1887

Abstract

Proteins directly involved in entry and dissemination of into epithelial cells are encoded by a virulence plasmid of 200 kb. A 30-kb region (designated the entry region) of this plasmid encodes components of a type III secretion (TTS) apparatus, substrates of this apparatus and their dedicated chaperones. During growth of bacteria in broth, expression of these genes is induced at 37 °C and the TTS apparatus is assembled in the bacterial envelope but is not active. Secretion is activated upon contact of bacteria with host cells and is deregulated in an mutant. The plasmid encodes four transcriptional regulators, VirF, VirB, MxiE and Orf81. VirF controls transcription of , whose product is required for transcription of entry region genes. MxiE, with the chaperone IpgC acting as a co-activator, controls expression of several effectors that are induced under conditions of secretion. Genes under the control of Orf81 are not known. The aim of this study was to define further the repertoires of virulence plasmid genes that are under the control of (i) the growth temperature, (ii) each of the known virulence plasmid-encoded transcriptional regulators (VirF, VirB, MxiE and Orf81) and (iii) the activity of the TTS apparatus. Using a macroarray analysis, the expression profiles of 71 plasmid genes were compared in the wild-type strain grown at 37 and 30 °C and in , , , , and mutants grown at 37 °C. Many genes were found to be under the control of VirB and indirectly of VirF. No alteration of expression of any gene was detected in the mutant. Expression of 13 genes was increased in the secretion-deregulated mutant in an MxiE-dependent manner. On the basis of their expression profile, substrates of the TTS apparatus can be classified into three categories: (i) those that are controlled by VirB, (ii) those that are controlled by MxiE and (iii) those that are controlled by both VirB and MxiE. The differential regulation of expression of TTS effectors in response to the TTS apparatus activity suggests that different effectors might be required at different times following contact of bacteria with host cells.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27639-0
2005-03-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/3/mic1510951.html?itemId=/content/journal/micro/10.1099/mic.0.27639-0&mimeType=html&fmt=ahah

References

  1. Adler, B., Sasakawa, C., Tobe, T., Makino, S., Komatsu, K. & Yoshikawa, M. ( 1989; ). A dual transcriptional activation system for the 230-kb plasmid genes coding for virulence-associated antigens of Shigella flexneri. Mol Microbiol 3, 627–635.[CrossRef]
    [Google Scholar]
  2. Allaoui, A., Mounier, J., Prevost, M. C., Sansonetti, P. J. & Parsot, C. ( 1992; ). icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread. Mol Microbiol 6, 1605–1616.[CrossRef]
    [Google Scholar]
  3. Bahrani, F. K., Sansonetti, P. J. & Parsot, C. ( 1997; ). Secretion of Ipa proteins by Shigella flexneri: inducer molecules and kinetics of activation. Infect Immun 65, 4005–4010.
    [Google Scholar]
  4. Beloin, C. & Dorman, C. J. ( 2003; ). An extended role for the nucleoid structuring protein H-NS in the virulence gene regulatory cascade of Shigella flexneri. Mol Microbiol 47, 825–838.[CrossRef]
    [Google Scholar]
  5. Beloin, C., McKenna, S. & Dorman, C. J. ( 2002; ). Molecular dissection of VirB, a key regulator of the virulence cascade of Shigella flexneri. J Biol Chem 277, 15333–15344.[CrossRef]
    [Google Scholar]
  6. Benjelloun-Touimi, Z., Sansonetti, P. J. & Parsot, C. ( 1995; ). SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion. Mol Microbiol 17, 123–135.[CrossRef]
    [Google Scholar]
  7. Benjelloun-Touimi, Z., Si Tahar, M., Montecucco, C., Sansonetti, P. J. & Parsot, C. ( 1998; ). SepA, the 110 kDa protein secreted by Shigella flexneri: two-domain structure and proteolytic activity. Microbiology 144, 1815–1822.[CrossRef]
    [Google Scholar]
  8. Berlutti, F., Casalino, M., Zagaglia, C., Fradiani, P. A., Visca, P. & Nicoletti, M. ( 1998; ). Expression of the virulence plasmid-carried apyrase gene (apy) of enteroinvasive Escherichia coli and Shigella flexneri is under the control of H-NS and the VirF and VirB regulatory cascade. Infect Immun 66, 4957–4964.
