The global pathogen Mycobacterium tuberculosis and other species in the suborder Corynebacterineae possess a distinctive outer membrane called the mycomembrane (MM). The MM is composed of mycolic acids, which are either covalently linked to an underlying arabinogalactan layer or incorporated into trehalose glycolipids that associate with the MM non-covalently. These structures are generated through a process called mycolylation, which is central to mycobacterial physiology and pathogenesis and is an important target for tuberculosis drug development. Current approaches to investigating mycolylation rely on arduous analytical methods that occur outside the context of a whole cell. Herein, we describe mycobacteria-specific chemical reporters that can selectively probe either covalent arabinogalactan mycolates or non-covalent trehalose mycolates in live mycobacteria. These probes, in conjunction with bioorthogonal chemistry, enable selective in situ detection of the major MM components.

Bacteria possess an extraordinary repertoire of cell envelope glycans that have critical physiological functions. Pathogenic bacteria have glycans that are essential for growth and virulence but are absent from humans, making them high-priority targets for antibiotic, vaccine, and diagnostic development. The advent of metabolic labeling with bioorthogonal chemical reporters and small-molecule fluorescent reporters has enabled the investigation and targeting of specific bacterial glycans in their native environments. These tools have opened the door to imaging glycan dynamics, assaying and inhibiting glycan biosynthesis, profiling glycoproteins and glycan-binding proteins, and targeting pathogens with diagnostic and therapeutic payload. These capabilities have been wielded in diverse commensal and pathogenic Gram-positive, Gram-negative, and mycobacterial speciesincluding within live host organisms. Here, we review the development and applications of chemical reporters for bacterial glycans, including peptidoglycan, lipopolysaccharide, glycoproteins, teichoic acids, and capsular polysaccharides, as well as mycobacterial glycans, including trehalose glycolipids and arabinan-containing glycoconjugates. We cover in detail how bacteria-targeting chemical reporters are designed, synthesized, and evaluated, how they operate from a mechanistic standpoint, and how this information informs their judicious and innovative application. We also provide a perspective on the current state and future directions of the field, underscoring the need for interdisciplinary teams to create novel tools and extend existing tools to support fundamental and translational research on bacterial glycans.

Mycobacteria have a distinctive glycolipid-rich outer membrane, the mycomembrane, which is a critical target for tuberculosis drug development. However, proteins that associate with the mycomembrane, or that are involved in its metabolism and host interactions, are not well-characterized. To facilitate the study of mycomembrane-related proteins, we developed photoactivatable trehalose monomycolate analogues that metabolically incorporate into the mycomembrane in live mycobacteria, enabling in vivo photo-cross-linking and click-chemistry-mediated analysis of mycolate-interacting proteins. When deployed in Mycobacterium smegmatis with quantitative proteomics, this strategy enriched over 100 proteins, including the mycomembrane porin (MspA), several proteins with known mycomembrane synthesis or remodeling functions (CmrA, MmpL3, Ag85, Tdmh), and numerous candidate mycolate-interacting proteins. Our approach is highly versatile, as it (i) enlists click chemistry for flexible protein functionalization; (ii) in principle can be applied to any mycobacterial species to identify endogenous bacterial proteins or host proteins that interact with mycolates; and (iii) can potentially be expanded to investigate protein interactions with other mycobacterial lipids. This tool is expected to help elucidate fundamental physiological and pathological processes related to the mycomembrane and may reveal novel diagnostic and therapeutic targets.

Protein O-mycoloylation is a unique post-translational lipidation that was recently discovered in Corynebacterium. We describe an alkyne-modified trehalose monomycolate chemical reporter that can metabolically tag O-mycoloylated proteins in C. glutamicum, enabling their detection and identification through click chemistry.