable suggestions, advice and help throughout my study. My fellow labmates and other friends who have helped me one-way or another during the course of my project are also greatly appreciated. Last, but not least, I would like to thank my family members, my father, my mother and my sister, who are always standing by me to give their utmost support. Their courage and consolidation is the most powerful strength and is never separated by the distance, which company me throughout my PhD process. i TABLE OF CONTENTS ACKNOWLEDGEMENTS·····························································································i TABLE OF CONTENTS································································································ii LIST OF PUBLICATIONS RELATED TO THIS STUDY········································ix LIST OF FIGURES ········································································································ x LIST OF TABLES ·······································································································xiii LIST OF ABBREVIATIONS······················································································ xiv SUMMARY ·················································································································· xvi Chapter I. Introduction ·································································································· I.1. Escherichia coli-the opportunistic infectious pathogen ······································ I.1.1. Pathogenic E. coli ··························································································· I.1.2. Epidemiology of EPEC ·················································································· I.1.3. Epidemiology of EHEC ················································································· I.2. Pathogenesis and virulence factors of EPEC and EHEC··································· I.2.1. EPEC pathogenesis and virulence factors···················································· I.2.1.1. Attaching and effacing histopathology ·················································· I.2.1.2. Localized Adherence (LA) and Bundle Forming Pili (BFP) ·············· 10 I.2.1.3. EAF plasmids ························································································ 11 I.2.1.4. Invasion·································································································· 12 ii I.2.1.5. Flagella··································································································· 13 I.2.1.6. Serine Protease - EspC·········································································· 14 I.2.1.7. Heat-stable enterotoxin (EAST1).························································ 14 I.2.2. EHEC pathogenesis and virulence factors ················································· 15 I.2.2.1. Shiga toxin ····························································································· 15 I.2.2.2. Intestinal adherence factors ································································· 16 I.2.2.3. Invasion·································································································· 17 I.2.2.4. Flagella··································································································· 18 I.2.2.5. pO157 plasmid······················································································· 19 I.2.2.6. Serine protease ······················································································ 19 1.2.2.6.1. EspP ································································································ 19 I.2.2.6.2. StcE ································································································· 20 I.2.2.7. Heat-stable enterotoxin (EAST1).························································ 21 I.2.2.8. Iron transport.······················································································· 22 I.2.3. Type Three Secretion System (TTSS) in EPEC and EHEC ····················· 22 I.2.3.1. Pathogenicity island (PAI)···································································· 23 I.2.3.2. Components of TTSS in EPEC and EHEC ········································· 24 I.2.3.2.1. Components of TTSS basal body ·················································· 25 I.2.3.2.2. TTSS translocators········································································· 26 I.2.3.2.3. LEE encoded adhesin····································································· 27 I.2.3.2.4. LEE encoded TTSS regulators······················································ 29 I.2.3.2.5. Switches of TTSS translocators and effectors ······························ 29 I.2.3.3.6. TTSS chaperons ············································································· 30 iii I.2.3.3.7. TTSS secreted Effectors································································· 30 I.3. Objectives ············································································································ 33 Chapter II. Common materials and methods······························································ 35 II.1. Common used medium and buffer··································································· 35 II.2. Bacteria strains and plasmids··········································································· 35 II.3. Tissue culture····································································································· 35 II.4. Molecular biology techniques··········································································· 36 II.4.1. Genomic DNA isolation·············································································· 36 II.4.2. Cloning DNA fragments and transformation into E. coli cells················ 36 II.4.3. Analysis of plasmid DNA ··········································································· 37 II.4.4. Plasmid DNA isolation and purification ··················································· 37 II.4.5. DNA sequencing ························································································· 38 II.4.6. Sequence analysis ······················································································· 38 II.4.7. Southern hybridization ·············································································· 39 II.5. Protein techniques····························································································· 40 II.5.1. Preparation of ECPs from E. coli strains ················································· 40 II.5.