The closely related mycobacterial lipids phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) are important virulence factors that mediate several aspects of pathogenesis.  In particular, PDIM has been shown to modulate macrophage phagosome maturation, inhibit antigen presentation, and is required to evade host toll-like receptor (TLR) signaling and subsequent antimicrobial immune responses. However, given their non-gene encoded nature, studies on their localization and mechanisms of action in vivo are lacking. While metabolic labeling approaches have succeeded in elucidating biological roles of trehalose containing glycolipids, no PDIM-specific precursor metabolites exist to harness this approach. Instead we’ve modernized a petroleum-ether extraction and reconstitution method for the removal and reincorporation of non-covalently attached mycomembrane lipids, including PDIM. This approach allowed us to isolate PDIM and to chemically modify the lipid to incorporate an azide handle (azido-DIM) prior to reconstituting the mycomembrane with azido-DIM. Using the zebrafish/<i>Mycobacterium marinum</i> model of Tuberculosis pathogenesis, we found that azido-DIM was able to rescue growth defects of PDIM-deficient bacteria comparable to the native lipid. We now had a pathogenic strain of mycobacteria where nearly all of its PDIM contained a reactive handle to further probe its mechanism of action. Bioorthogonal reaction with various cyclooctyne fluorophores enabled us to track azido-DIM during infection. We found that immediately following infection, azido-DIM began to spread away from the mycomembrane onto host membranes, and that this spreading was required for PDIM to prevent TLR-mediated immune responses. Finally, we found that the concentration of cholesterol in host membranes directly correlated with the amount of azido-DIM spreading. Treating zebrafish with the statin atorvastatin lead to a decrease in host cholesterol, a decrease in azido-DIM spreading, and the fish were resistant to mycobacterial infection. While it has long been appreciated that dyslipidemia is a risk factor for Tuberculosis incidence and that, on a population level, statins are associated with a decrease in incidence, our data suggests a potential molecular mechanism underlying these observations.<br/>

Uncovering molecular mechanisms of mycobacterial pathogenesis with bioorthogonal chemistry

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.