[Construction of hyaluronidase gene mutant of Enterococcus faecium and the function of hyaluronidase gene].

ABSTRACT

OBJECTIVE: To construct a hyaluronidase (hyl) gene mutant strain of Enterococcus faecium and to study the function of hyl gene. METHODS: An isogenic hyl-deficient mutant (*hyl) with suicide plasmid pTX4577 was constructed and screened by allelic replacement. The mutant strain and wild type strain TX2466 were cultured to observe the growth curves. Both *hyl and TX2466 strains were cultured in solid with kanamycin (KAN) to detect the tolerance to KAN. The study of virulence in mice peritonitis and rabbit endocarditis models in vivo were made. RESULTS: A mutant strain, *hyl, was selected by KAN and identified by PCR, pulsed field gel electrophoresis, and Southern blotting. The growth ability of the *hyl strain was remarkably reduced in comparison with the wide-type strain. The survival rate of the mice within 100 h after the inoculation of Enterococcus faecium. TX2466 was 0, significantly lower than that of the *hyl group (50%, P<0.01). The numbers of colonies in the aortic valve and ventricular wall vegetations of the TX2466 group rabbits were (3.04+/-0.63)x10(6) and (6.87+/-0.58)x10(6) CFU/g respectively, both significantly higher than those of the *hyl group [(0.21+/-0.06)x10(6) and (0.66+/-0.05)x10(6) CFU/g respective, both P<0.01]. CONCLUSION: Probably a major virulence factor of Enterococcus faecalis, hyl gene is important in the pathogenesis of this bacterium.