Investigating genetic regulators of Mycobacterium tuberculosis cell division and their role in persistent tuberculosis infection

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PROJECT SUMMARY/ABSTRACT Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has infected about one-quarter of the global population and kills over 1 million people every year. Mtb is an exceptionally successful human pathogen largely because the bacteria can adapt to withstand several different stresses in vivo and can enter a state of replicative quiescence where Mtb can persist for months to years. This presents major challenges to treating tuberculosis as many anti-tubercular drugs target bacterial replication and cell wall synthesis. Mtb have a unique mechanism of division where the bacteria grow asymmetrically and produce daughter cells that vary in size, growth rate, and cell wall composition. This contrasts with cell division processes employed by other well-studied bacteria, and Mtb possess distinct cellular machinery and regulatory pathways to coordinate their cell division. Thus, it is critical to understand Mtb’s unique mechanism of replication, how it is regulated, and how it contributes to bacterial pathogenesis. The overall goal of this proposal is to elucidate two novel regulatory networks that control Mtb cell division, and to understand the role this regulation plays during tuberculosis infection when Mtb slow or halt their growth. I hypothesize that the uncharacterized transcription factors WhiA and WhiB2 are essential regulators of cell division in Mtb, and that both proteins are required for maintaining bacterial viability in vivo, even during persistent tuberculosis infection when the bacteria enter a slow or non-replicative state. This proposal seeks to i) utilize novel CRISPR interference technology in Mtb to interrogate the function of two essential and previously uncharacterized genes, ii) define two regulons that are critical for regulating cell division in Mtb, and iii) determine the importance of these regulatory networks during persistent tuberculosis infection. These aims employ phenotypically relevant models of Mtb infection, including macrophage and mouse models of disease. Successful completion of this proposal will advance the field by elucidating how Mtb regulate cell division and provide insight into how these regulatory pathways contribute to Mtb’s persistence and survival in vivo. Understanding mechanisms of Mtb replication and persistence can aid with improving and developing therapeutics for treating tuberculosis.