Functional characterization of an essential mycobacterial protease

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, remains a global health threat due to its engimatic ability to withstand diverse environmental stresses in the host. The widespread phenomenon of antibiotic resistance can be at least partially attributed to Mtb’s incredible adaptibility. As such, an increased understanding of how this bacterium grows and survives under different conditions is ncessary to continue developing tools to prevent the spread of disease. In Chapter 1 of my dissertation, I present a brief overview of mycobacterial proteases. In Chapter 2, we explore the previously uncharacterized function of the predicted essential protease HtrA. We find that HtrA is essential and interacts with another essential protein of unknown function, LppZ. Loss of HtrA/LppZ leads to accumulation of the amidase Ami3, which is toxic when mannosylated. In the presence of HtrA/LppZ, Ami3 has a shorter half-life and accumulates to lower levels. These data suggest HtrA-LppZ blocks the toxicity of a cell wall enzyme. In Chapter 3, we explore another set of essential genes, FtsQLB, which we show to be critical for mediating proper mycobacterial cell division. We identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, adding to a previous body of work characterizing their partner FtsQ, and show that, as a set, these enzymes appear to function similarly to their homologs in E. coli. We then identify a number of previously undescribed septally-localized factors which could be involved in cell wall regulation, including SepIVA. Finally, in Chapter 4, we present preliminary findings on another conserved mycobacterial protease, FtsH. We show that FtsH is not essential for viability in Mycobacterium smegmatis or Mycobacterium tuberculosis. However, FtsH may play a small role in the viability of mycobacteria under conditions of oxidative stress. Additionally, we present candidate substrates for this protease. Characterizing the critical proteins that allow mycobacteria to survive extreme environments is a crucial pursuit in the push for new tubericidal agents. This work forms the foundation for future work on the stress-responsive growth patterns of mycobacteria, particularly through the lens of regulated proteolysis.