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ································································································································ 40 II.5.3. Visualization of protein bands/spots (Coomassie blue staining and silver staining)·················································································································· 41 II.5.4. Western blotting ························································································· 42 iv Chapter III. Comparative proteomics analysis of extracellular proteins of enterohemorrhagic and enteropathogenic Escherichia coli and their ······················· 44 Abstract······················································································································ 45 III.1. Introduction ····································································································· 46 III.2. Materials and methods ···················································································· 48 III.2.1. Bacterial strains and culture conditions·················································· 48 III.2.2. Proteins isolation and assay······································································ 49 III.2.3. One- and two-dimensional gel electrophoresis········································ 49 III.2.4. Tryptic in-gel digestion and MALDI-TOF MS analysis························· 50 III.3. Results and discussion ····················································································· 51 III.3.1. ECP production ························································································ 51 III.3.2. Extracellular Proteomes of EHEC and EPEC ········································ 59 III.3.3. Identification of Ler and IHF regulated proteins ··································· 63 III.3.4. Applications and conclusions ··································································· 67 Chapter IV. Characterization of EspB as a translocator and an effector and its involvement in EPEC autoaggregation········································································ 68 Abstract······················································································································ 69 IV.1 Introduction ······································································································ 70 IV.2. Materials and methods ···················································································· 72 IV.2.1. Bacterial strains and plasmids ································································· 72 IV.2.1. Fractionation of infected HeLa cells ························································ 73 IV.2.2. Invasion assay···························································································· 73 v IV.2.3. Examination of bacterial surface exposed structure by transmission electron microscopy (TEM) ·················································································· 74 IV.2.4. Phase contrast microscopy ······································································· 74 IV.2.5. Construction of plasmid for expression of EspB-TEM and β-lactamase based translocation assay······················································································ 75 IV.2.6. Edman N-terminal sequencing································································· 75 IV.3. Results··············································································································· 76 IV.3.1. Survey of the two forms of EspB······························································ 76 IV.3.2. Mutation of espB did not affect the secretion of other extracellular proteins··················································································································· 77 IV.3.3. Mutation of espB mutant abolished the translocation of effectors ········ 80 IV.3.4. EspB is translocated into infected HeLa cells·········································· 80 IV.3.5. EspB is involved in the autoaggregation·················································· 83 IV.3.6. EspB mutant does not form the autoaggregation ··································· 83 IV.3.7. ∆espB mutant showed less extracellular filamentous appendages········· 86 IV.3.8. ∆espB mutant has lower invasion ability ················································· 87 IV. 4. Discussion ········································································································ 89 IV. 5. Conclusion ······································································································· 92 Chapter V. Identification and Characterization of NleI, a New Non-LEE-encoded Effector of Enteropathogenic Escherichia coli (EPEC) ·············································· 93 Abstract······················································································································ 94 V.1. Introduction ······································································································· 95 vi V.2. Material and methods ······················································································· 98 V.2.1. Bacteria strains and culture conditions····················································· 98 V.2.2. Tissue culture conditions············································································ 98 V.2.3. Construction of deletion mutants and plasmids ······································· 98 V.2.4. Flow cytometric analysis ············································································ 99 V.2.5. 