1887

Abstract

is a re-emerging pathogen of molluscs that secretes a variety of extracellular products (ECPs), including a metalloprotease and a cytolysin/haemolysin. Previously, we reported that the haemolysin locus consists of two ORFs ( and ), similar to that of the homologous haemolysin genes ( and ) found in . Here, we demonstrate that the concomitant expression of both genes resulted in significantly higher haemolytic activity than the gene alone. In addition, we created a VthAB mutant strain of that was virtually devoid of haemolytic activity in liquid media. Interestingly, significant production of an additional haemolysin(s) was observed on blood plates. Moreover, we have previously reported that in , proteolytic and haemolytic activities are inversely produced during bacterial growth. Here, we study this correlation in more detail and present evidence that the VtpA metalloprotease inhibits haemolytic activity in culture supernatants, based on the following evidence: (i) loss of metalloprotease activity by either mutation or EDTA inhibition resulted in increased haemolytic activity; (ii) overexpression of the gene resulted in decreased haemolytic activity; (iii) purified VtpA metalloprotease directly diminished haemolytic activity by purified VthA haemolysin. Importantly, we found not only that gene expression remained high throughout growth but also that there were no dramatic differences in gene expression between the parent and VtpA mutant strains. Thus, our results strongly suggest that the metalloprotease directly targets its haemolysin.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.028605-0
2009-07-01
2019-10-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/7/2296.html?itemId=/content/journal/micro/10.1099/mic.0.028605-0&mimeType=html&fmt=ahah

References

  1. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Truhl, K. S. ( 1991; ). Current Protocols in Molecular Biology. New York: Wiley.
  2. Benitez, J. A., Silva, A. J. & Finkelstein, R. A. ( 2001; ). Environmental signals controlling production of hemagglutinin/protease in Vibrio cholerae. Infect Immun 69, 6549–6553.[CrossRef]
    [Google Scholar]
  3. Choi, H. K., Park, N. Y., Kim, D. I., Chung, H. J., Ryu, S. & Choi, S. H. ( 2002; ). Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of Vibrio vulnificus. J Biol Chem 277, 47292–47299.[CrossRef]
    [Google Scholar]
  4. Choi, M. H., Sun, H. Y., Park, R. Y., Bai, Y. H., Chung, Y. Y., Kim, C. M. & Shin, S. H. ( 2006; ). Human serum albumin enhances the hemolytic activity of Vibrio vulnificus. Biol Pharm Bull 29, 180–182.[CrossRef]
    [Google Scholar]
  5. Croxatto, A., Lauritz, J., Chen, C. & Milton, D. L. ( 2007; ). Vibrio anguillarum colonization of rainbow trout integument requires a DNA locus involved in exopolysaccharide transport and biosynthesis. Environ Microbiol 9, 370–382.[CrossRef]
    [Google Scholar]
  6. Datsenko, K. A. & Wanner, B. L. ( 2000; ). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.[CrossRef]
    [Google Scholar]
  7. Delston, R. B., Kothary, M. H., Shangraw, K. A. & Tall, B. D. ( 2003; ). Isolation and characterization of zinc-containing metalloprotease expressed by Vibrio tubiashii. Can J Microbiol 49, 525–529.[CrossRef]
    [Google Scholar]
  8. Elston, R. A., Hasegawa, H., Humphrey, K. L., Polyak, I. K. & Häse, C. C. ( 2008; ). Re-emergence of Vibrio tubiashii in bivalve shellfish aquaculture: severity, environmental drivers, geographic extent and management. Dis Aquat Organ 82, 119–134.[CrossRef]
    [Google Scholar]
