Mycobacterium tuberculosis (Mtb), the pathogen that causes tuberculosis (TB), is a member of the Corynebacterineae suborder, which comprises hundreds of important species. TB is a global health crisis with about 10 million new cases and 1.5 million deaths in 2017. The mycobacterial cell envelope includes a unique outer membrane, also known as the mycomembrane, which is the major defense barrier that confers intrinsic drug tolerance to Mtb. The mycomembrane is typified by long-chain (up to 100 carbons), α-branched, β-hydroxy fatty acids called mycolic acids, which are esterified to various acceptors, including trehalose, forming trehalose mono- and dimycolate (TMM and TDM), as well as protein serine residues, forming O-mycoloylated proteins. These structures are generated by a process called mycoloylation, which refers to the Ag85-catalyzed transfer of mycolic acid from the donor molecule TMM to the acceptor molecule. We recently reported an O-acylated TMM analogue containing a terminal alkyne group on the acyl chain (O-AlkTMM-C7), which allowed for metabolic incorporation of the alkyne handle into mycomembrane components and enabled their visualization using click chemistry. Here, we describe an expanded toolbox of O-acylated TMM analogues bearing alkyne, azide, trans-cyclooctene, and fluorescent tags, which allowed us to test the substrate tolerance of Ag85 enzymes at the cellular level. In addition, these compounds provide significantly expanded experimental versatility including one- or two-step cell labeling, live cell labeling and rapid cell labeling via tetrazene ligation. Moreover, these compounds allow for the labeling and analysis of mycomembrane components, including O-mycoloylated proteins. Thus, these compounds have potential for the specific detection and surface engineering of live Mtb, as well as for the identification of novel mycomembrane components that may be attractive targets for drug development.

O-acylated trehalose analogues for probing cellular processes and components in Corynebacterineae

2019 Fall National Meeting

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