1887

Abstract

The hyaluronate lyase (HL) gene of 8325-4 () was inactivated with the insertion of the erythromycin determinant, , from plasmid pE194. The  : :  mutation was introduced into via a temperature-sensitive shuttle vector, where it underwent homologous recombination with the wild-type (w.t.) allele. The insertion of in the chromosomal locus was confirmed by Southern blot hybridization and the loss of HL activity was demonstrated macroscopically by a plate assay. The importance of HL for pathogenicity was assessed by comparing the virulence of the HL mutant strain to that of the w.t. in an established mouse abscess model of infection. A significantly higher cell recovery was obtained from lesions infected with the w.t. strain compared to the lesions infected with the HL strain ( =0·01). Although the lesion areas from both groups were not significantly different (=0·9) they were of different morphology. A colorimetric assay was used to measure HL activity from culture supernatants of the 8325-4 strains w.t., WA250 () and PC1839 () grown in a chemically defined medium. HL activity reached a maximum in the w.t. strain during mid-exponential phase (=5 h) and while it showed a 16-fold decrease in the mutant it increased 35-fold in the mutant background. These results strongly suggest that HL is a virulence factor which is important in the early stages of subcutaneous infections.

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2004-06-01
2024-03-28
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References

  1. Adams C. A. 1999; Characterisation and potential exploitation of thermosensitive plasmids in Staphylococcus aureus. PhD thesis University of Leeds;
  2. Arvidson S. O. 1983; Extracellular enzymes from Staphylococcus aureus: hyaluronate lyase. In Staphylococci and Staphylococcal Infections pp. 749–752 Edited by Easmon C. S. F., Adlam C. London: Academic Press;
    [Google Scholar]
  3. Augustin J., Götz F. 1990; Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation. FEMS Microbiol Lett 66:203–208 [CrossRef]
    [Google Scholar]
  4. Augustin J., Rosenstein R., Wieland B., Schneider U., Schnell N., Engelke G., Entian K., Götz F. 1992; Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis. Eur J Biochem 204:1142–1154
    [Google Scholar]
  5. Bancroft D., Stevens A. 1996 Theory and Practice of Histological Techniques, 4th edn. New York & Edinburgh: Churchill Livingstone;
  6. 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]
  7. Chan P. F., Foster S. J. 1998; Role of SarA in virulence determinant production and environmental signal transduction in Staphylococcus aureus. J Bacteriol 180:6232–6241
    [Google Scholar]
  8. Chan P. F., Foster S. J., Ingham E., Clements M. O. 1998; The Staphylococcus aureus alternative sigma factor sigmaB controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model. J Bacteriol 180:6082–6089
    [Google Scholar]
  9. Choudhuri K. K., Chakrabarty A. N. 1969; Hyaluronate lyase activity of staphylococci. Indian J Exp Biol 7:183–185
    [Google Scholar]
  10. Devriese L. A. 1990; Staphylococci in healthy and diseased animals. Soc Appl Bacteriol Symp Ser 19:71S–80S
    [Google Scholar]
  11. Duran-Reynals F. 1933; Studies on a certain spreading factor existing in bacteria and its significance for bacterial invasiveness. J Exp Med 58:161–181 [CrossRef]
    [Google Scholar]
  12. Farrell A. M., Taylor D., Holland K. T. 1995; Cloning, nucleotide sequence determination and expression of the Staphylococcus aureus hyaluronate lyase gene. FEMS Microbiology Lett 130:81–85
    [Google Scholar]
  13. Fitzgerald S. N., Foster T. J. 2000; Molecular analysis of the tagF gene, encoding CDP-glycerol : poly(glycerophosphate) glycerophosphotranferase of Staphylococcus epidermidis ATCC 14990. J Bacteriol 182:1046–1052 [CrossRef]
    [Google Scholar]
  14. Foster T. J. 1998; Molecular genetic analysis of staphylococcal virulence. Methods Microbiol 27:433–454
    [Google Scholar]
  15. Greene C., McDevitt D., Francois P., Vaudaux P. E., Lew D. P., Foster T. J. 1995; Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes. Mol Microbiol 17:1143–1152 [CrossRef]
    [Google Scholar]
