Functional Dissection of Toxin-Antitoxin Systems in Mycobacterium tuberculosis

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DESCRIPTION (provided by applicant): Tuberculosis is a widespread and potent human pathogen--at least on third of the world's population is infected with Mycobacterium tuberculosis (Mtb) yielding an annual death toll estimated at two to three million. Although the subject of intense study, this infection is extremely complex and is able to cause either an active disease or persist in a latent state. Latency represents an adaptation that has enabled the efficient persistence of Mtb in the human population. Latent infections (where the bacilli are thought to persist in the host macrophage) can be reactivated, especially in immune compromised individuals. In fact, elevated tuberculosis rates--and an alarming number of multi-drug resistant Mtb strains--are being documented in regions of high HIV incidence. The molecular switches that trigger Mtb latency are poorly understood. The study of toxin-antitoxin (TA) systems/modules in Escherichia coli coupled with bioinformatic analysis of their abundance in Mtb has suggested a possible association between these modules and the characteristic slow growth and dormancy exhibited by Mtb in latent tuberculosis. Expression of TA toxins impart a dormant state and have been linked to bacterial persistence, two properties intrinsic to latent tuberculosis infection. The TA toxin-mediated dormant state was discovered during the study the one of the six chromosomally encoded TA toxins in E. coli called MazF (derived from the mazEF TA module). In contrast to E. coli and other free-living organisms, the Mtb genome harbors a remarkably high number of TA toxin modules. While E. coli possesses a single mazEF TA module, the Mtb genome contains eleven analogous modules. Since conversion of Mtb cells from an actively replicating state to a dormant-like state is thought to coincide with the latency phase of tuberculosis, and because MazF expression triggers a type of dormancy in E. coli, this R21 application tests the hypothesis that the multiple relatives of MazF unique to the Mtb genome contribute to dormancy of this organism, which may be critical to establishing the state of latency that is the hallmark of this pathogen. The goal of the first aim is to determine if either overexpression, or the absence, of individual MazF-mt TA toxins influences Mtb growth rates and viability. The goal of the second aim is to determine if deletion of one or more MazF counterparts influences Mtb survival in established in vitro models of Mtb non-replicating persistence PUBLIC HEALTH RELEVANCE: The molecular mechanisms that underlie the latency phase of Mtb infection are poorly understood. It is crucial to understand this process in detail since organisms in a dormant state are refractory to antibiotic treatment, confounding effective management of the disease and thus enabling continued disease transmission. The proposed experiments will help in the development of more effective therapeutics that inhibit the signals which trigger or maintain dormancy or slow growth, thus increasing the efficacy of conventional Mtb antibiotics.