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Volume 159, Issue Pt_11

RcsB determines the locus of enterocyte effacement (LEE) expression and adherence phenotype of O157 : H7 spinach outbreak strain TW14359 and coordinates bicarbonate-dependent LEE activation with repression of motility Free

Abstract

The 2006 US spinach outbreak of O157 : H7, characterized by unusually severe disease, has been attributed to a strain (TW14359) with enhanced pathogenic potential, including elevated virulence gene expression, robust adherence and the presence of novel virulence factors. This study proposes a mechanism for the unique virulence expression and adherence phenotype of this strain, and further expands the role for regulator RcsB in control of the locus of enterocyte effacement (LEE) pathogenicity island. Proteomic analysis of TW14359 revealed a virulence proteome consistent with previous transcriptome studies that included elevated levels of the LEE regulatory protein Ler and type III secretion system (T3SS) proteins, secreted T3SS effectors and Shiga toxin 2. Basal levels of the LEE activator and Rcs phosphorelay response regulator, RcsB, were increased in strain TW14359 relative to O157 : H7 strain Sakai. Deletion of eliminated inherent differences between these strains in expression, and in T3SS-dependent adherence. A reciprocating regulatory pathway involving RcsB and LEE-encoded activator GrlA was identified and predicted to co-ordinate LEE activation with repression of the flagellar regulator and motility. Overexpression of was shown to increase RcsB levels, but did not alter expression from promoters driving transcription. Expression of and RcsB was determined to increase in response to physiological levels of bicarbonate, and bicarbonate-dependent stimulation of the LEE was shown to be dependent on an intact Rcs system and activator . The results of this study significantly broaden the role for RcsB in enterohaemorrhagic virulence regulation.

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2013-11-01
2024-04-26
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References

  1. Abe A., Kenny B., Stein M., Finlay B. B.( 1997). Characterization of two virulence proteins secreted by rabbit enteropathogenic Escherichia coli, EspA and EspB, whose maximal expression is sensitive to host body temperature. Infect Immun 65:3547–3555[PubMed]
     [Google Scholar]
  2. Abe H., Tatsuno I., Tobe T., Okutani A., Sasakawa C.( 2002). Bicarbonate ion stimulates the expression of locus of enterocyte effacement-encoded genes in enterohemorrhagic Escherichia coli O157 : H7. Infect Immun 70:3500–3509 [View Article][PubMed]
     [Google Scholar]
  3. Abu-Ali G. S., Ouellette L. M., Henderson S. T., Whittam T. S., Manning S. D.( 2010a). Differences in adherence and virulence gene expression between two outbreak strains of enterohaemorrhagic Escherichia coli O157 : H7. Microbiology 156:408–419 [View Article][PubMed]
     [Google Scholar]
  4. Abu-Ali G. S., Ouellette L. M., Henderson S. T., Lacher D. W., Riordan J. T., Whittam T. S., Manning S. D.( 2010b). Increased adherence and expression of virulence genes in a lineage of Escherichia coli O157 : H7 commonly associated with human infections. PLoS ONE 5:e10167 [View Article][PubMed]
     [Google Scholar]
  5. Bergholz T. M., Wick L. M., Qi W., Riordan J. T., Ouellette L. M., Whittam T. S.( 2007a). Global transcriptional response of Escherichia coli O157 : H7 to growth transitions in glucose minimal medium. BMC Microbiol 7:97 [View Article][PubMed]
