1887

Abstract

is an opportunistic human pathogen that causes diarrhoea and extraintestinal infections, such as wound infection and septicaemia. An protease (AVP) from a biovar sobria strain, AeG1, was partially purified and characterized. Mature AVP hydrolysed casein but not elastin, and protease activity was inhibited by metalloprotease inhibitors. Nucleotide sequence analysis showed that AVP belongs to the thermolysin family of proteases. An AVP-deficient mutant was constructed to investigate the role of AVP in aerolysin activation. Western blot analysis using anti-aerolysin antisera revealed that proaerolysin (52 kDa) in the AVP-deficient mutant was not completely activated to mature aerolysin (47 kDa) as seen in the wild-type strain. The AVP-deficient mutant showed lower cytotoxic and haemolytic activities than wild type. AVP and proaerolysin had no haemolytic activity; however, activity appeared after incubating both proteins. Taken together, these results suggested that AVP plays an indirect role in virulence through activating aerolysin, which is an essential step for cytotoxic activity.

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2004-06-01
2024-04-20
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References

  1. Abrami L., Fivaz M., Decroly E., Seidah N. G., Jean F., Thomas G., Leppla S. H., Buckley J. T., van der Goot F. G. 1998; The pore-forming toxin proaerolysin is activated by furin. J Biol Chem 273:32656–32661 [CrossRef]
    [Google Scholar]
  2. Bagdasarian M., Bagdasarian M. M. 1994; Gene cloning and expression. In Methods for General and Molecular Bacteriology . pp 409–412 Edited by Gerhardt P., Murray R. G. E., Wood W. A., Krieg N. R. Washington, DC: American Society for Microbiology;
  3. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523 [CrossRef]
    [Google Scholar]
  4. Borrell N., Acinas S. G., Figueras M.-J., Martínez-Murcia A. J. 1997; Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes. J Clin Microbiol 35:1671–1674
    [Google Scholar]
  5. Cascón A., Yugueros J., Temprano A., Sánchez M., Hernanz C., Luengo J. M., Naharro G. 2000; A major secreted elastase is essential for pathogenicity of Aeromonas hydrophila . Infect Immun 68:3233–3241 [CrossRef]
    [Google Scholar]
  6. Fujii Y., Nomura T., Kanzawa H., Kameyama M., Yamanaka H., Akita M., Setsu K., Okamoto K. 1998; Purification and characterization of enterotoxin produced by Aeromonas sobria . Microbiol Immunol 42:703–714 [CrossRef]
    [Google Scholar]
  7. Häse C. C., Finkelstein R. A. 1993; Bacterial extracellular zinc-containing metalloproteases. Microbiol Rev 57:823–837
    [Google Scholar]
  8. Honda T., Lertpocasombat K., Hata A., Miwatani T., Finkelstein R. A. 1989; Purification and characterization of a protease produced by Vibrio cholerae non-O1 and comparison with a protease of V.cholerae O1. Infect Immun 57:2799–2803
    [Google Scholar]
  9. Howard S. P., Buckley J. T. 1985; Activation of the hole-forming toxin aerolysin by extracellular processing. J Bacteriol 163:336–340
    [Google Scholar]
  10. Izawa N., Hayashi K. 1996; Cloning and nucleotide sequencing of the aminopeptidase gene from Aeromonas caviae T-64. J Ferment Bioeng 82:544–548 [CrossRef]
    [Google Scholar]
  11. Janda J. M., Abbott S. L. 1998; Evolving concepts regarding the genus Aeromonas : an expanding panorama of species, disease presentations, and unanswered questions. Clin Infect Dis 27:332–344 [CrossRef]
    [Google Scholar]
  12. Kawakami K., Toma C., Honma Y. 2000; Cloning, sequencing and expression of the gene encoding the extracellular metalloprotease of Aeromonas caviae . Microbiol Immunol 44:341–347 [CrossRef]
