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. 1997 Sep;65(9):3686–3692. doi: 10.1128/iai.65.9.3686-3692.1997

Virulence plasmid instability in Shigella flexneri 2a is induced by virulence gene expression.

R Schuch 1, A T Maurelli 1
PMCID: PMC175525  PMID: 9284138

Abstract

Expression of the predominantly plasmid-encoded virulence regulon of Shigella flexneri 2a is induced by growth at 37 degrees C and repressed by growth at 30 degrees C. During growth at 37 degrees C, spontaneous S. flexneri mutants arise which have undergone virulence plasmid curing or rearrangement and no longer display the virulent phenotype. In the laboratory, the unstable nature of the virulence plasmid causes complete loss of virulence in a growing population. We have undertaken an analysis of virulence plasmid instability, classifying events which produced individual avirulent derivatives within a virulent population and identifying the factor(s) which controlled conversion. Multiplex PCR analysis of DNA obtained from spontaneous avirulent derivatives indicated that virF and virB were deleted or otherwise inactivated in over 97% of the isolates. The virF and virB loci encode regulatory proteins required for transcriptional activation of the virulence regulon. Inactivation of these key regulatory loci in the vast majority of avirulent derivatives which arose during growth at 37 degrees C suggested that virulence gene expression induced virulence plasmid instability. Consistent with this hypothesis, we observed stable virulence plasmid maintenance during growth of a wild-type strain at 30 degrees C where virulence gene expression was repressed. The virulence plasmid was also stably maintained in virF and virB mutants grown at 37 degrees C. Conversely, virulence plasmid destabilization was induced at 30 degrees C and accelerated at 37 degrees C through expression of VirF or VirB from multicopy plasmids. These results indicate that exposure of S. flexneri to conditions favoring induction of the virulent phenotype also favor its loss. The significance of this paradox of Shigella pathogenicity is discussed.

