Where Proteins Go to Die: Elucidating the Physiological and Therapeutic Significance of the Clp Protease Complex in Mycobacterium Tuberculosis

Abstract

Microbiologists have long focused on transcription as a main source of physiological regulation in bacterial adaptation. However, the time scale on which certain cellular responses must be coordinated dictates that a more rapid system be in place to deal with sudden environmental stresses. In eukaryotes, understanding the proteasome and ubiquitin-tagging has led to an appreciation for protein turnover as a mechanism for rapid adaptation. Like eukaryotes, bacteria possess several proteolytic complexes that degrade proteins into smaller polypeptides and amino acids. These enzymes were discovered as maintainers of protein quality control, through recognition of aberrant protein products, but recent studies have suggested that they play an active role in regulation of cell processes through degradation of endogenous proteins. Surprisingly, a genome wide screen for essential genes in Mycobacterium tuberculosis (Mtb) found numerous proteases to be essential for growth, providing evidence that degradative regulation may be critical for survival. One essential complex, Clp protease, was intriguing as it appeared to have a divergent structure in Mtb, and was largely dispensable for growth in most other organisms. In order to study the importance of protein turnover and degradative regulation in Mtb, I chose to study Clp as a model. I confirmed that Clp was required for normal growth in mycobacteria through targeted genetic engineering, and demonstrated that depletion of Clp was bactericidal. We hypothesized that a protease would be essential because it might prevent accumulation of toxic proteins or repressors of vital processes. To understand why Clp protease was so critical, I conducted proteomic analysis comparing wildtype and Clp-depleted cells to identify substrates of the protease. In line with our hypothesis, I identified WhiB1, a redox-sensitive transcriptional repressor. Blocking degradation of WhiB1 by Clp resulted in death, suggesting that the importance of Clp can be partially explained by its action on the repressor. Finally, taking advantage of known Clp-specific inhibitors in S. aureus, we showed that Clp could be targeted with small molecules in Mtb. The elucidation of novel drug targets and small molecules active against Mtb is crucial due to the overwhelming prevalence of the disease and rises in drug resistant forms.