Transcriptional Factor HpoR Regulates The Expression Of PDIM Synthetic Gene Cluster In Mycobacterium Bovis And The Pathogen-host Interaction

Mycobacterium tuberculosis has a unique cell wall structure that is closely related to its pathogenicity.Located on the cell surface,the phthiocerol dimycocerosate?PDIM?represents a typical component of the pathogenic mycobacterial cell wall and is required for cell wall integrity.PDIM has been recognized as one of the important virulence factors of M.tuberculosis.Its biosynthesis and transport play indispensable roles during infection and for the mycobacterial survival in host cells.However,very little is known about the transcriptional regulation of PDIM metabolic genes,and potential regulators have not been identified yet.In this study,M.bovis BCG was used as a model strain,and the transcription factor HpoR was first discovered to directly regulate the expression of PDIM gene cluster.The results are as follows:?1?Using EMSA and ChIP,we demonstrate that HpoR was found to specifically bind to the PDIM operon promoter bcg₂950p;?2?By designing and preparing bcg₂950p DNA fragments covering different lengths,EMSA experiments showed that HpoR specifically binds to the P1 segment of bcg₂950p,and further sequence analysis found that it contained a conserved reverse palindrome;?3??-galactosidase activity assay and real-time quantitative PCR?qRT-PCR?showed that HpoR is a transcriptional repressor and it represses the expression of target genes.When overexpressing HpoR,the expression of PDIM gene cluster is down-regulated,and HpoR knockout results in up-regulation of PDIM gene cluster expression;?4?Using HpoR recombinant strains to infect macrophages,it was found that knockout of HpoR enhanced the survival ability of BCG strains in macrophages.On the contrary,overexpressing HpoR decreased the mycobacterial survival in macrophages;?5?By comparing the secretion levels of cytokines in macrophages infected by different recombinant strains,it was found that HpoR knockout strains caused a significant increase in the secretion of pro-inflammatory cytokine IL-6;?6?Upon the mycobacterial infection,HpoR overexpressing BCG strain induced an increasingly apoptosis rate of macrophages and the ROS level.Taken together,HpoR was characteriaed as a new t transcriptional regulator for the expression of PDIM gene cluster in mycobacteria.Furthermore,we found that HpoR also affected the mycobacterium-host cells interaction.These findings provide important clues for further dissectingthe pathogenesis of Mycobacterium tuberculosis.