Thursday, April 18, 2024
4/18/2024
Abstract Tuberculosis (TB), a bacterial infection caused by Mycobacterium tuberculosis (Mtb), is now the leading cause of death by a single infectious agent. One reason for this is that subpopulations of Mtb cells can survive even lengthy chemotherapy, preventing cure of disease. These surviving cells are phenotypically tolerant, but not genetically resistant, to antibiotic therapy. Thus, the ability of genetically identical bacteria to display different phenotypes is a significant obstacle for the treatment of TB. A better understanding of the molecular mechanisms underlying this phenomenon could lead to therapeutic advances for TB and other mycobacterial infections. Much of the heterogeneity begins at mycobacterial cell division. Every time a mycobacterium divides it produces daughters with different characteristics. We have recently discovered that this process is genetically encoded. Deleting a single gene, which we have named lamA, leads to cells that grow and divide more symmetrically and are more uniformly susceptible to certain antibiotics. The function of LamA and how it mediates asymmetric growth and division are unknown. Here, we propose to investigate the molecular function of LamA. Our published and preliminary data show that LamA localizes to the site of division, where it inhibits the maturation of the new growth poles. In addition, we have connected LamA to the phosphorylation state of an essential peptidoglycan synthase, and have discovered that its own localization is regulated by phosphorylation. This leads to our hypothesis that LamA dynamically interrupts an unknown communication relay between the multi-protein complexes that accomplish division and elongation, in a phosphorylation- dependent manner. To test this model, we propose the following aims: (1) define the communication relay between the division and elongation complexes; and, (2) dynamically map the regulatory events that lead to asymmetry. Our innovation is to study a mycobacterial-specific protein that creates heterogeneity in a genetically identical population. We will do so by leveraging our expertise in advanced imaging techniques in combination with more traditional methods. Successful completion of these aims will lead to hypotheses about the function of LamA that can be tested with molecular and biochemical approaches. Further, our results will advance our understanding of the molecular basis of cell-division mediated heterogeneity, which has far- reaching consequences for the treatment of TB. For example, identifying the molecular mechanism(s) that leads to subpopulations of bacteria better able to survive antibiotic therapy will allow us to design drug strategies that target heterogeneity. Such interventions could treat TB more quickly, something that would greatly help reduce the global burden of TB disease.
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