     [Google Scholar]
  6. Bergholz T. M., Wick L. M., Qi W., Riordan J. T., Ouellette L. M., Whittam T. S.( 2007b). Global transcriptional response of Escherichia coli O157 : H7 to growth transitions in glucose minimal medium. BMC Microbiol 7:97 [View Article][PubMed]
     [Google Scholar]
  7. Bernadac A., Gavioli M., Lazzaroni J. C., Raina S., Lloubès R.( 1998). Escherichia coli tol-pal mutants form outer membrane vesicles. J Bacteriol 180:4872–4878[PubMed]
     [Google Scholar]
  8. Boulanger A., Francez-Charlot A., Conter A., Castanié-Cornet M. P., Cam K., Gutierrez C.( 2005). Multistress regulation in Escherichia coli: expression of osmB involves two independent promoters responding either to σS or to the RcsCDB His-Asp phosphorelay. J Bacteriol 187:3282–3286 [View Article][PubMed]
     [Google Scholar]
  9. Cascales E., Gavioli M., Sturgis J. N., Lloubès R.( 2000). Proton motive force drives the interaction of the inner membrane TolA and outer membrane pal proteins in Escherichia coli. Mol Microbiol 38:904–915 [View Article][PubMed]
     [Google Scholar]
  10. Castanié-Cornet M. P., Cam K., Bastiat B., Cros A., Bordes P., Gutierrez C.( 2010). Acid stress response in Escherichia coli: mechanism of regulation of gadA transcription by RcsB and GadE. Nucleic Acids Res 38:3546–3554 [View Article][PubMed]
     [Google Scholar]
  11. Chang A. C., Cohen S. N.( 1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156[PubMed]
     [Google Scholar]
  12. Clavel T., Lazzaroni J. C., Vianney A., Portalier R.( 1996). Expression of the tolQRA genes of Escherichia coli K-12 is controlled by the RcsC sensor protein involved in capsule synthesis. Mol Microbiol 19:19–25 [View Article][PubMed]
     [Google Scholar]
  13. Creasey E. A., Delahay R. M., Daniell S. J., Frankel G.( 2003). Yeast two-hybrid system survey of interactions between LEE-encoded proteins of enteropathogenic Escherichia coli. Microbiology 149:2093–2106 [View Article][PubMed]
     [Google Scholar]
  14. Datsenko K. A., Wanner B. L.( 2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645 [View Article][PubMed]
     [Google Scholar]
  15. Deng W., Puente J. L., Gruenheid S., Li Y., Vallance B. A., Vázquez A., Barba J., Ibarra J. A., O’Donnell P.& other authors ( 2004). Dissecting virulence: systematic and functional analyses of a pathogenicity island. Proc Natl Acad Sci U S A 101:3597–3602 [View Article][PubMed]
     [Google Scholar]
  16. Elliott S. J., Wainwright L. A., McDaniel T. K., Jarvis K. G., Deng Y. K., Lai L. C., McNamara B. P., Donnenberg M. S., Kaper J. B.( 1998). The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol Microbiol 28:1–4 [View Article][PubMed]
     [Google Scholar]
  17. Feldman M.( 1983). Gastric bicarbonate secretion in humans. Effect of pentagastrin, bethanechol, and 11,16,16-trimethyl prostaglandin E2. J Clin Invest 72:295–303 [View Article][PubMed]
     [Google Scholar]
  18. Francez-Charlot A., Laugel B., Van Gemert A., Dubarry N., Wiorowski F., Castanié-Cornet M. P., Gutierrez C., Cam K.( 2003). RcsCDB His-Asp phosphorelay system negatively regulates the flhDC operon in Escherichia coli. Mol Microbiol 49:823–832 [View Article][PubMed]
     [Google Scholar]
  19. Friedberg D., Umanski T., Fang Y., Rosenshine I.( 1999). Hierarchy in the expression of the locus of enterocyte effacement genes of enteropathogenic Escherichia coli. Mol Microbiol 34:941–952 [View Article][PubMed]
     [Google Scholar]
