Skip to main content
Infection and Immunity logoLink to Infection and Immunity
. 1997 Feb;65(2):774–782. doi: 10.1128/iai.65.2.774-782.1997

Secretion of Shigella flexneri Ipa invasins on contact with epithelial cells and subsequent entry of the bacterium into cells are growth stage dependent.

J Mounier 1, F K Bahrani 1, P J Sansonetti 1
PMCID: PMC176124  PMID: 9009341

Abstract

Upon contact with the surface of epithelial cells, Shigella flexneri secretes Ipa proteins through the Mxi-Spa type III secretion apparatus. Among the Ipa proteins, IpaB and IpaC form a soluble complex in the bacterial supernatant which appears to be sufficient to initiate the cellular rearrangements necessary to achieve bacterial entry. Here, we provide the first evidence that efficiency of bacterial entry into cells depends on the stage of bacterial growth. Bacteria in the early phase of exponential growth are six times more invasive than those in the stationary phase. The entry efficiency of the bacteria present on the cell surface appears to correlate with the percentage of those that are able to secrete their invasins. This suggests that the capacity to activate the Mxi-Spa apparatus is a major factor in the regulation of bacterial entry efficiency. Consistent with these observations, we have further shown that bacteria which have reached the stage of division secrete Ipa proteins more often than those that have not. Also, initial secretion occurs essentially in the area of the septation furrow. The Ipa proteins, secreted in the vicinity of the septation furrow, seem to initiate the early stages of reorganization of the host cell cytoskeleton.