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Selected References

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  1. Adler B., Sasakawa C., Tobe T., Makino S., Komatsu K., Yoshikawa M. A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri. Mol Microbiol. 1989 May;3(5):627–635. doi: 10.1111/j.1365-2958.1989.tb00210.x. [DOI] [PubMed] [Google Scholar]
  2. Andrews G. P., Maurelli A. T. mxiA of Shigella flexneri 2a, which facilitates export of invasion plasmid antigens, encodes a homolog of the low-calcium-response protein, LcrD, of Yersinia pestis. Infect Immun. 1992 Aug;60(8):3287–3295. doi: 10.1128/iai.60.8.3287-3295.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barzu S., Fontaine A., Sansonetti P., Phalipon A. Induction of a local anti-IpaC antibody response in mice by use of a Shigella flexneri 2a vaccine candidate: implications for use of IpaC as a protein carrier. Infect Immun. 1996 Apr;64(4):1190–1196. doi: 10.1128/iai.64.4.1190-1196.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baudry B., Maurelli A. T., Clerc P., Sadoff J. C., Sansonetti P. J. Localization of plasmid loci necessary for the entry of Shigella flexneri into HeLa cells, and characterization of one locus encoding four immunogenic polypeptides. J Gen Microbiol. 1987 Dec;133(12):3403–3413. doi: 10.1099/00221287-133-12-3403. [DOI] [PubMed] [Google Scholar]
  5. Bustos-Martinez J. A., Gomez-Eichelmann M. C. Frequency of IS1-mediated molecular events in different members of the family Enterobacteriaceae. J Bacteriol. 1987 Nov;169(11):4946–4949. doi: 10.1128/jb.169.11.4946-4949.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casey T. A., Moon H. W. Genetic characterization and virulence of enterotoxigenic Escherichia coli mutants which have lost virulence genes in vivo. Infect Immun. 1990 Dec;58(12):4156–4158. doi: 10.1128/iai.58.12.4156-4158.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chosa H., Makino S., Sasakawa C., Okada N., Yamada M., Komatsu K., Suk J. S., Yoshikawa M. Loss of virulence in Shigella strains preserved in culture collections due to molecular alteration of the invasion plasmid. Microb Pathog. 1989 May;6(5):337–342. doi: 10.1016/0882-4010(89)90075-2. [DOI] [PubMed] [Google Scholar]
  8. Colonna B., Casalino M., Fradiani P. A., Zagaglia C., Naitza S., Leoni L., Prosseda G., Coppo A., Ghelardini P., Nicoletti M. H-NS regulation of virulence gene expression in enteroinvasive Escherichia coli harboring the virulence plasmid integrated into the host chromosome. J Bacteriol. 1995 Aug;177(16):4703–4712. doi: 10.1128/jb.177.16.4703-4712.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Daskaleros P. A., Payne S. M. Characterization of Shigella flexneri sequences encoding congo red binding (crb): conservation of multiple crb sequences and role of IS1 in loss of the Crb+ phenotype. Infect Immun. 1986 Nov;54(2):435–443. doi: 10.1128/iai.54.2.435-443.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Daskaleros P. A., Payne S. M. Cloning the gene for Congo red binding in Shigella flexneri. Infect Immun. 1985 Apr;48(1):165–168. doi: 10.1128/iai.48.1.165-168.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guzman L. M., Belin D., Carson M. J., Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121–4130. doi: 10.1128/jb.177.14.4121-4130.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haider K., Azad A. K., Qadri F., Nahar S., Ciznar I. Role of plasmids in virulence-associated attributes and in O-antigen expression in Shigella dysenteriae type 1 strains. J Med Microbiol. 1990 Sep;33(1):1–9. doi: 10.1099/00222615-33-1-1. [DOI] [PubMed] [Google Scholar]
  13. Hale T. L., Formal S. B. Protein synthesis in HeLa or Henle 407 cells infected with Shigella dysenteriae 1, Shigella flexneri 2a, or Salmonella typhimurium W118. Infect Immun. 1981 Apr;32(1):137–144. doi: 10.1128/iai.32.1.137-144.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harris J. R., Wachsmuth I. K., Davis B. R., Cohen M. L. High-molecular-weight plasmid correlates with Escherichia coli enteroinvasiveness. Infect Immun. 1982 Sep;37(3):1295–1298. doi: 10.1128/iai.37.3.1295-1298.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hartman A. B., Ruiz M. M., Schultz C. L. Molecular analysis of variant plasmid forms of a bivalent Salmonella typhi-Shigella sonnei vaccine strain. J Clin Microbiol. 1991 Jan;29(1):27–32. doi: 10.1128/jcm.29.1.27-32.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hill W. E., Carlisle C. L. Loss of plasmids during enrichment for Escherichia coli. Appl Environ Microbiol. 1981 Apr;41(4):1046–1048. doi: 10.1128/aem.41.4.1046-1048.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hromockyj A. E., Maurelli A. T. Identification of Shigella invasion genes by isolation of temperature-regulated inv::lacZ operon fusions. Infect Immun. 1989 Oct;57(10):2963–2970. doi: 10.1128/iai.57.10.2963-2970.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hromockyj A. E., Tucker S. C., Maurelli A. T. Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1(Tyr)). Mol Microbiol. 1992 Aug;6(15):2113–2124. doi: 10.1111/j.1365-2958.1992.tb01385.x. [DOI] [PubMed] [Google Scholar]
