Invasion of HEp-2 cells by strains of of different virulence in relation to gastroenteritis Free

Abstract

Summary

Experiments to measure the invasiveness of seven strains of for HEp-2 cells showed that high inocula (100 bacteria/HEp-2 cell), as used by most workers to synchronise events and to increase the number of bacteria which invade, resulted in recovery of significantly less than 1% of the original inoculum after treatment with gentamicin to kill extracellular bacteria. Also, the cell culture medium became acidic, and microscopic examination of Giemsa-stained monolayers immediately following gentamicin treatment revealed high concentrations of bacteria associated with the cells. Moreover, with bacterium-cell interaction beyond 2 h, many HEp-2 cells became rounded, especially with virulent strains W118 and TML. Thus, the biological significance of the quantitative data was uncertain. The fall in pH and the rounding of HEp-2 cells were prevented by the use of a low (1:1) bacterium: cell ratio; but the recovery of bacteria after treatment with gentamicin was still lower than expected by microscopic examination. After treatment of cells with Triton X-100 to release bacteria, many remained bound to residual cell nuclei. Additional treatment with a rubber policeman, and vigorous pipetting to disperse aggregates of bacteria and cell debris, increased the recovery to 10% of the initial inoculum after interaction for 2 h, and 30–80% after 4 h, depending on the strain and experimental conditions. The pattern of invasiveness, but not the absolute count, was highly reproducible on different days and in different hands. However, after interaction exceeding 2 h, the distribution of bacteria was uneven, many cells having no associated organisms, others showing microcolonies. Either this variation does not happen with high inocula, or it is occluded by the high concentration of bacteria associated with the monolayer. Uptake of bacteria depends on the batch of fetal calf serum used in the cell culture medium. The bacterial phenotype is important: bacteria in early or mid log phase entered cells more efficiently, and bacteria grown in Hartley Digest Broth were significantly better at invading HEp-2 cells than those grown in Myosate Broth. Centrifuge-assisted inoculation of HEp-2 cells with bacteria may grossly distort the results, particularly with some avirulent strains.

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1991-12-01
2024-03-29
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References

