1887

Abstract

Shiga toxin-producing (STEC) cause severe human infections and their virulence abilities are not fully understood. Cattle are a key reservoir, and the terminal rectum is the principal site of bacterial carriage. Most STEC possess a pathogenicity island termed the locus of enterocyte effacement (LEE). Nonetheless, LEE-negative STEC have been associated with disease. We found that invasion of LEE-positive and LEE-negative strains was higher for human enterocytic cell lines and for undifferentiated Caco-2 cells. Intracellular bacteria could be detected as early as 5 min after infection and transmission electron microscopy showed bacteria within membrane-bound vacuoles. STEC invasion depended on actin microfilaments and protein kinases. Scanning electron microscopy revealed that bacterial entry was not associated with membrane ruffling. Absence of macropinocytosis or actin rearrangement at the entry points suggests a zipper-like entry mechanism. Disruption of the tight junction by EGTA enhanced invasion of Caco-2 monolayers, and bacterial invasion mostly proceeded through the basolateral pole of enterocytes. STEC persisted within Caco-2 cells for up to 96 h without cell death and bacterial viability increased after 48 h, suggesting intracellular multiplication. The relatively harmless intracellular localization of STEC can be an efficient strategy to prevent its elimination from the bovine intestinal tract.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.064204-0
2013-08-01
2019-11-21
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/8/1683.html?itemId=/content/journal/micro/10.1099/mic.0.064204-0&mimeType=html&fmt=ahah

References

  1. Barker J., Humphrey T. J., Brown M. W.. ( 1999;). Survival of Escherichia coli O157 in a soil protozoan: implications for disease. . FEMS Microbiol Lett 173:, 291–295. [CrossRef][PubMed]
    [Google Scholar]
  2. Basson M. D., Emenaker N. J., Rashid Z.. ( 1998;). Effects of modulation of tyrosine phosphorylation on brush border enzyme activity in human Caco-2 intestinal epithelial cells. . Cell Tissue Res 292:, 553–562. [CrossRef][PubMed]
    [Google Scholar]
  3. Beaulieu J. F.. ( 1992;). Differential expression of the VLA family of integrins along the crypt-villus axis in the human small intestine. . J Cell Sci 102:, 427–436.[PubMed]
    [Google Scholar]
  4. Beutin L., Kaulfuss S., Herold S., Oswald E., Schmidt H.. ( 2005;). Genetic analysis of enteropathogenic and enterohemorrhagic Escherichia coli serogroup O103 strains by molecular typing of virulence and housekeeping genes and pulsed-field gel electrophoresis. . J Clin Microbiol 43:, 1552–1563. [CrossRef][PubMed]
    [Google Scholar]
  5. Bono J. L., Keen J. E., Clawson M. L., Durso L. M., Heaton M. P., Laegreid W. W.. ( 2007;). Association of Escherichia coli O157:H7 tir polymorphisms with human infection. . BMC Infect Dis 7:, 98–105. [CrossRef][PubMed]
    [Google Scholar]
  6. Buchmeier N. A., Libby S. J.. ( 1997;). Dynamics of growth and death within a Salmonella typhimurium population during infection of macrophages. . Can J Microbiol 43:, 29–34. [CrossRef][PubMed]
    [Google Scholar]
  7. Caprioli A., Morabito S., Brugère H., Oswald E.. ( 2005;). Enterohaemorrhagic Escherichia coli: emerging issues on virulence and modes of transmission. . Vet Res 36:, 289–311. [CrossRef][PubMed]
    [Google Scholar]
  8. Chong Y., Fitzhenry R., Heuschkel R., Torrente F., Frankel G., Phillips A. D.. ( 2007;). Human intestinal tissue tropism in Escherichia coli O157 : H7–initial colonization of terminal ileum and Peyer’s patches and minimal colonic adhesion ex vivo. . Microbiology 153:, 794–802. [CrossRef][PubMed]
