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. 1996 Sep;64(9):3524–3531. doi: 10.1128/iai.64.9.3524-3531.1996

Requirement for exported proteins in secretion through the invasion-associated type III system of Salmonella typhimurium.

C M Collazo 1, J E Galán 1
PMCID: PMC174258  PMID: 8751894

Abstract

The inv and spa loci of Salmonella typhimurium encode a type III protein secretion system which is essential for the ability of this microorganism to gain access to cultured epithelial cells. These loci are located at centisome 63 in the Salmonella chromosome. We have carried out a functional analysis of several genes of these loci and have found that two exported proteins encoded in this region, InvJ and SpaO, are required for secretion through the invasion-associated type III secretion system. These findings suggest the existence of a hierarchy in the export process, since mutations in other targets of this secretory system have no effect on protein secretion. We have also shown that the spaO, spaP, spaQ, and spaR genes are required for protein secretion and for the ability of S. typhimurium to gain access to cultured epithelial cells. In addition, we investigated the ability of an invJ S. typhimurium mutant strain to present the SipB protein to the bacterial surface and demonstrated that, in contrast to Spa32, its putative Shigella homolog, InvJ is not involved in the surface presentation of the Sip proteins.

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Selected References

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  1. 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]
  2. Altmeyer R. M., McNern J. K., Bossio J. C., Rosenshine I., Finlay B. B., Galán J. E. Cloning and molecular characterization of a gene involved in Salmonella adherence and invasion of cultured epithelial cells. Mol Microbiol. 1993 Jan;7(1):89–98. doi: 10.1111/j.1365-2958.1993.tb01100.x. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bliska J. B., Galán J. E., Falkow S. Signal transduction in the mammalian cell during bacterial attachment and entry. Cell. 1993 Jun 4;73(5):903–920. doi: 10.1016/0092-8674(93)90270-z. [DOI] [PubMed] [Google Scholar]
  6. Collazo C. M., Zierler M. K., Galán J. E. Functional analysis of the Salmonella typhimurium invasion genes invl and invJ and identification of a target of the protein secretion apparatus encoded in the inv locus. Mol Microbiol. 1995 Jan;15(1):25–38. doi: 10.1111/j.1365-2958.1995.tb02218.x. [DOI] [PubMed] [Google Scholar]
  7. Donnenberg M. S., Yu J., Kaper J. B. A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells. J Bacteriol. 1993 Aug;175(15):4670–4680. doi: 10.1128/jb.175.15.4670-4680.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dreyfus G., Williams A. W., Kawagishi I., Macnab R. M. Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens. J Bacteriol. 1993 May;175(10):3131–3138. doi: 10.1128/jb.175.10.3131-3138.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Fields K. A., Plano G. V., Straley S. C. A low-Ca2+ response (LCR) secretion (ysc) locus lies within the lcrB region of the LCR plasmid in Yersinia pestis. J Bacteriol. 1994 Feb;176(3):569–579. doi: 10.1128/jb.176.3.569-579.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Galán J. E., Curtiss R., 3rd Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6383–6387. doi: 10.1073/pnas.86.16.6383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Galán J. E., Curtiss R., 3rd Distribution of the invA, -B, -C, and -D genes of Salmonella typhimurium among other Salmonella serovars: invA mutants of Salmonella typhi are deficient for entry into mammalian cells. Infect Immun. 1991 Sep;59(9):2901–2908. doi: 10.1128/iai.59.9.2901-2908.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Galán J. E. Molecular and cellular bases of Salmonella entry into host cells. Curr Top Microbiol Immunol. 1996;209:43–60. doi: 10.1007/978-3-642-85216-9_3. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Gough C. L., Genin S., Lopes V., Boucher C. A. Homology between the HrpO protein of Pseudomonas solanacearum and bacterial proteins implicated in a signal peptide-independent secretion mechanism. Mol Gen Genet. 1993 Jun;239(3):378–392. doi: 10.1007/BF00276936. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Hensel M., Shea J. E., Gleeson C., Jones M. D., Dalton E., Holden D. W. Simultaneous identification of bacterial virulence genes by negative selection. Science. 1995 Jul 21;269(5222):400–403. doi: 10.1126/science.7618105. [DOI] [PubMed] [Google Scholar]
  20. Hoiseth S. K., Stocker B. A. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature. 1981 May 21;291(5812):238–239. doi: 10.1038/291238a0. [DOI] [PubMed] [Google Scholar]
  21. Hsia R. C., Small P. L., Bavoil P. M. Characterization of virulence genes of enteroinvasive Escherichia coli by TnphoA mutagenesis: identification of invX, a gene required for entry into HEp-2 cells. J Bacteriol. 1993 Aug;175(15):4817–4823. doi: 10.1128/jb.175.15.4817-4823.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huang H. C., Lin R. H., Chang C. J., Collmer A., Deng W. L. The complete hrp gene cluster of Pseudomonas syringae pv. syringae 61 includes two blocks of genes required for harpinPss secretion that are arranged colinearly with Yersinia ysc homologs. Mol Plant Microbe Interact. 1995 Sep-Oct;8(5):733–746. doi: 10.1094/mpmi-8-0733. [DOI] [PubMed] [Google Scholar]