  9. Estes, R. M., Friedman, C. S., Elston, R. A. & Herwig, R. P. ( 2004; ). Pathogenicity testing of shellfish hatchery bacterial isolates on Pacific oyster Crassostrea gigas larvae. Dis Aquat Organ 58, 223–230.[CrossRef]
    [Google Scholar]
  10. Fan, J. J., Shao, C. P., Ho, Y. C., Yu, C. K. & Hor, L. I. ( 2001; ). Isolation and characterization of a Vibrio vulnificus mutant deficient in both extracellular metalloprotease and cytolysin. Infect Immun 69, 5943–5948.[CrossRef]
    [Google Scholar]
  11. Figueroa-Arredondo, P., Heuser, J. E., Akopyants, N. S., Morisaki, J. H., Giono-Cerezo, S., Enriquez-Rincon, F. & Berg, D. E. ( 2001; ). Cell vacuolation caused by Vibrio cholerae hemolysin. Infect Immun 69, 1613–1624.[CrossRef]
    [Google Scholar]
  12. Figurski, D. H. & Helinski, D. R. ( 1979; ). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 1648–1652.[CrossRef]
    [Google Scholar]
  13. Gómez-León, J., Villamil, L., Salger, S. A., Sallum, R. H., Remacha-Triviño, A., Leavitt, D. F. & Gómez-Chiarri, M. ( 2008; ). Survival of eastern oysters Crassostrea virginica from three lines following experimental challenge with bacterial pathogens. Dis Aquat Organ 79, 95–105.[CrossRef]
    [Google Scholar]
  14. Hasegawa, H., Lind, E. J., Boin, M. A. & Häse, C. C. ( 2008; ). The extracellular metalloprotease of Vibrio tubiashii is a major virulence factor for Pacific oyster (Crassostrea gigas) larvae. Appl Environ Microbiol 74, 4101–4110.[CrossRef]
    [Google Scholar]
  15. Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. ( 1988; ). Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene 70, 191–197.[CrossRef]
    [Google Scholar]
  16. Kim, S. Y., Lee, S. E., Kim, Y. R., Kim, C. M., Ryu, R. Y., Choy, H. E., Chung, S. S. & Rhee, J. H. ( 2003; ). Regulation of Vibrio vulnificus virulence by the LuxS quorum-sensing system. Mol Microbiol 48, 1647–1664.[CrossRef]
    [Google Scholar]
  17. Kim, M. Y., Park, R. Y., Choi, M. H., Sun, H. Y., Kim, C. M., Kim, S. Y., Rhee, J. H. & Shin, S. H. ( 2006; ). Swarming differentiation of Vibrio vulnificus downregulates the expression of the vvhBA hemolysin gene via the LuxS quorum-sensing system. J Microbiol 44, 226–232.
    [Google Scholar]
  18. 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.[CrossRef]
    [Google Scholar]
  19. Kothary, M. H., Delston, R. B., Curtis, S. K., McCardell, B. A. & Tall, B. D. ( 2001; ). Purification and characterization of vulnificolysin-like cytolysin produced by Vibrio tubiashii. Appl Environ Microbiol 67, 3707–3711.[CrossRef]
    [Google Scholar]
  20. Lee, S. E., Ryu, P. Y., Kim, S. Y., Kim, Y. R., Koh, J. T., Kim, O. J., Chung, S. S., Choy, H. E. & Rhee, J. H. ( 2004; ). Production of Vibrio vulnificus hemolysin in vivo and its pathogenic significance. Biochem Biophys Res Commun 324, 86–91.[CrossRef]
    [Google Scholar]
  21. 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.[CrossRef]
    [Google Scholar]
  22. Miller, J. H. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  23. Miyoshi, S., Fujii, S., Tomochika, K. & Shinoda, S. ( 1997; ). Some properties of nicked Vibrio vulnificus hemolysin. Microb Pathog 23, 235–239.[CrossRef]
    [Google Scholar]
  24. 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.[CrossRef]
    [Google Scholar]
  25. Paranjpye, R. N., Lara, C. J., Pepe, J. C., Pepe, C. M. & Strom, M. S. ( 1998; ). The type IV leader peptidase/N-methyltransferase of Vibrio vulnificus controls factors required for adherence to HEp-2 cells and virulence in iron-overloaded mice. Infect Immun 66, 5659–5668.