    [Google Scholar]
  9. Clements A., Smollett K., Lee S. F., Hartland E. L., Lowe M., Frankel G.. ( 2011;). EspG of enteropathogenic and enterohemorrhagic E. coli binds the Golgi matrix protein GM130 and disrupts the Golgi structure and function. . Cell Microbiol 13:, 1429–1439. [CrossRef][PubMed]
    [Google Scholar]
  10. Cobbold R. N., Hancock D. D., Rice D. H., Berg J., Stilborn R., Hovde C. J., Besser T. E.. ( 2007;). Rectoanal junction colonization of feedlot cattle by Escherichia coli O157:H7 and its association with supershedders and excretion dynamics. . Appl Environ Microbiol 73:, 1563–1568. [CrossRef][PubMed]
    [Google Scholar]
  11. Cossart P., Sansonetti P. J.. ( 2004;). Bacterial invasion: the paradigms of enteroinvasive pathogens. . Science 304:, 242–248. [CrossRef][PubMed]
    [Google Scholar]
  12. Dean-Nystrom E. A., Stoffregen W. C., Bosworth B. T., Moon H. W., Pohlenz J. F.. ( 2008;). Early attachment sites for Shiga-toxigenic Escherichia coli O157:H7 in experimentally inoculated weaned calves. . Appl Environ Microbiol 74:, 6378–6384. [CrossRef][PubMed]
    [Google Scholar]
  13. Denizot F., Lang R.. ( 1986;). Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. . J Immunol Methods 89:, 271–277. [CrossRef][PubMed]
    [Google Scholar]
  14. Donnenberg M. S., Donohue-Rolfe A., Keusch G. T.. ( 1990;). A comparison of HEp-2 cell invasion by enteropathogenic and enteroinvasive Escherichia coli. . FEMS Microbiol Lett 69:, 83–86. [CrossRef][PubMed]
    [Google Scholar]
  15. Dziva F., Mahajan A., Cameron P., Currie C., McKendrick I. J., Wallis T. S., Smith D. G. E., Stevens M. P.. ( 2007;). EspP, a Type V-secreted serine protease of enterohaemorrhagic Escherichia coli O157:H7, influences intestinal colonization of calves and adherence to bovine primary intestinal epithelial cells. . FEMS Microbiol Lett 271:, 258–264. [CrossRef][PubMed]
    [Google Scholar]
  16. Finlay B. B., Falkow S.. ( 1997;). Common themes in microbial pathogenicity revisited. . Microbiol Mol Biol Rev 61:, 136–169.[PubMed]
    [Google Scholar]
  17. Fitzhenry R. J., Stevens M. P., Jenkins C., Wallis T. S., Heuschkel R., Murch S., Thomson M., Frankel G., Phillips A. D.. ( 2003;). Human intestinal tissue tropism of intimin epsilon O103 Escherichia coli. . FEMS Microbiol Lett 218:, 311–316. [CrossRef][PubMed]
    [Google Scholar]
  18. Garcia-del Portillo F., Finlay B. B.. ( 1994;). Salmonella invasion of nonphagocytic cells induces formation of macropinosomes in the host cell. . Infect Immun 62:, 4641–4645.[PubMed]
    [Google Scholar]
  19. Grauke L. J., Kudva I. T., Yoon J. W., Hunt C. W., Williams C. J., Hovde C. J.. ( 2002;). Gastrointestinal tract location of Escherichia coli O157:H7 in ruminants. . Appl Environ Microbiol 68:, 2269–2277. [CrossRef][PubMed]
    [Google Scholar]
  20. Kaper J. B., Nataro J. P., Mobley H. L.. ( 2004;). Pathogenic Escherichia coli. . Nat Rev Microbiol 2:, 123–140. [CrossRef][PubMed]
    [Google Scholar]
  21. Karch H., Tarr P. I., Bielaszewska M.. ( 2005;). Enterohaemorrhagic Escherichia coli in human medicine. . Int J Med Microbiol 295:, 405–418. [CrossRef][PubMed]
    [Google Scholar]
  22. Kim J., Nietfeldt J., Benson A. K.. ( 1999;). Octamer-based genome scanning distinguishes a unique subpopulation of Escherichia coli O157:H7 strains in cattle. . Proc Natl Acad Sci U S A 96:, 13288–13293. [CrossRef][PubMed]
    [Google Scholar]
  23. Lim J. Y., Yoon J. W., Hovde C. J.. ( 2010;). A brief overview of Escherichia coli O157:H7 and its plasmid O157. . J Microbiol Biotechnol 20:, 5–14.[PubMed]
    [Google Scholar]
