Post-Translational Regulation via Clp Protease Is Critical for Survival of Mycobacterium tuberculosis

Abstract

Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology.

The Mycobacterium tuberculosis Clp Gene Regulator Is Required for in Vitro Reactivation from Hypoxia-induced Dormancy

“…Thus far, RpsL has been identified as a Clp protease target under normal culturing conditions (27). Upon expression profile analysis, we found no expression of rpsL within Mtb at early stages or reaeration, whereas there was increased expression of rpsL at later stages of reaeration within the mutant (Fig.…”

“…Interestingly, B , rpsL, dnaK, and Rv3334 have been identified as Clp protease substrates (27). As such, upon day 1 of hypoxia, these genes were induced in Mtb but were not induced at later time points (Fig.…”

“…Mtb achieves this by invoking the expression of multiple stress induced sigma factor-anti-sigma factor networks, eukaryoticlike protein kinases, two-component regulatory systems, and transcriptional activators and repressors (22, 30 -34). In addition to transcriptional regulation, it is increasingly being recognized that post-translational regulation of gene expression is a key component of Mtb phase variation (27). Thus, several dozen toxins encoded by the Mtb genome contain the potential to target and degrade specific transcripts and are regulated by their cognate antitoxin pairs yet exhibit promiscuous binding affinities (35,36).…”

“…Although a majority of Clp protease substrates are currently unknown in Mtb, a small subset of substrates have been identified using a conditional knock down under normal culturing conditions (27). Interestingly, B , rpsL, dnaK, and Rv3334 have been identified as Clp protease substrates (27).…”

The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host

“…Raju et al identified 132 and 107 putative ClpP substrates in M. tuberculosis and Mycobacterium smegmatis, respectively, by quantitative proteomic profiling of the wild-type and ClpP knockdown strains (40). The two ClpP homologs (ClpP1 and ClpP2) in M. tuberculosis and M. smegmatis form the ClpP proteolytic core and are essential for mycobacterial survival in vitro and during infection of mice (33,40,41).…”