1887

Abstract

The GacS/GacA two-component signal transduction system regulates virulence, biofilm formation and symbiosis in species. The present study investigated this regulatory pathway in a human pathogen that causes life-threatening disease associated with the consumption of raw oysters and wound infections. Small non-coding RNAs (, , and ) commonly regulated by the GacS/GacA pathway were decreased (<0.0003) in a CMCP6 Δ : :  mutant compared with the wild-type parent, and expression was restored by complementation of the deletion mutation Of the 20 genes examined by RT-PCR, significant reductions in the transcript levels of the mutant in comparison with the wild-type strain were observed only for genes related to motility (), stationary phase () and protease () (=0.04, 0.01 and 0.002, respectively). Swimming motility, flagellation and opaque colony morphology indicative of capsular polysaccharide (CPS) were unchanged in the mutant, while cytotoxicity, protease activity, CPS phase variation and the ability to acquire iron were decreased compared with the wild-type (<0.01). The role of in virulence of was also demonstrated by significant impairment in the ability of the mutant strain to cause either skin (<0.0005) or systemic infections (<0.02) in subcutaneously inoculated, non-iron-treated mice. However, the virulence of the mutant was equivalent to that of the wild-type in iron-treated mice, demonstrating that the GacA pathway in regulates the virulence of this organism in an iron-dependent manner.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.043422-0
2010-12-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/12/3722.html?itemId=/content/journal/micro/10.1099/mic.0.043422-0&mimeType=html&fmt=ahah

References

  1. Alice A. F., Naka H., Crosa J. H. 2008; Global gene expression as a function of the iron status of the bacterial cell: influence of differentially expressed genes in the virulence of the human pathogen Vibrio vulnificus . Infect Immun 76:4019–4037
    [Google Scholar]
  2. Amako K., Okada K., Miake S. 1984; Evidence for the presence of a capsule in Vibrio vulnificus . J Gen Microbiol 130:2741–2743
    [Google Scholar]
  3. Babitzke P., Romeo T. 2007; CsrB sRNA family: sequestration of RNA-binding regulatory proteins. Curr Opin Microbiol 10:156–163
    [Google Scholar]
  4. Baker C. S., Morozov I., Suzuki K., Romeo T., Babitzke P. 2002; CsrA regulates glycogen biosynthesis by preventing translation of glgC in Escherichia coli . Mol Microbiol 44:1599–1610
    [Google Scholar]
  5. Blake P. A., Merson M. H., Weaver R. E., Hollis D. G., Heublein P. C. 1979; Disease caused by a marine Vibrio. Clinical characteristics and epidemiology. N Engl J Med 300:1–5
    [Google Scholar]
  6. Chatzidaki-Livanis M., Hubbard M. A., Gordon K., Harwood V. J., Wright A. C. 2006a; Genetic distinctions among clinical and environmental strains of Vibrio vulnificus . Appl Environ Microbiol 72:6136–6141
    [Google Scholar]
  7. Chatzidaki-Livanis M., Jones M. K., Wright A. C. 2006b; Genetic variation in the Vibrio vulnificus group 1 capsular polysaccharide operon. J Bacteriol 188:1987–1998
    [Google Scholar]
  8. DePaola A., Capers G. M., Alexander D. 1994; Densities of Vibrio vulnificus in the intestines of fish from the U.S. Gulf Coast. Appl Environ Microbiol 60:984–988
    [Google Scholar]
  9. DePaola A., Nordstrom J. L., Dalsgaard A., Forslund A., Oliver J., Bates T., Bourdage K. L., Gulig P. A. 2003; Analysis of Vibrio vulnificus from market oysters and septicemia cases for virulence markers. Appl Environ Microbiol 69:4006–4011