  1. Lindquist BL, Lebenthal E, Lee P-C, Stinson MW, Merrick JM. Adherence of Salmonella typhimurium to small-intestinal enterocytes of the rat. Infect Immun 1987; 55:3044–3050
    [Google Scholar]
  2. Small PLC, Isberg RR, Falkow S. Comparison of the ability of enteroinvasive Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Yersinia enterocolitica to enter and replicate within HEp-2 cells. Infect Immun 1987; 55:1674–1679
    [Google Scholar]
  3. Finlay BB, Falkow S. Comparisons of the invasion strategies used by Salmonella choleraesuis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication. Biochimie 1988; 70:1089–1099
    [Google Scholar]
  4. Finlay BB, Gumbiner B, Falkow S. Penetration of Salmonella through a polarized Madin-Darby canine kidney epithelial cell monolayer. J Cell Biol 1988; 107:221–230
    [Google Scholar]
  5. Finlay BB, Starnbach MN, Francis CL. et al. Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell layer. Mol Microbiol 1988; 2:757–766
    [Google Scholar]
  6. Finlay BB, Heffron F, Falkow S. Epithelial cell surfaces induce Salmonella proteins required for bacterial adherence and invasion. Science 1989; 243:940–943
    [Google Scholar]
  7. Elsinghorst EA, Baron LS, Kopecko DJ. Penetration of human intestinal epithelial cells by Salmonella: molecular cloning and expression of Salmonella typhi invasion determinants in Escherichia coli. Proc Natl Acad Sci USA 1989; 86:5173–5177
    [Google Scholar]
  8. Miller I, Maskell D, Hormaeche C, Johnson K, Pickard D, Dougan G. The isolation of orally attenuated Salmonella typhimurium following TnpAoA mutagenesis. Infect Immun 1989; 57:2758–2763
    [Google Scholar]
  9. Worton KJ, Candy DCA, Wallis TS. et al. Studies on early association of Salmonella typhimurium with intestinal mucosa in vivo and in vitro: relationship to virulence. J Med Microbiol 1989; 29:283–294
    [Google Scholar]
  10. Gahring LC, Heffron F, Finlay BB, Falkow S. Invasion and replication of Salmonella typhimurium in animal cells. Infect Immun 1990; 58:443–448
    [Google Scholar]
  11. Galân JE, Curtiss R. Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proc Natl Acad Sci USA 1989; 86:6383–6387
    [Google Scholar]
  12. Galân JE, Curtiss R. Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supereoiling. Infect Immun 1990; 58:1879–1885
    [Google Scholar]
  13. Ernst RK, Dombroski DM, Merrick JM. Anaerobiosis, type 1 fimbriae, and growth phase are factors that affect invasion of HEp-2 cells by Salmonella typhimurium. Infect Immun 1990; 58:2014–2016
    [Google Scholar]
  14. Lee CA, Falkow S. The ability of Salmonella to enter mammalian cells is affected by bacterial growth state. Proc Natl Acad Sci USA 1990; 87:4304–308
    [Google Scholar]
  15. Fields PI, Swanson RV, Haidaris CG, Heffron F. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc Natl Acad Sci USA 1986; 83:5189–5193
    [Google Scholar]
  16. Fields PI, Groisman EA, Heffron F. A Salmonella locus that controls resistance to microbicidal proteins from phagocytic cells. Science 1989; 243:1059–1062
    [Google Scholar]
  17. Miller SI, Kukral AM, Mekalanos JJ. A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence. Proc Natl Acad Sci USA 1989; 86:5054–5058
    [Google Scholar]
  18. Groisman EA, Chiao E, Lipps CJ, Heffron F. Salmonella typhimurium phoP virulence gene is a transcriptional regulator. Proc Natl Acad Sci USA 1989; 86:7077–7081
    [Google Scholar]
  19. Groisman EA, Saier MH. Salmonella virulence: new clues to intramacrophage survival. TIBS 1990; 15:30–33
    [Google Scholar]
  20. Giannella RA, Formal SB, Dammin GJ, Collins H. Pathogenesis of salmonellosis. Studies of fluid secretion, mucosal invasion, and morphologic reaction in the rabbit ileum. J Clin Invest 1973; 52:441–53
    [Google Scholar]
  21. Wallis TS, Starkey WG, Stephen J, Haddon SJ, Osborne MP, Candy DCA. Enterotoxin production by Salmonella typhimurium strains of different virulence. J Med Microbiol 1986; 21:19–23
    [Google Scholar]
  22. Stephen J, Wallis TS, Starkey WG, Candy DCA, Osborne MP, Haddon SJ. Salmonellosis: in retrospect and prospect. Evered D, Whelan J. eds Microbial toxins and diarrhoeal disease. Ciba Foundation Symposium 112 London: Pitman; 1985175–192
    [Google Scholar]
  23. Miller SI, Mekalanos J J. Constitutive expression of the PhoP regulon attenuates Salmonella virulence and survival within macrophages. J Bacterial 1990; 172:2485–2490
    [Google Scholar]
  24. Wallis TS, Starkey WG, Stephen J, Haddon SJ, Osborne MP, Candy DCA. The nature and role of mucosal damage in relation to Salmonella typhimurium-’mduced fluid secretion in the rabbit ileum. J Med Microbiol 1986; 22:39–19
    [Google Scholar]
  25. Wallis TS, Hawker RJH, Candy DCA. et al. Quantification of the leucocyte influx into rabbit ileal loops induced by strains of Salmonella typhimurium of different virulence. J Med Microbiol 1989; 30:149–156
    [Google Scholar]
  26. Wallis TS, Vaughan ATM, Clarke GJ. et al. The role of leucocytes in the induction of fluid secretion by Salmonella typhimurium. J Med Microbiol 1990; 31:27–35
    [Google Scholar]
  27. Clarke GJ, Qi G-M, Wallis TS. et al. Expression of an antigen in strains of Salmonella typhimurium which reacts with antibodies to cholera toxin. J Med Microbiol 1988; 25:139–146
    [Google Scholar]
  28. Qi G-M, Clarke GJ, Wallis TS, Stephen J. The influence of cultural conditions on the expression in Salmonella typhimurium of an antigen related to cholera toxin. J Med Microbiol 1989; 30:213–217
    [Google Scholar]
  29. Candy DCA, Stephen J. Salmonella. Farthing MJG, Keusch GT. eds Enteric infection. Mechanisms, manifestations and management London: Chapham and Hall Medical; 1989289–298
    [Google Scholar]
  30. Shaw JH, Falkow S. Model for invasion of human tissue culture cells by Neisseria gonorrhoeae. Infect Immun 1988; 56:1625–1632
    [Google Scholar]
  31. Takeuchi A. Electron microscope studies of experimental salmonella infection. I. Penetration into the intestinal epithelium by Salmonella typhimurium. Am J Pathol 1967; 50:109–136
    [Google Scholar]
  32. Takeuchi A, Sprinz H. Electron microscope studies of experimental salmonella infection in the preconditioned guinea pig. II. Response of the intestinal mucosa to invasion by Salmonella typhimurium. Am J Pathol 1967; 51:137–161
    [Google Scholar]
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