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. Significance The 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.
Nathan Roberge, Paankhi Dave, Véronique L. Taylor et al.· bioRxiv· 0 citations
ABSTRACT Iron acquisition is a crucial step for bacterial survival that shapes bacterial fitness and ecological interactions within microbial communities. Pseudomonas aeruginosa, an opportunistic clinical and environmental bacterium, relies on high-affinity siderophores such as pyoverdines to scavenge iron. Understanding these pathways provides vital insights into siderophore specificity, bacterial resource competition, and iron metabolism in bacteria. Here, we report vacidobactin A (VacA), a siderophore produced by the soil bacterium Variovorax paradoxus, identified through a screen of natural product extracts targeting a clinical multi-drug-resistant strain of P. aeruginosa. Rather than functioning as a direct antimicrobial agent, VacA impairs the growth of P. aeruginosa by imposing iron starvation. This is observed exclusively in strains that are unable to produce pyoverdine, their native siderophore. To determine whether VacA-mediated inhibition resulted from restricted access to iron, we heterologously expressed a TonB-dependent transporter from V. paradoxus in a pyoverdine- and pyochelin-deficient P. aeruginosa strain. Transporter expression enabled utilization of VacA-bound iron and restored growth, demonstrating that the antagonistic effect of VacA is governed by selective siderophore recognition and uptake rather than iron sequestration alone. Additionally, VacA synergized with thiostrepton, which hijacks pyoverdine receptors to enter the cell and inhibit protein synthesis. This investigation demonstrates how environmental microorganisms exploit siderophore specificity to compete for iron and establish antagonistic relationships within microbial communities. IMPORTANCE Iron acquisition is fundamental to bacterial survival and shapes ecological interactions within microbial communities, yet the mechanistic principles governing siderophore specificity and iron competition remain incompletely understood. Here, we demonstrate that vacidobactin A, a siderophore produced by Variovorax paradoxus, suppresses P. aeruginosa growth by limiting iron availability, particularly in strains deficient in pyoverdine production. By demonstrating that heterologous expression of a TonB-dependent transporter restores iron utilization from vacidobactin A, we establish that siderophore-mediated antagonism is governed by selective recognition and uptake pathways. Furthermore, the synergy between vacidobactin A and thiostrepton illustrates how siderophore systems integrate with other cellular vulnerabilities. These findings illuminate fundamental principles of bacterial resource competition and highlight how environmental microorganisms exploit iron metabolism as a competitive strategy, advancing our understanding of microbial community dynamics and bacterial physiology. Iron acquisition is fundamental to bacterial survival and shapes ecological interactions within microbial communities, yet the mechanistic principles governing siderophore specificity and iron competition remain incompletely understood. Here, we demonstrate that vacidobactin A, a siderophore produced by Variovorax paradoxus, suppresses P. aeruginosa growth by limiting iron availability, particularly in strains deficient in pyoverdine production. By demonstrating that heterologous expression of a TonB-dependent transporter restores iron utilization from vacidobactin A, we establish that siderophore-mediated antagonism is governed by selective recognition and uptake pathways. Furthermore, the synergy between vacidobactin A and thiostrepton illustrates how siderophore systems integrate with other cellular vulnerabilities. These findings illuminate fundamental principles of bacterial resource competition and highlight how environmental microorganisms exploit iron metabolism as a competitive strategy, advancing our understanding of microbial community dynamics and bacterial physiology.
Manpreet Kaur, Derek C. K. Chan, Hodan Wardere et al.· Microbiology spectrum· 0 citations
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