  20. Gauger E. J., Leatham M. P., Mercado-Lubo R., Laux D. C., Conway T., Cohen P. S.( 2007). Role of motility and the flhDC operon in Escherichia coli MG1655 colonization of the mouse intestine. Infect Immun 75:3315–3324 [View Article][PubMed]
     [Google Scholar]
  21. Grant A. J., Farris M., Alefounder P., Williams P. H., Woodward M. J., O’Connor C. D.( 2003). Co-ordination of pathogenicity island expression by the BipA GTPase in enteropathogenic Escherichia coli (EPEC). Mol Microbiol 48:507–521 [View Article][PubMed]
     [Google Scholar]
  22. Guzman L. M., Belin D., Carson M. J., Beckwith J.( 1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130[PubMed]
     [Google Scholar]
  23. Huang L. H., Syu W. J.( 2008). GrlA of enterohemorrhagic Escherichia coli O157 : H7 activates LEE1 by binding to the promoter region. J Microbiol Immunol Infect 41:9–16[PubMed]
     [Google Scholar]
  24. Iyoda S., Watanabe H.( 2004). Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157 : H7 to HEp-2 cells. Microbiology 150:2357–2371 [View Article][PubMed]
     [Google Scholar]
  25. Iyoda S., Koizumi N., Satou H., Lu Y., Saitoh T., Ohnishi M., Watanabe H.( 2006). The GrlR-GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli. J Bacteriol 188:5682–5692 [View Article][PubMed]
     [Google Scholar]
  26. Kenny B., Abe A., Stein M., Finlay B. B.( 1997). Enteropathogenic Escherichia coli protein secretion is induced in response to conditions similar to those in the gastrointestinal tract. Infect Immun 65:2606–2612[PubMed]
     [Google Scholar]
  27. Krin E., Danchin A., Soutourina O.( 2010). RcsB plays a central role in H-NS-dependent regulation of motility and acid stress resistance in Escherichia coli. Res Microbiol 161:363–371 [View Article][PubMed]
     [Google Scholar]
  28. Kulasekara B. R., Jacobs M., Zhou Y., Wu Z., Sims E., Saenphimmachak C., Rohmer L., Ritchie J. M., Radey M.& other authors ( 2009). Analysis of the genome of the Escherichia coli O157 : H7 2006 spinach-associated outbreak isolate indicates candidate genes that may enhance virulence. Infect Immun 77:3713–3721 [View Article][PubMed]
     [Google Scholar]
  29. Laaberki M. H., Janabi N., Oswald E., Repoila F.( 2006). Concert of regulators to switch on LEE expression in enterohemorrhagic Escherichia coli O157 : H7: interplay between Ler, GrlA, HNS and RpoS. Int J Med Microbiol 296:197–210 [View Article][PubMed]
     [Google Scholar]
  30. Leatham M. P., Stevenson S. J., Gauger E. J., Krogfelt K. A., Lins J. J., Haddock T. L., Autieri S. M., Conway T., Cohen P. S.( 2005). Mouse intestine selects nonmotile flhDC mutants of Escherichia coli MG1655 with increased colonizing ability and better utilization of carbon sources. Infect Immun 73:8039–8049 [View Article][PubMed]
     [Google Scholar]
  31. Lio J. C., Syu W. J.( 2004). Identification of a negative regulator for the pathogenicity island of enterohemorrhagic Escherichia coli O157 : H7. J Biomed Sci 11:855–863[PubMed]
     [Google Scholar]
  32. Livak K. J., Schmittgen T. D.( 2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods 25:402–408 [View Article][PubMed]
     [Google Scholar]
  33. Manning S. D., Motiwala A. S., Springman A. C., Qi W., Lacher D. W., Ouellette L. M., Mladonicky J. M., Somsel P., Rudrik J. T.& other authors ( 2008). Variation in virulence among clades of Escherichia coli O157 : H7 associated with disease outbreaks. Proc Natl Acad Sci U S A 105:4868–4873 [View Article][PubMed]
     [Google Scholar]
  34. McDaniel T. K., Kaper J. B.( 1997). A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. Mol Microbiol 23:399–407 [View Article][PubMed]