2-D SDS-PAGE and proteomics ······························································ 100 V.2.6. Fractionation of infected HeLa cells························································ 100 V.2.7. Expression and Immunoblot analysis······················································ 101 V.2.8. Translocation assay ·················································································· 102 V.2.9. Fluorescence microscopy for observation of translocation, transfection and actin condensation························································································ 102 V.3. Results ·············································································································· 106 V.3.1. Identification of NleI from EPEC sepL and sepD mutants ···················· 106 V.3.2. NleI is located within a prophage-associated island in EPEC ··············· 107 V.3.3. NleI is a secreted protein and the secretion of NleI is TTSS-dependent113 V.3.4. NleI is translocated into the host cells ····················································· 116 V.3.5. CesT is involved in the translocation but not the stabilization of NleI · 119 V.3.6. NleI is localized in the host cytoplasm and membrane ·························· 119 V.3.7. NleI is regulated by SepD but not regulated by Ler and SepL at the transcriptional level····························································································· 124 V.3.8. NleI is not involved in the filopodia and pedestal formation ················· 125 V.4. Discussion········································································································· 128 V.5. Conclusion········································································································ 130 vii Chapter VI. General conclusions and future directions ··········································· 132 VI.1. General conclusions ······················································································· 132 VI.2. Future directions···························································································· 134 Reference ····················································································································· 137 viii LIST OF PUBLICATIONS RELATED TO THIS STUDY 1. Li, M., I. Rosenshine, S.L. Tung, X.H. Wang, D. Friedberg, C.L. Hew, and K.Y. Leung. 2004. Comparative proteomics analysis of extracellular proteins of enterohemorrhagic and enteropathogenic Escherichia coli and their ihf and ler mutants. Appl. Environ. Microbiol. 70:5274-5282. 2. Li, M., I. Rosenshine, H.B. Yu, C. Nadler, E. Mills, C.L. Hew, and K.Y. Leung. Identification and characterization of NleI, a new non-LEE-encoded effector of enteropathogenic Escherichia coli (EPEC). (Microbes and Infection. 2006. Accepted.) 3. Li, M., I. Rosenshine, and K. Y. Leung. Characterization of EspB as a translocator and an effector and its involvement in EPEC autoaggregation. (In preparation) ix 96. Hamada, D., T. Kato, T. Ikegami, K. N. Suzuki, M. Hayashi, Y. Murooka, T. Honda, and I. Yanagihara. 2005. EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein. FEBS J. 272:756-768. 97. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580. 98. Hantke, K., Nicholson, G., Rabsch, W., and Winkelmann, G. 2003. Salmochelins, siderophores of Salmonella enterica and uropathogenic Escherichia coli strains, are recognized by the outer membrane receptor IroN. Proc. Natl. Acad. Sci. USA 100: 3677-3682. 99. Hasman, H., T. Chakraborty, and P. Klemm. 1999. Antigen-43-mediated autoaggregation of Escherichia coli is blocked by fimbriation. J. Bacteriol. 181:4834-4841. 100. Hayashi, T., K. Makino, M. Ohnishi, K. Kurokawa, K. Ishii, K. Yokoyama, C. G. Han, E. Ohtsubo, K. Nakayama, T. Murata, M. Tanaka, T. Tobe, T. Iida, H. Takami, T. Honda, C. Sasakawa, N. Ogasawara, T. Yasunaga, S. Kuhara, T. Shiba, M. Hattori, and H. Shinagawa. 2001. Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Res. 8:11-22. 101. Heath, P. T., N. K. Nik. Yusoff, and C. J. Baker. 2003. Neonatal meningitis Archives of Disease in Childhood Fetal and Neonatal Edition. 88:173-178. 102. Hedberg, C. W., S. J. Savarino, J.M. Besser, C. J. Paulus, V. M. Thelen, L. J. Myers, D. N. Cameron, T. J. Barrett, L. B. Kaper, and M. T. Osterholm. 1997. An outbreak of foodborne illness caused by Escherichia coli O39:NM, an agent not fitting into the existing scheme for classifying diarrheogenic E. coli. J. Infect. Dis. 176:1625-1628. 103. Hill, S. M., A. D. Phillips, and J. A. Walker-Smith. 1991. Enteropathogenic Escherichia coli and life threatening chronic diarrhoea. Gut 32:154-158. 104. Hiroyuki, A., T. Ichiro, T. Toru, O. Akiko, and S. Chihiro. 2002. Bicarbonate ion stimulates the expression of locus of enterocyte effacement-encoded genes in enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 70:3500-3509. 147 105. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989. Sitedirected mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59. 106. Hoffman, P. S., and R. A. Garduno. 1999. Surface-associated heat shock proteins of Legionella pneumophila and Helicobacter pylori: roles in pathogenesis and immunity. Infect. Dis. Obstet. Gynecol. 7:58-63. 107. Huang, A., J. Friesen, and J. L. Brunton. 1987. Characterization of a bacteriophage that carries the genes for production of Shiga-like toxin in Escherichia coli. J. Bacteriol. 169:4308-4312. 108. Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62:379-433. 109. Ide, T., S. Laarmann, L. Greune, H. Schillers, H. Oberleithner, and M. A. Schmidt. 2001. Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli. Cell. Microbiol. 3: 669-679. 110. Jackson, R. W., E. Athanassopoulos, G. Tsiamis, J. W. Mansfield, A. Sesma, D. L. Arnold, M. J. Gibbon, J. Murillo, J. D. Taylor, and A. Vivian.1999. Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola. Proc. Natl. Acad. Sci. USA 96:10875-10880. 111. Jarvis, K. G., J. A. Giron, A. E. Jerse, T. K. McDaniel, M. S. Donnenberg, and J. B. Kaper. 1995. Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. Proc. Natl. Acad. Sci. USA 92:7996-8000. 112. Jarvis, K. G., and J. B. Kaper. 