Intra-strain and inter-strain heterogeneity shape phage-host interactions and phenotypic adaptation in <i>Pseudomonas aeruginosa</i>

The efficacy of phage therapy relies on a precise understanding of phage-host interactions, particularly how phages recognize bacterial surface receptors and how these interactions shape host adaptation. In <i>Pseudomonas aeruginosa</i>, the O-antigen of lipopolysaccharide (LPS) is a common but incompletely characterized determinant of phage susceptibility. Here, we used an O-antigen-specific lytic podophage, phipa9, to prove receptor plasticity in <i>P. aeruginosa</i> strain ZS-PA-11. Phage-resistant mutants arose through either a point mutation in <i>gtaB</i> (phipa9-G), encoding UTP-glucose-1-phosphate uridylyltransferase, or a large chromosomal deletion (411 kb) (phipa9-B) encompassing <i>gtaB</i>. Functional analyses revealed that <i>gtaB</i> is essential for O-antigen synthesis, phage adsorption, and infection. Disruption of <i>gtaB</i> altered the LPS profile, reduced motility, and promoted biofilm formation. Both resistant mutants (phipa9-G and phipa9-B) exhibited attenuated virulence in <i>Galleria mellonella</i>, with the 411 kb deletion mutant showing a more pronounced reduction, underscoring the contribution of O-antigen and deleted loci to bacterial virulence. Comparative assays with another O-antigen-specific phage, phipa10, revealed distinct host ranges and inhibition profiles, highlighting receptor-dependent diversity and possible roles of intracellular defenses. Together, these findings demonstrate that natural variation in O-antigen biosynthetic pathways governs phage susceptibility and bacterial physiology. By uncovering <i>gtaB</i>-mediated intra-strain heterogeneity that restricts phage infection while reshaping host phenotypes, this work underscores the dual role of surface receptors in mediating phage-host dynamics and provides mechanistic insight for the rational design of therapeutic phages targeting <i>P. aeruginosa</i>.IMPORTANCEEffective phage therapy against multidrug-resistant <i>Pseudomonas aeruginosa</i> requires understanding how surface receptors govern susceptibility and resistance. We show that <i>gtaB</i>-mediated loss of the O-antigen blocks phage adsorption, alters bacterial physiology, and promotes population heterogeneity through pleiotropic phenotypic alterations. These multifaceted consequences reveal that phage resistance is not binary but instead reprograms bacterial adaptation and virulence. Comparison of two O-antigen-dependent phages with distinct host specificities further demonstrates that natural receptor variation critically shapes infection outcomes. Thus, receptor-based resistance represents both a barrier and an opportunity: while it limits phage efficacy, it can also attenuate virulence and expose new vulnerabilities. Recognizing these trade-offs is essential for designing phage therapies that both eradicate pathogens and harness the evolutionary costs of resistance.