  23. Hwang I., Lim S. M., Shaw P. D. Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines. J Bacteriol. 1992 Mar;174(6):1923–1931. doi: 10.1128/jb.174.6.1923-1931.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jones B. D., Falkow S. Identification and characterization of a Salmonella typhimurium oxygen-regulated gene required for bacterial internalization. Infect Immun. 1994 Sep;62(9):3745–3752. doi: 10.1128/iai.62.9.3745-3752.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kaniga K., Bossio J. C., Galán J. E. The Salmonella typhimurium invasion genes invF and invG encode homologues of the AraC and PulD family of proteins. Mol Microbiol. 1994 Aug;13(4):555–568. doi: 10.1111/j.1365-2958.1994.tb00450.x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Kaniga K., Tucker S., Trollinger D., Galán J. E. Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells. J Bacteriol. 1995 Jul;177(14):3965–3971. doi: 10.1128/jb.177.14.3965-3971.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  29. Lee C. A., Jones B. D., Falkow S. Identification of a Salmonella typhimurium invasion locus by selection for hyperinvasive mutants. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1847–1851. doi: 10.1073/pnas.89.5.1847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Li J., Ochman H., Groisman E. A., Boyd E. F., Solomon F., Nelson K., Selander R. K. Relationship between evolutionary rate and cellular location among the Inv/Spa invasion proteins of Salmonella enterica. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7252–7256. doi: 10.1073/pnas.92.16.7252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Mills D. M., Bajaj V., Lee C. A. A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome. Mol Microbiol. 1995 Feb;15(4):749–759. doi: 10.1111/j.1365-2958.1995.tb02382.x. [DOI] [PubMed] [Google Scholar]
  34. Miras I., Hermant D., Arricau N., Popoff M. Y. Nucleotide sequence of iagA and iagB genes involved in invasion of HeLa cells by Salmonella enterica subsp. enterica ser. Typhi. Res Microbiol. 1995 Jan;146(1):17–20. doi: 10.1016/0923-2508(96)80267-1. [DOI] [PubMed] [Google Scholar]
  35. Mulholland V., Hinton J. C., Sidebotham J., Toth I. K., Hyman L. J., Pérombelon M. C., Reeves P. J., Salmond G. P. A pleiotropic reduced virulence (Rvi-) mutant of Erwinia carotovora subspecies atroseptica is defective in flagella assembly proteins that are conserved in plant and animal bacterial pathogens. Mol Microbiol. 1993 Jul;9(2):343–356. doi: 10.1111/j.1365-2958.1993.tb01695.x. [DOI] [PubMed] [Google Scholar]
  36. Obert S., O'Connor R. J., Schmid S., Hearing P. The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex. Mol Cell Biol. 1994 Feb;14(2):1333–1346. doi: 10.1128/mcb.14.2.1333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pang T., Bhutta Z. A., Finlay B. B., Altwegg M. Typhoid fever and other salmonellosis: a continuing challenge. Trends Microbiol. 1995 Jul;3(7):253–255. doi: 10.1016/s0966-842x(00)88937-4. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Sansonetti P. J. Molecular and cellular biology of Shigella flexneri invasiveness: from cell assay systems to shigellosis. Curr Top Microbiol Immunol. 1992;180:1–19. doi: 10.1007/978-3-642-77238-2_1. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Stoker N. G., Fairweather N. F., Spratt B. G. Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene. 1982 Jun;18(3):335–341. doi: 10.1016/0378-1119(82)90172-x. [DOI] [PubMed] [Google Scholar]
  43. Straley S. C., Skrzypek E., Plano G. V., Bliska J. B. Yops of Yersinia spp. pathogenic for humans. Infect Immun. 1993 Aug;61(8):3105–3110. doi: 10.1128/iai.61.8.3105-3110.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Strathmann M., Hamilton B. A., Mayeda C. A., Simon M. I., Meyerowitz E. M., Palazzolo M. J. Transposon-facilitated DNA sequencing. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1247–1250. doi: 10.1073/pnas.88.4.1247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Uchiya K., Tobe T., Komatsu K., Suzuki T., Watarai M., Fukuda I., Yoshikawa M., Sasakawa C. Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading. Mol Microbiol. 1995 Jul;17(2):241–250. doi: 10.1111/j.1365-2958.1995.mmi_17020241.x. [DOI] [PubMed] [Google Scholar]
  46. Van Gijsegem F., Genin S., Boucher C. Conservation of secretion pathways for pathogenicity determinants of plant and animal bacteria. Trends Microbiol. 1993 Aug;1(5):175–180. doi: 10.1016/0966-842x(93)90087-8. [DOI] [PubMed] [Google Scholar]
  47. Van Gijsegem F., Gough C., Zischek C., Niqueux E., Arlat M., Genin S., Barberis P., German S., Castello P., Boucher C. The hrp gene locus of Pseudomonas solanacearum, which controls the production of a type III secretion system, encodes eight proteins related to components of the bacterial flagellar biogenesis complex. Mol Microbiol. 1995 Mar;15(6):1095–1114. doi: 10.1111/j.1365-2958.1995.tb02284.x. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  50. Wang R. F., Kushner S. R. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene. 1991 Apr;100:195–199. [PubMed] [Google Scholar]
  51. 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]
  52. Watarai M., Tobe T., Yoshikawa M., Sasakawa C. Disulfide oxidoreductase activity of Shigella flexneri is required for release of Ipa proteins and invasion of epithelial cells. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4927–4931. doi: 10.1073/pnas.92.11.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zierler M. K., Galán J. E. Contact with cultured epithelial cells stimulates secretion of Salmonella typhimurium invasion protein InvJ. Infect Immun. 1995 Oct;63(10):4024–4028. doi: 10.1128/iai.63.10.4024-4028.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

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