Mechanisms On The Lipid Biosynthesis Regulation Of Mycobacterium Tuberculosis And Pathogen-host Interaction Triggered By MpbR

Mycobacterium tuberculosis is an important intracellular pathogen causing human and animal tuberculosis.It possesses unique cellular envelope components that significantly contribute to bacterial escape from host immune surveillance and the adaptation of the pathogen to the host environment during infection.Several studies suggested that phosphatidylinositol mannoside（PIM）and its derivatives are important molecules implicated in host-pathogen interactions and modulating host immune in the course of tuberculosis.However,the biosynthetic regulation of these specific lipids and its effect on the bacterial fate in the infected host remain unclear.In this study,using Mycobacterium smegmatis mc²155 strain as a model,we first identified a conserved transcriptional factor Mpb R participating in the mycobacterial PIM biosynthesis regulation.Further,we dissected the regulation pathway of PIM biosynthesis in M.tuberculosis and investigated the effect of the transcriptional regulation on mycobacterial escape from host immune.The details are listed below:（1）A transcriptional factor Mpb R affecting mycobacterial colony morphology and biofilm formation was identified based on transposon library screening.A transposon library containing over 30,000 mutants of M.smegmatis was produced using TM4 phage-delivery of transposons.Based on visual inspection of changes in colony morphologies,180 mutants with smooth colonies were isolated from the library.A hypothetical transcriptional factor encoded by mpb R is conserved in several mycobacterial species,such as M.smegmatis and M.tuberculosis,and remarkably affects the colony morphology and significantly enhances the mycobacterial biofilm formation.（2）Two target transporter genes（Mra1696/97）of Mpb R inhibit the biofilm formation of mycobacteria.Transcriptomic assays revealed that Mpb R acts as an inhibitor and negatively regulates the expression of two hypothetical transporter genes（Mra1696/97）encoded by the same operon.A 44-bp DNA binding motif recognized by Mpb R in Mra1697p was identified by EMSA and DNaseⅠfootprinting assays,and the interaction between Mpb R and Mra1697p was confirmed by B1H and Ch IP assays in vivo.Futher q RT-PCR andβ-galactosidase activity assays confirmed the negative regulation of Mpb R on Mra1696/97 expression.Furthermore,the two transporter genes were shown to affect the biofilm formation of M.tuberculosis H37Ra.（3）Mpb R reduces Ac₁PIM₂and Ac₂PIM₂accumulation through regulating expression of Mra1696/97.Lipidomic assays were performed to compare the differential lipid profiles of five recombinant M.tuberculosis H37Ra strains.Compared with those in the wild-type strain,the contents of two acylated PIM lipids,Ac₁PIM₂and Ac₂PIM₂,were reduced in mpb R-overexpressing and Mra1696/97-deleted M.tuberculosis H37Ra strains.By contrast,Ac₁PIM₂and Ac₂PIM₂were accumulated in mpb R-deleted and Mra1696/97-overexpressing strains.This result suggests that Mpb R may inhibit the accumulation of two cell envelope lipid components in M.tuberculosis through reducing the Mra1696 and Mra1697 expression.（4）Mpb R-triggered lipid metabolism significantly affects host cell immune responses induced by M.tuberculosis.Transcriptomic assays revealed that Mpb R reduces some immune genes expression including Il1b,Ccl2,and Nos2 in macrophages infected with the recombinant M.tuberculosis H37Ra strains.CBA assays confirmed that Mpb R inhibits IL-1β,IL-6,and MCP-1 secretion of infected macrophages.And Mpb R could reduce reactive oxygen species and NO production,and prohibits macrophages apoptosis and the inflammatory responses of macrophages during M.tuberculosis infection.（5）Mpb R enhances M.tuberculosis survival in the host.Utilizing two macrophages models,we found that Mpb R can enhance the survival of M.tuberculosis in macrophages.In further animal experiments,we showed that,compared with the wild-type strain,Mpb R overexpression increased M.tuberculosis burdens in the lungs and inflammation of infected mice.In summary,Mpb R has been identified as a mycobacterial PIM biosynthesis regulator,which negatively regulates two transporter genes Mra1696/97 expression.Mpb R reduces the Ac₁PIM₂and Ac₂PIM₂accumulation in mycobacterial cells and enhances the biofilm formation of mycobacteria.Mpb R can trigger the mycobacterium to inhibit the secretion of inflammatory cytokines,the production of reactive oxygen species and NO during infection,which ultimately prohibits cell apoptosis and enhances the survival of M.tuberculosis in macrophages and mice.These findings provide new insights into the regulation of mycobacterial lipid metabolism and its correlation with pathogenesis of M.tuberculosis.