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
2019-10-15
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