    [Google Scholar]
  10. Duffy B. K., Defago G. 2000; Controlling instability in gacS - gacA regulatory genes during inoculant production of Pseudomonas fluorescens biocontrol strains. Appl Environ Microbiol 66:3142–3150
    [Google Scholar]
  11. Gander R. M., LaRocco M. T. 1989; Detection of piluslike structures on clinical and environmental isolates of Vibrio vulnificus . J Clin Microbiol 27:1015–1021
    [Google Scholar]
  12. Gonzalez Chavez R., Alvarez A. F., Romeo T., Georgellis D. 2010; The physiological stimulus for the BarA sensor kinase. J Bacteriol 192:2009–2012
    [Google Scholar]
  13. Goodier R. I., Ahmer B. M. 2001; SirA orthologs affect both motility and virulence. J Bacteriol 183:2249–2258
    [Google Scholar]
  14. Grossart H. P., Steward G. F., Martinez J., Azam F. 2000; A simple, rapid method for demonstrating bacterial flagella. Appl Environ Microbiol 66:3632–3636
    [Google Scholar]
  15. Gulig P. A., Bourdage K. L., Starks A. M. 2005; Molecular pathogenesis of Vibrio vulnificus . J Microbiol 43, (Spec. No):118–131
    [Google Scholar]
  16. Gulig P. A., Tucker M. S., Thiaville P. C., Joseph J. L., Brown R. N. 2009; USER friendly cloning coupled with chitin-based natural transformation enables rapid mutagenesis of Vibrio vulnificus . Appl Environ Microbiol 75:4936–4949
    [Google Scholar]
  17. Guzman L. M., Belin D., Carson M. J., Beckwith J. 1995; Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130
    [Google Scholar]
  18. Hammer B. K., Tateda E. S., Swanson M. S. 2002; A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila . Mol Microbiol 44:107–118
    [Google Scholar]
  19. Han B., Pain A., Johnstone K. 1997; Spontaneous duplication of a 661 bp element within a two-component sensor regulator gene causes phenotypic switching in colonies of Pseudomonas tolaasii , cause of brown blotch disease of mushrooms. Mol Microbiol 25:211–218
    [Google Scholar]
  20. Heeb S., Haas D. 2001; Regulatory roles of the GacS/GacA two-component system in plant-associated and other Gram-negative bacteria. Mol Plant Microbe Interact 14:1351–1363
    [Google Scholar]
  21. Hülsmann A., Rosche T. M., Kong I. S., Hassan H. M., Beam D. M., Oliver J. D. 2003; RpoS-dependent stress response and exoenzyme production in Vibrio vulnificus . Appl Environ Microbiol 69:6114–6120
    [Google Scholar]
  22. Jeong K. C., Jeong H. S., Rhee J. H., Lee S. E., Chung S. S., Starks A. M., Escudero G. M., Gulig P. A., Choi S. H. 2000; Construction and phenotypic evaluation of a Vibrio vulnificus vvpE mutant for elastolytic protease. Infect Immun 68:5096–5106
    [Google Scholar]
  23. Jonas K., Edwards A. N., Simm R., Romeo T., Romling U., Melefors O. 2008; The RNA binding protein CsrA controls cyclic di-GMP metabolism by directly regulating the expression of GGDEF proteins. Mol Microbiol 70:236–257
    [Google Scholar]
  24. Jones M. K., Oliver J. D. 2009; Vibrio vulnificus : disease and pathogenesis. Infect Immun 77:1723–1733
    [Google Scholar]
  25. Jones M. K., Warner E. B., Oliver J. D. 2008; csrA inhibits the formation of biofilms by Vibrio vulnificus . Appl Environ Microbiol 74:7064–7066
    [Google Scholar]
  26. Kim Y. R., Rhee J. H. 2003; Flagellar basal body flg operon as a virulence determinant of Vibrio vulnificus . Biochem Biophys Res Commun 304:405–410
    [Google Scholar]
  27. Kim Y. R., Lee S. E., Kim C. M., Kim S. Y., Shin E. K., Shin D. H., Chung S. S., Choy H. E., Progulske-Fox A. other authors 2003; Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients. Infect Immun 71:5461–5471
