Mechanism Of Mycobacterium Bovis PPE13-induced IL-1? Secretion

Mycobacterium bovis,which belongs to intracellular Gram-positive bacteria,can cause humans and mammals tuberculosis through respiratory tract.With the continuous occurrence of HIV and Mycobacterium infection and drug-resistant strains,the diagnosis and treatment of drug-resistant tuberculosis is becoming more and more difficult.Therefore,the molecular mechanism of Mycobacterium infection is particularly important for the diagnosis and treatment of tuberculosis.It has been reported that PE/PPE family members play an important role during Mycobacterium bovis infection in host cells,which is thought to be the main source of bacterial virulence and antigen shift.To investigate the role of PPE13 in Mycobacterium bovis infecting host cells,the recombinant Mycobacterium smegmatis expressing PPE13 was constructed by molecular cloning technology in the study.Firstly,the gene ppe13 was amplified by PCR and inserted into the vector p MN437.The recombinant vector was transformed into Mycobacterium smegmatis.Western blot analysis showed that PPE13 could express in Mycobacterium smegmatis and locates in cell wall.Growth curve analysis of Mycobacterium smegmatis indicated that PPE13 does not influence the growth of bacteria in vivo.We used the recombinant Mycobacterium smegmatis Ms_PPE13 expressing PPE13 to infect murine macrophages,the result showed that that PPE13 did not affect the survival of Mycobacterium smegmatis in macrophages,but it could promote the death of infected macrophages.The secretion of IL-1? can cause cell membrane rupture thus causing cell death,and PPE13 can significantly promote the secretion of IL-1? caused by Mycobacterium smegmatis,which is dependent on the activation of Caspase-1.In addition,activation of Caspase-1 is regulated by inflammasome.We found that PPE13 can promote the expression of NLRP3,which is a component of NLRP3 inflammasome,and induce the oligomerization of ASC.MCC950,a specific inhibitor of NLRP3 inflammaasome,can significantly inhibit PPE13-induced IL-1?secretion,which indicates that PPE13 can activate the NLRP3inflammasomeIn conclusion,the recombinant Mycobacterium smegmatis expressing PPE13 was successfully constructed.PPE13 can induce pyroptosis and IL-1? secretion in macrophages by activating NLRP3 inflammasome.These results provide important fundations for the understanding of the pathogenity of Mycobacterium bovis.