  24. Low J. C., McKendrick I. J., McKechnie C., Fenlon D., Naylor S. W., Currie C., Smith D. G., Allison L., Gally D. L.. ( 2005;). Rectal carriage of enterohemorrhagic Escherichia coli O157 in slaughtered cattle. . Appl Environ Microbiol 71:, 93–97. [CrossRef][PubMed]
    [Google Scholar]
  25. Lowe R. M. S., Baines D., Selinger L. B., Thomas J. E., McAllister T. A., Sharma R.. ( 2009;). Escherichia coli O157:H7 strain origin, lineage, and Shiga toxin 2 expression affect colonization of cattle. . Appl Environ Microbiol 75:, 5074–5081. [CrossRef][PubMed]
    [Google Scholar]
  26. Luck S. N., Bennett-Wood V., Poon R., Robins-Browne R. M., Hartland E. L.. ( 2005;). Invasion of epithelial cells by locus of enterocyte effacement-negative enterohemorrhagic Escherichia coli. . Infect Immun 73:, 3063–3071. [CrossRef][PubMed]
    [Google Scholar]
  27. 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. [CrossRef][PubMed]
    [Google Scholar]
  28. McCormick B. A., Nusrat A., Parkos C. A., D’Andrea L., Hofman P. M., Carnes D., Liang T. W., Madara J. L.. ( 1997;). Unmasking of intestinal epithelial lateral membrane beta1 integrin consequent to transepithelial neutrophil migration in vitro facilitates inv-mediated invasion by Yersinia pseudotuberculosis. . Infect Immun 65:, 1414–1421.[PubMed]
    [Google Scholar]
  29. McCracken V. J., Lorenz R. G.. ( 2001;). The gastrointestinal ecosystem: a precarious alliance among epithelium, immunity and microbiota. . Cell Microbiol 3:, 1–11. [CrossRef][PubMed]
    [Google Scholar]
  30. 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]
  31. McNally A., Roe A. J., Simpson S., Thomson-Carter F. M., Hoey D. E., Currie C., Chakraborty T., Smith D. G., Gally D. L.. ( 2001;). Differences in levels of secreted locus of enterocyte effacement proteins between human disease-associated and bovine Escherichia coli O157. . Infect Immun 69:, 5107–5114. [CrossRef][PubMed]
    [Google Scholar]
  32. Meichtri L., Miliwebsky E., Gioffré A., Chinen I., Baschkier A., Chillemi G., Guth B. E. C., Masana M. O., Cataldi A.. & other authors ( 2004;). Shiga toxin-producing Escherichia coli in healthy young beef steers from Argentina: prevalence and virulence properties. . Int J Food Microbiol 96:, 189–198. [CrossRef][PubMed]
    [Google Scholar]
  33. Nart P., Naylor S. W., Huntley J. F., McKendrick I. J., Gally D. L., Low J. C.. ( 2008;). Responses of cattle to gastrointestinal colonization by Escherichia coli O157:H7. . Infect Immun 76:, 5366–5372. [CrossRef][PubMed]
    [Google Scholar]
  34. Naylor S. W., Low J. C., Besser T. E., Mahajan A., Gunn G. J., Pearce M. C., McKendrick I. J., Smith D. G., Gally D. L.. ( 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. [CrossRef][PubMed]
    [Google Scholar]
  35. Naylor S. W., Roe A. J., Nart P., Spears K., Smith D. G. E., Low J. C., Gally D. L.. ( 2005;). Escherichia coli O157 : H7 forms attaching and effacing lesions at the terminal rectum of cattle and colonization requires the LEE4 operon. . Microbiology 151:, 2773–2781. [CrossRef][PubMed]
    [Google Scholar]
  36. Oelschlaeger T. A., Barrett T. J., Kopecko D. J.. ( 1994;). Some structures and processes of human epithelial cells involved in uptake of enterohemorrhagic Escherichia coli O157:H7 strains. . Infect Immun 62:, 5142–5150.[PubMed]
    [Google Scholar]
  37. Paton J. C., Paton A. W.. ( 1998;). Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. . Clin Microbiol Rev 11:, 450–479.[PubMed]
    [Google Scholar]
  38. Perdomo J. J., Gounon P., Sansonetti P. J.. ( 1994;). Polymorphonuclear leukocyte transmigration promotes invasion of colonic epithelial monolayer by Shigella flexneri. . J Clin Invest 93:, 633–643. [CrossRef][PubMed]