    [Google Scholar]
  13. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  14. Leung K. Y., Stevenson R. M. W. 1988; Tn5-induced protease-deficient strains of Aeromonas hydrophila with reduced virulence for fish. Infect Immun 56:2639–2644
    [Google Scholar]
  15. Loewy A. G., Santer U. V., Wieczorek M., Blodgett J. K., Jones S. W., Cheronis J. C. 1993; Purification and characterization of a novel zinc-proteinase of Aeromonas hydrophila . J Biol Chem 268:9071–9078
    [Google Scholar]
  16. Matthews B. W. 1988; Structural basis of the action of thermolysin and related zinc peptidases. Acc Chem Res 21:333–340 [CrossRef]
    [Google Scholar]
  17. Miyazato T., Toma C., Nakasone N., Yamamoto K., Iwanaga M. 2003; Molecular analysis of VcfQ protein involved in Vibrio cholerae type IV pilus biogenesis. J Med Microbiol 52:283–288 [CrossRef]
    [Google Scholar]
  18. Miyoshi S., Shinoda S. 2000; Microbial metalloproteases and pathogenesis. Microbes Infect 2:91–98 [CrossRef]
    [Google Scholar]
  19. Nomura T., Fujii Y., Okamoto K. 1999; Secretion of haemolysin of Aeromonas sobria as protoxin and contribution of the propeptide region removed from the protoxin to the proteolytic stability of the toxin. Microbiol Immunol 43:29–38 [CrossRef]
    [Google Scholar]
  20. Ochman H., Gerber A. S., Hartl D. L. 1988; Genetic applications of an inverse polymerase chain reaction. Genetics 120:621–623
    [Google Scholar]
  21. Rivero O., Anguita J., Paniagua C., Naharro G. 1990; Molecular cloning and characterization of an extracellular protease gene from Aeromonas hydrophila . J Bacteriol 172:3905–3908
    [Google Scholar]
  22. Rivero O., Anguita J., Mateos D., Paniagua C., Naharro G. 1991; Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila . FEMS Microbiol Lett 81:1–8 [CrossRef]
    [Google Scholar]
  23. Sha J., Kozlova E. V., Chopra A. K. 2002; Role of various enterotoxins in Aeromonas hydrophila -induced gastroenteritis: generation of enterotoxin gene-deficient mutants and evaluation of their enterotoxic activity. Infect Immun 70:1924–1935 [CrossRef]
    [Google Scholar]
  24. Simon R., Priefer U., Pühler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/technology 11:784–791
    [Google Scholar]
  25. Toma C., Ichinose Y., Iwanaga M. 1999; Purification and characterization of an Aeromonas caviae metalloprotease that is related to the Vibrio cholerae hemagglutinin/protease. FEMS Microbiol Lett 170:237–242 [CrossRef]
    [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354 [CrossRef]
    [Google Scholar]
  27. Vila J., Ruiz J., Gallardo F., Vargas M., Soler L., Figueras M. J., Gascon J. 2003; Aeromonas spp.and traveler's diarrhoea: clinical features and antimicrobial resistance. Emerg Infect Dis 9:552–555 [CrossRef]
    [Google Scholar]
  28. Vipond R., Bricknell I. R., Durant E., Bowden T. J., Ellis A. E., Smith M., MacIntyre S. 1998; Defined deletion mutants demonstrate that the major secreted toxins are not essential for the virulence of Aeromonas salmonicida . Infect Immun 66:1990–1998
    [Google Scholar]
  29. Wang J.-T., Fang C.-T., Hsueh P.-R., Chang S.-C., Lun K.-T. 2000; Spontaneous bacterial empyema caused by Aeromonas veronii biotype sobria. Diagn Microbiol Infect Dis 37:271–273 [CrossRef]
    [Google Scholar]
  30. Whayeb S. A., Yamamoto K., Castillo M.-E. Y., Tojo H., Honda T. 1996; Lysophospholipase L2 of Vibrio cholerae O1 affects cholera toxin production. FEMS Immunol Med Microbiol 15:9–15 [CrossRef]
    [Google Scholar]
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