Study On The Mechanism Of Mycobacterium Tuberculosis Escape From Host Innate Immune Defense Mediated By PE12 Protein

Tuberculosis is a chronic,consumptive,zoonotic disease caused by infection with Mycobacterium tuberculosis(MTB).In 2021,there were 10.6 million new cases of TB worldwide,resulting in an estimated 1.3 million deaths.Until the coronavirus(COVID-19)pandemic,TB was the leading cause of death from a single infectious agent,ranking above HIV/AIDS.The worsening multidrug-resistant TB remains a serious global public health problem.Therefore,clarifying the interaction mechanism between MTB and host provides new targets and theoretical basis for the development of antituberculosis drugs and vaccines.Macrophages are the main immune cells of host’s innate immune defense against MTB infection.During MTB infection,macrophages express a variety of pattern recognition receptors,such as TLRs and NLRs,which participate in the recognition and uptake of MTB and thus initiate the defense function of various innate immune cells.Among them,NLRP3 of the NLRs family plays an important role in the resistance to MTB infection.On the other hand,MTB escapes the host innate immune killing by inhibiting the activation of NLRP3 inflammasome,but the detailed mechanism is still unclear.In this study,the recombinant Mycobacterium smegmatis(Msg)expressing of Mycobacterium tuberculosis PE/PPE family proteins were constructed,and we found that PE12-Msg can inhibit the secretion of proinflammatory cytokines IL-1β and TNF-α in murine peritoneal macrophages.Studies showed that the PE12 protein did not affect the growth rate of Msg,but it changed the morphology of Msg colony.High purity PE12 protein was obtained by expression of E.coli and purifying it through affinity chromatography.An anti-PE12 polyclonal antibody was prepared by immunizing New Zealand white rabbit.The results of subcellular localization showed that PE12 protein was localized on the cell wall and membrane of Mycobacterium tuberculosis and was an extracellular protein.Using pAIN-Msg and PE12-Msg,and MTB H37 Rv,H37RvΔ PE12 and H37RvΔ PE12::PE12infected murine peritoneal macrophages.It was found that PE12 protein can not only inhibit the transcription and secretion of proinflammatory cytokines IL-1β 、 IL-6 、 IL-12 and TNF-α in macrophages but also suppress the transcription and expression of NLRP3 in macrophages.Additionally,it promoted intracellular colonization of mycobacteria by inhibiting macrophage death.To study the interaction between PE12 protein and host protein,the Pulldown assay was used to screen host proteins interacting with PE12 protein.The result showed that there was an interaction between the PE12 protein and TLR4 protein.After treatment with the TLR4 inhibitor TAK242 in mouse peritoneal macrophages,the inhibitory effect of PE12 protein on IL-1β transcription and release in macrophages,NLRP3 transcription and expression in macrophages,LDH release in macrophages,and promotion effect on the intracellular colonization of mycobacteria was lost.These results indicated that PE12 protein mediates MTB escape from the killing of macrophage through TLR4.The 6-to 8-week-old C57BL/6 mice were infected with recombinant Msg pAIN-Msg and PE12-Msg via tail vein injection.The PE12 protein promoted the colonization of Msg in the lungs,liver,and spleen of mice by inhibiting the m RNA transcription level in the lungs and spleen and serum release level of IL-1β of infected mice.The pathological and histopathological results showed that PE12 protein promote pathological injury of lungs,liver and spleen of mice infected with recombinant Msg.In conclusion,PE12 protein not only inhibited the transcription and release of inflammatory cytokines but also mediated the killing of MTB escape macrophages through TLR4.Additionally,it promoted the colonization of mycobacteria in mouse organs and causes serious pathological injury.This study provided a basis for the analysis of the interaction mechanism between MTB and host,and provided a new drug target for development of anti-TB drugs.