Targeting Knock Out Of Leptospira Interrogans Flagellum-associated FliN Gene And Pathogenic Function Alteration Of The Mutant

Background and ObjectiveLeptospira interrogans is recognized as one of the causative agents of leptospirosis causing nanukayami fever in both animals and humans within the worldwide range,whose pathogenic mechanism still remains to be answered.The result shows that L.interrogans with different toxicity can adhere to host cell using one or two terminals,which is just the location of L.interrogans flagellum terminal knob of basal body.The basal body,which containⅢsecretion system,not only undertake to output flagellin when bacteria split and flagellum grow,but also help some factors related to bacterial adhesion,invasiveness and toxicity to release through T3SS.L.interrogans fliN gene is comment as a flagellar motor switch protein,but its sequence has the common motif Pf:SpoA with E.coli fliN gene and Yersinia pestis yscQ gene.E.coli fliN gene belongs to T3SS and its product plays an important role during bacterial adhesion and intrusion,while yscQ is a virulence protein about invasiveness in Yersinia T3SS. Therefore it seems quite possible that L.interrogans fliN has similar functions with E. coli fliN and Yersinia yscQ.In this research,we use gene knockout technique to construct L.interrogans mutant strain with inactive fliN gene.By comparing the wild strain and mutant strain as well as the differences between secreting virulent,adhering cell and inducing cell apoptosis,we study the pathogenic mechnism of L.interrogans fliN gene.Methods Target gene knock out technique based on homologious sequence recombinance of suicide plasmid and chromosome was applied to construct fliN unexpressed mutant from L.interrogans serovar Lai strain Lai.PCR,sequencing and Western Blot assay 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,e,cytotoxicity of the culture supernatant to murine mononuclear-macrophage like cell line(J774A.1),adhering to and inducing apoptosis of J774A.1 cells of fliN~- mutant.Results All the results of PCR,sequencing and Western blot assay confirmed a successful construction of fliN~- mutant,and this mutant could growth as well as propagate in 100μg/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 that that of wild strain.Furthermore,both the necrotic and apoptotic rates(30.6%and 22.7%) induced by fliN~- mutant were lower than those (48.2%and 35.2%) acused by wild strain.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 suicide plasmid can be used to sduty the pathogenic mechnism of target gene products of L.interrogans.