    [Google Scholar]
  39. Pizarro-Cerdá J., Cossart P.. ( 2006;). Bacterial adhesion and entry into host cells. . Cell 124:, 715–727. [CrossRef][PubMed]
    [Google Scholar]
  40. 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. [CrossRef][PubMed]
    [Google Scholar]
  41. Rogers T. J., Thorpe C. M., Paton A. W., Paton J. C.. ( 2012;). Role of lipid rafts and flagellin in invasion of colonic epithelial cells by Shiga-toxigenic Escherichia coli O113:H21. . Infect Immun 80:, 2858–2867. [CrossRef][PubMed]
    [Google Scholar]
  42. Rosa A. C. P., Vieira M. A. M., Tibana A., Gomes T. A. T., Andrade J. R. C.. ( 2001;). Interactions of Escherichia coli strains of non-EPEC serogroups that carry eae and lack the EAF and stx gene sequences with undifferentiated and differentiated intestinal human Caco-2 cells. . FEMS Microbiol Lett 200:, 117–122. [CrossRef][PubMed]
    [Google Scholar]
  43. Scheutz F., Nielsen E. M., Frimodt-Møller J., Boisen N., Morabito S., Tozzoli R., Nataro J. P., Caprioli A.. ( 2011;). Characteristics of the enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli O104:H4 strain causing the outbreak of haemolytic uraemic syndrome in Germany, May to June 2011. . Euro Surveill 16:, 19889.[PubMed]
    [Google Scholar]
  44. Sharma R., Stanford K., Louie M., Munns K., John S. J., Zhang Y., Gannon V., Chui L., Read R.. & other authors ( 2009;). Escherichia coli O157:H7 lineages in healthy beef and dairy cattle and clinical human cases in Alberta, Canada. . J Food Prot 72:, 601–607.[PubMed]
    [Google Scholar]
  45. Sheng H., Wang J., Lim J. Y., Davitt C., Minnich S. A., Hovde C. J.. ( 2011;). Internalization of Escherichia coli O157:H7 by bovine rectal epithelial cells. . Front Microbiol 2:, 1–10. [CrossRef][PubMed]
    [Google Scholar]
  46. Stevens M. P., van Diemen P. M., Dziva F., Jones P. W., Wallis T. S.. ( 2002;). Options for the control of enterohaemorrhagic Escherichia coli in ruminants. . Microbiology 148:, 3767–3778.[PubMed]
    [Google Scholar]
  47. Veiga E., Guttman J. A., Bonazzi M., Boucrot E., Toledo-Arana A., Lin A. E., Enninga J., Pizarro-Cerdá J., Finlay B. B.. & other authors ( 2007;). Invasive and adherent bacterial pathogens co-opt host clathrin for infection. . Cell Host Microbe 2:, 340–351. [CrossRef][PubMed]
    [Google Scholar]
  48. Vlisidou I., Dziva F., La Ragione R. M., Best A., Garmendia J., Hawes P., Monaghan P., Cawthraw S. A., Frankel G.. & other authors ( 2006;). Role of intimin-Tir interactions and the Tir-cytoskeleton coupling protein in the colonization of calves and lambs by Escherichia coli O157:H7. . Infect Immun 74:, 758–764. [CrossRef][PubMed]
    [Google Scholar]
  49. Yamamoto D., Hernandes R. T., Blanco M., Greune L., Schmidt M. A., Carneiro S. M., Dahbi G., Blanco J. E., Mora A.. & other authors ( 2009;). Invasiveness as a putative additional virulence mechanism of some atypical Enteropathogenic Escherichia coli strains with different uncommon intimin types. . BMC Microbiol 9:, 146–156. [CrossRef][PubMed]
    [Google Scholar]
  50. Yang Z., Kovar J., Kim J., Nietfeldt J., Smith D. R., Moxley R. A., Olson M. E., Fey P. D., Benson A. K.. ( 2004;). Identification of common subpopulations of non-sorbitol-fermenting, beta-glucuronidase-negative Escherichia coli O157:H7 from bovine production environments and human clinical samples. . Appl Environ Microbiol 70:, 6846–6854. [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.064204-0
Loading
/content/journal/micro/10.1099/mic.0.064204-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error