Full Text

The Full Text of this article is available as a PDF (475.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adam T., Arpin M., Prévost M. C., Gounon P., Sansonetti P. J. Cytoskeletal rearrangements and the functional role of T-plastin during entry of Shigella flexneri into HeLa cells. J Cell Biol. 1995 Apr;129(2):367–381. doi: 10.1083/jcb.129.2.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allaoui A., Sansonetti P. J., Parsot C. MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri lpa invasins. Mol Microbiol. 1993 Jan;7(1):59–68. doi: 10.1111/j.1365-2958.1993.tb01097.x. [DOI] [PubMed] [Google Scholar]
  3. Allaoui A., Sansonetti P. J., Parsot C. MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologous to YscJ, a secretion factor of the Yersinia Yop proteins. J Bacteriol. 1992 Dec;174(23):7661–7669. doi: 10.1128/jb.174.23.7661-7669.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Allaoui A., Woestyn S., Sluiters C., Cornelis G. R. YscU, a Yersinia enterocolitica inner membrane protein involved in Yop secretion. J Bacteriol. 1994 Aug;176(15):4534–4542. doi: 10.1128/jb.176.15.4534-4542.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Barzu S., Nato F., Rouyre S., Mazie J. C., Sansonetti P., Phalipon A. Characterization of B-cell epitopes on IpaB, an invasion-associated antigen of Shigella flexneri: identification of an immunodominant domain recognized during natural infection. Infect Immun. 1993 Sep;61(9):3825–3831. doi: 10.1128/iai.61.9.3825-3831.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Baudry B., Kaczorek M., Sansonetti P. J. Nucleotide sequence of the invasion plasmid antigen B and C genes (ipaB and ipaC) of Shigella flexneri. Microb Pathog. 1988 May;4(5):345–357. doi: 10.1016/0882-4010(88)90062-9. [DOI] [PubMed] [Google Scholar]
  8. Bergman T., Erickson K., Galyov E., Persson C., Wolf-Watz H. The lcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium. J Bacteriol. 1994 May;176(9):2619–2626. doi: 10.1128/jb.176.9.2619-2626.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bernardini M. L., Fontaine A., Sansonetti P. J. The two-component regulatory system ompR-envZ controls the virulence of Shigella flexneri. J Bacteriol. 1990 Nov;172(11):6274–6281. doi: 10.1128/jb.172.11.6274-6281.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bernardini M. L., Mounier J., d'Hauteville H., Coquis-Rondon M., Sansonetti P. J. Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin. Proc Natl Acad Sci U S A. 1989 May;86(10):3867–3871. doi: 10.1073/pnas.86.10.3867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bliska J. B., Guan K. L., Dixon J. E., Falkow S. Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1187–1191. doi: 10.1073/pnas.88.4.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clerc P., Sansonetti P. J. Entry of Shigella flexneri into HeLa cells: evidence for directed phagocytosis involving actin polymerization and myosin accumulation. Infect Immun. 1987 Nov;55(11):2681–2688. doi: 10.1128/iai.55.11.2681-2688.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cornelis G., Laroche Y., Balligand G., Sory M. P., Wauters G. Yersinia enterocolitica, a primary model for bacterial invasiveness. Rev Infect Dis. 1987 Jan-Feb;9(1):64–87. doi: 10.1093/clinids/9.1.64. [DOI] [PubMed] [Google Scholar]
  14. Dehio C., Prévost M. C., Sansonetti P. J. Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of cortactin by a pp60c-src-mediated signalling pathway. EMBO J. 1995 Jun 1;14(11):2471–2482. doi: 10.1002/j.1460-2075.1995.tb07244.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Eichelberg K., Ginocchio C. C., Galán J. E. Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins. J Bacteriol. 1994 Aug;176(15):4501–4510. doi: 10.1128/jb.176.15.4501-4510.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Galán J. E., Ginocchio C., Costeas P. Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family. J Bacteriol. 1992 Jul;174(13):4338–4349. doi: 10.1128/jb.174.13.4338-4349.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ginocchio C. C., Olmsted S. B., Wells C. L., Galán J. E. Contact with epithelial cells induces the formation of surface appendages on Salmonella typhimurium. Cell. 1994 Feb 25;76(4):717–724. doi: 10.1016/0092-8674(94)90510-x. [DOI] [PubMed] [Google Scholar]
  18. Ginocchio C., Pace J., Galán J. E. Identification and molecular characterization of a Salmonella typhimurium gene involved in triggering the internalization of salmonellae into cultured epithelial cells. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5976–5980. doi: 10.1073/pnas.89.13.5976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goldberg M. B., Bârzu O., Parsot C., Sansonetti P. J. Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement. J Bacteriol. 1993 Apr;175(8):2189–2196. doi: 10.1128/jb.175.8.2189-2196.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Groisman E. A., Ochman H. Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri. EMBO J. 1993 Oct;12(10):3779–3787. doi: 10.1002/j.1460-2075.1993.tb06056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Guan K. L., Dixon J. E. Protein tyrosine phosphatase activity of an essential virulence determinant in Yersinia. Science. 1990 Aug 3;249(4968):553–556. doi: 10.1126/science.2166336. [DOI] [PubMed] [Google Scholar]
  22. Hermant D., Ménard R., Arricau N., Parsot C., Popoff M. Y. Functional conservation of the Salmonella and Shigella effectors of entry into epithelial cells. Mol Microbiol. 1995 Aug;17(4):781–789. doi: 10.1111/j.1365-2958.1995.mmi_17040781.x. [DOI] [PubMed] [Google Scholar]
  23. High N., Mounier J., Prévost M. C., Sansonetti P. J. IpaB of Shigella flexneri causes entry into epithelial cells and escape from the phagocytic vacuole. EMBO J. 1992 May;11(5):1991–1999. doi: 10.1002/j.1460-2075.1992.tb05253.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kaniga K., Trollinger D., Galán J. E. Identification of two targets of the type III protein secretion system encoded by the inv and spa loci of Salmonella typhimurium that have homology to the Shigella IpaD and IpaA proteins. J Bacteriol. 1995 Dec;177(24):7078–7085. doi: 10.1128/jb.177.24.7078-7085.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Labigne-Roussel A. F., Lark D., Schoolnik G., Falkow S. Cloning and expression of an afimbrial adhesin (AFA-I) responsible for P blood group-independent, mannose-resistant hemagglutination from a pyelonephritic Escherichia coli strain. Infect Immun. 1984 Oct;46(1):251–259. doi: 10.1128/iai.46.1.251-259.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Lee C. A., Falkow S. The ability of Salmonella to enter mammalian cells is affected by bacterial growth state. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4304–4308. doi: 10.1073/pnas.87.11.4304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ling C. M., Overby L. R. Prevalence of hepatitis B virus antigen as revealed by direct radioimmune assay with 125 I-antibody. J Immunol. 1972 Oct;109(4):834–841. [PubMed] [Google Scholar]