     [Google Scholar]
  35. McKee M. L., O’Brien A. D.( 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[PubMed]
     [Google Scholar]
  36. Mellies J. L., Barron A. M., Carmona A. M.( 2007). Enteropathogenic and enterohemorrhagic Escherichia coli virulence gene regulation. Infect Immun 75:4199–4210 [View Article][PubMed]
     [Google Scholar]
  37. Michino H., Araki K., Minami S., Takaya S., Sakai N., Miyazaki M., Ono A., Yanagawa H.( 1999). Massive outbreak of Escherichia coli O157 : H7 infection in schoolchildren in Sakai City, Japan, associated with consumption of white radish sprouts. Am J Epidemiol 150:787–796 [View Article][PubMed]
     [Google Scholar]
  38. Miller J.( 1972). Assay of β-galactosidase. Experiments in Molecular Genetics352–355 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  39. Mitra A., Fay P. A., Morgan J. K., Vendura K. W., Versaggi S. L., Riordan J. T.( 2012). Sigma factor N, liaison to an ntrC and rpoS dependent regulatory pathway controlling acid resistance and the LEE in enterohemorrhagic Escherichia coli. PLoS ONE 7:e46288 [View Article][PubMed]
     [Google Scholar]
  40. Murphy K. C., Campellone K. G.( 2003). Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol Biol 4:11 [View Article][PubMed]
     [Google Scholar]
  41. Nataro J. P., Kaper J. B.( 1998). Diarrheagenic Escherichia coli. Clin Microbiol Rev 11:142–201[PubMed]
     [Google Scholar]
  42. Neupane M., Abu-Ali G. S., Mitra A., Lacher D. W., Manning S. D., Riordan J. T.( 2011). Shiga toxin 2 overexpression in Escherichia coli O157 : H7 strains associated with severe human disease. Microb Pathog 51:466–470 [View Article][PubMed]
     [Google Scholar]
  43. Perna N. T., Mayhew G. F., Pósfai G., Elliott S., Donnenberg M. S., Kaper J. B., Blattner F. R.( 1998). Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157 : H7. Infect Immun 66:3810–3817[PubMed]
     [Google Scholar]
  44. Pescaretti M. L., Morero R., Delgado M. A.( 2009). Identification of a new promoter for the response regulator rcsB expression in Salmonella enterica serovar Typhimurium. FEMS Microbiol Lett 300:165–173 [View Article][PubMed]
     [Google Scholar]
  45. Rangel J. M., Sparling P. H., Crowe C., Griffin P. M., Swerdlow D. L.( 2005). Epidemiology of Escherichia coli O157 : H7 outbreaks, United States, 1982–2002. Emerg Infect Dis 11:603–609 [View Article][PubMed]
     [Google Scholar]
  46. Riley L. W., Remis R. S., Helgerson S. D., McGee H. B., Wells J. G., Davis B. R., Hebert R. J., Olcott E. S., Johnson L. M.& other authors ( 1983). Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med 308:681–685 [View Article][PubMed]
     [Google Scholar]
  47. Riordan J. T., Tietjen J. A., Walsh C. W., Gustafson J. E., Whittam T. S.( 2010). Inactivation of alternative sigma factor 54 (RpoN) leads to increased acid resistance, and alters locus of enterocyte effacement (LEE) expression in Escherichia coli O157 : H7. Microbiology 156:719–730 [View Article][PubMed]
     [Google Scholar]
  48. Rivera F. E., Miller H. K., Kolar S. L., Stevens S. M. Jr, Shaw L. N.( 2012). The impact of CodY on virulence determinant production in community-associated methicillin-resistant Staphylococcus aureus. Proteomics 12:263–268 [View Article][PubMed]
     [Google Scholar]
  49. Schauer D. B., Falkow S.( 1993). Attaching and effacing locus of a Citrobacter freundii biotype that causes transmissible murine colonic hyperplasia. Infect Immun 61:2486–2492[PubMed]
     [Google Scholar]
