The Activity And Pathogenicity Of MAP1068 Protease Of Mycobacterium Avium Subsp. Paratuberculosis

Paratuberculosis is a chronic consumptive disease caused by Mycobacterium avium subsp.Paratuberculosis(MAP).This disease is mainly prevalent in ruminants,with dairy cows and cattle being the most susceptible.Cattle infected with MAP are characterized by persistent diarrhea,progressive emaciation,thickening of intestinal mucosa and formation of wrinkles,so MAP is also called paratuberculous enteritis.Since the first report of paratuberculosis in 1895,it has been discovered in many countries and now is spreading all over the world,this disease was first discovered in China in 1975.Paratuberculosis has great adverse impacts on the breeding industry,especially dairy and beef cattle breeding industry.The milk yield and beef quality of infected cows were affected to varying degrees.The MAP1068 protein is a highly conserved and species-differentiated protein,and bioinformatics predicts that the MAP1068 protein belongs to a kind of cystein protease.Many studies on pathogenic microorganisms(such as Streptococcus suis)include studies on cysteine proteases.Previous studies have found that cysteine protease played an important role in the invasion of pathogenic microorganisms into the host,especially in the process of microorganisms colonizing in the host and evading host immunity.In this study,the recombinant plasmid pET28a-MAP1068 was constructed,expressed and purified in E.coli.The anti-MAP1068 polyclonal antibody was prepared by BALB/c mice.The optimal substrate required for the activity of MAP1068 protein was screened,and it was confirmed that the divalent metal ions could increase the enzymatic activity of MAP1068 protein,and the optimal temperature and optimal pH value were determined.Site-directed mutagenesis revealed that the C195,H238 and D258 amino acid residues played catalytic roles in MAP1068,especially D258 played a decisive role for the protease activity of MAP1068.To investigate the pathogenicity of MAP1068 protease in mice,the pMV261 shuttle vector was transformed into Mycobacterium smegmatis and recombinant M.smegmatis(pMV261/MS)was constructed;The map1068 gene was cloned into the pMV261 vector and recombinant plasmids were transformed into M.smegmatis toconstruct recombinant M.smegmatis(MAP1068/MS).The recombinant strain D258A/MS was constructed by site-directed mutagenesis using the recombinant plasmid pMV261-MAP1068 as a template.The mice were infected by three recombinant strains(pMV261/MS,MAP1068/MS and D258A/MS)for pathogenicity studies.Pathological results showed that D258A/MS and MAP1068/MS had obvious pathological damage to liver,spleen and lung of mice.Cytokine detection in serum showed that MAP1068 protease before and after mutation can cause different changes in cytokines,leading to varying degrees of inflammatory response.The results showed that MAP1068 protein has the characteristics of protease as a kind of extracellular protease and has certain pathogenicity.After site-directed mutagenesis,its activity will be partially lost,but the pathogenicity of mice has no obvious change.This experiment provides a new way of thinking for the pathogenesis of M.tuberculosis and lays a foundation for scientific prevention and treatment of paratuberculosis.