The Corynebacterineae suborder of bacteria comprises hundreds of species, many of which are of high significance to human health, notably the causative agent of tuberculosis, Mycobacterium tuberculosis. These bacteria are characterized by presence of a glycolipid-rich barrier, known as the mycomembrane that is predicted to enlist a substantial proteome to balance various functions in the cell. However, due to the structural complexity of the mycobacterial envelope and lack of effective tools, the proteomic composition is poorly defined. We developed a chemical strategy that permits selective labeling of mycomembrane proteins in living cells followed by analysis on the whole-proteome level. Trehalose analogues were used to metabolically install photoactivatable and click chemistry enabling groups. When deployed in the model organism M. smegmatis in conjunction with label-free quantitative proteomics, this strategy identified approximately 100 proteins that were statistically over-represented in probe-treated, UV-exposed bacteria, including the known mycomembrane porin MspA and other proteins with known mycomembrane synthesis or remodeling functions, including antigen 85 (Ag85) mycoloyltransferases, and trehalose dimycolate hydrolase (Tdmh). Additional proteins of known and unknown function were identified as candidate mycomembrane proteins, which are of considerable interest for future structural and functional investigation. This strategy is applicable to the discovery and characterization of mycomembrane proteins across the Corynebacterineae suborder and will serve as a valuable tool in the pursuit of urgently needed antibiotics, diagnostic, and vaccines for tuberculosis and related diseases.

Development of clickable photoaffinity probes to identify outer membrane proteins in mycobacteria

2019 Fall National Meeting

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