Minimalist Tetrazine N-Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion.

N-Acetyl muramic acid (NAM) probes containing alkyne or azide groups are commonly used to investigate aspects of cell wall synthesis because of their small size and ability to incorporate into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions are not compatible with live cells, and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction rates are modest and, therefore, not as desirable for tracking the temporal alterations of bacterial cell growth, remodeling, and division. Alternatively, the tetrazine-trans-cyclooctene ligation (Tz-TCO), which is the fastest known bioorthogonal reaction and not cytotoxic, allows for rapid live-cell labeling of PG at biologically relevant time scales and concentrations. Previous work to increase reaction kinetics on the PG surface by using tetrazine probes was limited because of low incorporation of the probe. Described here are new approaches to construct a minimalist tetrazine (Tz)-NAM probe utilizing recent advancements in asymmetric tetrazine synthesis. This minimalist Tz-NAM probe was successfully incorporated into pathogenic and commensal bacterial PG where fixed and rapid live-cell, no-wash labeling was successful in both free bacterial cultures and in coculture with human macrophages. Overall, this probe allows for expeditious labeling of bacterial PG, thereby making it an exceptional tool for monitoring PG biosynthesis for the development of new antibiotic screens. The versatility and selectivity of this probe will allow for real-time interrogation of the interactions of bacterial pathogens in a human host and will serve a broader utility for studying glycans in multiple complex biological systems.

Investigating Peptidoglycan Recycling Pathways in Tannerella forsythia with N-Acetylmuramic Acid Bioorthogonal Probes.

The human oral microbiome is the second largest microbial community in humans, harboring over 700 bacterial species, which aid in digestion and protect from growth of disease-causing pathogens. One such oral pathogen, Tannerella forsythia, along with other species, contributes to the pathogenesis of periodontitis. T. forsythia is unable to produce its own N-acetylmuramic acid (NAM) sugar, essential for peptidoglycan biosynthesis and therefore must scavenge NAM from other species with which it cohabitates. Here, we explore the recycling potential of T. forsythia for NAM uptake with a bioorthogonal modification into its peptidoglycan, allowing for click-chemistry-based visualization of the cell wall structure. Additionally, we identified NAM recycling enzyme homologues in T. forsythia that are similar to the enzymes found in Pseudomonas putida. These homologues were then genetically transformed into a laboratory safe Escherichia coli strain, resulting in the efficient incorporation of unnatural NAM analogues into the peptidoglycan backbone and its visualization, alone or in the presence of human macrophages. This strain will be useful in further studies to probe NAM recycling and peptidoglycan scavenging pathways of T. forsythia and other cohabiting bacteria.