Bacteria in the suborder Corynebacterineae—including the pathogens that cause tuberculosis, leprosy, and diphtheria—are characterized by a unique outer membrane that is rich in mycolic acids (long-chain (C26-C90), α-branched, β-hydroxy fatty acids), hence its moniker “mycomembrane.” The mycomembrane is critical to bacterial virulence and persistence, as it hosts immunoactive glycolipids and provides a robust defensive barrier that protects the bacterium from external assaults. The major components of the mycomembrane are arabinogalactan mycolate (AGM), which is covalently linked to peptidoglycan, and trehalose dimycolate (TDM), which associates non-covalently with the mycomembrane. In addition, there exist mycomembrane-associated proteins which participate in pore-forming activities to allow diffusion of water soluble molecules, among other functions. Recently, some of these proteins were also discovered to be modified with mycolic acids. Modification of the AGM, TDM, and proteins with mycolic acid (a process termed as mycoloylation) occurs via mycoloyltransferase-catalyzed transfer of a mycoloyl group from a common precursor—trehalose monomycolate (TMM)—to the hydroxyl-bearing acceptor substrates AG, TMM, and polypeptides, respectively. Despite the relevance of mycoloylated proteins to mycobacteria, methods currently used to investigate them occur outside the context of a whole cell and cannot provide whole proteome-level information about this post-translational modification. In this presentation, we report an approach that exploits endogenous mycobacteria-specific biosynthetic pathways utilizing a trehalose monomycolate mimic with a click chemistry tag that enables the probing of the mycomembrane, including mycoloylated proteins. This chemical reporter enabled the detection and identification of the known O-mycoloylated proteins PorA and PorH, as well as several other candidate O-mycoloylated proteins, including the outer-membrane porin protein PorB. This strategy will facilitate future efforts to identify and characterize O-mycoloylated proteins in Corynebacterium and related genera.

Trehalose monomycolate mimic chemical reporter: A strategy for detecting, profiling and identifying O-mycoloylated proteins in mycobacterium

255th National Meeting (2018)

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