1887

Abstract

of both biotypes produce a soluble Zn-dependent metalloprotease: haemagglutinin/protease (Hap), encoded by . Hap has been shown to have mucinolytic and cytotoxic activity. These activities are likely to play an important role in the pathogenesis of cholera and the reactogenicity of attenuated vaccine strains. Production of Hap requires transcriptional activation by the HapR regulator and is repressed by glucose. The present study shows that mucin purified from two sources, bile salts, and growth at 37 °C enhanced Hap protease production. Analysis of and promoter fusions with the gene showed both promoters to be activated in a cell-density-dependent pattern. Glucose repressed and mucin induced the promoter by a HapR-independent mechanism. Bile had no effect on either or promoter activity. Expression of was required for vibrios to translocate through a mucin-containing gel. These results suggest Hap to play an important role in cholera pathogenesis by promoting mucin gel penetration, detachment and spreading of infection along the gastrointestinal tract.

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2003-07-01
2020-01-25
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References

  1. Atsumi T., Maekawa Y., Yamada T., Kawagishi I., Imae Y., Homma M.. 1996; Effect of viscosity on swimming by the lateral and polar flagella of Vibrio alginolyticus . J Bacteriol178:5024–5026
    [Google Scholar]
  2. Benitez J. A., Spelbrink R. G., Silva A. J., Phillips T. E., Stanley C. M., Boesman-Finkelstein M., Finkelstein R. A.. 1997; Adherence of Vibrio cholerae to cultured differentiated human intestinal cells: an in vitro colonization model. Infect Immun65:3474–3477
    [Google Scholar]
  3. Benitez J. A., Garcia L., Silva A. J.. 16 other authors 1999; Preliminary assessment of the safety and immunogenicity of a new CTXΦ-negative hemagglutinin/protease-defective El Tor strain as a cholera vaccine candidate. Infect Immun67:539–545
    [Google Scholar]
  4. Benitez J. A., Silva A. J., Finkelstein R. A.. 2001; Environmental signals controlling production of hemagglutinin/protease in Vibrio cholerae . Infect Immun69:6549–6553
    [Google Scholar]
  5. Booth B. A., Boesman-Finkelstein M., Finkelstein R. A.. 1983; Vibrio cholerae soluble hemagglutinin/protease is a metalloenzyme. Infect Immun42:639–644
    [Google Scholar]
  6. Booth B. A., Boesman-Finkelstein M., Finkelstein R. A.. 1984; Vibrio cholerae hemagglutinin/protease nicks cholera enterotoxin. Infect Immun45:558–560
    [Google Scholar]
  7. Brosius J., Ullrich A., Raker M. A., Gray A., Dull T. J., Gutell R. R., Noller H. F.. 1981; Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon in Escherichia coli . Plasmid6:112–118
    [Google Scholar]
  8. Brown I., Häse C.. 2001; Flagellum-independent surface migration of Vibrio cholerae and Escherichia coli . J Bacteriol183:3784–3790
    [Google Scholar]
  9. Chiang S. L., Taylor R. K., Koomey M., Mekalanos J. J.. 1995; Single amino acid substitutions in the N-terminus of Vibrio cholerae TcpA affect colonization, autoagglutination, and serum resistance. Mol Microbiol17:1133–1142
    [Google Scholar]
  10. Denkin S. M., Nelson D. R.. 1999; Induction of protease activity in Vibrio anguillarum by gastrointestinal mucus. Appl Environ Microbiol65:3555–3560
    [Google Scholar]
  11. Finkelstein R. A., Hanne L. F.. 1982; Purification and characterization of the soluble hemagglutinin (cholera lectin) produced by Vibrio cholerae . Infect Immun36:1199–1208
    [Google Scholar]
