Sequential receptor engagement dictates the broad host range and fitness trade-offs of <i>Salmonella</i> phage PSA5-1

ABSTRACT The global rise of multidrug-resistant (MDR) Salmonella spp. necessitates the development of alternative antimicrobials, yet the molecular determinants of phage host range and the collateral effects of bacterial resistance remain poorly defined. Here, we characterize PSA5-1, a T4-like phage, and decipher the hierarchical two-step receptor recognition governing its infection of Salmonella enterica . We demonstrate that PSA5-1 utilizes the outer-membrane porin C (OmpC) as a primary receptor required for reversible adsorption, while the lipopolysaccharide (LPS) inner core is required for productive infection. Selection for PSA5-1 resistance reveals distinct evolutionary trajectories: whereas ompC deletion confers resistance with minimal fitness costs, disruption of the LPS core (via rfaF ) triggers profound physiological remodeling and enhanced biofilm formation. Comparative genomics further indicates that host susceptibility is governed by OmpC sequence conservation and O-antigen diversity, with additional contributions from intracellular defense systems. By mapping the interplay between receptor engagement and resistance-associated trade-offs, our findings provide a mechanistic framework for the rational design of precision phage-based interventions against enteric pathogens. IMPORTANCE The increasing prevalence of multidrug-resistant Salmonella spp. necessitates the development of precision antimicrobial alternatives to antibiotics, such as bacteriophage therapy. However, the successful clinical application of phages is often hampered by the rapid evolution of bacterial resistance and complex host defense mechanisms. In this study, we dissect the infection logic of the phage PSA5-1, identifying the outer-membrane protein C–lipopolysaccharide inner core dependency as a critical determinant enabling sequential receptor engagement during genome delivery. By mapping the genetic determinants of susceptibility, ranging from receptor variation to intracellular immunity, we highlight the evolutionary trade-offs Salmonella must navigate to evade infection. These findings provide a mechanistic blueprint for selecting phages that exploit conserved surface targets, ultimately aiding in the design of therapeutic cocktails that are robust against bacterial resistance.