Construction Of The Variant Strain Containing The Enterococcal Cytolysin Gene

Objectives:The purpose of this study was to construct a EF A₇ cyl mutant of enterococcus and investigate the virulence of hemolysin, so as to understand the pathogenic mechanisms of enterococcal hemolysin and establish the foundation to diagnose and control relevant diseases.Methods:The mutant gene of cyl was constructed by transforming the plasmid pCGC which contained the complete cytolysin gene cluster to the deficient mutant of enterococcus faecalis EF A₇. After the hemolysis and resistant phenotype were identified, we established mouse peritonitis models and compared LD50,survival percentage,the number of blood leukocytes,the TNF-αlevel in peritoneal active cytokine in peritonitis at 6 hour, observed the histological changes of internal organ between EF A₇ cyl mutant group and EF A₇ group.Results:1. The mutant gene of cyl was constructed by electroporation. After screen of spectinomycin, the EF A₇ cyl mutant was identified by PCR and the phenotype of hemolysis, and about a 1 kb band was clearly showed in agarose gel electrophoresis.2. In the mouse peritonitis models The 50% lethal dose (LD50) of EF A₇ cyl mutant was 100 times lower than enterococcus faecalis EF A₇.The survival percentage of EF A₇ cyl mutant groups was 0%, while EF A₇ groups was 83.33% at same inoculum in mouse peritonitis, the differences have significance.The changes of blood leukocytes numbers of EF A₇ cyl mutant groups were go up to (11.60±0.28)×109 /L and (14.60±0.36)×109 /L at 6 hour and 12 hour. while EF A₇ groups were go up to (7.47±0.17)×109 /L and (8.15±0.15)×109 /L, the differences have significance.The TNF-αlevel of EF A₇ cyl mutant in peritoneal active TNF-αin peritonitis at 6 hour was 104.70±3.73 ng/ml. while EF A₇ groups was 32.86±0.97 ng/ml, the differences have significance.H-E staining showed that the inflammatory cell infiltration was found in internal organ of EF A₇ cyl mutant groups. Conclusions:1.The EF A₇ cyl mutant of E.faecalis was constructed successfully and named EF A₇ cyl mutant.2. Cyl gene is probably a major virulence factor of Enterococcus and plays an important role in the mouse peritonitis model.