Identification And Function Analysis Of Virulence Gene Hyl Of Enterococcus Faecium

Background:Enterococci are Gram-positive bacteria, normal inhabitants of the alimentary tract of humans and other animals. Enterococcus faecium generally is considered to be a species with little virulence, at least in some measure, because it was found to be responsible for only 10% of enterococcal infections in human. However, in the past decade, the relative percentage of enterococcal infections attributed to E. faecium has greatly increased, in some instances to 30%～40% of enterococcal infections. Vancomycin resistant enterococci, most of which are E. faecium, are now responsible for 25% of enterococcal infections that occur in ICU in the United States.Taken together, these data suggest that some E. faecium strains may be accumulating determinants that increase their virulence. However,the mechanisms of pathogenesis and virulence factor of E. faecium are not yet well understood,and the development of multidrug resistance in enterococci has made it more difficult to treat enterococcal infections. How to cure E. faecacium infection is always the focus of research in the infection disease domain. Oral immune treatment, may be a new treatment which is inexpensive, safe and practical. It is suitable to mass immunization among people with high risk and it has good prospect of development.Hyaluronidase has been proposed as one of virulence factors in Streptococcus pyogenes, Staphylococcus aureus, and Streptococcus pneumoniae. Enterococcus belonged to the D group Streptococcus initially and was divided into a new group later but many characteristics of it were similar to Streptococcus. Rice et al use BLAST to search a ORF is 1662 bp in Enterococcus faecium. It revealed high similarity to hyaluronidases from S. pyogenes and named hylEfm. In order to study the role of hyl in the virulence and competence of E.faecium, the hyl gene deficient mutant of E. faecium was constructed. It's function was studied in vivo and vitro base on its hereditary character didn't change and provid scientific basis for prevention and cure the infection of E. faecium.Objective:The hyl gene mutant of E. faecium was constructed to study its pathogenic function by growth curve of the mutant and compared with the TX2466 in vitro and animal models in vivo. In base of gene mutant analysis, then constructing a recombinant hyl gene expressed plasmid of E.faecium and expressing recombinant Hyl protein in E. coli. To explore the immune response and anti-bacteria effect after infection in mice fed orally with Hyl protein. Methods:The hyl gene in E. faecium TX2466 were disrupted by using a suicide vector pTX4577 containing internal fragments to construct the hyl mutant. The hyl mutant was identified by PCR , pulsed field gel electrophoresis(PFGE), and Southern blot. The growth ability of the mutant was compared with the TX2466 in vitro at 37℃. The virulence of the mutant was compared with TX2466 by mouse peritonitis and rabbit endocarditis models in vivo. Then hyl gene was amplified by PCR and was inserted into a prokaryotic expression vector pQE-30. Then the vector was translated into E.coli DH5 to expressing Hyl fusion protein. It's immunoreactivity was analyzed by western blot. The fusion protein purified by Ni+-column was fed mice by oral immunization. The changes of antigen-specific antibody in the serum, feces and mucosal fluid were detected by ELISA assay. The effect of anti-bacteria was evaluated after infected with E. faecium TX2466.Results:After screen of Kanamycin, the hyl gene mutant was identified by PCR, PFGE and Southern blot.The result indicated that the growth ability of mutant was remarkably reduced in comparison with that of the wide-type strain. The 50% lethal dose (LD50) of hyl mutant was 7 times higher than that of wide type TX2466. The survival percentage of hyl mutant groups was 50%, while TX2466 groups was 0 at same inoculum in mouse peritonitis, the differences have significant. The changes of blood leukocytes of hyl mutant groups were lower than that of TX2466 at 6 hour and 12 hour. So were the TNF-αlevel in peritoneal fluid and active TNF-a in peritonitis at 6 hour. The colony counts of aortic valve and ventricular wall vegetation of hyl mutant groups were (2.11±0.59)×105 cfu/g and (6.59±0.53)×105 cfu/g respective, while TX2466 groups were (3.04±0.63)×106 cfu/g and (6.87±0.58)×106 cfu/g respective in rabbit endocarditis, the differences have significant too. After PCR the lenghth of hyl gene was sequenced as 1662 bp, its fusion protein encoded polypeptides of 553 amino acid residues. The relative molecular weight was 60 000, by analysis of SDS-PAGE. The level of soluble expression product was about 38% of total cell protein. After affinity chromatography, the purity of fusion protein was above 90%. Western blot analysis confirmed that fusion protein could be specicifically recognized by the anti-TX2466 serum. The serum IgA , serum IgG,faeces sIgA and Intestmucosal fluid sIgA of Hyl groups were 0.365±0.048,0.431±0.064,0.743±0.056 and 1.112±0.113 respective, while the antibody of control groups were 0.051±0.013,0.098±0.019,0.102±0.032 and 0.187±0.051 respective, the differences have significant. The average survival time was longer than that of control too after infected with TX2466.Conclusion:The hyl mutant of E. faecium was constructed successfully.hyl gene is important in the pathogenesis of E.faeciums. It is probably a major virulence factor of E. faecium. The expressing vector pQE-hyl of hyl gene was constructed successfully too. Hyl fusion protein of expression could induce effective mucosal immune response and produced higher levels of sIgA by oral immunization. The recombinant Hyl fusion protein could be used as an oral vaccine candidate antigen for prevention of E. faecium.