1996. Secretion of extracellular proteins by enterohemorrhagic Escherichia coli via a putative type III secretion system. Infect. Immun. 64:4826-4829. 113. Jepson, M. A., S. Pellegrin, L. Peto, D. N. Banbury, A. D. Leard, H. Mellor, and B. Kenny. 2003. Synergistic roles for the Map and Tir effector molecules in mediating uptake of enteropathogenic Escherichia coli (EPEC) into non-phagocytic cells. Cell. Microbiol. 5:773-783. 148 114. Jerse, A. E., J. Yu, B. D. Tall, and J. B. Kaper. 1990. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc. Natl. Acad. Sci. USA 87:7839-7843. 115. Johnson, J. R. 1991. Virulence factors in Escherichia coli urinary tract infection. Clin. Microbiol. Rev. 4:80-128. 116. Johnson, R. P., R. C. Clarke, J. B. Wilson, S. C. Read, K. Rahn, S. A. Renwick, K. A. Sandhu, D. Alves, M. A. Karmali, H. Lior, S. A. McEwen, J. S. Spika, and C. L. Gyles. 1996. Growing concerns and recent outbreaks involving nonO157:H7 serotypes of verotoxigenic Escherichia coli. J. Food Prot. 59:11121122. 117. Kalogeraki, V. S., and S. C. Winans. 1997. Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. Gene 188:69-75. 118. Kanack, K. J., J. A. Crawford, I. Tatsuno, M. Karmali, and J. B. Kaper, 2005. SepZ/EspZ is secreted and translocated into HeLa cells by the enteropathogenic Escherichia coli type III secretion system. Infect. Immun. 73:4327-4337. 119. Kaper, J. B., and J. Hacker (eds). 1999. Pathogenicity Islands and Other Mobile Virulence Elements. Washington, DC: Am. Soc. Microbiol. 120. Karmali M. A. 1989. Infection by verocytotoxin-producing Escherichia coli. Clin. Microbiol. Rev. 2:15-38. 121. Kenny, B., and B. B. Finlay. 1995. Protein secretion by enteropathogenic Escherichia coli is essential for transducing signals to epithelial cells. Proc. Natl. Acad. Sci. USA 92:7991-7995. 122. Kenny, B., R. DeVinney, M. Stein, D. J. Reinscheid, E. A. Frey, and B. B. Finlay. 1997. Enteropathogenic Eshcerichia coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell 91:511-520. 123. Kenny, B., and M. Jepson. 2000. Targeting of an enteropathogenic Escherichia coli (EPEC) effector protein to host mitochondria. Cell. Microbiol. 2:579-590. 124. Khursigara, C., M. Abul-Milh, B. Lau, J. A. Giron, C. A. Lingwood, and D. E. Foster. 2001. Enteropathogenic Escherichia coli virulence factor bundle-forming 149 pilus has a binding specificity for phosphatidylethanolamine. Infect. Immun. 69:6573-6579. 125. Klapproth, J. M., M. S. Donnenberg, J. M. Abraham, H. L. T. Mobley, and S. P. James. 1995. Products of enteropathogenic Escherichia coli inhibit lymphocyte activation and lymphokine production. Infect.Immun. 63:2248-2254. 126. Klapproth, J. M., M. S. Donnenberg, J. M. Abraham, and S. P. James. 1996. Products of enteropathogenic E. coli inhibit lymphokine production by gastrointestinal lymphocytes. Am.J.Physiol.Gastrointest.Liver Physiol. 271:841-848. 127. Knappstein, S., T. Ide, M. A. Schmidt, and G. Heusipp. 2004. Alpha 1antitrypsin binds to and interferes with functionality of EspB from atypical and typical enteropathogenic Escherichia coli strains. Infect. Immun. 72:4344-4350. 128. Knutton, S., M. M. McConnell, B. Rowe, and A. S. McNeish. 1989a. Adhesion and ultrastructural properties of human enterotoxigenic Escherichia coli producing colonization factor antigens III and IV. Infect. Immun. 57:3364-3371. 129. Knutton, S., T. Baldwin, P. H. Williams, and A. S. McNeish. 1989b. Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect. Immun. 57:1290-1298. 130. Knutton, S., I. Rosenshine, M. J. Pallen, I. Nisan, B. C. Neves, C. Bain, C. Wolff, G. Dougan, and G. Frankel. 1998. A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells. EMBO J. 17:2166-2176. 131. Kodama, T., Y. Akeda, G. Kono, A. Takahashi, K. Imura, T. Iida, and T. Honda. 2002. The EspB protein of enterohaemorrhagic Escherichia coli interacts directly with β-catenin. Cell. Microbiol. 4:213-222. 132. Krause, G., S. Zimmermann, and L. Beutin. 2005. Investigation of domestic animals and pets as a reservoir for intimin-(eae) gene positive Escherichia coli types. Vet. Microbiol. 106:87-95. 133. Kresse, A. U., M. Rohde, and C. A. Guzman. 1999. The EspD protein of enterohemorrhagic Escherichia coli is required for the formation of bacterial 150 surface appendages and is incorporated in the cytoplasmic membranes of target cells. Infect. Immun. 67: 4834-4842. 134. Kresse, A. U., F. Beltrametti, A. Muller, F. Ebel, and C. A. Guzman. 2000. Characterization of SepL of enterohemorrhagic Escherichia coli. J. Bacteriol. 182:6490-6498. 135. Lathem, W. W., T. E. Grys, S. E. Witowski, A. G. Torres, J. B. Kaper, P. I. Tarr, and R. A. Welch. 2002. StcE, a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor. Mol. Microbiol. 45:277-288. 136. Lathem, W. W., T. Bergsbaken, and R. A. Welch. 2004. Potentiation of C1 esterase inhibitor by StcE, a metalloprotease secreted by Escherichia coli O157:H7. J. Exp. Med. 199: 1077-1087. 137. Law, D., and J. Kelly. 1995. Use of heme and hemoglobin by Escherichia coli O157 and other Shiga-like-toxin-producing E. coli serogroups. Infect. Immun. 63:700-702. 138. Leomil, L A. F. G. de Castro, G. Krause, H. Schmidt and L. Beutin. 2005. Characterization of two major groups of diarrheagenic Escherichia coli O26 strains which are globally spread in human patients and domestic animals of different species. FEMS Microbiol. Lett. 249:335-342. 139. Levine, M. M, J. B. Kaper, R. E. Black and M. L. Clements. 1983. New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development. Microbiol. Rev. 47:510-550. 140. Levine, M. M., and R. Edelman. 1984. Enteropathogenic Escherichia coli of classic serotypes associated with infant diarrhea: epidemiology and pathogenesis. Epidemiol. Rev. 6:31-51. 141. Li, M., I. Rosenshine, S. L. Tung, X. H. Wang, D. Friedberg, C. L. Hew, and K. Y. Leung, 2004. Comparative proteomic analysis of extracellular proteins of enterohemorrhagic and enteropathogenic Escherichia coli strains and their ihf and ler mutants. Appl. Environ. Microbiol. 70:5274-5282. 142. Luck, S. N., V. Bennett-Wood, R. Poon, R. M. Robins-Browne and E. L. Hartland. 