Abstract
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 diverse bacterial species via their encoded Type IV secretion systems (T4SS). Here, we characterize the single-stranded RNA bacteriophage (ssRNA phage) PRR1, which selectively targets AMR ESKAPEE pathogens carrying the IncP plasmid RP4, and assess its ability to inhibit conjugation. Using cryo-electron microscopy, we first resolved the mature PRR1 virion at 3.45 Å resolution revealing two phage maturation protein (Mat)-RNA interactions within the 3' untranslated region (UTR) - 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-crosslinked PRR1 were 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.