Secretin-interacting plug proteins prevent antibiotic influx during type IV pilus assembly in Pseudomonas aeruginosa

Type IV pili (T4P) are important virulence factors that mediate host attachment and other pathogenic functions. In Gram-negative bacteria, T4P are assembled from pilin subunits at the inner membrane (IM) and extend through the outer membrane (OM) via secretin channels. Although essential for T4P function, secretin complexes can impair the OM permeability barrier, potentially allowing entry of toxic compounds. The mechanisms that prevent such influx remain poorly understood. Here, we identify SlkA and SlkB (PA5122 and PA5123) as periplasmic proteins that interact with the T4P secretin channel and block antibiotic influx. Our data indicate that these proteins function as physical plugs sealing the channel until the IM complex docks and pilus assembly begins. These findings demonstrate that Slk proteins and the IM complex function redundantly to maintain OM barrier integrity, and that their interaction with the secretin channel represents a promising target for antibiotic potentiation.