    [Google Scholar]
  28. Kim C. M., Park R. Y., Chun H. J., Kim S. Y., Rhee J. H., Shin S. H. 2007; Vibrio vulnificus metalloprotease VvpE is essentially required for swarming. FEMS Microbiol Lett 269:170–179
    [Google Scholar]
  29. Kim Y. R., Lee S. E., Kook H., Yeom J. A., Na H. S., Kim S. Y., Chung S. S., Choy H. E., Rhee J. H. 2008; Vibrio vulnificus RTX toxin kills host cells only after contact of the bacteria with host cells. Cell Microbiol 10:848–862
    [Google Scholar]
  30. Kinscherf T. G., Willis D. K. 1999; Swarming by Pseudomonas syringae B728a requires gacS ( lemA ) and gacA but not the acyl-homoserine lactone biosynthetic gene ahlI . J Bacteriol 181:4133–4136
    [Google Scholar]
  31. Kreger A. S., Gray L. D., Testa J. 1984; Protection of mice against Vibrio vulnificus disease by vaccination with surface antigen preparations and anti-surface antigen antisera. Infect Immun 45:537–543
    [Google Scholar]
  32. Kulkarni P. R., Cui X., Williams J. W., Stevens A. M., Kulkarni R. V. 2006; Prediction of CsrA-regulating small RNAs in bacteria and their experimental verification in Vibrio fischeri . Nucleic Acids Res 34:3361–3369
    [Google Scholar]
  33. Lapouge K., Schubert M., Allain F. H., Haas D. 2008; Gac/Rsm signal transduction pathway of γ -proteobacteria: from RNA recognition to regulation of social behaviour. Mol Microbiol 67:241–253
    [Google Scholar]
  34. Lee J.-H., Rho J. B., Park K.-J., Kim C. B., Han Y.-S., Choi S. H., Lee K.-H., Park S.-J. 2004; Role of flagellum and motility in pathogenesis of Vibrio vulnificus . Infect Immun 72:4905–4910
    [Google Scholar]
  35. Lee J. H., Kim M. W., Kim B. S., Kim S. M., Lee B. C., Kim T. S., Choi S. H. 2007; Identification and characterization of the Vibrio vulnificus rtxA essential for cytotoxicity in vitro and virulence in mice. J Microbiol 45:146–152
    [Google Scholar]
  36. Lenz D. H., Miller M. B., Zhu J., Kulkarni R. V., Bassler B. L. 2005; CsrA and three redundant small RNAs regulate quorum sensing in Vibrio cholerae . Mol Microbiol 58:1186–1202
    [Google Scholar]
  37. Litwin C. M., Rayback T. W., Skinner J. 1996; Role of catechol siderophore synthesis in Vibrio vulnificus virulence. Infect Immun 64:2834–2838
    [Google Scholar]
  38. Liu M. Y., Romeo T. 1997; The global regulator CsrA of Escherichia coli is a specific mRNA-binding protein. J Bacteriol 179:4639–4642
    [Google Scholar]
  39. Liu M. Y., Yang H., Romeo T. 1995; The product of the pleiotropic Escherichia coli gene csrA modulates glycogen biosynthesis via effects on mRNA stability. J Bacteriol 177:2663–2672
    [Google Scholar]
  40. Liu M., Alice A. F., Naka H., Crosa J. H. 2007; The HlyU protein is a positive regulator of rtxA1 , a gene responsible for cytotoxicity and virulence in the human pathogen Vibrio vulnificus . Infect Immun 75:3282–3289
    [Google Scholar]
  41. Livak K. J., Schmittgen T. D. 2001; Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔ C t method. Methods 25:402–408
    [Google Scholar]
  42. McDougald D., Rice S. A., Kjelleberg S. 2001; SmcR-dependent regulation of adaptive phenotypes in Vibrio vulnificus . J Bacteriol 183:758–762
    [Google Scholar]
  43. Meibom K. L., Blokesch M., Dolganov N. A., Wu C. Y., Schoolnik G. K. 2005; Chitin induces natural competence in Vibrio cholerae . Science 310:1824–1827
    [Google Scholar]
  44. Miyoshi N., Miyoshi S., Sugiyama K., Suzuki Y., Furuta H., Shinoda S. 1987; Activation of the plasma kallikrein-kinin system by Vibrio vulnificus protease. Infect Immun 55:1936–1939