    [Google Scholar]
  26. Park, K. S., Ono, T., Rokuda, M., Jang, M. H., Okada, K., Iida, T. & Honda, T. ( 2004; ). Functional characterization of two type III secretion systems of Vibrio parahaemolyticus. Infect Immun 72, 6659–6665.[CrossRef]
    [Google Scholar]
  27. 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. Vaccine 14, 1517–1522.[CrossRef]
    [Google Scholar]
  28. Saltikov, C. W. & Newman, D. K. ( 2003; ). Genetic identification of a respiratory arsenate reductase. Proc Natl Acad Sci U S A 100, 10983–10988.[CrossRef]
    [Google Scholar]
  29. Senoh, M., Okita, Y., Shinoda, S. & Miyoshi, S. ( 2008; ). The crucial amino acid residue related to inactivation of Vibrio vulnificus hemolysin. Microb Pathog 44, 78–83.[CrossRef]
    [Google Scholar]
  30. Shao, C. P. & Hor, L. I. ( 2000; ). Metalloprotease is not essential for Vibrio vulnificus virulence in mice. Infect Immun 68, 3569–3573.[CrossRef]
    [Google Scholar]
  31. Shao, C. P. & Hor, L. I. ( 2001; ). Regulation of metalloprotease gene in Vibrio vulnificus by a Vibrio harveyi LuxR homologue. J Bacteriol 183, 1369–1375.[CrossRef]
    [Google Scholar]
  32. Shin, S. H., Sun, H. Y., Choi, M. H., Park, R. Y., Bai, Y. H., Kim, C. M., Kim, S. Y., Kim, Y. R., Lee, S. E. & Rhee, J. H. ( 2005; ). Inactivation of Vibrio vulnificus hemolysin by oligomerization but not proteolysis. Biol Pharm Bull 28, 1294–1297.[CrossRef]
    [Google Scholar]
  33. Shinoda, S. ( 1999; ). Protein toxins produced by pathogenic vibrios. J Nat Toxins 8, 259–269.
    [Google Scholar]
  34. Spaink, H. P., Okker, R. J. H., Wijffelman, C. A., Pees, E. & Lugtenberg, B. J. J. ( 1987; ). Promoters in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI. Plant Mol Biol 9, 27–39.[CrossRef]
    [Google Scholar]
  35. Su, J. H., Chang, M. C., Lee, Y. S., Tseng, I. C. & Chuang, Y. C. ( 2004; ). Cloning and characterization of the lipase and lipase activator protein from Vibrio vulnificus CKM-1. Biochim Biophys Acta 1678, 7–13.[CrossRef]
    [Google Scholar]
  36. Takahashi, K. G., Nakamura, A. & Mori, K. ( 2000; ). Inhibitory effects of ovoglobulins on bacillary necrosis in larvae of the pacific oyster, Crassostrea gigas. J Invertebr Pathol 75, 212–217.[CrossRef]
    [Google Scholar]
  37. Tubiash, H. S., Chanley, P. E. & Leifson, E. ( 1965; ). Bacillary necrosis, a disease of larval and juvenile bivalve mollusks. I. Etiology and epizootiology. J Bacteriol 90, 1036–1044.
    [Google Scholar]
  38. Wyckoff, E. E., Mey, A. R. & Payne, S. M. ( 2007; ). Iron acquisition in Vibrio cholerae. Biometals 20, 405–416.[CrossRef]
    [Google Scholar]
  39. Yamamoto, K., Wright, A. C., Kaper, J. B. & Morris, J. G., Jr ( 1990; ). The cytolysin gene of Vibrio vulnificus: sequence and relationship to the Vibrio cholerae El Tor hemolysin gene. Infect Immun 58, 2706–2709.
    [Google Scholar]
  40. Zhang, X. H. & Austin, B. ( 2005; ). Haemolysins in Vibrio species. J Appl Microbiol 98, 1011–1019.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.028605-0
Loading
/content/journal/micro/10.1099/mic.0.028605-0
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