Tail spike protein evolution enables phage Psm140 to target Salmonella ShdA and overcome host resistance.

The growing challenge of bacterial antibiotic resistance has heightened attention in phage therapy. However, the rapid evolution of phage resistance in bacteria poses a major obstacle to its clinical application, making the development of strategies to overcome bacterial resistance a critical research priority. In this study, we investigated the coevolutionary dynamics between Salmonella enterica sm140 and its lytic phage, Psm140. Our previous work revealed that bacterial resistance to Psm140 arises through mutations in the rfbD gene, disrupting O-antigen synthesis and preventing phage recognition. Building on this finding, we employed laboratory-directed evolution to generate an evolved phage variant, PRsm1-20, capable of reinfecting resistant bacteria. Genomic analysis identified 18 nonsynonymous mutations in the tail spike protein gene (ORF63), enabling PRsm1-20 to utilize the outer membrane protein (OMP) ShdA as an alternative receptor while retaining lipopolysaccharide (LPS) binding capability. Genetic knockout and complementation assays confirmed ShdA as the essential receptor for PRsm1-20 infection. These findings elucidate a molecular mechanism by which phage tail spike protein evolution overcomes host resistance, providing new insights for the development of engineered phage-based antimicrobial therapies.