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.

Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)‐catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne‐modified TMM analogue (O‐AlkTMM‐C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane. Here, we describe the synthesis and evaluation of a toolbox of TMM‐based reporters bearing alkyne, azide, trans‐cyclooctene, and fluorescent tags. These compounds gave further insight into the substrate tolerance of mycoloyltransferases (e.g., Ag85s) in a cellular context and they provide significantly expanded experimental versatility by allowing one‐ or two‐step cell labeling, live cell labeling, and rapid cell labeling via tetrazine ligation. Such capabilities will facilitate research on mycomembrane composition, biosynthesis, and dynamics. Moreover, because TMM is exclusively metabolized by Corynebacterineae, the described probes may be valuable for the specific detection and cell‐surface engineering of Mycobacterium tuberculosis and related pathogens. We also performed experiments to establish the dependence of probe incorporation on mycoloyltransferase activity, results from which suggested that cellular labeling is a function not only of metabolic incorporation (and likely removal) pathway(s), but also accessibility across the envelope. Thus, whole‐cell labeling experiments with TMM reporters should be carefully designed and interpreted when envelope permeability may be compromised. On the other hand, this property of TMM reporters can potentially be exploited as a convenient way to probe changes in envelope integrity and permeability, facilitating drug development studies.

The mycobacterial outer membrane, or mycomembrane, is essential for the viability and virulence of Mycobacterium tuberculosis and related pathogens. The mycomembrane is a dynamic structure, whose chemical composition and biophysical properties can change during stress to give an advantage to the bacterium. However, the mechanisms that govern mycomembrane remodeling and their significance to mycobacterial pathogenesis are still not well characterized. Recent studies have shown that trehalose dimycolate (TDM), a major glycolipid of the mycomembrane, is broken down by the mycobacteria-specific enzyme TDM hydrolase (Tdmh) in response to nutrient deprivation, a process which appears to modulate the mycomembrane to increase nutrient acquisition, but at the expense of stress tolerance. Tdmh activity thus balances the growth of M. tuberculosis during infection in a manner that is contingent upon host immunity. Current methods to probe Tdmh activity are limited, impeding the development of inhibitors and the investigation of the role of Tdmh in bacterial growth and persistence. Here, we describe the synthesis and evaluation of FRET-TDM, which is a fluorescence-quenched analogue of TDM that is designed to fluoresce upon hydrolysis by Tdmh and potentially other trehalose ester-degrading hydrolases involved in mycomembrane remodeling. We found that FRET-TDM was efficiently activated in vitro by recombinant Tdmh, generating a 100-fold increase in fluorescence. FRET-TDM was also efficiently activated in the presence of whole cells of Mycobacterium smegmatis and M. tuberculosis , but the observed signal was predominantly Tdmh-independent, suggesting that physiological levels of Tdmh are low and that other mycobacterial enzymes also hydrolyze the probe. The latter notion was confirmed by employing a native protein gel-based fluorescence assay to profile FRET-TDM-activating enzymes from M. smegmatis lysates. On the other hand, FRET-TDM was capable of detecting the activity of Tdmh in cells when it was overexpressed. Together, our data demonstrate that FRET-TDM is a convenient and sensitive in vitro probe of Tdmh activity, which will be beneficial for Tdmh enzymatic characterization and inhibitor screening. In more complex samples, for example, live cells or cell lysates, FRET-TDM can serve as a tool to probe Tdmh activity at elevated enzyme levels, and it may facilitate the identification and characterization of related hydrolases that are involved in mycomembrane remodeling. Our study also provides insights as to how the structure of FRET-TDM or related fluorogenic probes can be optimized to achieve improved specificity and sensitivity for detecting mycobacteria.

Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green-emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose-based fluorogenic probe featuring a molecular rotor turn-on fluorophore with bright farred emission (RMR-Tre). RMR-Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no-wash, low-background fluorescence detection of live mycobacteria. Aided by the red-shifted molecular rotor fluorophore, RMR-Tre exhibited up to a 100fold enhancement in M. tuberculosis labeling compared to existing fluorogenic trehalose probes. We show that RMR-Tre reports on M. tuberculosis drug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.