2005. Invasion of epithelial cells by locus of enterocyte effacementnegative enterohemorrhagic Escherichia coli. Infect. Immun. 73: 3063-3071. 151 143. Luo, W., and M. S. Donnenberg. 2006. Analysis of the Function of Enteropathogenic Escherichia coli EspB by Random Mutagenesis. Infect. Immun. 74:810-820. 144. Luo, Y., E. A. Frey, R. A. Pfuetzner, A. L. Creagh, D. G. Knoechel, C. A. Haynes, B. B. Finlay, and N. C. Strynadka. 2000. Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex. Nature. 405:10731077. 145. Malstrom,C. and S. James. 1998. Inhibition of murine splenic and mucosal lymphocyte function by enteric bacterial products. Infect. Immun. 66:3120-3127. 146. Marchés, O., S. Wiles, F. Dziva, R. M. La Ragione, S. Schüller, A. Best, A. D. Phillips, E. L. Hartland, M. J. Woodward, M. P. Stevens, and G. Frankel. 2005. Characterization of two non-locus of enterocyte effacement-encoded type IIItranslocated effectors, NleC and NleD, in attaching and effacing pathogens. Infect. Immun. 73:8411-8417. 147. Marchés, O., T. N. Ledger, M. Boury, M. Ohara, X. Tu, F. Goffaux, J. Mainil, I. Rosenshine, M. Sugai, J. De Rycke, and E. Oswald. 2003. Enteropathogenic and enterohaemorrhagic Escherichia coli deliver a novel effector called Cif, which blocks cell cycle G2/M transition. Mol. Microbiol. 50:1553-1567. 148. Matsuzawa, T., A. Kuwae, S. Yoshida, C. Sasakawa, and A. Abe. 2004. Enteropathogenic Escherichia coli activates the RhoA signalling pathway via the stimulation of GEF-H1. EMBO J. 23:3570-3582. 149. Matsuzawa T, Kuwae A, and Abe A. 2005. Enteropathogenic Escherichia coli type III effectors EspG and EspG2 alter epithelial paracellular permeability. Infect. Immun. 73:6283-6289. 150. McDaniel, T. K., K. G. Jarvis, M. S. Donnenberg, and J. B. Kaper. 1995. A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc. Natl. Acad. Sci. USA 92:1664-1668. 151. McKee, M. L., and A. D. O’Brien. 1995. Investigation of enterohemorrhagic Escherichia coli O157:H7 adherence characteristics and invasion potential reveals a new attachment pattern shared by intestinal E. coli. Infect. Immun. 63:2070-2074. 152 152. McNamara, B. P., A. Koutsouris, C. B. O’Connell, J. P. Nougayrede, M. S. Donnenberg, and G. Hecht. 2001. Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function. J. Clin. Invest. 107:621-629. 153. Mellies, J. L., S. J. Elliott, V. Sperandio, M. S. Donnenberg, and J. B. Kaper. 1999. The Per regulon of enteropathogenic Escherichia coli: identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler). Mol. Microbiol. 33: 296-306. 154. Mellies, J. L., F. Navarro-Garcia, I. Okeke, J. Frederickson, J. P. Nataro, and J. B. Kaper. 2001. espC pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin. Infect. Immun. 69:315-324. 155. Miller, J. H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. 156. Miller, V. L., and J. J. Mekalanos. 1988. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170: 25752583. 157. Mills, M., and S. M. Payne. 1995. Genetics and regulation of heme iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7. J. Bacteriol. 177:3004-3009. 158. Moon, H. W., S. C. Whipp, R. A. Argenzio, M. M. Levine, and R. A. Giannella. 1983. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect. Immun. 41:1340-1351. 159. Mulvey, M. A., J. D. Schilling, J. J. Martinez, and S. J. Hultgren. 2000. Bad bugs and beleaguered bladders: interplay between uropathogenic Escherichia coli and innate host defenses. Proc. Natl. Acad. Sci. U S A. 97:8829-8835. 160. Mundy, R., L. Petrovska, K. Smollett, N. Simpson, R. K. Wilson, J. Yu, X. Tu, I. Rosenshine, S. Clare, G. Dougan, and G. Frankel. 2004. Identification of a novel Citrobacter rodentium type III secreted protein, EspI, and roles of this and other secreted proteins in infection. Infect. Immun. 72:2288-2302. 153 161. Mustaftschie, S., J. Vasse, and G. Truchet. 1982. Exostructures of Rhizobium meliloti. FMS Micriobiol. Lett. 13: 171-175. 162. Nakazato, G., C. Gyles, K. Ziebell, R. Keller, L. R. Trabulsi, T. A. T. Gomes, K. Irino, W. D. Da Silveira, and A. F. P. De Castro. 2004. Attaching and effacing Escherichia coli isolated from dogs in Brazil: characteristics and serotypic relationship to human enteropathogenic E. coli (EPEC). Vet. Microbiol. 101:269277. 163. Nash, H. A. 1996. The HU and IHF proteins: accessory factors for complex protein-DNA assemblies, p. 149. In E. C. C. Lin and A. C. Lynch (ed.), Regulation of Gene Expression in Escherichia coli. Austin, TX: Chapman, Hall. 164. Nataro, J. P., K. O. Maher, P. Mackie, and J. B. Kaper. 1987. Characterization of plasmids encoding the adherence factor of enteropathogenic Escherichia coli. Infect. Immun. 55:2370-2377. 165. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201. 166. Navarro-Garcia, F., A. Canizalez-Roman, B. Q. Sui, J. P. Nataro and Y. Azamar. 2004. The serine protease motif of EspC from enteropathogenic Escherichia coli produces epithelial damage by a mechanism different from that of pet toxin from enteroaggregative E. coli. Infect Immun 72: 3609-3621. 167. Naylor, S. W., J. C. Low, T. E. Besser, A. Mahajan, G. J. Gunn, M. C. Pearce, I. J. McKendrick, D. G. Smith, and D. L. Gally. 2003. Lymphoid follicle-dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli O157:H7 in the bovine host. Infect. Immun. 71: 1505–1512. 168. Neves, B.C., R. Mundy, L. Petrovska, G. Dougan, S. Knutton, and G. Frankel. 2003. CesD2 of enteropathogenic Escherichia coli is a second chaperone for the type III secretion translocator protein EspD. Infect. Immun. 71:2130-2141. 169. Newman, J. V., B. A. Zabel, S. S. Jha, and D. B. Schauer. 1999. Citrobacter rodentium espB is necessary for signal transduction and for infection of laboratory mice. Infect. Immun. 67:6019-6025. 154 170. Nougayrede, J. P., P. J. Fernandes, and M. S. Donnenberg. 2003. Adhesion of enteropathogenic Escherichia coli to host cells. Cell. Microbiol. 5:359-372. 171. O’Brien, A. D., V. L. Tesh, A. Donohue-Rolfe, M. P. Jackson, S. Olsnes, K. Sandvig, A. A. Lindberg, and G. T. Keusch. 1992. Shiga toxin: biochemistry, genetics, mode of action, and role in pathogenesis. Curr. Top. Microbial. Immunol. 180:65-94. 172. O’Connell, C. B., E. A. Creasey, S. Knutton, S. Elliott, L. J. Crowther, W. Luo, M. J. Albert, J. B. Kaper, G. Frankel, and M. S. Donnenberg. 