    [Google Scholar]
  9. Bernardini, M. L., Mounier, J., d'Hauteville, H., Coquis-Rondon, M. & Sansonetti, P. J. ( 1989; ). 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 86, 3867–3871.[CrossRef]
    [Google Scholar]
  10. Buchrieser, C., Glaser, P., Rusniok, C., Nedjari, H., d'Hauteville, H., Kunst, F., Sansonetti, P. & Parsot, C. ( 2000; ). The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri. Mol Microbiol 38, 760–771.[CrossRef]
    [Google Scholar]
  11. Buysse, J. M., Hartman, A. B., Strockbine, N. & Venkatesan, M. ( 1995; ). Genetic polymorphism of the ipaH multicopy antigen gene in Shigella spps and enteroinvasive Escherichia coli. Microb Pathog 19, 335–349.
    [Google Scholar]
  12. Cornelis, G. R., Boland, A., Boyd, A. P., Geuijen, C., Iriarte, M., Neyt, C., Sory, M. P. & Stainier, I. ( 1998; ). The virulence plasmid of Yersinia, an antihost genome. Microbiol Mol Biol Rev 62, 1315–1352.
    [Google Scholar]
  13. Darwin, K. H. & Miller, V. L. ( 2000; ). The putative invasion protein chaperone SicA acts together with InvF to activate the expression of Salmonella typhimurium virulence genes. Mol Microbiol 35, 949–960.[CrossRef]
    [Google Scholar]
  14. Darwin, K. H. & Miller, V. L. ( 2001; ). Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium. EMBO J 20, 1850–1862.[CrossRef]
    [Google Scholar]
  15. Day, W. A., Jr & Maurelli, A. T. ( 2001; ). Shigella flexneri LuxS quorum-sensing system modulates virB expression but is not essential for virulence. Infect Immun 69, 15–23.[CrossRef]
    [Google Scholar]
  16. Demers, B., Sansonetti, P. J. & Parsot, C. ( 1998; ). Induction of type III secretion in Shigella flexneri is associated with differential control of transcription of genes encoding secreted proteins. EMBO J 17, 2894–2903.[CrossRef]
    [Google Scholar]
  17. d'Hauteville, H., Khan, S., Maskell, D. J. & 7 other authors ( 2002; ). Two msbB genes encoding maximal acylation of lipid A are required for invasive Shigella flexneri to mediate inflammatory rupture and destruction of the intestinal epithelium. J Immunol 168, 5240–5251.[CrossRef]
    [Google Scholar]
  18. Dorman, C. J. & Porter, M. E. ( 1998; ). The Shigella virulence gene regulatory cascade: a paradigm of bacterial gene control mechanisms. Mol Microbiol 29, 677–684.[CrossRef]
    [Google Scholar]
  19. Dorman, C. J., McKenna, S. & Beloin, C. ( 2001; ). Regulation of virulence gene expression in Shigella flexneri, a facultative intracellular pathogen. Int J Med Microbiol 291, 89–96.[CrossRef]
    [Google Scholar]
  20. Durand, J. M. B., Dagberg, B., Uhlin, B. E. & Bjork, G. R. ( 2000; ). Transfer RNA modification, temperature and DNA superhelicity have a common target in the regulatory network of the virulence of Shigella flexneri: the expression of the virF gene. Mol Microbiol 35, 924–935.[CrossRef]
    [Google Scholar]
  21. Egile, C., d'Hauteville, H., Parsot, C. & Sansonetti, P. J. ( 1997; ). SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri. Mol Microbiol 23, 1063–1073.[CrossRef]
    [Google Scholar]
  22. Eichelberg, K. & Galan, J. E. ( 1999; ). Differential regulation of Salmonella typhimurium type III secreted proteins by pathogenicity island 1 (SPI-1)-encoded transcriptional activators InvF and HilA. Infect Immun 67, 4099–4105.
    [Google Scholar]
  23. Francis, M. S., Lloyd, S. A. & Wolf-Watz, H. ( 2001; ). The type III secretion chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis. Mol Microbiol 42, 1075–1093.[CrossRef]
    [Google Scholar]
  24. Francis, M. S., Wolf-Watz, H. & Forsberg, A. ( 2002; ). Regulation of type III secretion systems. Curr Opin Microbiol 5, 166–172.[CrossRef]
    [Google Scholar]
  25. Goldberg, M. B. & Theriot, J. A. ( 1995; ). Shigella flexneri surface protein Icsa is sufficient to direct actin-based motility. Proc Natl Acad Sci U S A 92, 6572–6576.[CrossRef]
    [Google Scholar]
  26. Hueck, C. J. ( 1998; ). Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62, 379–433.
    [Google Scholar]
  27. Jin, Q., Yuan, Z., Xu, J. & 30 other authors ( 2002; ). Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157. Nucleic Acids Res 30, 4432–4441.[CrossRef]
    [Google Scholar]
  28. Kane, C. D., Schuch, R., Day, W. A., Jr & Maurelli, A. T. ( 2002; ). MxiE regulates intracellular expression of factors secreted by the Shigella flexneri 2a type III secretion system. J Bacteriol 184, 4409–4419.[CrossRef]
    [Google Scholar]
  29. Lambert de Derouvroit, C., Sluiters, C. & Cornelis, G. R. ( 1992; ). Role of the transcriptional activator, VirF, and temperature in the expression of the pYV plasmid genes of Yersinia enterocolitica. Mol Microbiol 6, 395–409.[CrossRef]
    [Google Scholar]
  30. Lan, R. T., Stevenson, G. & Reeves, P. R. ( 2003; ). Comparison of two major forms of the Shigella virulence plasmid pINV: positive selection is a major force driving the divergence. Infect Immun 71, 6298–6306.[CrossRef]
    [Google Scholar]
  31. Lett, M. C., Sasakawa, C., Okada, N., Sakai, T., Makino, S., Yamada, M., Komatsu, K. & Yoshikawa, M. ( 1989; ). virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the VirG protein and determination of the complete coding sequence. J Bacteriol 171, 353–359.
    [Google Scholar]
  32. Maurelli, A. T. & Sansonetti, P. J. ( 1988; ). Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence. Proc Natl Acad Sci U S A 85, 2820–2824.[CrossRef]
    [Google Scholar]
  33. Maurelli, A. T., Blackmon, B. & Curtiss, R., III ( 1984; ). Temperature-dependent expression of virulence genes in Shigella species. Infect Immun 43, 195–201.
    [Google Scholar]
  34. Mavris, M., Page, A. L., Tournebize, R., Demers, B., Sansonetti, P. & Parsot, C. ( 2002a; ). Regulation of transcription by the activity of the Shigella flexneri type III secretion apparatus. Mol Microbiol 43, 1543–1553.[CrossRef]
    [Google Scholar]
  35. Mavris, M., Sansonetti, P. J. & Parsot, C. ( 2002b; ). Identification of the cis-acting site involved in activation of promoters regulated by activity of the type III secretion apparatus in Shigella flexneri. J Bacteriol 184, 6751–6759.[CrossRef]
    [Google Scholar]
  36. McKenna, S., Beloin, C. & Dorman, C. J. ( 2003; ). In vitro DNA-binding properties of VirB, the Shigella flexneri virulence regulatory protein. FEBS Lett 545, 183–187.[CrossRef]
    [Google Scholar]
  37. Menard, R., Sansonetti, P. & Parsot, C. ( 1994a; ). The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD. EMBO J 13, 5293–5302.
    [Google Scholar]
  38. Menard, R., Sansonetti, P., Parsot, C. & Vasselon, T. ( 1994b; ). Extracellular association and cytoplasmic partitioning of the IpaB and IpaC invasins of S. flexneri. Cell 79, 515–525.[CrossRef]
    [Google Scholar]
  39. Michiels, T., Vanooteghem, J. C., Lambert de Derouvroit, C., China, B., Gustin, A., Boudry, P. & Cornelis, G. R. ( 1991; ). Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enterocolitica. J Bacteriol 173, 4994–5009.
    [Google Scholar]
  40. Miller, V. L. ( 2002; ). Connections between transcriptional regulation and type III secretion? Curr Opin Microbiol 5, 211–215.[CrossRef]
    [Google Scholar]
  41. Nakata, N., Sasakawa, C., Okada, N., Tobe, T., Fukuda, I., Suzuki, T., Komatsu, K. & Yoshikawa, M. ( 1992; ). Identification and characterization of virK, a virulence-associated large plasmid gene essential for intercellular spreading of Shigella flexneri. Mol Microbiol 6, 2387–2395.[CrossRef]
    [Google Scholar]
  42. Niebuhr, K., Jouihri, N., Allaoui, A., Gounon, P., Sansonetti, P. J. & Parsot, C. ( 2000; ). IpgD, a protein secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE and implicated in entry focus formation. Mol Microbiol 38, 8–19.[CrossRef]
    [Google Scholar]
  43. Ogawa, M., Suzuki, T., Tatsuno, I., Abe, H. & Sasakawa, C. ( 2003; ). IcsB, secreted via the type III secretion system, is chaperoned by IpgA and required at the post-invasion stage of Shigella pathogenicity. Mol Microbiol 48, 913–931.[CrossRef]
    [Google Scholar]
  44. Page, A. L., Fromont-Racine, M., Sansonetti, P., Legrain, P. & Parsot, C. ( 2001; ). Characterization of the interaction partners of secreted proteins and chaperones of Shigella flexneri. Mol Microbiol 42, 1133–1145.[CrossRef]
    [Google Scholar]
  45. Page, A. L., Sansonetti, P. & Parsot, C. ( 2002; ). Spa15 of Shigella flexneri, a third type of chaperone in the type III secretion pathway. Mol Microbiol 43, 1533–1542.[CrossRef]
    [Google Scholar]
  46. Parsot, C., Menard, R., Gounon, P. & Sansonetti, P. J. ( 1995; ). Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures. Mol Microbiol 16, 291–300.[CrossRef]
    [Google Scholar]
  47. Pettersson, J., Nordfelth, R., Dubinina, E., Bergman, T., Gustafsson, M., Magnusson, K. E. & Wolf-Watz, H. ( 1996; ). Modulation of virulence factor expression by pathogen target cell contact. Science 273, 1231–1233.[CrossRef]
    [Google Scholar]
  48. Porter, M. E. & Dorman, C. J. ( 1997; ). Differential regulation of the plasmid-encoded genes in the Shigella flexneri virulence regulon. Mol Gen Genet 256, 93–103.[CrossRef]
    [Google Scholar]
  49. Porter, M. E. & Dorman, C. J. ( 2002; ). In vivo DNA-binding and oligomerization properties of the Shigella flexneri AraC-like transcriptional regulator VirF as identified by random and site-specific mutagenesis. J Bacteriol 184, 531–539.[CrossRef]
    [Google Scholar]
  50. Prosseda, G., Fradiani, P. A., Di Lorenzo, M., Falconi, M., Micheli, G., Casalino, M., Nicoletti, M. & Colonna, B. ( 1998; ). A role for H-NS in the regulation of the virF gene of Shigella and enteroinvasive Escherichia coli. Res Microbiol 149, 15–25.[CrossRef]
    [Google Scholar]
  51. Prosseda, G., Falconi, M., Giangrossi, M., Gualerzi, C. O., Micheli, G. & Colonna, B. ( 2004; ). The virF promoter in Shigella: more than just a curved DNA stretch. Mol Microbiol 51, 523–537.[CrossRef]
    [Google Scholar]
  52. Rimpilainen, M., Forsberg, A. & Wolfwatz, H. ( 1992; ). A novel protein, LcrQ, involved in the low-calcium response of Yersinia pseudotuberculosis shows extensive homology to YopH. J Bacteriol 174, 3355–3363.
    [Google Scholar]
  53. Sakai, T., Sasakawa, C., Makino, S. & Yoshikawa, M. ( 1986; ). DNA sequence and product analysis of the virfF locus responsible for Congo red binding and cell invasion in Shigella flexneri 2a. Infect Immun 54, 395–402.
    [Google Scholar]
  54. Sakai, T., Sasakawa, C. & Yoshikawa, M. ( 1988; ). Expression of four virulence antigens of Shigella flexneri is positively regulated at the transcriptional level by the 30 kilodalton VirF protein. Mol Microbiol 2, 589–597.[CrossRef]
    [Google Scholar]
  55. Santapaola, D., Casalino, M., Petrucca, A., Presutti, C., Zagaglia, C., Berlutti, F., Colonna, B. & Nicoletti, M. ( 2002; ). Enteroinvasive Escherichia coli virulence-plasmid-carried apyrase (apy) and ospB genes are organized as a bicistronic operon and are subject to differential expression. Microbiology 148, 2519–2529.
    [Google Scholar]
  56. Sasakawa, C., Komatsu, K., Tobe, T., Suzuki, T. & Yoshikawa, M. ( 1993; ). Eight genes in region 5 that form an operon are essential for invasion of epithelial cells by Shigella flexneri 2a. J Bacteriol 175, 2334–2346.
    [Google Scholar]
  57. Shere, K. D., Sallustio, S., Manessis, A., D'Aversa, T. G. & Goldberg, M. B. ( 1997; ). Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin-based motility. Mol Microbiol 25, 451–462.[CrossRef]
    [Google Scholar]
  58. Taniya, T., Mitobe, J., Nakayama, S., Mingshan, Q., Okuda, K. & Watanabe, H. ( 2003; ). Determination of the InvE binding site required for expression of IpaB of the Shigella sonnei virulence plasmid: involvement of a ParB boxA-like sequence. J Bacteriol 185, 5158–5165.[CrossRef]
    [Google Scholar]
  59. Tobe, T., Nagai, S., Okada, N., Adler, B., Yoshikawa, M. & Sasakawa, C. ( 1991; ). 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 5, 887–893.[CrossRef]
    [Google Scholar]
  60. Tobe, T., Yoshikawa, M., Mizuno, T. & Sasakawa, C. ( 1993; ). Transcriptional control of the invasion regulatory gene virB of Shigella flexneri: activation by Virf and repression by H-NS. J Bacteriol 175, 6142–6149.
    [Google Scholar]
  61. Uchiya, K., Tobe, T., Komatsu, K., Suzuki, T., Watarai, M., Fukuda, I., Yoshikawa, M. & Sasakawa, C. ( 1995; ). Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading. Mol Microbiol 17, 241–250.[CrossRef]
    [Google Scholar]
  62. Venkatesan, M. M., Goldberg, M. B., Rose, D. J., Grotbeck, E. J., Burland, V. & Blattner, F. R. ( 2001; ). Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri. Infect Immun 69, 3271–3285.[CrossRef]
    [Google Scholar]
  63. Watanabe, H., Arakawa, E., Ito, K. I., Kato, J. I. & Nakamura, A. ( 1990; ). Genetic analysis of an invasion region by use of a Tn3-lac transposon and identification of a second positive regulator gene, invE, for cell invasion of Shigella sonnei: significant homology of InvE with ParB of plasmid P1. J Bacteriol 172, 619–629.
    [Google Scholar]
  64. Wei, J., Goldberg, M. B., Burland, V. & 14 other authors ( 2003; ). Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T. Infect Immun 71, 2775–2786.[CrossRef]
    [Google Scholar]
  65. Wing, H. J., Yan, A. W., Goldman, S. R. & Goldberg, M. B. ( 2004; ). Regulation of IcsP, the outer membrane protease of the Shigella actin tail assembly protein IcsA, by virulence plasmid regulators VirF and VirB. J Bacteriol 186, 699–705.[CrossRef]
    [Google Scholar]
  66. Wulff-Strobel, C. R., Williams, A. W. & Straley, S. C. ( 2002; ). LcrQ and SycH function together at the Ysc type III secretion system in Yersinia pestis to impose a hierarchy of secretion. Mol Microbiol 43, 411–423.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27639-0
Loading
/content/journal/micro/10.1099/mic.0.27639-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error