Metabolic determinants of Mtb virulence, vulnerability and variation

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ABSTRACT - Metabolic determinants of Mtb virulence, vulnerability and variation Mycobacterium tuberculosis (Mtb) has emerged as the world's most deadly pathogen based in large part on the highly unusual biological and chemical properties of its cell envelope. Comprised of a distinctive hydrophobic outer mycolate membrane, anchored to an underlying complex of polysaccharide and peptidoglycan polymers, the Mtb envelope serves as both the primary interface with, and barrier to, the human host. In human tuberculosis (TB) disease the Mtb envelope mediates a years-long standoff, and serves as the barrier to all anti-mycobacterial drugs. Yet, knowledge of its native composition, variation and regulation of drug entry remains fragmentary. This team of applicants has created new genetic and metabolomic tools to comprehensively dissect and analyze the metabolite and lipid components of the Mtb envelope on an organism-wide basis across a large set of clinical isolates. Moreover, this TBRU proposes to provide the first descriptions of cell envelope variation among isolates from human patients and identify key determinants of its virulence and barrier to drug action that could inform the development of better diagnostics and therapeutics. Structures of new molecules will first be determined using synthetic chemistry and mass spectrometry. The genes encoding these metabolites will then be identified and functionally validated using new genome-scale CRISPR interference technologies, assays for penetration into the cell envelope, and genetically defined mouse models of in vivo growth. Using mass spectrometry, we will solve the structures of up to 250 surface barrier lipids and more than 41 gene-lipid pairs that dominate in cell envelope variation among patients. Patient-derived Mtb strains will be obtained from clinical samples collected at our field sites in Masiphumelele, South Africa, where we will implement clinically relevant technology for detection of live Mtb in exhaled (non- coughed) human bioaerosols. Studies of barrier function place special emphasis on rifampicin as a model compound due to its clinical importance as a frontline drug and role as a defining element of drug resistant TB. The ability to analyze patient urine and serum has further resulted in the discovery of new biomarkers of disease activity and response to drug therapy, motivating linked translational efforts to advance the development of non-sputum based, real time point-of-care diagnostic tests. This highly interactive group of scientists thus seeks to provide better drugs and diagnostic tests, as well as a deep and durable scientific foundation for understanding of the Mtb envelope, especially the particular genes and molecules that control active remodeling, drug action and human host response.