2004. SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD. Mol. Microbiol. 52:1613-1625. 173. Oelschlaeger, T. A., T. J. Barrett, and D. J. Kopecko. 1994. Some structures and processes of human epithelial cells involved in uptake of enterohemorrhagic Escherichia coli O157:H7 strains. Infect. Immun. 62:5142-5150. 174. Oswald, E., H. Schmidt, S. Morabito, H. Karch, O. Marches, and A. Caprioli. 2000. Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli: characterization of a new intimin variant. Infect. Immun. 68:64-71. 175. Pappin, D. J. C., P. Hojrup, and A. J. Bleasby. 1993. Rapid identification of proteins by peptide-mass fingerprinting. Current. Biol. 3:327-332. 176. Paton, J.C., and A.W. Paton. 1998. Pathogenesis and diagnosis of Shiga toxinproducing Escherichia coli infections. Clin. Microbiol. Rev. 11:450-479. 177. Perna, N. T., G. F. Mayhew, G. Posfai, S. J. Elliott, M. S. Donnenberg, J. B. Kaper, and F. R. Blattner. 1998. Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 66:3810-3817. 178. Perna, N. T., G. Plunkett III, V. Burland, B. Mau, J. D. Glasner, D. J. Rose, G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J. Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis, A. Lim, E. T. Dimalanta, K. D. Potamousis, J. Apodaca, T. S. Anantharaman, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409:529-533. 179. Phillips, A. D., and G. Frankel. 2000. Intimin-mediated tissue specificity in 155 enteropathogenic Escherichia coli interaction with woman intestinal organ cultures. J. Infect. Dis. 181: 1496–1500. 180. Phillips, A. D., S. Navabpour, S. Hicks, G. Dougan, T. Wallis, and G. Frankel. 2000. Enterohaemorrhagic Escherichia coli O157:H7 target Peyer’s patches in humans and cause attaching/effacing lesions in both human and bovine intestine. Gut 47: 377–381. 181. Polotsky, Y. E., E. M. Dragunskaya, V. G. Seliverstova, T. A. Avdeeva, M. G. Chakhutinskaya, I. Ke´tyi, A. Verte´nyi, B. Ralovich, L. Emody, I. Ma´lovics, N. V. Safonova, E. S. Snigirevskaya, and E. I. Karyagina. 1977. Pathogenic effect of enterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhea. Acta Microbiol. Acad. Sci. Hung. 24:221-236. 182. Pozidis, C., A. Chalkiadaki, A. Gomez-Serrano, H. Stahlberg, I. Brown, A. P. Tampakaki, A. Lustig, G. Sianidis, A. S. Politou, A. Engel, N. J. Panopoulos, J. Mansfield, A. P. Pugsley, S. Karamanou, A. Economou. 2003. Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization. J. Biol. Chem. 278:25816-25824. 183. Prigent-Combaret, C., E. Brombacher, O. Vidal, A. Ambert, P. Lejeune, P. Landini, and C. Dorel. 2001. Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene. J. Bacteriol. 183:7213-7223. 184. Puente, J. L., D. Bieber, S. W. Ramer, W. Murray, and G. K. Schoolnik. 1996. The bundle-forming pili of enteropathogenic Escherichia coli: transcriptional regulation by environmental signals. Mol. Microbiol. 20:87-100. 185. Pugsley, A. P. 1993. The complete general secretory pathway in gram-negative bacteria. Microbiol. Rev. 57:50-108. 186. Rice, P. A., S. W. Yang, K. Mizuuchi, and H. A. Nash. 1996. Crystal structure of an IHF-DNA complex: a protein induced DNA U-turn. Cell 87:1295-1306. 187. Rosenshine, I., S. Ruschkowski, and B. B. Finlay. 1996. Expression of attaching/effacing activity by enteropathogenic Escherichia coli depends on growth phase, temperature, and protein synthesis upon contact with epithelial cells. Infect. Immun. 64:966-973. 156 188. Rubires, X., F. Saigí, N. Piqué, N. Climent, N., S. Merino, S. Albertí, J. M. Tomas, and M. Regue. 1997. A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb50 mutation of Escherichia coli K-12 derivatives. J. Bacteriol. 179:7581-7586. 189. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 190. Schauer, D. B., and S. Falkow. 1993. Attaching and effacing locus of a Citrobacter freundii biotype that causes transmissible murine colonic hyperplasia. Infect. Immun. 61:2486-2492. 191. Schembri, M. A., G. Christiansen, and P. Klemm. 2001. FimH-mediated autoaggregation of Escherichia coli. Mol. Microbiol. 41:1419-1430. 192. Schlosser-Silverman, E., M. Elgrably-Weiss, I. Rosenshine, R. Kohen, and S. Altuvia. 2000. Characterization of Escherichia coli DNA lesions generated within J774 macrophages. J. Bacteriol. 182:5225-5230. 193. Schmidt, H., H. Karch, and L. Beutin. 1994. The large-sized plasmids of enterohemorrhagic Escherichia coli O157 strains encode hemolysins which are presumably members of the E. coli a-hemolysin family. FEMS Microbiol. Lett. 117:189-196. 194. Schmidt H, Beutin L, Karch H. 1995. Molecular analysis of the plasmid-encoded hemolysin of Escherichia co/i 0157:H7 strain EDL 933. Infect. Immun. 63:1055-l 061. 195. Schmidt, H., and H. Karch. 1996a. Enterohemolytic phenotypes and genotypes of Shiga toxin-producing Escherichia co/i 0111 strains from patients with diarrhea and hemolytic uremic syndrome. J. Clin. Microbial. 34:2364-2367. 196. Schmidt, H., C. Kernbach, and H. Karch. 1996b. Analysis of the EHEC hly operon and its location in the physical map of the large plasmid of enterohaemorrhagic Escherichia coli O157:H7. Microbiology 142:907-914. 197. Schmidt H, B. Henkel, and H. Karch. 1997. A gene cluster closely related to type II secretion pathway operons of Gram-negative bacteria is located on a large 157 plasmid of enterohemorrhagic Escherichia co/f 0167 strains. FEMS Microbial. Lett. 148:265-272. 198. Scotland, S. M., S. Knutton, B. Said, and B. Rowe. 1994. Adherence to Caco-2 cells of Vero cytotoxin-producing strains of Escherichia coli belonging to serogroups other than O157, p. 257–260. In M. A. Karmali and A. G. Goglio (ed.), Recent advances in verocytotoxin-producing Escherichia coli infections. Elsevier Science B.V., Amsterdam, The Netherlands. 199. Shaw R. K., K. Smollett, J. Cleary, J. Garmendia, A. Straatman-Iwanowska, G. Frankel, and S. Knutton. 2005. Enteropathogenic Escherichia coli type III effectors EspG and EspG2 disrupt the microtubule network of intestinal epithelial cells. Infect. Immun. 73:4385-4390. 200. Shaw, R. K., S. Daniell, F. Ebel, G. Frankel, and S. Knutton. 2001. EspA filament-mediated protein translocation into red blood cells. Cell. Microbiol. 