Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Sep;175(18):5899–5906. doi: 10.1128/jb.175.18.5899-5906.1993

Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells.

R Ménard 1, P J Sansonetti 1, C Parsot 1
PMCID: PMC206670  PMID: 8376337

Abstract

A 31-kb fragment of the large virulence plasmid of Shigella flexneri is necessary for bacterial entry into epithelial cells in vitro. One locus of this fragment encodes the IpaA, -B, -C, and -D proteins, which are the dominant antigens of the humoral immune response during shigellosis. To address the role of the ipa genes, which are clustered in an operon, we constructed a selectable cassette that does not affect transcription of downstream genes and used this cassette to inactivate the ipaB, ipaC, and ipaD genes. Each of these nonpolar mutants was defective in entry and lysis of the phagocytic vacuole but was not impaired in adhesion to the cells. We showed that, like IpaB and IpaC, IpaD is secreted into the culture supernatant and that none of these proteins is necessary for secretion of the other two. This result differentiates the Ipa proteins, which direct the entry process, from the Mxi and Spa proteins, which direct secretion of the Ipa proteins. Moreover, lack of either IpaB or IpaD resulted in the release of larger amounts of the other Ipa polypeptides into the culture medium, which indicates that, in addition to their role in invasion, IpaB and IpaD are each involved in the maintenance of the association of the Ipa proteins with the bacterium.

Full text

PDF
5899

Images in this article

5902Image
on p.5902
5903Image
on p.5903
5903Image
on p.5903
5904Image
on p.5904

Selected References

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

  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. Allaoui A., Mounier J., Prévost M. C., Sansonetti P. J., Parsot C. icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread. Mol Microbiol. 1992 Jun;6(12):1605–1616. doi: 10.1111/j.1365-2958.1992.tb00885.x. [DOI] [PubMed] [Google Scholar]
  3. Allaoui A., Ménard R., Sansonetti P. J., Parsot C. Characterization of the Shigella flexneri ipgD and ipgF genes, which are located in the proximal part of the mxi locus. Infect Immun. 1993 May;61(5):1707–1714. doi: 10.1128/iai.61.5.1707-1714.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Andrews G. P., Hromockyj A. E., Coker C., Maurelli A. T. Two novel virulence loci, mxiA and mxiB, in Shigella flexneri 2a facilitate excretion of invasion plasmid antigens. Infect Immun. 1991 Jun;59(6):1997–2005. doi: 10.1128/iai.59.6.1997-2005.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Båga M., Norgren M., Normark S. Biogenesis of E. coli Pap pili: papH, a minor pilin subunit involved in cell anchoring and length modulation. Cell. 1987 Apr 24;49(2):241–251. doi: 10.1016/0092-8674(87)90565-4. [DOI] [PubMed] [Google Scholar]
  10. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Gaillard J. L., Berche P., Frehel C., Gouin E., Cossart P. Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell. 1991 Jun 28;65(7):1127–1141. doi: 10.1016/0092-8674(91)90009-n. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Isberg R. R., Voorhis D. L., Falkow S. Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell. 1987 Aug 28;50(5):769–778. doi: 10.1016/0092-8674(87)90335-7. [DOI] [PubMed] [Google Scholar]
  15. Jones C. H., Jacob-Dubuisson F., Dodson K., Kuehn M., Slonim L., Striker R., Hultgren S. J. Adhesin presentation in bacteria requires molecular chaperones and ushers. Infect Immun. 1992 Nov;60(11):4445–4451. doi: 10.1128/iai.60.11.4445-4451.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  22. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mills J. A., Buysse J. M., Oaks E. V. Shigella flexneri invasion plasmid antigens B and C: epitope location and characterization with monoclonal antibodies. Infect Immun. 1988 Nov;56(11):2933–2941. doi: 10.1128/iai.56.11.2933-2941.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mounier J., Vasselon T., Hellio R., Lesourd M., Sansonetti P. J. Shigella flexneri enters human colonic Caco-2 epithelial cells through the basolateral pole. Infect Immun. 1992 Jan;60(1):237–248. doi: 10.1128/iai.60.1.237-248.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
  26. Oaks E. V., Hale T. L., Formal S. B. Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella spp. Infect Immun. 1986 Jul;53(1):57–63. doi: 10.1128/iai.53.1.57-63.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Pál T., Hale T. L. Plasmid-associated adherence of Shigella flexneri in a HeLa cell model. Infect Immun. 1989 Aug;57(8):2580–2582. doi: 10.1128/iai.57.8.2580-2582.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Sansonetti P. J., Ryter A., Clerc P., Maurelli A. T., Mounier J. Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infect Immun. 1986 Feb;51(2):461–469. doi: 10.1128/iai.51.2.461-469.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  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. Trieu-Cuot P., Courvalin P. Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III. Gene. 1983 Sep;23(3):331–341. doi: 10.1016/0378-1119(83)90022-7. [DOI] [PubMed] [Google Scholar]
  35. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES