M. tuberculosis infection is an ongoing global health crisis that requires new drugs or vaccines for effective control. Although many individual genes have been shown to be important for M. tuberculosis pathogenesis in the mouse, we still understand relatively little about how M. tuberculosis responds to varied host environments at the molecular level. The Rip1 (Rv2869c) intramembrane protease is a member of the Site two protease (S2P) class of intramembrane metalloproteases which cleave substrate proteins within transmembrane domains. We have previously shown that Rip1 is a critical determinant of M. tuberculosis growth and persistence in mice and controls cell envelope composition through transcriptional regulation of lipid biosynthetic genes. Further preliminary data presented here identifies three anti-sigma factors as Rip1 substrates: anti-SigK (RskA), anti-SigL (RslA), and anti-SigM (RsmA). Accordingly, we show that SigK, SigL, and SigM target genes are not activated in the rip1 strain, demonstrating that loss of Rip1 inactivates three downstream transcriptional programs. In addition, we show that Rip1 is required for the regulation of iron responsive genes in response to iron limitation, a response that also requires SigL and SigM. Based on this data, we advance and test the major hypothesis that the severe attenuation of the Rip1 null strain is due to simultaneous inactivation of SigK, SigL, SigM regulons. To test this hypothesis, we propose a two year project to define the genetic and biochemical characteristics of the Rip1 pathway outlined in the specific aims below. We will determine the contribution of SigK/anti-sigK, SigL/anti-sigL, and SigM/anti-sigM to Rip1 dependent transcriptional regulation by transcriptional profiling of defined mutant strains in high and low iron. We will analyze anti-sigma factor cleavage in the same conditions using western blotting using anti-sigma factor antibodies in wild type and Rip1 deficient strains. Finally, we will define the molecular basis for site one cleavage of anti-sigma factors by genetically ablating candidate site one proteases and testing anti-sigma factor cleavage in these mutant strains. .
Relevance This project seeks to understand the function of the Rip1 protease of M. tuberculosis in the pathogenesis of Tuberculosis. Detailed understanding of the Rip1 pathway will validate this protease as a drug target and further our understanding of this important global pathogen.
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