    [Google Scholar]
  45. Motes M. L., DePaola A., Cook D. W., Veazey J. E., Hunsucker J. C., Garthright W. E., Blodgett R. J., Chirtel S. J. 1998; Influence of water temperature and salinity on Vibrio vulnificus in Northern Gulf and Atlantic Coast oysters ( Crassostrea virginica . Appl Environ Microbiol 64:1459–1465
    [Google Scholar]
  46. Mukhopadhyay S., Audia J. P., Roy R. N., Schellhorn H. E. 2000; Transcriptional induction of the conserved alternative sigma factor RpoS in Escherichia coli is dependent on BarA, a probable two-component regulator. Mol Microbiol 37:371–381
    [Google Scholar]
  47. Paranjpye R. N., Strom M. S. 2005; A Vibrio vulnificus type IV pilin contributes to biofilm formation, adherence to epithelial cells, and virulence. Infect Immun 73:1411–1422
    [Google Scholar]
  48. Paranjpye R. N., Johnson A. B., Baxter A. E., Strom M. S. 2007; Role of type IV pilins of Vibrio vulnificus in persistence in oysters, Crassostrea virginica . Appl Environ Microbiol 73:5041–5044
    [Google Scholar]
  49. Romeo T. 1998; Global regulation by the small RNA-binding protein CsrA and the non-coding RNA molecule CsrB. Mol Microbiol 29:1321–1330
    [Google Scholar]
  50. Rozen S., Skaletsky H. 2000; Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386
    [Google Scholar]
  51. Sánchez-Contreras M., Martin M., Villacieros M., O'Gara F., Bonilla I., Rivilla R. 2002; Phenotypic selection and phase variation occur during alfalfa root colonization by Pseudomonas fluorescens F113. J Bacteriol 184:1587–1596
    [Google Scholar]
  52. Shao C. P., Hor L. I. 2000; Metalloprotease is not essential for Vibrio vulnificus virulence in mice. Infect Immun 68:3569–3573
    [Google Scholar]
  53. Simon R., Priefer U., Pühler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Nat Biotechnol 1:784–791
    [Google Scholar]
  54. Simpson L. M., Oliver J. D. 1983; Siderophore production by Vibrio vulnificus . Infect Immun 41:644–649
    [Google Scholar]
  55. Simpson L. M., White V. K., Zane S. F., Oliver J. D. 1987; Correlation between virulence and colony morphology in Vibrio vulnificus . Infect Immun 55:269–272
    [Google Scholar]
  56. Srivastava M., Tucker M. S., Gulig P. A., Wright A. C. 2009; Phase variation, capsular polysaccharide, pilus and flagella contribute to uptake of Vibrio vulnificus by the Eastern oyster ( Crassostrea virginica . Environ Microbiol 11:1934–1944
    [Google Scholar]
  57. Stewart B. J., McCarter L. L. 2003; Lateral flagellar gene system of Vibrio parahaemolyticus . J Bacteriol 185:4508–4518
    [Google Scholar]
  58. Suzuki K., Wang X., Weilbacher T., Pernestig A.-K., Melefors O., Georgellis D., Babitzke P., Romeo T. 2002; Regulatory circuitry of the CsrA/CsrB and BarA/UvrY systems of Escherichia coli . J Bacteriol 184:5130–5140
    [Google Scholar]
  59. Tamplin M. L., Capers G. M. 1992; Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica , exposed to seawater disinfected with UV light. Appl Environ Microbiol 58:1506–1510
    [Google Scholar]
  60. Teplitski M., Goodier R. I., Ahmer B. M. 2003; Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella . J Bacteriol 185:7257–7265
    [Google Scholar]
  61. Teplitski M., Al-Agely A., Ahmer B. M. 2006; Contribution of the SirA regulon to biofilm formation in Salmonella enterica serovar Typhimurium. Microbiology 152:3411–3424
    [Google Scholar]
  62. Timmermans J., Van Melderen L. 2010; Post-transcriptional global regulation by CsrA in bacteria. Cell Mol Life Sci 67:2897–2908
