Chemical biology tools to probe bacterial glycans

Abstract

Bacterial cells are covered by a complex carbohydrate coat of armor that allows bacteria to thrive in a range of environments. As a testament to the importance of bacterial glycans, effective and heavily utilized antibiotics including penicillin and vancomycin target and disrupt the bacterial glycocalyx. Despite their importance, the study of bacterial glycans lags far behind their eukaryotic counterparts. Bacterial cells use a large palette of monosaccharides to craft glycans, leading to molecules that are significantly more complex than eukaryotic glycans and that are refractory to study. Fortunately, chemical tools designed to probe bacterial glycans have yielded insights into these molecules, their structures, their biosynthesis, and their functions.

Keywords: Azides; Bacterial glycans; Bioorthogonal chemistry; Carbohydrates; Click chemistry; Lectins; Metabolic glycan labeling.

Figure 1. Overview of bacterial cell envelope glycans and major approaches to study them discussed in this review.

(a) The Gram-negative bacterial cell envelope contains a variety of exclusively bacterial glycans including peptidoglycan, lipopolysaccharide (LPS), capsular polysaccharide (CPS), and, for some bacteria, glycosylated proteins (glycoproteins). (b) Bacterial glycans can be studied using an indirect two-step metabolic labeling approach in which they are first metabolically labeled with an unnatural sugar bearing a bioorthogonal functional group (e.g., azide) and then detected in a second step via bioorthogonal chemistry (e.g., strain-promoted azide–alkyne cycloaddition). (c) Direct metabolic labeling with a sugar bearing a detectable probe such as a fluorophore presents an alternative to indirect labeling. (d) Proteins that bind to carbohydrates (e.g., lectins, antibodies) can be used to detect and study bacterial glycans.

Figure 2. Approaches to probe bacterial glycans in physiologically relevant settings.

(a) Bacteria-host interactions can be probed in cell culture models to understand the role of glycans in eliciting immune recognition and cytokine production from host cells (left). Direct metabolic labeling with sugars bearing antibody-recruiting molecules (e.g., trehalose-dinitrophenyl conjugates) can be used to induce antibody binding to and immune recognition of bacterial targets (right). (b) Glycans on the surface of gut microbiota isolated from human stool samples can be probed using a two-step metabolic labeling approach in which they are first metabolically labeled with an azidosugar and then detected in a second step via strain-promoted azide–alkyne cycloaddition (top). Alternatively, lectin-based approaches can be used to bind glycan epitopes on microbiome constituents and then enrich and identify species presenting those epitopes (bottom).

Figure 3. Insights into bacterial glycan biosynthesis, recycling, and degradation.

(a) The bacterial glycan life cycle begins with monosaccharide activation to produce sugar-nucleotide donors, followed by glycan construction by a series of glycosyltransferases (GTs) that catalyze monosaccharide addition to growing glycans in a directional manner, and en bloc glycan transfer to yield fully elaborated glycoconjugates. Cell envelope and cell wall glycans can be tailored by modifying enzymes (e.g., glycosidases, acetyltransferases) and ultimately degraded and recycled by glycosyl hydrolases. Recent advances have yielded (b) nucleotidyltransferases with enhanced substrate flexibility to facilitate access to sugar-nucleotide donors and ease glycan production in vitro, (c) metabolic labeling-based screens to identify genes encoding glycosyltransferases that play a role in glycan biosynthesis, (d) probes to detect and identify glycosyl hydrolase activity, and (e) metabolic labeling-based methods to track glycan biosynthetic intermediates within membrane domains to yield insight into directional glycan biosynthesis.

Figure 4

Expedient synthesis of rare bacterial sugar scaffolds opens the door to create novel chemical biology tools by adopting precedented design principles.