  16. Grenier D., Michaud J. 1993; Evidence for the absence of hyaluronidase activity in Porphyromonas gingivalis. J Clin Microbiol 31:1913–1915
    [Google Scholar]
  17. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580 [CrossRef]
    [Google Scholar]
  18. Horinouchi S., Weisblum B. 1982; Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics. J Bacteriol 150:804–814
    [Google Scholar]
  19. Horsburgh M. J., Aish J. L., White I. J., Shaw L., Lithgow J. K., Foster S. J. 2002; σB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4. J Bacteriol 184:5457–5467 [CrossRef]
    [Google Scholar]
  20. Jeljaszewicz J. 1983; Infections caused by staphylococci. Infection 11: Suppl 2S109–S11 [CrossRef]
    [Google Scholar]
  21. Kreiswirth B. N., Lofdahl S., Betley M. J., Schlievert P. M., Bergdoll M. S., Novick R. P., O'Reilly M. 1983; The toxic shock syndrome exotoxins structural gene is not detectably transmitted by a prophage. Nature 305:680–685
    [Google Scholar]
  22. Li S., Kelly S. J., Lamani E., Ferraroni M., Jedrzejas J. 2000; Structural basis of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. EMBO J 19:1228–1240 [CrossRef]
    [Google Scholar]
  23. Lindberg M., Sjöström J.-E., Johansson T. 1972; Transformation of chromosomal and plasmid characters in Staphylococcus aureus. J Bacteriol 109:844–847
    [Google Scholar]
  24. Lindsay J. A., Foster S. J. 1999; Interactive regulatory pathways control virulence determinant production and stability in response to environmental conditions in Staphylococcus aureus. Mol Gen Genet 262:323–331 [CrossRef]
    [Google Scholar]
  25. Linker A., Meyer K., Hoffman P. 1955; The production of unsaturated uronides by bacterial hyaluronidases. J Biol Chem 219:13–25
    [Google Scholar]
  26. Luchansky J. B., Benson A. K., Atherly A. G. 1989; Construction, transfer and properties of a novel temperature-sensitive integrable plasmid for genomic analysis of Staphylococcus aureus. Mol Microbiol 3:65–78 [CrossRef]
    [Google Scholar]
  27. Morfeldt E., Janzon C., Arvidson S., Löfdahl S. 1988; Cloning of a chromosomal locus (exp) which regulates the expression of several exoprotein genes in Staphylococcus aureus. Mol Gen Genet 211:435–440 [CrossRef]
    [Google Scholar]
  28. Novick R. P. 1967; Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus.. Virology 33:155–166 [CrossRef]
    [Google Scholar]
  29. Novick R. P. 1991; Genetic systems in staphylococci. Methods Enzymol 204:587–636
    [Google Scholar]
  30. Pattee P. A., Neveln D. S. 1975; Transformation analysis of three linkage groups in Staphylococcus aureus. J Bacteriol 145:479–488
    [Google Scholar]
  31. Pritchard D. G., Lin B. 1993; Group B streptococcal neuraminidase is actually a hyaluronidase. Infect Immun 61:3234–3239
    [Google Scholar]
  32. Pritchard D. G., Trent J. O., Zhang P., Egan M. L., Baker J. R. 2000; Characterisation of the active site of group B streptococcal hyaluronate lyase. Proteins Struct Funct Genet 40:126–134 [CrossRef]
    [Google Scholar]
  33. Reissig J. L., Strominger J. L., Leloir L. F. 1955; A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 217:959–966
    [Google Scholar]
  34. Rogers H. J. 1954; The rate of formation of hyaluronidase, coagulase and total extracellular protein by strains of Staphylococcus aureus. J Gen Microbiol 10:209–220 [CrossRef]
    [Google Scholar]
  35. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  36. Taylor D., Holland K. T. 1989; Amino acid requirements for the growth and production of some exocellular products of Staphylococcus aureus. J Appl Bacteriol 66:319–329 [CrossRef]
    [Google Scholar]
  37. Taylor D., Holland K. T. 1991; Differential regulation of toxic shock syndrome toxin-l and hyaluronate lyase production by Staphylococcus aureus. In Zentralbattl Bacteriologie: International Journal of Medical Microbiology Supplement 21 pp. 209–212Edited by Jeljaszewicz J., Ciborowski P. Stuttgart: Gustav Fischer Verlag;
    [Google Scholar]
  38. Villafane R., Bechhofer D. H., Narayanan C. S., Dubnau D. 1987; Replication control genes of plasmid pE194. J Bacteriol 169:4822–4829
    [Google Scholar]
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