The bacteriophage-encoded regulator PemR attenuates <i>Pseudomonas aeruginosa</i> virulence by hijacking quorum sensing and metabolism

The global challenge of multidrug-resistant <i>Pseudomonas aeruginosa</i> demands innovative anti-virulence approaches. We characterize PemR, a novel transcriptional regulator encoded by <i>P. aeruginosa</i> phage PAYQ66, which orchestrates multimodal virulence attenuation in <i>P. aeruginosa</i>. Biochemical analyses demonstrated that PemR directly binds to the <i>mvfR</i> promoter, resulting in significant repression of this key quorum-sensing regulator. This repression, in turn, induces profound metabolic reprogramming by redirecting metabolic flux away from PQS biosynthesis toward catechol accumulation. PemR globally attenuates virulence phenotypes, including pyocyanin production, rhamnolipid synthesis, motility, and biofilm formation. Transcriptomic profiling further reveals that PemR upregulates <i>rsmA</i> to suppress the type VI secretion system, thereby potentially modulating the host's interaction with competing bacteria in polymicrobial environments. The motility analysis shows that PemR suppresses bacterial swimming, swarming, and twitching at gene expression levels. Overall, infection models demonstrate that PemR significantly inhibits bacterial virulence. As the first reported phage-encoded transcriptional hijacker of the <i>mvfR</i> regulon, PemR concurrently disrupts quorum sensing and bacterial pathogenicity, offering a strategic blueprint for novel anti-virulence therapeutics targeting multidrug-resistant pathogens.IMPORTANCEThis study identifies a novel bacteriophage-encoded regulator PemR that simultaneously disrupts multiple virulence pathways in the opportunistic pathogen <i>P. aeruginosa</i>. By hijacking a key bacterial quorum-sensing (QS) system and reprogramming host metabolism, PemR significantly reduces pathogenicity without killing the bacteria. This work reveals a sophisticated strategy phages use to manipulate their hosts and provides a promising blueprint for developing next-generation anti-virulence therapeutics. Such approaches aim to disarm dangerous bacteria rather than eliminate them, potentially slowing the emergence of antibiotic resistance and offering new strategies against multidrug-resistant infections.