A novel phage depolymerase ORF45 attenuates Vibrio parahaemolyticus virulence in Litopenaeus vannamei by degrading capsular polysaccharides

Vibrio parahaemolyticus ( V. parahaemolyticus ) is an important pathogen threatening aquaculture health and seafood safety, and phage depolymerases are considered promising antibiotic alternatives. In this study, the putative depolymerase ORF45 was identified from the genome of V. parahaemolyticus phage PP270 and characterized by bioinformatic analysis, recombinant expression, and functional assays. ORF45 showed low similarity to known depolymerases, suggesting that it may represent a previously uncharacterized Vibrio phage depolymerase. Recombinant ORF45 formed typical expanding halos on lawns of the host strain VP270 and retained activity under pH 3–10, 4–50 °C, and 0.8%–5% NaCl. Functional assays showed that ORF45 degraded VP270 capsular polysaccharides (CPS) in a dose-dependent manner, significantly inhibited biofilm formation, and disrupted mature biofilms, but did not directly kill bacteria or damage cell membranes. ORF45 pretreatment enhanced the adsorption of phage PP270 to VP270 and improved late-stage phage-mediated bacterial inhibition. In addition, ORF45 retained depolymerase activity against some phage-resistant receptor-deficient mutants. Further analysis showed that ORF45-treated VP270 was more susceptible to killing by Litopenaeus vannamei ( L. vannamei ) hemolymph, exhibited reduced cytotoxicity toward Caco-2 cells, and showed attenuated pathogenicity in an L. vannamei infection model. Immune-related gene expression and humoral immune enzyme activity analyses indicated that ORF45 altered host immune responses and promoted bacterial clearance by removing CPS. Overall, ORF45 is a stable and novel phage depolymerase with potential application in the sustainable control of V. parahaemolyticus infections.