  12. Finkelstein R. A., Boesman-Finkelstein M., Holt P.. 1983; Vibrio cholerae hemagglutinin/lectin/protease hydrolyzes fibronectin and ovomucin: F. M. Burnet revisited. Proc Natl Acad Sci U S A80:1092–1095
    [Google Scholar]
  13. Fullner K. J., Lencer W. I., Mekalanos J. J.. 2001; Vibrio cholerae -induced cellular responses of polarized T84 intestinal epithelial cells are dependent on production of cholera toxin and the RTX toxin. Infect Immun69:6310–6317
    [Google Scholar]
  14. Ganguly U., Greennough W. B. III. 1975; Adenosine 3′5′-cyclic monophosphate in Vibrio cholerae . Infect Immun11:343–349
    [Google Scholar]
  15. Gardel C. L., Mekalanos J. J.. 1996; Alterations in Vibrio cholerae motility phenotypes correlate with changes in virulence factor expression. Infect Immun64:2246–2255
    [Google Scholar]
  16. Gupta S., Chowdhury R.. 1997; Bile affects production of virulence factors and motility of Vibrio cholerae . Infect Immun65:1131–1134
    [Google Scholar]
  17. Häse C. C.. 2001; Analysis of the role of flagellar activity in virulence gene expression in Vibrio cholerae . Microbiology147:831–837
    [Google Scholar]
  18. Häse C. C., Finkelstein R. A.. 1991; Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain. J Bacteriol173:3311–3317
    [Google Scholar]
  19. Huet C., Sahuquillo-Merino L., Coudrier E., Louvard S.. 1987; Absorptive and mucus secreting subclones isolated from a muiltipotent intestinal cell line (HT29) provide new models for cell polarity and terminal differentiation. J Cell Biol105:345–358
    [Google Scholar]
  20. Jobling M. G., Holmes R. K.. 1997; Characterization of hapR , a positive regulator of the Vibrio cholerae HA/protease gene hap , and its identification as a functional homologue of the Vibrio harveyi luxR gene. Mol Microbiol26:1023–1034
    [Google Scholar]
  21. Jordan N., Newton J., Pearson J., Allen A.. 1998; A novel method for the visualization of the in situ mucus layer in rat and man. Clin Sci95:97–106
    [Google Scholar]
  22. Kolb A., Busby S., Buc H., Garges S., Adhya S.. 1993; Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem62:749–795
    [Google Scholar]
  23. Lee S. H., Hava D. L., Waldor M. K., Camilli A.. 1999; Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection. Cell99:625–634
    [Google Scholar]
  24. Lehker M. W., Sweeney D.. 1999; Trichomonads invasion of the mucous layer requires adhesins, mucinases, and motility. Sex Transm Infect75:231–238
    [Google Scholar]
  25. Mel S. F., Fullner K. J., Wimer-Mackin S., Lencer W. I., Mekalanos J. J.. 2000; Association of protease activity in Vibrio cholerae vaccine strains with decrease in transcellular epithelial resistance of polarized T84 intestinal cells. Infect Immun68:6487–6492
    [Google Scholar]
  26. Miller J. H.. 1971; Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  27. Miller M. B., Skorupski K., Lenz D. H., Taylor R. K., Bassler B. L.. 2002; Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae . Cell110:303–314
    [Google Scholar]
  28. Miyoshi S., Shinoda S.. 2000; Microbial metalloproteases and pathogenesis. Microbes Infect2:91–98
    [Google Scholar]
  29. Nagamune K., Yamamoto K., Naka A., Matsuyama J., Miwatani T., Honda T.. 1996; In vitro proteolytic processing and activation of the recombinant precursor of El Tor cytolysin/hemolysin (Pro-HlyA) of Vibrio cholerae by soluble hemagglutinin/protease of V . cholerae , trypsin, and other proteases. Infect Immun64:4655–4658
    [Google Scholar]
  30. O'Toole R., Lundberg S., Fredriksson S., Jansson A., Nilsson B., Wolf-Watz H.. 1999; The chemotactic response of Vibrio anguillarum to fish intestinal mucus is mediated by a combination of multiple mucus components. J Bacteriol181:4308–4317