  19. Labrec E. H., Schneider H., Magnani T. J., Formal S. B. EPITHELIAL CELL PENETRATION AS AN ESSENTIAL STEP IN THE PATHOGENESIS OF BACILLARY DYSENTERY. J Bacteriol. 1964 Nov;88(5):1503–1518. doi: 10.1128/jb.88.5.1503-1518.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Maurelli A. T., Baudry B., d'Hauteville H., Hale T. L., Sansonetti P. J. Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri. Infect Immun. 1985 Jul;49(1):164–171. doi: 10.1128/iai.49.1.164-171.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maurelli A. T., Blackmon B., Curtiss R., 3rd Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid. Infect Immun. 1984 Jan;43(1):397–401. doi: 10.1128/iai.43.1.397-401.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maurelli A. T., Blackmon B., Curtiss R., 3rd Temperature-dependent expression of virulence genes in Shigella species. Infect Immun. 1984 Jan;43(1):195–201. doi: 10.1128/iai.43.1.195-201.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mills J. A., Venkatesan M. M., Baron L. S., Buysse J. M. Spontaneous insertion of an IS1-like element into the virF gene is responsible for avirulence in opaque colonial variants of Shigella flexneri 2a. Infect Immun. 1992 Jan;60(1):175–182. doi: 10.1128/iai.60.1.175-182.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ménard R., Dehio C., Sansonetti P. J. Bacterial entry into epithelial cells: the paradigm of Shigella. Trends Microbiol. 1996 Jun;4(6):220–226. doi: 10.1016/0966-842X(96)10039-1. [DOI] [PubMed] [Google Scholar]
  25. Oaks E. V., Wingfield M. E., Formal S. B. Plaque formation by virulent Shigella flexneri. Infect Immun. 1985 Apr;48(1):124–129. doi: 10.1128/iai.48.1.124-129.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. SERENY B. Experimental shigella keratoconjunctivitis; a preliminary report. Acta Microbiol Acad Sci Hung. 1955;2(3):293–296. [PubMed] [Google Scholar]
  27. Sansonetti P. J., Kopecko D. J., Formal S. B. Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect Immun. 1982 Mar;35(3):852–860. doi: 10.1128/iai.35.3.852-860.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sansonetti P. J., Kopecko D. J., Formal S. B. Shigella sonnei plasmids: evidence that a large plasmid is necessary for virulence. Infect Immun. 1981 Oct;34(1):75–83. doi: 10.1128/iai.34.1.75-83.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sasakawa C., Kamata K., Sakai T., Makino S., Yamada M., Okada N., Yoshikawa M. Virulence-associated genetic regions comprising 31 kilobases of the 230-kilobase plasmid in Shigella flexneri 2a. J Bacteriol. 1988 Jun;170(6):2480–2484. doi: 10.1128/jb.170.6.2480-2484.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sasakawa C., Kamata K., Sakai T., Murayama S. Y., Makino S., Yoshikawa M. Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri. Infect Immun. 1986 Feb;51(2):470–475. doi: 10.1128/iai.51.2.470-475.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sizemore D. R., Branstrom A. A., Sadoff J. C. Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization. Science. 1995 Oct 13;270(5234):299–302. doi: 10.1126/science.270.5234.299. [DOI] [PubMed] [Google Scholar]
  32. Surgalla M. J., Beesley E. D. Congo red-agar plating medium for detecting pigmentation in Pasteurella pestis. Appl Microbiol. 1969 Nov;18(5):834–837. doi: 10.1128/am.18.5.834-837.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tobe T., Nagai S., Okada N., Adler B., Yoshikawa M., Sasakawa C. Temperature-regulated expression of invasion genes in Shigella flexneri is controlled through the transcriptional activation of the virB gene on the large plasmid. Mol Microbiol. 1991 Apr;5(4):887–893. doi: 10.1111/j.1365-2958.1991.tb00762.x. [DOI] [PubMed] [Google Scholar]
  34. Tobe T., Yoshikawa M., Mizuno T., Sasakawa C. Transcriptional control of the invasion regulatory gene virB of Shigella flexneri: activation by virF and repression by H-NS. J Bacteriol. 1993 Oct;175(19):6142–6149. doi: 10.1128/jb.175.19.6142-6149.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Van de Verg L., Herrington D. A., Murphy J. R., Wasserman S. S., Formal S. B., Levine M. M. Specific immunoglobulin A-secreting cells in peripheral blood of humans following oral immunization with a bivalent Salmonella typhi-Shigella sonnei vaccine or infection by pathogenic S. sonnei. Infect Immun. 1990 Jun;58(6):2002–2004. doi: 10.1128/iai.58.6.2002-2004.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vilette D., Ehrlich S. D., Michel B. Transcription-induced deletions in Escherichia coli plasmids. Mol Microbiol. 1995 Aug;17(3):493–504. doi: 10.1111/j.1365-2958.1995.mmi_17030493.x. [DOI] [PubMed] [Google Scholar]
  37. Watanabe H., Nakamura A. Large plasmids associated with virulence in Shigella species have a common function necessary for epithelial cell penetration. Infect Immun. 1985 Apr;48(1):260–262. doi: 10.1128/iai.48.1.260-262.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zagaglia C., Casalino M., Colonna B., Conti C., Calconi A., Nicoletti M. Virulence plasmids of enteroinvasive Escherichia coli and Shigella flexneri integrate into a specific site on the host chromosome: integration greatly reduces expression of plasmid-carried virulence genes. Infect Immun. 1991 Mar;59(3):792–799. doi: 10.1128/iai.59.3.792-799.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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