A broad-spectrum phage-encoded mechanism to disarm bacterial type IV filaments.

Bacteria-specific viruses, or phages, can modify host cell physiology to thwart competitors. The Pseudomonas aeruginosa-specific phage DMS3 encodes Aqs1, a protein inhibitor of type IV pilus (T4P) function. T4P are important virulence factors widely distributed in bacteria and archaea, but they are also common P. aeruginosa phage receptors. Disabling these structures therefore prevents host cell recognition by other phages that leverage these filaments for infection. Aqs1 disrupts pilus production by binding to PilB, the hexameric ATPase required to power T4P filament extension, though several mechanistic details remain unclear. We show that Aqs1 has broad-spectrum activity and can disrupt T4P ATPase-dependent surface motility, DNA uptake, and protein secretion by binding to PilB homologues in a variety of bacteria. Aqs1 inhibits multiple PilB orthologues by binding to a conserved solvent-exposed hydrophobic patch on its N2-domain, distal to the active site. Binding to this region destabilizes the hexamer, preventing PilB accumulation near T4P machines. We report that a flexible linker segment connecting the PilB N-terminal domains functionally interacts with the Aqs1-binding N2-patch, and that this interaction is required to promote PilB inter-subunit contact. The function of the linker represents a novel element of T4P ATPase allosteric regulation which has been exploited by phages to disrupt diverse functions. The Aqs1 mode-of-action therefore provides a design template for broad-spectrum inhibitors against diverse bacterial virulence factors.