Biological Function of Prophage-Related Gene Cluster Δ VpaChn25_RS25055 ~Δ VpaChn25_0714 of Vibrio parahaemolyticus CHN25.

Vibrio parahaemolyticus is the primary foodborne pathogen known to cause gastrointestinal infections in humans. Nevertheless, the molecular mechanisms of V. parahaemolyticus pathogenicity are not fully understood. Prophages carry virulence and antibiotic resistance genes commonly found in Vibrio populations, and they facilitate the spread of virulence and the emergence of pathogenic Vibrio strains. In this study, we characterized three such genes, VpaChn25_0713 , VpaChn25_0714 , and VpaChn25_RS25055 , within the largest prophage gene cluster in V. parahaemolyticus CHN25. The deletion mutants Δ VpaChn25_RS25055 , Δ VpaChn25_0713 , Δ VpaChn25_0714 , and Δ VpaChn25_RS25055-0713-0714 were derived with homologous recombination, and the complementary mutants Δ VpaChn25_0713 -com, Δ VpaChn25_0714 -com, Δ VpaChn25_RS25055 -com, Δ VpaChn25_RS25055-0713-0714 -com were also constructed. In the absence of the VpaChn25_RS25055 , VpaChn25_0713 , VpaChn25_0714 , and VpaChn25_RS25055-0713-0714 genes, the mutants showed significant reductions in low-temperature survivability and biofilm formation ( p < 0.001). The Δ VpaChn25_0713 , Δ VpaChn25_RS25055 , and Δ VpaChn25_RS25055-0713-0714 mutants were also significantly defective in swimming motility ( p < 0.001). In the Caco-2 model, the above four mutants attenuated the cytotoxic effects of V. parahaemolyticus CHN25 on human intestinal epithelial cells ( p < 0.01), especially the Δ VpaChn25_RS25055 and Δ VpaChn25_RS25055-0713-0714 mutants. Transcriptomic analysis showed that 15, 14, 8, and 11 metabolic pathways were changed in the Δ VpaChn25_RS25055 , Δ VpaChn25_0713 , Δ VpaChn25_0714 , and Δ VpaChn25_RS25055-0713-0714 mutants, respectively. We labeled the VpaChn25_RS25055 gene with superfolder green fluorescent protein (sfGFP) and found it localized at both poles of the bacteria cell. In addition, we analyzed the evolutionary origins of the above genes. In summary, the prophage genes VpaChn25_0713 , VpaChn25_0714 , and VpaChn25_RS25055 enhance V. parahaemolyticus CHN25's survival in the environment and host. Our work improves the comprehension of the synergy between prophage-associated genes and the evolutionary process of V. parahaemolyticus .