3:213222. 201. Shevchenko, A., M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68:850-858. 202. Shimizu, T., K. Shima, K. Yoshino, K. Yonezawa, T. Shimizu, and H. Hayashi. 2002. Proteome and transcriptome analysis of the virulence genes regulated by the VirR/VirS system in Clostridium perfringens. J. Bacteriol. 184:2587-2594. 203. Siegler, R. L., P. M. Griffin, T. J. Barrett, and N. A. Strockbine. 1993. Recurrent hemolytic uremic syndrome secondary to Escherichia coli O157:H7 infection. Pediatrics 91:666-668. 204. Siegler, R. L. 1995. The hemolytic uremic syndrome. Pediatr. Clin. North. Am. 42:1505-1529. 205. Sohel, I., J. L. Puente, S. W. Ramer, D. Bieber, C. Y. Wu, and G. K. Schoolnik. 1996. Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis. J. Bacteriol. 178:2613-2628. 206. Stein, M., B. Kenny, M. A. Stein, and B. B. Finlay. 1996. Characterization of EspC, a 110-kilodalton protein secreted by enteropathogenic Escherichia coli which 158 is homologous to members of the immunoglobulin A protease-like family of secreted proteins. J. Bacteriol. 178:6546-6554. 207. Stone, K. D., H. Z. Zhang, L. K. Carlson, and M. S. Donnenberg. 1996. A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus. Mol. Microbiol. 20:325-337. 208. Tacket, C. O., M. B. Sztein, G. Losonsky, A. Abe, B. B. Finlay, B. P. McNamara, G. T. Fantry, S. P. James, J. P. Nataro, M. M. Levine, and M. S. Donnenberg, 2000. Role of EspB in experimental human enteropathogenic Escherichia coli infection. Infect. Immun. 68:3689-3695. 209. Tampakaki, A., V. Fadouloglou, A. Gazi, N. Panopoulos, and M. Kokkinidis. 2004. Conserved features of type III secretion. Cell. Microbiol. 6:805-816. 210. Taylor, C. J., A. Hart, R. M. Batt, C. McDougall, and L. McLean. 1986. Ultrastructural and biochemical changes in human jejunal mucosa associated with enteropathogenic Escherichia coli (O111) infection. J. Pediatr. Gastroenterol. Nutr. 5:70-73. 211. Taylor, K. A., C. B. O’Connell, P. W. Luther, and M. S. Donnenberg. 1998. The EspB protein of enteropathogenic Escherichia coli is targeted to the cytoplasm of infected HeLa cells. Infect. Immun. 66:5501-5507. 212. Thomas, N.A., W. Deng , J. L. Puente, E. A. Frey, C. K. Yip, N. C. Strynadka, and B. B. Finlay. 2005. CesT is a multi-effector chaperone and recruitment factor required for the efficient type III secretion of both LEE- and non-LEE-encoded effectors of enteropathogenic Escherichia coli. Mol Microbiol. 57:1762-1779. 213. Tobe, T., and C. Sasakawa. 2002. Species-specific cell adhesion of enteropathogenic Escherichia coli is mediated by type IV bundle-forming pili. Cell. Microbiol. 4:29-42. 214. Tomson, F.L., V. K. Viswanathan, K. J. Kanack, R. P. Kanteti, K. V. Straub, M. Menet, J. B. Kaper, and G. Hecht. 2005. Enteropathogenic Escherichia coli EspG disrupts microtubules and in conjunction with Orf3 enhances perturbation of the tight junction barrier. Mol. Microbiol. 56:447-464. 215. Torres, A. G., and S. M. Payne. 1997. Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7. Mol. Microbiol. 23:825-833. 159 216. Totten, P. A., J. C. Lara, and S. Lory. 1990. The rpoNrpoN gene product of Pseudomonas is required for expression of diverse genes, including the flagellin gene. J. Bacteriol. 172:389-396. 217. Tu, X., I. Nisan, C. Yona, E. Hanski, and I. Rosenshine. 2003. EspH, a new cytoskeleton-modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli. Mol. Microbiol. 47:595-606. 218. Ueno, T., K. Oosawa, and S. Aizawa. 1994. Domain structures of the MS ring component protein (FliF) of the flagellar basal body of Salmonella typhimurium. J. Mol. Biol. 236:546-555. 219. Ulshen, M. H., and J. L. Rollo. 1980. Pathogenesis of Escherichia coli gastroenteritis in man—another mechanism. N. Engl. J. Med. 302:99-101. 220. Vallance B. A., C. Chan, M. L. Robertson, and B. B. Finlay. 2002. Enteropathogenic and enterohemorrhagic Escherichia coli infections: emerging themes in pathogenesis and prevention. Can. J. Gastroenterol. 16:771-778. 221. Viljanen, M. K., T. Peltola, S. Y. T. Junnila, L. Olkkonen, H. Ja¨rvinen, M. Kuistila, and P. Huovinen. 1990. Outbreak of diarrhoea due to Escherichia coli O111:B4 in schoolchildren and adults: association of Vi antigen-like reactivity. Lancet. 336:831-834. 222. Viswanathan, V. K., A. Koutsouris, S. Lukic, M. Pilkinton, I. Simonovic, M. Simonovic, and G. Hecht. 2004. Comparative analysis of EspF from enteropathogenic and enterohemorrhagic Escherichia coli in alteration of epithelial barrier. Infect. Immun. 72:3218-3227. 223. Wachter, C., C. Beinke, M. Mattes, and M. A. Schmidt. 1999. Insertion of EspD into epithelial target cell membranes by infecting enteropathogenic Escherichia coli. Mol. Microbiol. 31:1695-1707. 224. Wai, S. N., A. Takade, and K. Amako. 1996. The hydrophobic surface protein layer of enteroaggregative Escherichia coli strains. FEMS Microbiol. Lett. 135:1722. 225. Wainwright, L. A., and J. B. Kaper. 1998. EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli. Mol. Microbiol. 27:1247-1260. 160 226. Waldor M. K., and J. J. Mekalanos. 1996. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science. 272:1910-1914. 227. Warawa, J., B. B. Finlay, and B. Kenny. 1999. Type III secretion-dependent hemolytic activity of enteropathogenic Escherichia coli. Infect. Immun. 67: 55385540. 228. Wolff, C., I. Nisan, E. Hanski, G. Frankel, and I. Rosenshine. 1998. Protein translocation into host epithelial cells by infecting enteropathogenic Escherichia coli. Mol. Microbiol. 28:143-155. 229. Wu, S. X., and R. Q. Peng. 1992. Studies on an outbreak of neonatal diarrhea caused by EPEC O127:H6 with plasmid analysis restriction analysis and outer membrane protein determination. Acta Paediatr. Scand. 81:217-221. 230. Yaron, S., G. L. Kolling, L. Simon, and K. R. Matthews. 2000. Vesicle-mediated transfer of virulence genes from Escherichia coli O157:H7 to other enteric bacteria. Appl. Environ. Microbiol. 66:4414-4420. 231. Yip, C. K., T. G. Kimbrough, H. B. Felise, M. Vuckovic, N. A. Thomas, R. A. Pfuetzner, E. A. Frey, B. B. Finlay, S. I. Miller, and N. C. Strynadka. 2005. Structural characterization of the molecular platform for type III secretion system assembly. Nature. 