Signal Transduction across the M. Tuberculosis Cell Envelope via Regulated Intramembrane Proteolysis

Rip1 as a critical virulence determinant of M. tuberculosis

Present model of the Rip1 pathwayPresent model of the Rip1 pathway. The Rip1 protease has four transmembrane domains (pictured in red). The specific host-derived activating signals for the Rip1 pathway are not known, but the pathway is critical for M. tuberculosis survival within the host. Rip1 has four known substrates, the anti-Sigma factors for three ECF sigma factors — SigD, SigK, SigL, and SigM. Multiple downstream pathways are controlled by Rip1 and its substrates. We also have strong evidence for SigKLMD-independent arms of the Rip1 pathway (not pictured), suggesting additional substrates.

Signal transduction across membranes through regulated intramembrane proteolysis is conserved throughout all domains of life. In the SREBP pathway of human cells, the membrane-bound SREBP transcription factors undergo sequential cleavage by site-one and site-two proteases (S1P and S2P, respectively), with human S2P being the founding member of the zinc metalloprotease family of intramembrane proteases. RseP, an E. coli S2P, cleaves the anti-Sigma factor for SigE and thereby also regulates transcription in response to environmental signals, specifically unfolded outer membrane proteins. Our original characterization of the Rip1 (Rv2869c) pathway in M. tuberculosis was based on our suspicion that S2Ps in M. tuberculosis might be important for virulence and regulation of lipid biosynthesis. (1)The M. tuberculosis Δrip1 strain has the following phenotypes:

  • M. tuberculosis Drip1 titers in mouse lung are 100-fold lower than wild type during acute infection and 10,000-fold lower during chronic infection. Both of these phenotypes are complemented by the wild type copy of rip1.
  • Altered biosynthesis of extractable mycolic acids
  • Altered transcription of lipid biosynthetic genes, including genes encoding mycolic acid biosynthetic enzymes.

These results established the Rip1 pathway as an important virulence determinant through transcriptional control of downstream target genes. The phenotype of the Rip1 knockout is among the most severe attenuation phenotypes for a single gene mutant of M. tuberculosis. We have identified three anti-Sigma factor substrates of Rip1, anti-SigK, L, and M, which regulate multiple downstream target genes that mediate oxidant defense and iron storage. (2) These findings suggest a model in which the Rip1 protease controls multiple ECF sigma factor pathways. However, further data from the lab indicates that the phenotype of an M. tuberculosis ΔsigKLM triple mutant in mice does not phenocopy the severe attenuation of Δrip, strongly indicating that Rip1 controls additional pathways independent of SigKLM that are important for virulence. We are actively investigating the additional downstream pathways controlled by Rip1, the upstream activators of each arm of the pathway, and additional components of the Rip1 signal transduction system. Our present model of the Rip1 pathway is shown in the figure to the right.

Rip1 as a model system for S2P substrate choice and regulation

Adapter protein model of Rip1 substrate specificityAdapter protein model of Rip1 substrate specificity. — Ppr1 binds to both the Rip1 PDZ domain and the C terminus of the RsmA Rip1 substrate. Rip1 is tethered to RsmA by Ppr1 through the Rip1-PDZ, thereby holding Rip1 inactive. S1 cleavage of RsmA, which is not inhibited by Ppr1, releases the inhibitory effect of Ppr1 on the PDZ domain, allowing Rip1 cleavage. The model predicts additional unidentified substrate-specific adapters that tether Rip1 to Anti-SigK and Anti-SigL.

In addition to its critical role in M. tuberculosis pathogenesis and signal transduction, Rip1 also serves as a fertile model system for S2P biology. S2Ps often have multiple substrates, but the mechanisms of substrate recognition and specificity are not well understood. 
“We are using the Rip1 pathway as a model to investigate the following principles of S2P regulation:

  1. How is the S2P held inactive until site-1 cleavage has activated the pathway?

  2. In the absence of a conserved proteolytic cleavage site, how do S2Ps identify physiologic substrates while avoiding nonspecific cleavage of transmembrane segments.

  3. Our work in this area has identified a family of proteins that bind directly to the Rip1 PDZ domain. We have characterized one of these proteins, Ppr1, and shown that is bridges between Rip1 and one its substrates, anti-SigM. These results suggests that Rip1 is tethered to its substrates via an adapter protein and that distinct Rip1 signaling complexes exist. As many S2Ps have PDZ domains, adapter proteins similar to Ppr1 may exist in other systems.