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Audette, Rebecca

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Audette

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Rebecca

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Audette, Rebecca

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2017 - 20192

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Author's Publications: search.filters.isAuthorOfPublication.09d4730d-ae49-493c-ad96-90d587966578

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    Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity
    (2017) Rego, E. Hesper; Audette, Rebecca; Rubin, Eric
    Summary Paragraph While microorganisms are often studied as populations, the behavior of single, individual cells can have profound consequences. For example, tuberculosis, caused by the bacterial pathogen Mycobacterium tuberculosis, requires months of antibiotic therapy even though the bulk of the bacterial population rapidly dies. Shorter courses lead to high rates of relapse because subpopulations of bacilli can survive despite being genetically identical to those that are easily killed 1. In fact, mycobacteria create variability every time a cell divides, producing daughter cells with different sizes and growth rates 2, 3. The mechanism(s) that underlie this high-frequency variation and how variability relates to survival of the population are unknown. Here we show that mycobacteria actively create heterogeneity. Using a fluorescent reporter and a FACS-based transposon screen, we find that deletion of lamA, a gene of previously unknown function, decreases the amount of heterogeneity in the population by decreasing asymmetric polar growth. LamA has no known homologs in other organisms, but is highly conserved across mycobacterial species. We find that LamA is a member of the mycobacterial division complex (“the divisome”). It inhibits growth at nascent new poles, creating asymmetry in polar growth. The kinetics of killing individual cells that lack lamA are more uniform and more rapid with rifampicin and certain drugs that target the cell wall. Our results show that mycobacteria encode a non-conserved protein that controls the pattern of cell growth, resulting in a population that is both heterogeneous and better able to survive antibiotic pressure.
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    Large-Scale Chemical–genetics Yields New M. Tuberculosis Inhibitor Classes
    (Springer Science and Business Media LLC, 2019-06-19) LaVerriere, Emily; Meyer, Elisabeth; Kawate, Tomohiko; Gomez, James; Gardner, Michelle; Cigarroa Kennedy, Sofia; Wakabayashi, Shoko; Watson, Christopher; Fitzgerald, Michael; Johnson, Eachan; Office, Emma; Stanley, Mary; Audette, Rebecca; Bandyopadhyay, Nirmalya; Betancourt, Natalia; Delano, Kayla; Da Silva, Israel; Davis, Joshua; Gallo, Christina; Golas, Aaron; Guinn, Kristine; Korn, Rebecca; McConnell, Jennifer; Moss, Caitlin; Murphy, Kenan; Nietupski, Raymond; Papavinasasundaram, Kadamba; Pinkham, Jessica; Pino, Paula; Proulx, Megan; Ruecker, Nadine; Song, Naomi; Thompson, Matthew; Trujillo, Carolina; Metcalf-Wallach, Joshua; Ioerger, Thomas; Lander, Eric; Hubbard, Brian; Serrano-Wu, Michael; Ehrt, Sabine; Rubin, Eric; Sassetti, Christopher; Schnappinger, Dirk; Hung, Deborah
    New antibiotics are needed to combat rising resistance, with new Mycobacterium tuberculosis (Mtb) drugs of highest priority. Conventional whole-cell and biochemical antibiotic screens have failed. We developed a novel strategy termed PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) in which we screen compounds against pools of strains depleted for essential bacterial targets. We engineered strains targeting 474 Mtb essential genes and screened pools of 100-150 strains against activity-enriched and unbiased compounds libraries, probing >8.5-million chemical-genetic interactions. Primary screens identified >10-fold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insight. We identified >40 novel compounds targeting DNA gyrase, cell wall, tryptophan, folate biosynthesis, and RNA polymerase, as well as inhibitors of a novel target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating PROSPECT’s ability to yield inhibitors against novel targets which would have eluded conventional drug discovery.