435:702-707. 232. Yip, C. K., and N. C. Strynadka. 2006. New structural insights into the bacterial type III secretion system. Trends. Biochem. Sci. 31:223-230. 233. Yona-Nadler, C., T. Umanski, S. Aizawa, D. Friedberg, and I. Rosenshine. 2003. Integration host factor (IHF) mediates repression of flagella in enteropathogenic and enterohaemorrhagic Escherichia coli. Microbiology 149:877884. 234. Yoshioka, K, K. Yagi, N. Moriguchi. 1999. Clinical features and treatment of children with hemolytic uremic syndrome caused by enterohemorrhagic Escherichia coli O157:H7 infection: experience of an outbreak in Sakai City, 1996. Pediatr. Int. 41:223-227. 235. Yu, J., and J. B. Kaper. 1992. Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7. Mol. Microbiol. 6:411-417. 161 236. Zhang, H. Z., S. Lory, and M. S. Donnenberg. 1994. A plasmid-encoded prepilin peptidase gene from enteropathogenic Escherichia coli. J. Bacteriol. 176:6885-6891. 237. Zhang, H. Z., and M. S. Donnenberg. 1996. DsbA is required for stability of the type IV pilin of enteropathogenic Escherichia coli. Mol. Microbiol. 21:787-797. 238. Zhou, X., J. A. Giron, A. G. Torres, J. A. Crawford, E. Negrete, S. N. Vogel, and J. B. Kaper. 2003. Flagellin of enteropathogenic Escherichia coli stimulates interleukin-8 production in T84 cells. Infect. Immun. 71:2120-2129. 239. Zhu, C., J. Harel, M. Jacques, C. Desautels, M. S. Donnenberg, M. Beaudry, and J. M. Fairbrother. 1994. Virulence properties and attachingeffacing activity of Escherichia coli O45 from swine postweaning diarrhea. Infect. Immun. 62:41534159. 162 [...]... attaching and effacing (A/E) lesions on the epithelial cells EPEC and EHEC utilize the type III secretion systems (TTSSs) encoded on the locus of enterocyte effacement (LEE) to secrete and translocate several effectors into host cells The effectors may subvert the host signaling transduction pathways and consequently cause diseases To further understand the pathogenesis and dissect the virulence of EPEC and. .. the diagnostics and therapeutics for these pathogens Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are two of the most common human pathogens which constitute a significant risk to human health worldwide The present study attempts to investigate and understand the pathogenesis of EPEC and EHEC Approaches for understanding the pathogenesis of EPEC and EHEC, which... infection This study has established a proteomics platform for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnosis of these pathogens The discovery of new secreted proteins and new effectors by comparative proteomics approach has provided valuable information on new virulence factors in EPEC and EHEC These results further supported the notion that... pathogenic E coli and there have been significant advances in our understanding of the pathogenesis of E coli infection Urinary tract infections are a serious health problem affecting millions of people each year Most infections of the urinary tract are caused by one type of bacteria called 2 uropathogenic E coli (UPEC) and are by far the second most common type of bacteria infection in the body (Foxman... proteins and phage related proteins These novel proteins provided new candidates for exploiting potential virulence factors of EPEC and EHEC One of the major secreted proteins, EspB, was found to have two dominant forms in extracellular proteomics profiles of EPEC and EHEC The two forms of EspB were shown to remain unmodified at their N-terminus and they were unphosphorylated With a translocation signal... type strain and the espB mutant 79 Fig IV.4 Western blot analysis of Triton X-100 insoluble and soluble fractions of HeLa cells after infected with the EPEC wild type and the espB mutant and detected with anti-Tir antibody 79 Fig IV.5 Demonstration of the translocation of EPEC EspB proteins into live HeLa cells by using TEM-1 fusions and fluorescence microscopy 82 Fig IV.6 Autoaggregation of EPEC in... and on the EAF plasmid PerA is homologue to an AraC family of bacteria regulators and activate the expression of the down stream gene perC The production of PerC can increase the expression of the chromosomal eae (E coli attaching 11 and effacing) and espB (E coli secreted protein B) genes, as well as that of genes encoding 20 kD, 33 kD and 50 kD outer membrane proteins (Gomez-Duarte et al., 1995) The. .. utilizes the hemoglobin and heme released from lysed erythrocytes (Hantke et al., 2003) These virulence factors function jointly causing the infection and inflammatory symptoms in the urinary tract Meningitis is the inflammation of the membranes covering the brain and spinal cord Though multiple organisms may cause meningitis, E coli is the leading agent responsible for around 20% of the neonatal meningitis... 1990) These pedestal structures can extend up to 10 mm out from the epithelial cell in pseudopod like structures (Moon et al., 1983) It is observed that the composition of the A/E lesion contained high concentrations of polymerized filamentous actin (Knutton et al., 1989) This observation led to the development of the fluorescent-actin staining (FAS) test which enabled the screening of clones and mutants,... lesion observed in the ileum after oral inoculation of gnotobiotic piglets Note the intimate attachment of the bacteria to the enterocyte membrane with disruption of the apical cytoskeleton Note the loss of microvilli and the formation of a cup-like pedestal to which the bacterium is intimately attached (highlighted by circle) Reprinted from Baldini et al., 1983b 9 Fig I.2 Genetic organization of the . PROTEOMICS APPROACH ON THE IDENTIFICATION OF VIRULENCE FACTORS OF ENTEROPATHOGENIC AND ENTEROHEMORRHAGIC ESCHERICHIA COLI (EPEC AND EHEC) AND FURTHER CHARACTERIZATION OF TWO EFFECTORS: ESPB. understand their mechanisms of infection and to develop the diagnostics and therapeutics for these pathogens. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). information on new virulence factors in EPEC and EHEC. These results further supported the notion that EPEC and EHEC may use multiple virulence factors to exploit the host cells and many factors