Targeting knock out of Leptospira interrogans flagellum-associated fliN gene and pathogenic function alteration of the mutant

Abstract

Objective To determine the pathogenicity of product expressed by Leptospira interrogans flagellum-associated fliN gene belonging to type Ⅲ secretion system.Methods Target gene knock out technique based on homologious sequence recombinant of suicide plasmid and chromosome was applied to construct FliN unexpressed mutant from L.interrogans serovar Lai strain 56601.PCR,sequencing and Western blot were used to identify FliN-mutant.Motility test,MTT,Fontana silver staining method and flow cytometry were respectively performed to determine the alterations of migration ability,cytotoxicity of the culture supematant to murine mononuclear-macrophage like cell line (J774A.1),adhering to and inducing apoptosis ofJ774A.1 cells of FliN-mutant.Results All the results of PCR,sequencing and Western blot confirmed a successful construction of FliN-mutant,and this mutant could grow as well as propagate in 100 μg/ml ampicillin contained Korthof medium.The diameters (2-3 mm)of FliN-mutant's colonies on semisolid Korthof medium were remarkbly less than those (6-8 mm)of wild strain,indicating mobility absence of the mutant.The supernatant of FliN-mutant culture treating J774A.1 cells for 72 h showed cytotoxicity,but the cytotoxicity was significantly lower than that of wild strain (P＜0.05).The adhering ratio (25.5%)of FliN-mutant co-incubating with J774A.1 cells for 60 min was also obviously lower than that of wild strain.Furthermore,beth the necrotic and apoptotic rates (30.6%and 22.7%)induced by FliN-mutant were lower than those (48.2%and 35.2%)caused by wild strain (P＜0.05).Conclusion The product of fliN gene displays a close relationship with adhering cells,secreting virulent factors and inducing cell apoptosis of L.interrogans.The gene knock out technique based on suicide plasmid can be used to study the pathogenic mechanism of target gene products of L.interrogans. Key words: Leptospira interrogans; fliN gene; Gene knock out; Mutant; Pathogenicity