    [Google Scholar]
  63. Wei B. L., Brun-Zinkernagel A. M., Simecka J. W., Pruss B. M., Babitzke P., Romeo T. 2001; Positive regulation of motility and flhDC expression by the RNA-binding protein CsrA of Escherichia coli . Mol Microbiol 40:245–256
    [Google Scholar]
  64. Weilbacher T., Suzuki K., Dubey A. K., Wang X., Gudapaty S., Morozov I., Baker C. S., Georgellis D., Babitzke P., Romeo T. 2003; A novel sRNA component of the carbon storage regulatory system of Escherichia coli . Mol Microbiol 48:657–670
    [Google Scholar]
  65. Whistler C. A., Ruby E. G. 2003; GacA regulates symbiotic colonization traits of Vibrio fischeri and facilitates a beneficial association with an animal host. J Bacteriol 185:7202–7212
    [Google Scholar]
  66. Whistler C. A., Corbell N. A., Sarniguet A., Ream W., Loper J. E. 1998; The two-component regulators GacS and GacA influence accumulation of the stationary-phase sigma factor σ S and the stress response in Pseudomonas fluorescens Pf-5. J Bacteriol 180:6635–6641
    [Google Scholar]
  67. Whistler C. A., Koropatnick T. A., Pollack A., McFall-Ngai M. J., Ruby E. G. 2007; The GacA global regulator of Vibrio fischeri is required for normal host tissue responses that limit subsequent bacterial colonization. Cell Microbiol 9:766–778
    [Google Scholar]
  68. Winson M. K., Swift S., Fish L., Throup J. P., Jorgensen F., Chhabra S. R., Bycroft B. W., Williams P., Stewart G. S. 1998; Construction and analysis of luxCDABE -based plasmid sensors for investigating N -acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol Lett 163:185–192
    [Google Scholar]
  69. Wright A. C., Morris J. G. Jr 1991; The extracellular cytolysin of Vibrio vulnificus : inactivation and relationship to virulence in mice. Infect Immun 59:192–197
    [Google Scholar]
  70. Wright A. C., Simpson L. M., Oliver J. D. 1981; Role of iron in the pathogenesis of Vibrio vulnificus infections. Infect Immun 34:503–507
    [Google Scholar]
  71. Wright A. C., Simpson L. M., Richardson K., Maneval D. R. Jr, Olever J. D., Morris J. G. Jr 1986; Siderophore production and outer membrane proteins of selected Vibrio vulnificus strains under conditions of iron limitation. FEMS Microbiol Lett 35:255–260
    [Google Scholar]
  72. Wright A. C., Simpson L. M., Oliver J. D., Morris J. G. Jr 1990; Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus . Infect Immun 58:1769–1773
    [Google Scholar]
  73. Wright A. C., Hill R. T., Johnson J. A., Roghman M. C., Colwell R. R., Morris J. G. Jr 1996; Distribution of Vibrio vulnificus in the Chesapeake Bay. Appl Environ Microbiol 62:717–724
    [Google Scholar]
  74. Wright A. C., Powell J. L., Tanner M. K., Ensor L. A., Karpas A. B., Morris J. G. Jr, Sztein M. B. 1999; Differential expression of Vibrio vulnificus capsular polysaccharide. Infect Immun 67:2250–2257
    [Google Scholar]
  75. Wright A. C., Powell J. L., Kaper J. B., Morris J. G. Jr 2001; Identification of a group 1-like capsular polysaccharide operon for Vibrio vulnificus . Infect Immun 69:6893–6901
    [Google Scholar]
  76. Yildiz F. H., Liu X. S., Heydorn A., Schoolnik G. K. 2004; Molecular analysis of rugosity in a Vibrio cholerae O1 El Tor phase variant. Mol Microbiol 53:497–515
    [Google Scholar]
  77. Yoshida S., Ogawa M., Mizuguchi Y. 1985; Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus . Infect Immun 47:446–451
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.043422-0
Loading
/content/journal/micro/10.1099/mic.0.043422-0
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