Functional Investigation With Serine Protease MAP3292c Of Mycobacterium Avium Subsp. Paratuberculosis

Paratuberculosis is a chronic irreversible consumptive disease of ruminants caused by Mycobacterium avium subsp.paratuberculosis(MAP),characterized by chronic diarrhea,progressive extenuation,and hyperplastic enteritis.It is widespread worldwide and causes serious economic losses to the cattle and dairy industries.Therefore,it is an urgent need to study the pathogenic mechanism of MAP and provide strong scientific support for vaccine development and diagnosis and treatment of paratuberculosis.This study predicted serine protease gene map3292 c in MAP by bioinformatics.MAP3292 c protein was obtained by PCR amplification,construction of expression vector and prokaryotic expression and purification.The results showed that MAP3292 c protein has serine protease activity.The optimal temperature and pH of MAP3292 c protein were determined to be 41 ° C and 9.0 by casein quantitative method,which confirmed that calcium ion could promote the activity of MAP3292 c protease.Site-directed mutagenesis studies showed that the 63 H,87D and 238 S amino acid residues play catalytic roles in MAP3292 c protease,especially the 238 S site plays a decisive role in its activity.And MAP3292 c protease can also destroy its bactericidal activity by degrading LL-37.map3292c gene was cloned into a pMV261 shuttle vector and recombinant plasmids were transformed into Mycobacterium smegmatis(MS)cells and recombinant MS MAP3292c/MS was constructed.In order to determine the subcellular localization of MAP3292 c protein,and differential centrifugation was used to separate MAP3292c/MS strain subcellular fractions of cell lysates(CL),culture filtrate(CF),cell wall(CW),cytoplasm(CP)and cell membrane(CM).Using polyclonal antibody against MAP3292 c protein,we determined that the MAP3292 c protein was mainly localized in the cell wall,and secreted into the culture supernatant,and localized in a small amount on the cell membrane by the Western Blotting.In order to study the pathogenicity of MAP3292 c protein,recombinant plasmid pMV261-MAP3292 c was used as template to construct a serine protease mutant recombinant S238A/MS,and 5-week-old BALB/c mice was infected with MAP3292c/MS and S238A/MS recombinant strains,respectively,and pMV261/MS was used as control.The results showed that MAP3292 c protease can promote the colonization ability of MS,and cause damage of liver,spleen and lung,and promote the release of inflammatory cytokines IL-1?,IL-6 and IL-10,TNF-? in mice.This study not only enriched the pathogenic mechanism of MAP,but also laid the foundation for the treatment and drug development of paratuberculosis.