Tracking Surface Protein Assembly and Peptidoglycan Biosynthesis in Fusobacterium nucleatum.

Fusobacterium nucleatum is a Gram-negative, obligate anaerobe and oncogenic oral bacterium that employs a diverse array of surface-exposed adhesins to colonize extra-oral tissues. Among these, Fusobacterial adhesin A (FadA) is a well-characterized virulence factor known for its ability to target epithelial and endothelial tight junctions, disrupt barrier integrity, and activate pro-tumorigenic signaling pathways implicated in colorectal cancer. FadA also facilitates placental and fetal colonization, contributing to adverse pregnancy outcomes such as preterm birth and stillbirth. While its functional roles in bacterial colonization and pathogenicity are increasingly recognized, the spatial and temporal dynamics of FadA assembly on the bacterial surface-and its relationship to cell wall biogenesis-remain poorly understood. Here, we present a fluorescence-based labeling approach using fluorescent D-amino acids (FDAAs) to simultaneously track peptidoglycan biosynthesis and the localization of FadA in F. nucleatum. This methodology provides a versatile platform for investigating the dynamic interplay between virulence factor localization and cell envelope remodeling.