  29. Makino S., Sasakawa C., Kamata K., Kurata T., Yoshikawa M. A genetic determinant required for continuous reinfection of adjacent cells on large plasmid in S. flexneri 2a. Cell. 1986 Aug 15;46(4):551–555. doi: 10.1016/0092-8674(86)90880-9. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Maurelli A. T., Sansonetti P. J. Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2820–2824. doi: 10.1073/pnas.85.8.2820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Michiels T., Vanooteghem J. C., Lambert de Rouvroit C., China B., Gustin A., Boudry P., Cornelis G. R. Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enterocolitica. J Bacteriol. 1991 Aug;173(16):4994–5009. doi: 10.1128/jb.173.16.4994-5009.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ménard R., Prévost M. C., Gounon P., Sansonetti P., Dehio C. The secreted Ipa complex of Shigella flexneri promotes entry into mammalian cells. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1254–1258. doi: 10.1073/pnas.93.3.1254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ménard R., Sansonetti P. J., Parsot C. Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol. 1993 Sep;175(18):5899–5906. doi: 10.1128/jb.175.18.5899-5906.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ménard R., Sansonetti P., Parsot C. The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD. EMBO J. 1994 Nov 15;13(22):5293–5302. doi: 10.1002/j.1460-2075.1994.tb06863.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ménard R., Sansonetti P., Parsot C., Vasselon T. Extracellular association and cytoplasmic partitioning of the IpaB and IpaC invasins of S. flexneri. Cell. 1994 Nov 4;79(3):515–525. doi: 10.1016/0092-8674(94)90260-7. [DOI] [PubMed] [Google Scholar]
  37. Parsot C., Ménard R., Gounon P., Sansonetti P. J. Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures. Mol Microbiol. 1995 Apr;16(2):291–300. doi: 10.1111/j.1365-2958.1995.tb02301.x. [DOI] [PubMed] [Google Scholar]
  38. Parsot C., Sansonetti P. J. Invasion and the pathogenesis of Shigella infections. Curr Top Microbiol Immunol. 1996;209:25–42. doi: 10.1007/978-3-642-85216-9_2. [DOI] [PubMed] [Google Scholar]
  39. Pegues D. A., Hantman M. J., Behlau I., Miller S. I. PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion. Mol Microbiol. 1995 Jul;17(1):169–181. doi: 10.1111/j.1365-2958.1995.mmi_17010169.x. [DOI] [PubMed] [Google Scholar]
  40. Phalipon A., Arondel J., Nato F., Rouyre S., Mazie J. C., Sansonetti P. J. Identification and characterization of B-cell epitopes of IpaC, an invasion-associated protein of Shigella flexneri. Infect Immun. 1992 May;60(5):1919–1926. doi: 10.1128/iai.60.5.1919-1926.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rosqvist R., Forsberg A., Wolf-Watz H. Intracellular targeting of the Yersinia YopE cytotoxin in mammalian cells induces actin microfilament disruption. Infect Immun. 1991 Dec;59(12):4562–4569. doi: 10.1128/iai.59.12.4562-4569.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rosqvist R., Håkansson S., Forsberg A., Wolf-Watz H. Functional conservation of the secretion and translocation machinery for virulence proteins of yersiniae, salmonellae and shigellae. EMBO J. 1995 Sep 1;14(17):4187–4195. doi: 10.1002/j.1460-2075.1995.tb00092.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rosqvist R., Magnusson K. E., Wolf-Watz H. Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. EMBO J. 1994 Feb 15;13(4):964–972. doi: 10.1002/j.1460-2075.1994.tb06341.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Salmond G. P., Reeves P. J. Membrane traffic wardens and protein secretion in gram-negative bacteria. Trends Biochem Sci. 1993 Jan;18(1):7–12. doi: 10.1016/0968-0004(93)90080-7. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Sansonetti P. J., Mounier J., Prévost M. C., Mège R. M. Cadherin expression is required for the spread of Shigella flexneri between epithelial cells. Cell. 1994 Mar 11;76(5):829–839. doi: 10.1016/0092-8674(94)90358-1. [DOI] [PubMed] [Google Scholar]
  47. Sasakawa C., Adler B., Tobe T., Okada N., Nagai S., Komatsu K., Yoshikawa M. Functional organization and nucleotide sequence of virulence Region-2 on the large virulence plasmid in Shigella flexneri 2a. Mol Microbiol. 1989 Sep;3(9):1191–1201. doi: 10.1111/j.1365-2958.1989.tb00269.x. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Sasakawa C., Komatsu K., Tobe T., Suzuki T., Yoshikawa M. Eight genes in region 5 that form an operon are essential for invasion of epithelial cells by Shigella flexneri 2a. J Bacteriol. 1993 Apr;175(8):2334–2346. doi: 10.1128/jb.175.8.2334-2346.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Venkatesan M. M., Buysse J. M., Kopecko D. J. Characterization of invasion plasmid antigen genes (ipaBCD) from Shigella flexneri. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9317–9321. doi: 10.1073/pnas.85.23.9317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Venkatesan M. M., Buysse J. M. Nucleotide sequence of invasion plasmid antigen gene ipaA from Shigella flexneri 5. Nucleic Acids Res. 1990 Mar 25;18(6):1648–1648. doi: 10.1093/nar/18.6.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Venkatesan M. M., Buysse J. M., Oaks E. V. Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus. J Bacteriol. 1992 Mar;174(6):1990–2001. doi: 10.1128/jb.174.6.1990-2001.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Watarai M., Tobe T., Yoshikawa M., Sasakawa C. Contact of Shigella with host cells triggers release of Ipa invasins and is an essential function of invasiveness. EMBO J. 1995 Jun 1;14(11):2461–2470. doi: 10.1002/j.1460-2075.1995.tb07243.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Woestyn S., Allaoui A., Wattiau P., Cornelis G. R. YscN, the putative energizer of the Yersinia Yop secretion machinery. J Bacteriol. 1994 Mar;176(6):1561–1569. doi: 10.1128/jb.176.6.1561-1569.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wu H., Parsons J. T. Cortactin, an 80/85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex. J Cell Biol. 1993 Mar;120(6):1417–1426. doi: 10.1083/jcb.120.6.1417. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES