1887

Abstract

The type III secretion system encoded by pathogenicity island 2 (SPI-2) is essential for the intracellular survival and replication of . The expression of SPI-2 genes is dependent on a two-component regulatory system, SsrA (SpiR)/SsrB, encoded in the SPI-2 region. This paper shows that SlyA regulates transcription of the sensor kinase SsrA by binding to the promoter, indicating that SlyA is directly involved in the regulation of SPI-2 gene expression. A structure model of the SlyA dimer in complex with DNA was constructed. The model of SlyA indicated that its structure is very similar to that of other MarR family proteins. Based on this model, site-directed mutagenesis of residues located in the winged-helix region required for DNA binding and in the -helices of the N-terminal and C-terminal regions required for dimerization of the SlyA protein was performed to identify the residues that are critical for SlyA function. Nine mutants of SlyA with single substitutions were unable to activate transcription . These mutant SlyA proteins revealed that the residues Leu-63, Val-64, Arg-65, Leu-67, Leu-70, Arg-86 and Lys-88 within the winged-helix region are required for DNA binding, and residues Leu-12 and Leu-126 within the -helices of the N-terminal and C-terminal regions are required for efficient dimer formation. A mutant strain carrying a plasmid expressing SlyA derivatives containing mutations at these amino acid positions did not exhibit restored SlyA function in infected HeLa cells, thereby confirming the structural and functional relationships of the SlyA protein.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.29259-0
2007-02-01
2020-08-10
Loading full text...

Full text loading...

/deliver/fulltext/micro/153/2/548.html?itemId=/content/journal/micro/10.1099/mic.0.29259-0&mimeType=html&fmt=ahah

References

  1. Alekshun M. N., Levy S. B.. 1997; Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob Agents Chemother41:2067–2075
    [Google Scholar]
  2. Alekshun M. N., Levy S. B., Mealy T. R., Seaton B. A., Head J. F.. 2001; The crystal structure of MarR, a regulator of multiple antibiotic resistance, at 2.3 Å resolution. Nat Struct Biol8:710–714[CrossRef]
    [Google Scholar]
  3. Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J.. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res25:3389–3402[CrossRef]
    [Google Scholar]
  4. Baker D., Sali A.. 2001; Protein structure prediction and structural genomics. Science294:93–96[CrossRef]
    [Google Scholar]
  5. Berman H. M., Westbrook J., Feng Z., Gilliland G., Bhat T. N., Weissig H., Shindyalov I. N., Bourne P. E.. 2000; The Protein Data Bank. Nucleic Acids Res28:235–242[CrossRef]
    [Google Scholar]
  6. Beuzon C. R., Unsworth K. E., Holden D. W.. 2001; In vivo genetic analysis indicates that PhoP-PhoQ and the Salmonella pathogenicity island 2 type III secretion system contribute independently to Salmonella enterica serovar Typhimurium virulence. Infect Immun69:7254–7261[CrossRef]
    [Google Scholar]
  7. Bijlsma J. J., Groisman E. A.. 2005; The PhoP/PhoQ system controls the intramacrophage type three secretion system of Salmonella enterica . Mol Microbiol57:85–96[CrossRef]
    [Google Scholar]
  8. Brumell J. H., Goosney D. L., Finlay B. B.. 2002; SifA, a type III secreted effector of Salmonella typhimurium , directs Salmonella -induced filament (Sif) formation along microtubules. Traffic3:407–415[CrossRef]
    [Google Scholar]
  9. Buchmeier N., Bossie S., Chen C. Y., Fang F. C., Guiney D. G., Libby S. J.. 1997; SlyA, a transcriptional regulator of Salmonella typhimurium , is required for resistance to oxidative stress and is expressed in the intracellular environment of macrophages. Infect Immun65:3725–3730
    [Google Scholar]
  10. Cirillo D. M., Valdivia R. H., Monack D. M., Falkow S.. 1998; Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol Microbiol30:175–188[CrossRef]
    [Google Scholar]
  11. Daniels J. J., Autenrieth I. B., Ludwig A., Goebel W.. 1996; The gene slyA of Salmonella typhimurium is required for destruction of M cells and intracellular survival but not for invasion or colonization of the murine small intestine. Infect Immun64:5075–5084
    [Google Scholar]
  12. Deiwick J., Nikolaus T., Erdogan S., Hensel M.. 1999; Environmental regulation of Salmonella pathogenicity island 2 gene expression. Mol Microbiol31:1759–1773[CrossRef]
    [Google Scholar]
  13. De Silva R. S., Kovacikova G., Lin W., Taylor R. K., Skorupski K., Kull F. J.. 2005; Crystal structure of the virulence gene activator AphA from Vibrio cholerae reveals it is a novel member of the winged helix transcription factor superfamily. J Biol Chem280:13779–13783[CrossRef]
    [Google Scholar]
  14. Egland P. G., Harwood C. S.. 1999; BadR, a new MarR family member, regulates anaerobic benzoate degradation by Rhodopseudomonas palustris in concert with AadR, an Fnr family member. J Bacteriol181:2102–2109
    [Google Scholar]
  15. Evans K., Adewoye L., Poole K.. 2001; MexR repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa : identification of MexR binding sites in the mexA-mexR intergenic region. J Bacteriol183:807–812[CrossRef]
    [Google Scholar]
  16. Feng X., Oropeza R., Kenney L. J.. 2003; Dual regulation by phospho-OmpR of ssrA/B gene expression in Salmonella pathogenicity island 2. Mol Microbiol48:1131–1143[CrossRef]
    [Google Scholar]
  17. Feng X., Walthers D., Oropeza R., Kenney L. J.. 2004; The response regulator SsrB activates transcription and binds to a region overlapping OmpR binding sites at Salmonella pathogenicity island 2. Mol Microbiol54:823–835[CrossRef]
    [Google Scholar]
  18. Frishman D., Argos P.. 1995; Knowledge-based protein secondary structure assignment. Proteins23:566–579[CrossRef]
    [Google Scholar]
  19. Gallois A., Klein J. R., Allen L. A., Jones B. D., Nauseef W. M.. 2001; Salmonella pathogenicity island 2-encoded type III secretion system mediates exclusion of NADPH oxidase assembly from the phagosomal membrane. J Immunol166:5741–5748[CrossRef]
    [Google Scholar]
  20. Garmendia J., Beuzon C. R., Ruiz-Albert J., Holden D. W.. 2003; The roles of SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system. Microbiology149:2385–2396[CrossRef]
    [Google Scholar]
  21. Gotoh H., Okada N., Kim Y. G., Shiraishi K., Hirami N., Haneda T., Kurita A., Kikuchi Y., Danbara H.. 2003; Extracellular secretion of the virulence plasmid-encoded ADP-ribosyltransferase SpvB in Salmonella . Microb Pathog34:227–238[CrossRef]
    [Google Scholar]
  22. Guignot J., Caron E., Beuzon C., Bucci C., Kagan J., Roy C., Holden D. W.. 2004; Microtubule motors control membrane dynamics of Salmonella -containing vacuoles. J Cell Sci117:1033–1045[CrossRef]
    [Google Scholar]
  23. Hensel M.. 2000; Salmonella pathogenicity island 2. Mol Microbiol36:1015–1023[CrossRef]
    [Google Scholar]
  24. Hensel M., Shea J. E., Waterman S. R., Mundy R., Nikolaus T., Banks G., Vazquez-Torres A., Gleeson C., Fang F. C., Holden D. W.. 1998; Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages. Mol Microbiol30:163–174[CrossRef]
    [Google Scholar]
  25. Heroven A. K., Nagel G., Tran H. J., Parr S., Dersch P.. 2004; RovA is autoregulated and antagonizes H-NS-mediated silencing of invasin and rovA expression in Yersinia pseudotuberculosis . Mol Microbiol53:871–888[CrossRef]
    [Google Scholar]
  26. Hong M., Fuangthong M., Helmann J. D., Brennan R. G.. 2005; Structure of an OhrR- ohrA operator complex reveals the DNA binding mechanism of the MarR family. Mol Cell20:131–141[CrossRef]
    [Google Scholar]
  27. Jones D. T.. 1999; Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol292:195–202[CrossRef]
    [Google Scholar]
  28. Kaneko A., Mita M., Sekiya K., Matsui H., Kawahara K., Danbara H.. 2002; Association of a regulatory gene, slyA with a mouse virulence of Salmonella serovar Choleraesuis. Microbiol Immunol46:109–113[CrossRef]
    [Google Scholar]
  29. Kawakami T., Kaneko A., Okada N., Imajoh-Ohmi S., Nonaka T., Matsui H., Kawahara K., Danbara H.. 1999; TTG as the initiation codon of Salmonella slyA , a gene required for survival within macrophages. Microbiol Immunol43:351–357[CrossRef]
    [Google Scholar]
  30. Laskowski R. A., MacArthur M. W., Moss D. S., Thornton J. M.. 1993; procheck: a program to check the stereochemical quality of protein structures. J Appl Cryst26:283–291[CrossRef]
    [Google Scholar]
  31. Lee A. K., Detweiler C. S., Falkow S.. 2000; OmpR regulates the two-component system SsrA-SsrB in Salmonella pathogenicity island 2. J Bacteriol182:771–781[CrossRef]
    [Google Scholar]
  32. Libby S. J., Goebel W., Ludwig A., Buchmeier N., Bowe F., Fang F. C., Guiney D. G., Songer J. G., Heffron F.. 1994; A cytolysin encoded by Salmonella is required for survival within macrophages. Proc Natl Acad Sci U S A91:489–493[CrossRef]
    [Google Scholar]
  33. Lim D., Poole K., Strynadka N. C.. 2002; Crystal structure of the MexR repressor of the mexRAB-oprM multidrug efflux operon of Pseudomonas aeruginosa . J Biol Chem277:29253–29259[CrossRef]
    [Google Scholar]
  34. Linehan S. A., Rytkonen A., Yu X. J., Liu M., Holden D. W.. 2005; SlyA regulates function of Salmonella pathogenicity island 2 (SPI-2) and expression of SPI-2-associated genes. Infect Immun73:4354–4362[CrossRef]
    [Google Scholar]
  35. Liu Y., Manna A., Li R., Martin W. E., Murphy R. C., Cheung A. L., Zhang G.. 2001; Crystal structure of the SarR protein from Staphylococcus aureus . Proc Natl Acad Sci U S A98:6877–6882[CrossRef]
    [Google Scholar]
  36. Ludwig A., Tengel C., Bauer S., Bubert A., Benz R., Mollenkopf H. J., Goebel W.. 1995; SlyA, a regulatory protein from Salmonella typhimurium , induces a haemolytic and pore-forming protein in Escherichia coli . Mol Gen Genet249:474–486[CrossRef]
    [Google Scholar]
  37. Menard R., Sansonetti P. J., Parsot C.. 1993; Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol175:5899–5906
    [Google Scholar]
  38. Meresse S., Unsworth K. E., Habermann A., Griffiths G., Fang F., Martinez-Lorenzo M. J., Waterman S. R., Gorvel J. P., Holden D. W.. 2001; Remodelling of the actin cytoskeleton is essential for replication of intravacuolar Salmonella . Cell Microbiol3:567–577[CrossRef]
    [Google Scholar]
  39. Miao E. A., Freeman J. A., Miller S. I.. 2002; Transcription of the SsrAB regulon is repressed by alkaline pH and is independent of PhoPQ and magnesium concentration. J Bacteriol184:1493–1497[CrossRef]
    [Google Scholar]
  40. Miki T., Okada N., Danbara H.. 2004; Two periplasmic disulfide oxidoreductases, DsbA and SrgA, target outer membrane protein SpiA, a component of the Salmonella pathogenicity island 2 type III secretion system. J Biol Chem279:34631–34642[CrossRef]
    [Google Scholar]
  41. Miller J. H.. 1992; A Short Course in Bacterial Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  42. Navarre W. W., Halsey T. A., Walthers D., Frye J., McClelland M., Potter J. L., Kenney L. J., Gunn J. S., Fang F. C.. other authors 2005; Co-regulation of Salmonella enterica genes required for virulence and resistance to antimicrobial peptides by SlyA and PhoP/PhoQ. Mol Microbiol56:492–508[CrossRef]
    [Google Scholar]
  43. Norte V. A., Stapleton M. R., Green J.. 2003; PhoP-responsive expression of the Salmonella enterica serovar Typhimurium slyA gene. J Bacteriol185:3508–3514[CrossRef]
    [Google Scholar]
  44. Ochman H., Soncini F. C., Solomon F., Groisman E. A.. 1996; Identification of a pathogenicity island required for Salmonella survival in host cells. Proc Natl Acad Sci U S A93:7800–7804[CrossRef]
    [Google Scholar]
  45. Ogata K., Umeyama H.. 2000; An automatic homology modeling method consisting of database searches and simulated annealing. J Mol Graph Model18:258–272 305–256
    [Google Scholar]
  46. Revell P. A., Miller V. L.. 2000; A chromosomally encoded regulator is required for expression of the Yersinia enterocolitica inv gene and for virulence. Mol Microbiol35:677–685
    [Google Scholar]
  47. Shea J. E., Hensel M., Gleeson C., Holden D. W.. 1996; Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium . Proc Natl Acad Sci U S A93:2593–2597[CrossRef]
    [Google Scholar]
  48. Shi Y., Latifi T., Cromie M. J., Groisman E. A.. 2004; Transcriptional control of the antimicrobial peptide resistance ugtL gene by the Salmonella PhoP and SlyA regulatory proteins. J Biol Chem279:38618–38625[CrossRef]
    [Google Scholar]
  49. Simon R., Priefer U., Puhler A.. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology1:784–791[CrossRef]
    [Google Scholar]
  50. Spory A., Bosserhoff A., Goebel W., Ludwig A., von Rhein C.. 2002; Differential regulation of multiple proteins of Escherichia coli and Salmonella enterica serovar Typhimurium by the transcriptional regulator SlyA. J Bacteriol184:3549–3559[CrossRef]
    [Google Scholar]
  51. Stapleton M. R., Norte V. A., Read R. C., Green J.. 2002; Interaction of the Salmonella typhimurium transcription and virulence factor SlyA with target DNA and identification of members of the SlyA regulon. J Biol Chem277:17630–17637[CrossRef]
    [Google Scholar]
  52. Steele-Mortimer O., Brumell J. H., Knodler L. A., Meresse S., Lopez A., Finlay B. B.. 2002; The invasion-associated type III secretion system of Salmonella enterica serovar Typhimurium is necessary for intracellular proliferation and vacuole biogenesis in epithelial cells. Cell Microbiol4:43–54[CrossRef]
    [Google Scholar]
  53. Takeda-Shitaka M., Nojima H., Takaya D., Kanou K., Iwadate M., Umeyama H.. 2004a; Evaluation of homology modeling of the severe acute respiratory syndrome (SARS) coronavirus main protease for structure based drug design. Chem Pharm Bull52:643–645[CrossRef]
    [Google Scholar]
  54. Takeda-Shitaka M., Takaya D., Chiba C., Tanaka H., Umeyama H.. 2004b; Protein structure prediction in structure based drug design. Curr Med Chem11:551–558[CrossRef]
    [Google Scholar]
  55. Takeda-Shitaka M., Terashi G., Takaya D., Kanou K., Iwadate M., Umeyama H.. 2005; Protein structure prediction in CASP6 using chimera and fams. Proteins61:122–127[CrossRef]
    [Google Scholar]
  56. Takeda-Shitaka M., Terashi G., Chiba C., Takaya D., Umeyama H.. 2006; fams complex: a fully automated homology modeling system for protein complex structures. Med Chem2:191–201[CrossRef]
    [Google Scholar]
  57. Terashi G., Takeda-Shitaka M., Takaya D., Komatsu K., Umeyama H.. 2005; Searching for protein-protein interaction sites and docking by the methods of molecular dynamics, grid scoring, and the pairwise interaction potential of amino acid residues. Proteins60:289–295[CrossRef]
    [Google Scholar]
  58. Thomson N. R., Cox A., Bycroft B. W., Stewart G. S., Williams P., Salmond G. P.. 1997; The Rap and Hor proteins of Erwinia, Serratia and Yersinia : a novel subgroup in a growing superfamily of proteins regulating diverse physiological processes in bacterial pathogens. Mol Microbiol26:531–544[CrossRef]
    [Google Scholar]
  59. Tobe T., Schoolnik G. K., Sohel I., Bustamante V. H., Puente J. L.. 1996; Cloning and characterization of bfpTVW , genes required for the transcriptional activation of bfpA in enteropathogenic Escherichia coli . Mol Microbiol21:963–975[CrossRef]
    [Google Scholar]
  60. Tran H. J., Heroven A. K., Winkler L., Spreter T., Beatrix B., Dersch P.. 2005; Analysis of RovA, a transcriptional regulator of Yersinia pseudotuberculosis virulence that acts through antirepression and direct transcriptional activation. J Biol Chem280:42423–42432[CrossRef]
    [Google Scholar]
  61. Valdivia R. H., Falkow S.. 1997; Fluorescence-based isolation of bacterial genes expressed within host cells. Science277:2007–2011[CrossRef]
    [Google Scholar]
  62. Vazquez-Torres A., Xu Y., Jones-Carson J., Holden D. W., Lucia S. M., Dinauer M. C., Mastroeni P., Fang F. C.. 2000; Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase. Science287:1655–1658[CrossRef]
    [Google Scholar]
  63. Waterman S. R., Holden D. W.. 2003; Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system. Cell Microbiol5:501–511[CrossRef]
    [Google Scholar]
  64. Watson P. R., Paulin S. M., Bland A. P., Libby S. J., Jones P. W., Wallis T. S.. 1999; Differential regulation of enteric and systemic salmonellosis by slyA . Infect Immun67:4950–4954
    [Google Scholar]
  65. Worley M. J., Ching K. H., Heffron F.. 2000; Salmonella SsrB activates a global regulon of horizontally acquired genes. Mol Microbiol36:749–761
    [Google Scholar]
  66. Wu R. Y., Zhang R. G., Zagnitko O., Dementieva I., Maltzev N., Watson J. D., Laskowski R., Gornicki P., Joachimiak A.. 2003; Crystal structure of Enterococcus faecalis SlyA-like transcriptional factor. J Biol Chem278:20240–20244[CrossRef]
    [Google Scholar]
  67. Wyborn N. R., Stapleton M. R., Norte V. A., Roberts R. E., Grafton J., Green J.. 2004; Regulation of Escherichia coli hemolysin E expression by H-NS and Salmonella SlyA. J Bacteriol186:1620–1628[CrossRef]
    [Google Scholar]
  68. Yu X. J., Ruiz-Albert J., Unsworth K. E., Garvis S., Liu M., Holden D. W.. 2002; SpiC is required for secretion of Salmonella Pathogenicity Island 2 type III secretion system proteins. Cell Microbiol4:531–540[CrossRef]
    [Google Scholar]
  69. Zaharik M. L., Vallance B. A., Puente J. L., Gros P., Finlay B. B.. 2002; Host-pathogen interactions: host resistance factor Nramp1 up-regulates the expression of Salmonella pathogenicity island-2 virulence genes. Proc Natl Acad Sci U S A99:15705–15710[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.29259-0
Loading
/content/journal/micro/10.1099/mic.0.29259-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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