    [Google Scholar]
  31. Phillips T. E., Frisch E. B.. 1990; Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line. Analysis using double labeling with lectins. Histochemistry93:311–317
    [Google Scholar]
  32. Phillips T. E., Huet C., Bilbo P. R., Podolsky D. K., Louvard D., Neutra M. R.. 1988; Human intestinal goblet cell in monolayer culture: characterization of a mucus-secreting subclone derived from HT29 colon adenocarcinoma cell line. Gastroenterology94:1390–1403
    [Google Scholar]
  33. Robert A., Silva A., Benitez J. A., Rodriguez B. L., Fando R., Campos J., Sengupta D. K., Boesman-Finkelstein M., Finkelstein R. A.. 1996; Tagging a Vibrio cholerae El Tor candidate vaccine strain by disruption of its hemagglutinin/protease gene using a novel reporter enzyme: Clostridium thermocellum endoglucanase A. Vaccine14:1517–1522
    [Google Scholar]
  34. Rothmel R. D., Shinabarger D., Parsek M., Aldrich T., Chakrabarty A. M.. 1991; Functional analysis of the Pseudomonas putida regulatory protein CatR: transcriptional studies and determination of the CatR DNA binding site by hydroxyl-radical footprinting. J Bacteriol173:4717–4724
    [Google Scholar]
  35. Sandkvist M., Michel L. O., Hough L. P., Morales V. M., Bagdasarian M., Koomkey M., DiRita V. J., Bagdasarian M.. 1997; General secretion pathway ( eps ) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae . J Bacteriol179:6994–7003
    [Google Scholar]
  36. Sheikh J., Czeczulin J. R., Harrington S., Hicks S., Henderson I. R., Le Bouguenec C., Gounon P., Phillips A., Nataro J. P.. 2002; A novel dispersin protein in enteroaggregative Escherichia coli . J Clin Invest110:1329–1337
    [Google Scholar]
  37. Skorupski K., Taylor R. K.. 1997a; Control of the ToxR virulence regulon in Vibrio cholerae by environmental stimuli. Mol Microbiol25:1003–1009
    [Google Scholar]
  38. Skorupski K., Taylor R. K.. 1997b; Cyclic AMP and its receptor protein negatively regulate the coordinate expression of cholera toxin and toxin co-regulated pilus in Vibrio cholerae . Proc Natl Acad Sci U S A94:265–270
    [Google Scholar]
  39. Stanley C. M., Phillips T. E.. 1999; Selective secretion and replenishment of discrete mucin glycoforms from intestinal goblet cells. Am J Physiol227:G191–G200
    [Google Scholar]
  40. Wibenmeyer J. A., Provenzano D., Landry C. F., Klose K. E., Delcour H.. 2002; Vibrio cholerae OmpU and OmpT porins are differentially affected by bile. Infect Immun70:121–126
    [Google Scholar]
  41. Wu Z., Milton D., Nybon P., Sjo A., Magnusson K-E.. 1996; Vibrio cholerae hemagglutinin/protease (HA/protease) causes morphological changes in cultured epithelial cells and perturbs their paracellular barrier function. Microb Pathog21:111–123
    [Google Scholar]
  42. Wu Z., Nybom P., Magnusson K.-E.. 2000; Distinct effects of Vibrio cholerae hemagglutinin/protease on the structure and localization of the tight junction-associated proteins occludin and ZO-1. Cell Microbiol2:11–17
    [Google Scholar]
  43. Yamamoto T., Yokota T.. 1988; Electron microscopic study of Vibrio cholerae O1 adherence to the mucus coat and villus surface in the human small intestine. Infect Immun56:2753–2759
    [Google Scholar]
  44. Zhu J., Miller M. B., Vance R. E., Dziejman M., Bassler B. L., Mekalanos J. J.. 2002; Quorum-sensing regulators control virulence gene expression in Vibrio cholerae . Proc Natl Acad Sci U S A99:3129–3134
    [Google Scholar]
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