Spatial positioning of cell wall-anchored virulence factors in Gram-positive bacteria

► Cell wall-anchored surface proteins promote pathogenesis in Gram-positive bacteria. ► 3D imaging highlights non-random spatio-temporal distributions of surface virulence factors. ► Listerial sorted proteins shift from helical to polar sites in response to environmental changes. ► Signal peptides in coccal surface proteins direct specific localization patterns. ► Gene expression, wall dynamics and protein sorting compartmentalize the bacterial envelope.