Person:

Schweber, Jessica

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 goal of this work was to investigate whether a ubiquitous bacterial protein, peptidyl-tRNA hydrolase (Pth), would be an appropriate candidate for target-based drug discovery research directed against pathogenic M. tuberculosis. Many successful antibiotics target protein synthesis machinery to arrest bacterial growth. Hydrolysis of peptidyl-tRNA by Pth is an important action during recovery from stalled protein synthesis. To determine whether an attack on Pth would damage M. tuberculosis, several Pth depletion strains were created. A tightly regulated knockdown strain confirmed essentiality of the protein for normal growth. Loosely regulated partial knockdown (hypomorph) strains were created for use in drug-susceptibility testing which showed that depletion of Pth induced hypersensitivity to macrolide antibiotics erythromycin, clarithromycin, and azithromycin. These drugs are comparatively ineffective against M. tuberculosis and are not used in first line treatment regimens. Pth was shown to be an attractive candidate for drug discovery research not only because it is essential but because abrogation of Pth function may sensitize this pathogen to an entire class of well-characterized drugs not typically used to combat M. tuberculosis.