Why WhiB1: Manipulation of Clp System to Determine Role of WhiB1 in Regulating Mycobacterial Cell Division

Abstract

Mycobacterium tuberculosis (Mtb) is the causal agent of tuberculosis (TB), a major public health issue in the world. In the 2015 WHO report, there were approximately 10.4 million new cases of TB worldwide and 1.4 million TB deaths. Moreover, the rising number of multi-drug-resistant (MDR) and extensively drug-resistant (XDR) TB cases highlights the urgent demand for new TB drugs. In Mtb, the Clp system is essential and provides a promising target for new antibiotics. In this thesis, we identified the preferred cleavage sites of ClpP1P2 and synthesized new peptide boronate inhibitors that are specific to mycobacteria. Several compounds inhibited ClpP1P2 activity in vitro and growth of both Mtb and Mycobacterium smegmatis (Msm) by specifically targeting ClpP1P2 proteolytic activity. As more compounds inhibiting the Clp system are identified in mycobacteria, there is a growing need to understand the precise function of the mycobacterial Clp system. We hypothesized that Clp essentiality stems from its role as a proteolytic regulator of substrates that become toxic when accumulated. To determine the substrates of mycobacterial Clp, we conducted quantitative proteomics to identify proteins that accumulate upon depletion of the Clp protease. One identified substrate was WhiB1, an essential transcriptional regulator whose function was largely unknown. To further understand WhiB1 as a Clp substrate, we utilized peptide boronates and genetic depletions of Clp components. Blocking the degradation sequence of WhiB1 resulted in toxic accumulation. We found that the transcriptional regulatory function of WhiB1 was responsible for the toxicity of WhiB1 accumulation. RNAseq identified essential genes involved in mycobacterial cell division that were repressed by WhiB1, namely sepF, ftsZ, and mtrAB. WhiB1 accumulation decreased levels of FtsZ in cells and caused elongation, branching, and loss of FtsZ ring formation. Taken together, we conclude that Clp essentiality is in part due to its role in degrading WhiB1, a cell division regulator.