Roberge, N. | bioRxiv Science

bioRxiv2026

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

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 to prevent host cell recognition by other phages that leverage these filaments for infection. Aqs1 disrupts T4P by binding to the hexameric ATPase PilB, required to power pilus filament extension, though several mechanistic details remain unclear. We show that Aqs1 has broad-spectrum activity and can disrupt T4P function in a variety of Gram negative bacteria. This protein inhibits PilB by binding to a solvent-exposed hydrophobic patch on the N2-domain, distal to the active site. Binding destabilizes the hexamer, preventing PilB accumulation at T4P machines. Aqs1 likely disrupts PilB oligomerization by displacing a flexible linker segment between the PilB N1- and N2-domains required for inter-subunit contact. Together, the Aqs1 mode of action provides a design template for broad-spectrum inhibitors of diverse bacterial virulence factors. SignificanceThe phage-encoded protein Aqs1 disables type IV pilus (T4P) production in Pseudomonas aeruginosa by targeting the hexameric ATPase responsible for assembling pilus fibers. We show that despite originating from a P. aeruginosa-specific phage, Aqs1 can also disable T4 ATPase-dependent phenotypes across other pathogenic bacteria and homologous systems. Mechanistically, Aqs1 binds to a conserved patch on the PilB N2-domain away from the active site. Binding here breaks apart the PilB oligomer, preventing it from acting on T4P machines. Aqs1 binding at the N2-domain patch likely displaces a flexible PilB linker segment that binds to this site to stabilize the hexamer. Our work highlights a novel and conserved PilB allosteric site which is exploited by the phage-encoded protein Aqs1 to disrupt diverse T4 systems in multiple bacteria.

microbiology↗

bioRxiv2025

Twitching motility suppressors reveal a role for FimX in type IV pilus extension dynamics

In Pseudomonas aeruginosa, retractable protein filaments called type IV pili (T4P) facilitate surface adherence, sensing, and directional movement known as twitching motility. T4P are necessary for the bacteria to engage in surface-associated behaviors, including establishing acute infections. Pilus extension is driven by the hexameric ATPase, PilB, at the base of the T4P nanomachine in coordination with various protein regulatory effectors. The cyclic-di-GMP binding protein, FimX, works with PilB to mediate normal extension processes, though how this effector controls pilus assembly remains unclear. To explore the role of FimX in T4P function, we leveraged the significant {Delta}fimX twitching motility deficit to screen for mutants capable of overcoming this phenotype. We identified suppressor mutations that increase twitching in {Delta}fimX background, mapping primarily to cyclic-AMP homeostatic machinery or to PilB, the FimX target. Distinct suppressor mutations in PilB increased ATP hydrolysis in vitro and this activity was subject to modulation by FimX. Using microscopy to monitor the extension dynamics of fluorescently labelled T4P, we showed that {Delta}fimX mutants produce slow-to-extend, short pili, a phenotype that is rescued by mutations enhancing PilB ATP hydrolysis and/or re-introduction of FimX. Together, these data implicate FimX as a regulator of PilB enzymatic function, potentially enabling P. aeruginosa to fine-tune pilus extension dynamics in response to environmental cues. SummaryType IV pili enable Pseudomonas aeruginosa to attach to surfaces, move (twitch), and form biofilms. Pilus extension is powered by the motor protein PilB, which is regulated by other factors, including FimX, a protein that binds cyclic-di-GMP. Although FimX is important for twitching, how it influences PilB was unclear. We deleted fimX, which severely reduces motility, and searched for mutants that regained movement. We identified two types: some had mutations in PilB that increased its ATPase activity, allowing it to function without FimX, while others affected the cyclic-AMP signaling pathway and increased overall production of pilus components, showing that motility can also be improved through changes in quantity versus quality. Our results suggest that FimX normally fine-tunes PilB enzymatic activity, enabling dynamic control of pilus extension in response to surface signals. This work helps explain how P. aeruginosa adapts to different environments, a process crucial for infection and biofilm development.

microbiology↗