The diagnosis and treatment of tuberculosis is complicated by the fact that Mycobacterium tuberculosis (M. tuberculosis) can cause both clinically active infection and clinically inapparent or latent infection, during which bacteria evade the host immune system and persist in the human host for decades. During both active and latent infection, a subpopulation of bacteria likely exists in a non-replicating state, refractory to traditional antibiotics. We propose to understand the physiology of this state by identifying essential functions in M. tuberculosis necessary for establishing and maintaining this non-replicating state. Because it is difficult to study essential gene functions using classical genetics given inadequate genetic tools for M. tuberculosis, our approach is to use two complementary methods, genomics and chemical biology, facilitating the study of genes essential under in vitro culture conditions. We propose a systematic screen of a comprehensive arrayed transposon knockout library to identify genetic mutants and a forward chemical genetics screen to identify small molecules affecting the viability of carbon-starved bacteria. We hypothesize that a subset of the genetic mutants and small molecules identified will have targets in metabolic or regulatory pathways important in other models of non-replication, including a stochastic drug tolerance model, an anaerobic model and a stationary phase model, and that these targets are also important in establishing and maintaining a persistent infection in vivo. The outlined research proposal will provide the candidate with training in molecular biology and genetics techniques relevant to M. tuberculosis, chemical biology, the application of genomics techniques to microbiology, and most importantly, experimental design and scientific critical thinking. These skills, combined with the outlined career development plan, will prepare the candidate for a career as an independent physician-scientist in the field of M. tuberculosis pathogenesis. The work will be conducted at the Broad Institute of Harvard University and MIT under the mentorship of Dr. Deborah Hung, Assistant Professor of Microbiology at Harvard Medical School, and Dr. Eric Rubin, Professor of Immunology and Infectious Diseases at the Harvard School of Public Health.
Despite the use of potent anti-TB agents like isoniazid (INH) and rifampin, the global incidence of tuberculosis has been increasing over the last 25 years, driven by HIV, increasing emergence of drug resistant strains, immigration and the persistent nature of infection. Successful treatment of infection typically requires a complex treatment regimen over at least six months. The long duration of therapy is likely due in part to the presence of non-replicating bacteria within the human host. This proposal aims to contribute to our understanding of the non-replicating state of M. tuberculosis and offer new potential targets for antimicrobial therapies against this disease.
|Grant, Sarah Schmidt; Wellington, Samantha; Kawate, Tomohiko et al. (2016) Baeyer-Villiger Monooxygenases EthA and MymA Are Required for Activation of Replicating and Non-replicating Mycobacterium tuberculosis Inhibitors. Cell Chem Biol 23:666-77|
|Grant, Sarah Schmidt; Kawate, Tomohiko; Nag, Partha P et al. (2013) Identification of novel inhibitors of nonreplicating Mycobacterium tuberculosis using a carbon starvation model. ACS Chem Biol 8:2224-34|
|Stanley, Sarah A; Kawate, Tomohiko; Iwase, Noriaki et al. (2013) Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium tuberculosis by targeting FadD32. Proc Natl Acad Sci U S A 110:11565-70|
|Grant, Sarah Schmidt; Hung, Deborah T (2013) Persistent bacterial infections, antibiotic tolerance, and the oxidative stress response. Virulence 4:273-83|
|Grant, Sarah Schmidt; Kaufmann, Benjamin B; Chand, Nikhilesh S et al. (2012) Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals. Proc Natl Acad Sci U S A 109:12147-52|
|Stanley, Sarah A; Grant, Sarah Schmidt; Kawate, Tomohiko et al. (2012) Identification of novel inhibitors of M. tuberculosis growth using whole cell based high-throughput screening. ACS Chem Biol 7:1377-84|