Chemical Probes and Strategies to Study Mycobacterial Cell Envelope Assembly

Abstract

The cell envelope of Mycobacterium tuberculosis (Mtb) is central to its pathogenicity, immune evasion, and intrinsic drug resistance. While the importance of its glycan components is well recognized, their structural intricacies have hindered efforts to directly perturb and investigate their function. In this work, we discuss chemical approaches to study and manipulate mycobacterial cell envelope biosynthesis. Specifically, we present biosynthetic glycan labeling strategies that leverage the activity of native glycosyltransferases to probe arabinogalactan and mannose-containing glycolipids. Building upon prior work using lipid-linked probes to label mycobacterial arabinan, Chapter 2 details the development of azido-functionalized farnesyl phosphoryl mannose (AzFPM) probes that mimic native polyprenyl-phosphoryl donors and selectively label mannose-containing glycolipids in live mycobacteria. Chapter 3 showcases how these probes enable glycan substructure-specific labeling and biochemical enrichment of glycolipids across Corynebacterium glutamicum, Mycobacterium smegmatis, and Mtb. This strategy provides a platform to study glycolipid dynamics in wild-type cells, a task previously hindered by the lack of selective labeling tools. In Chapter 4, we further interrogate endogenous glycan biosynthesis by applying biosynthetic labeling probes in C. glutamicum. Perturbation of arabinan structure by probe incorporation led to impaired cell wall integrity and growth defects. In glycosyltransferase deletion strains, altered probe incorporation patterns revealed enzyme-specific roles in glycan assembly and architecture. Beyond novel labeling strategies, Chapter 5 describes the development of targeted inhibitors of galactan biosynthesis, an essential yet underexplored component of the mycobacterial cell wall. We employed a prodrug strategy to inhibit UDP-galactopyranose mutase (UGM), which catalyzes the committed step in Galf production. To overcome delivery challenges, we designed amide prodrugs activated intracellularly by amidases. One prodrug exhibited improved efficacy in Mtb, providing a promising lead for antibiotic development. Collectively, these studies establish biosynthetic labeling and targeted galactan inhibition as powerful tools for dissecting the structure and function of the mycobacterial cell envelope, offering new avenues for developing chemical probes and therapeutics against tuberculosis.