  50. Shin S., Castanie-Cornet M. P., Foster J. W., Crawford J. A., Brinkley C., Kaper J. B.( 2001). An activator of glutamate decarboxylase genes regulates the expression of enteropathogenic Escherichia coli virulence genes through control of the plasmid-encoded regulator, Per. Mol Microbiol 41:1133–1150 [View Article][PubMed]
     [Google Scholar]
  51. Simons R. W., Houman F., Kleckner N.( 1987). Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53:85–96 [View Article][PubMed]
     [Google Scholar]
  52. Sperandio V., Mellies J. L., Nguyen W., Shin S., Kaper J. B.( 1999). Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli. Proc Natl Acad Sci U S A 96:15196–15201 [View Article][PubMed]
     [Google Scholar]
  53. Stout V., Torres-Cabassa A., Maurizi M. R., Gutnick D., Gottesman S.( 1991). RcsA, an unstable positive regulator of capsular polysaccharide synthesis. J Bacteriol 173:1738–1747[PubMed]
     [Google Scholar]
  54. Sturny R., Cam K., Gutierrez C., Conter A.( 2003). NhaR and RcsB independently regulate the osmCp1 promoter of Escherichia coli at overlapping regulatory sites. J Bacteriol 185:4298–4304 [View Article][PubMed]
     [Google Scholar]
  55. Tauschek M., Yang J., Hocking D., Azzopardi K., Tan A., Hart E., Praszkier J., Robins-Browne R. M.( 2010). Transcriptional analysis of the grlRA virulence operon from Citrobacter rodentium. J Bacteriol 192:3722–3734 [View Article][PubMed]
     [Google Scholar]
  56. Tobe T., Ando H., Ishikawa H., Abe H., Tashiro K., Hayashi T., Kuhara S., Sugimoto N.( 2005). Dual regulatory pathways integrating the RcsC-RcsD-RcsB signalling system control enterohaemorrhagic Escherichia coli pathogenicity. Mol Microbiol 58:320–333 [View Article][PubMed]
     [Google Scholar]
  57. Tree J. J., Wolfson E. B., Wang D., Roe A. J., Gally D. L.( 2009). Controlling injection: regulation of type III secretion in enterohaemorrhagic Escherichia coli. Trends Microbiol 17:361–370 [View Article][PubMed]
     [Google Scholar]
  58. Umanski T., Rosenshine I., Friedberg D.( 2002). Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli. Microbiology 148:2735–2744[PubMed]
     [Google Scholar]
  59. Uzzau S., Figueroa-Bossi N., Rubino S., Bossi L.( 2001). Epitope tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci U S A 98:15264–15269 [View Article][PubMed]
     [Google Scholar]
  60. Venkatesh G. R., Kembou Koungni F. C., Paukner A., Stratmann T., Blissenbach B., Schnetz K.( 2010). BglJ-RcsB heterodimers relieve repression of the Escherichia coli bgl operon by H-NS. J Bacteriol 192:6456–6464 [View Article][PubMed]
     [Google Scholar]
  61. Vizcaíno J. A., Côté R., Reisinger F., Foster J. M., Mueller M., Rameseder J., Hermjakob H., Martens L.( 2009). A guide to the Proteomics Identifications Database proteomics data repository. Proteomics 9:4276–4283 [View Article][PubMed]
     [Google Scholar]
  62. Wehland M., Bernhard F.( 2000). The RcsAB box. Characterization of a new operator essential for the regulation of exopolysaccharide biosynthesis in enteric bacteria. J Biol Chem 275:7013–7020 [View Article][PubMed]
     [Google Scholar]
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RcsB determines the locus of enterocyte effacement (LEE) expression and adherence phenotype of Escherichia coli O157 : H7 spinach outbreak strain TW14359 and coordinates bicarbonate-dependent LEE activation with repression of motility
Microbiology 159, 2342 (2013); https://doi.org/10.1099/mic.0.070201-0
/content/journal/micro/10.1099/mic.0.070201-0
/content/journal/micro/10.1099/mic.0.070201-0
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