Bacterial and Molecular Determinants of Mycobacterial Impermeability

Project Summary The typical course of treatment for uncomplicated Mycobacterium tuberculosis infection comprises four antibiotics and lasts for at least six months. The impermeability of the multi-layered M. tuberculosis cell envelope has long been linked to the organism’s intrinsically-poor drug susceptibility. While the outer ‘myco’ membrane is hypothesized to be the primary barrier to accessing antibiotic targets in the peptidoglycan or cytoplasm, other facets of the envelope likely contribute. Moreover, compared to the model organism Escherichia coli and to mammalian cells, the field has only a rudimentary understanding of the kinds of compounds that can permeate M. tuberculosis. Based on published and preliminary data, the PIs hypothesize that M. tuberculosis impermeability is a consequence of envelope composition as well as the target location and chemical structure of the compound. A major hurdle to testing this hypothesis is the lack of high-throughput tools for identifying bacterial and molecular factors that control compound permeation across envelope layers. In this proposal, The PIs develop and deploy two complementary methods for defining the bacterial and molecular determinants of M. tuberculosis impermeability. They will first test the relative importance of various M. tuberculosis factors in molecule gate-keeping. Next, they will globally determine the M. tuberculosis genes that contribute to mycomembrane impermeability. Finally, they will comprehensively determine the structural motifs associated with M. tuberculosis mycomembrane permeation or lack thereof. Successful completion of these aims will lay the foundation for medicinal chemistry efforts to improve M. tuberculosis uptake of both existing drugs and newly-discovered compounds. The methods that the PIs use to achieve these aims are easily ported to species for which envelope permeability is also treatment-limiting, e.g., non-tuberculous mycobacteria (NTMs) and Gram-negatives.