Chemical Reporters for Bacterial Glycans: Development and Applications

Abstract

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.

Rediscovery of 4‐Trehalosamine as a Biologically Stable, Mass‐Producible, and Chemically Modifiable Trehalose Analog

“…An increasing number of published studies have reported on the staining of mycobacteria with labeled trehaloses, including 4-fluorescent-and 4-azido-trehaloses. [41][42][43][44][45][46][47] Mycobacteria have a thick cell wall with an outer membrane composed of several major lipids, including trehalose monomycolate (TMM) and trehalose dimycolate (TDM). TMM and TDM are synthesized in mycobacteria in processes involving multiple enzymes, transporters, and trehalose incorporation.…”

“…[48] Trehalose analogs, including fluorescently labeled ones, could be reportedly incorporated into TMM and TDM instead of trehalose. [41][42][43][44][45][46][47] Certain types of mycobacteriosis, such as that caused by multidrug-resistant tuberculosis and nontuberculous mycobacteria, are currently regarded as serious health problems worldwide. [49,50] The labeled trehalose probes could be new helpful tools to detect the pathogens of these mycobacterioses or investigate cell wall structures and functions as well as biosynthesis-related enzymes and transporters to develop new antibiotics and treatments.…”

“…Since the report of Backus et al, [41] several fluorescent trehaloses have been developed to stain mycobacteria. [42][43][44][45][46][47] The currently available good probes are more advantageous for staining living cells than the probes investigated in this study. However, it was demonstrated that the C-4 of trehalose could be modified to create probes for mycobacterial staining, and 4-trehalosamine would be a good material for the development of better probes in the future.…”

Metabolic labeling of the bacterial peptidoglycan by functionalized glucosamine

“…Fluorescent labeling of PG-precursors and their use in vitro and in cells provided important insights into the mode of action of antibiotics and the PG biosynthesis by PBPs and their regulation ( Breukink et al., 2003 ; Hasper et al., 2004 , 2006 ; van Dam et al., 2007 , 2009 ; Olrichs et al., 2011 ). A recent review on chemical reporters for bacterial glycans provides a nice “panoramic view” of the advances in labeling bacteria ( Banahene et al., 2022 ).

Figure 1 GlcNAc in the peptidoglycan biosynthesis pathway GlcNAc is first converted into UDP-GlcNAc from which it is transferred onto Lipid I to form Lipid II.

…”

Multiscale Invasion Assay for Probing Macrophage Response to Gram-Negative Bacteria

“…In parallel, novel methods have emerged to study the bacterial cell wall. Work has been done previously to label various aspects of PG including small fluorophores coupled to D-amino acids, NAG and NAM sugar probes, and near-infrared radiation (NIR) fluorogenic probes ( Wodzanowski et al, 2020 ; Banahene et al, 2021 ). Because synthetic fragments MDP and MTP both contain the NAM sugar, it is believed that NAM plays an important structural role in fragment identification and subsequent infection clearing by macrophages.…”