Tuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains a serious global health threat that kills over a million people per year worldwide. While antibiotic treatments have existed for some time, they are often not successful. The necessity for an extended treatment course and the development of resistance to commonly prescribed antibiotics during treatment are major challenges that need to be overcome to improve patient outcomes. Chromosomal mutations due to the action of error-prone DNA polymerases are a major driver of mutagenesis in Mtb and therefore the emergence of antibiotic resistance. Error-prone polymerases are involved in a DNA damage tolerance process known as translesion synthesis. Typically, error-prone polymerases are tightly regulated so that they are not active during DNA replication. However, they become activated under stress conditions, such as those experienced during antibiotic treatment. In recent work from our laboratory, we demonstrated that within the model bacterium Escherichia coli, the activation of the error- prone polymerase Pol IV requires an interaction with single-stranded DNA binding protein (SSB), which acts to locally concentrate Pol IV near sites of DNA damage. Importantly, ablation of this interaction substantially reduced Pol IV-mediated translesion synthesis and mutagenesis. In this proposal we will test whether the three Pol IV homologs in Mtb interact with MtSSB. Next, we will identify mutations that ablate this putative interaction without affecting other molecular interactions or polymerase activity. Finally, we will introduce these mutations into Mycobacterium smegmatis, a non-pathogenic model system of Mtb, and test whether these strains are sensitized to DNA damage agents. If successful, these studies will identify a novel molecular interaction to potentially target therapeutically to suppress the emergence of antibiotic resistance in Mtb.
Despite the development of therapeutics, tuberculosis remains a major global health crisis. Treatment requires prolonged exposure to antibiotics, often resulting in the emergence of antibiotic resistant strains. This proposal seeks to test whether Mycobacterium tuberculosis homologs of the error-prone polymerase Pol IV interact with single-stranded DNA binding protein and whether ablating this interaction might reduce antibiotic resistance.
