The cryo-EM structure of bacteriophage PRR1 and its role in conjugation inhibition

The global rise of antimicrobial resistance (AMR) demands innovative strategies to limit the spread of multidrug-resistant bacteria. Conjugative plasmids, particularly those in the incompatibility group P (IncP), play a central role in disseminating resistance genes across bacterial species via their encoded type IV secretion system (T4SS). Here, we characterize the single-stranded RNA (ssRNA) bacteriophage (ssRNA phage) PRR1, which selectively targets bacteria carrying the IncP plasmid RP4, including many Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli (ESKAPEE) pathogens, and assess its ability to inhibit conjugation. Using cryo-electron microscopy, we resolved the mature PRR1 virion at 3.45 Å resolution, revealing two phage maturation protein (Mat)-RNA interactions within the 3' untranslated region: a conserved interaction (Mat-U1) and a novel interaction (Mat-V1) for ssRNA phages. To characterize the PRR1-RP4 pilus interaction, we performed alanine-scanning mutagenesis and pinpointed four critical TrbC pilin residues (S12, W13, S72, and R77) for infection. Computational modeling revealed that these residues are located near the termini of the pilin at the phage-pilus interface. Notably, native and non-infectious, UV-cross-linked PRR1 was sufficient to block RP4 transfer, indicating conjugation inhibition does not require a complete infection cycle. Finally, combining PRR1 and antibiotic treatment yielded nine unique phage-resistant mutants within T4SS-associated genes on the RP4 plasmid. Eight of these mutants nearly abolished conjugation, while the trbE frameshift mutant retained ~30% of wild-type efficiency, which is pivotal to clarifying the relationship between phage infection and pilus function. Collectively, these results establish ssRNA phages as specific T4SS plasmid-targeting agents and underscore their potential to limit horizontal gene transfer in AMR pathogens.IMPORTANCEAntimicrobial resistance (AMR) spreads rapidly through horizontal gene transfer, largely driven by conjugative plasmids. Despite their central role, few strategies exist to directly block plasmid transfer. Here, we show that the IncP plasmid-dependent ssRNA phage PRR1 can inhibit the spread of antibiotic resistance genes by targeting the RP4 T4SS pilus. Structural and mutational analyses reveal previously unrecognized RNA packaging interactions and identify four pilin residues critical for infection. Remarkably, non-infectious PRR1 particles alone are sufficient to block conjugation, offering inhibition without the selective pressure from phage replication. Almost all PRR1-resistant RP4 mutants lost or had severely reduced plasmid transfer, while the remaining mutant is critical for studying the link between T4SS function and phage infection. These results highlight ssRNA phages as precise agents for limiting AMR gene dissemination.