Screening Of New Mycobacterium Tuberculosis Effector Proteins And Characterization Of MhpA-host Cell Interaction

Mycobacterium tuberculosis(Mtb)is a bacterial pathogen that causes tuberculosis.It can evade the host immune protection and enters into dormant state in the host,which finally results in tuberculosis.It is known that effective proteins of a pathogen usually play critically important roles during infection and pathogenesis.However,very few effective proteins of Mtb have been identified and little is known about the interaction bewteen effective proteins and host cells in Mtb.Here,we extensively screened and found a batch of new Mtb secreted proteins and studied the interaction between a new secreted protein(designated as MhpA)and macrophages.There are two main results:(1)Using Mycobacterium smegmatis as a model expression strain,we extensively screened and compared the regulatory effects of 446 potential secretory proteins of Mtb on cytokine release by infected macrophages.Cytometric Beads Array(CBA)was used to determine the secretion of several important cytokines in the culture medium supernatant of RAW264.7 cells and THP-1 cells infected by the recombinant strain of M.smegmatis.After several rounds of screenings,33 secreted proteins were found to significantly affect the secretion of several important cytokines in RAW264.7,and 32 proteins were shown to affect the secretion in THP-1.In addition,20 significantly affected the secretion of important cytokines both in RAW264.7 and THP-1 cells.(2)The new secreted protein MhpA was shown to significantly promote the release of IL-6 and enhance the mycobacterial survival in host cells.Firstly,we successfully constructed H37 Ra knockout strain and its complementary strain of mhpA homologous gene.Subsequently,by comparing the wild-type strain,knockout strain and complementary strain,we found that the expression levels of IL-6 in RAW264.7 by mhpA knockout strain were significantly decreased,indicating that mhpA may promote the release of IL-6 upon the mycobacterial infection with macrophages.Further,we found that the necrosis degree of macrophage infected by mhpA knockout strain was significantly lower than those of the infected cells by wild-type strain or complementary strain.Finally,if compared with wild-type strains and complementary strains,the intracellular survival rate of mhpA-knockout strains was significantly reduced in THP-1,indicating that mhpA was beneficial to the mycobacterial survival in host cells.These findings provide important clues for further dissecting the molecular interaction mechanism between Mtb and host cells.