The Functional Study Of Mycobacterium Avium Subsp.Paratuberculosis GDSL Lipase MAP＿2739

Paratuberculosis is a chronic wasting infectious disease of many ruminants caused by infection with Mycobacterium avium subsp.paratuberculosis(MAP),characterised by intractable diarrhoea,progressive wasting,thickening of the intestinal mucosa and formation of folds.MAP is an intracellular parasitic bacterium that,due to its long-term presence in host cells,is not only able to escape the killing of host immune defences,but also to develop a latent state of MAP infection.Therefore,it is imperative to conduct research on the pathogenic mechanisms of MAP in order to find effective prevention strategies and new means of treating MAP infections.In this study,a bioinformatics prediction method was applied to search and analyse the conserved amino acid residues in the MAP genome,and four conserved structural domains of-GDSL-lipase were found in the amino acid sequence of the protein encoded by MAP＿2739,suggesting that MAP＿2739 protein is the lipase of MAP based on the description in the NCBI database.The MAP＿2739 protein was obtained by PCR amplification,expression vector construction,prokaryotic expression and purification studies,and its protease activity was measured.The results showed that MAP＿2739 has lipase activity,and the optimum p H value of MAP＿2739 protein was determined to be 9.0.The lipase activity showed a positive correlation with temperature(25℃～83℃),while the application of pasteurization(65℃,30 min or 83℃,15 s)to treat MAP＿2739 protein further verified that the lipase activity was enhanced under high temperature conditions instead.and confirmed that nickel and manganese ions promoted the protease activity of MAP＿2739.The application of site-directed mutagenesis to MAP＿2739 demonstrated that amino acid residues 204 D and 46 S are the active centre of GDSL lipase MAP＿2739 and play a decisive role in lipase activity.The recombinant Mycobacterium smegmatis rMsg-2739 was constructed by connecting the MAP＿2739 gene to the p MV-261 Mycobacterium shuttle vector and electrotransforming competent Mycobacterium smegmatis(Msg).The GDSL lipase MAP＿2739 belongs to the extracellular protein of MAP and can significantly change the single colony morphology of Mycobacterium smegmatis after heterologous overexpression.The single mutant strains of rMsg-S46 A and rMsg-D204 A,and the double mutant strain of rMsg-D204AS46 A were used to infect mouse peritoneal primary macrophages,and the results of colony counting showed that GDSL lipase MAP＿2739significantly enhanced the survival of recombinant Mycobacterium discoideum in mouse peritoneal macrophages.Application of confocal microscopy showed that MAP＿2739 could colocalize with the lipid raft structural marker protein,caveolin-1,suggesting that MAP＿2739 could bind to lipid rafts in macrophage cell membranes.Transmission electron microscopy showed that GDSL lipase MAP＿2739 promoted the escape of Mycobacterium smegmatis from phagosomal vesicles and significantly caused damage to mouse peritoneal macrophages by the bacteriophage.The pathogenicity of recombinant Mycobacterium smegmatis rMsg-2739,rMsg-D204AS46 A and rMsg-261 was evaluated in mice infected with C57BL/6J by intraperitoneal injection route.The results showed that the GDSL lipase MAP＿2739 promoted the colonization of Mycobacterium smegmatis in mouse organs and caused different degrees of pathological damage to the liver and spleen,and also promoted the release of the inflammatory cytokines TNF-α,IFN-γ and IL-6 in mice.In summary,the study confirmed that MAP＿2739 is an extracellular GDSL lipase of MAP and elucidated the pathogenic mechanism of MAP＿2739 by promoting the escape of Mycobacterium smegmatis from the phagosomes of macrophages,thereby enhancing its survival in the host and ultimately the persistent MAP infection.This study not only provides an understanding of the pathogenic mechanism of MAP,but also lays the foundation for treatment and drug development for paratuberculosis.