Chemical Probes to Reveal the Assembly and Dynamics of Wall Teichoic Acids

Wall teichoic acids (WTAs) are anionic glycopolymers that shape the Gram-positive bacterial cell envelope, guiding critical processes such as surface recognition, peptidoglycan assembly, and cell division. Despite their central roles, few tools exist to monitor WTAs in their native context. Here, we introduce azido-ribitol (RibAz) derivatives that enter the WTA biosynthetic pathway in Staphylococcus aureus via a salvage-like mechanism and incorporate into the poly(ribitol phosphate) backbone. The resulting RibAz probes enable WTA fluorescence imaging without cell wall digestion or genetic manipulation. Using RibAz probes with super-resolution microscopy, we mapped WTA localization in cells. Our data show that WTAs are produced at the division septum, resolving a longstanding question about the spatial organization of WTA synthesis during cell division. RibAz probes provide a generalized chemical approach for interrogating WTA function, abundance, and dynamics, and establish a foundation for mechanistic studies of cell wall biogenesis. Such data can inform antibiotic discovery efforts to combat antimicrobial resistance.