The impact of the phoP gene on Pseudomonas plecoglossicida virulence and host immune responses in Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂.

Pseudomonas plecoglossicida is a significant pathogen in aquaculture, capable of infecting various teleost fish and causing visceral white spot disease, which leads to substantial economic losses. The PhoP/PhoQ two-component system plays a central role in virulence regulation in Gram-negative bacteria; however, its specific function in P. plecoglossicida remains unclear. To elucidate the role of the phoP gene in the pathogenicity of this bacterium, we used hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂) as an infection model to compare the pathogenicity and host immune responses among the wild-type (NZBD9), phoP deletion (ΔphoP), and complemented (C-ΔphoP) strains. In artificial infection experiments, the ΔphoP strain exhibited significantly attenuated virulence, with an LD50 value of 4.082 × 104, which was 13.7 times higher than that of the wild-type strain. Furthermore, the deletion of phoP reduced bacterial load in the spleen and alleviated histopathological damage in the spleen, head kidney, and intestine. RNA-seq analysis of spleen tissue at 4 days post-infection identified 1768 differentially expressed genes (DEGs), including 704 up-regulated and 1064 down-regulated genes. GO and KEGG enrichment analyses revealed that these DEGs were primarily involved in immune-related processes and pathways, including TLR signaling, NF-κB activation, PI3K-Akt signaling, cytokine-cytokine receptor interaction, and cell adhesion molecules. Temporal expression profiling via qRT-PCR demonstrated that key immune genes (including tlr1, tlr5, tlr13, traf5, nf-κb, mcp1, cd3, and il10) were generally down-regulated or exhibited delayed and weakened activation in ΔphoP-infected fish, indicating a subdued and dysregulated immune response. These results suggest that the phoP gene is essential for maintaining full virulence in P. plecoglossicida, modulating host immunity by suppressing critical inflammatory and immune signaling pathways. Our study provides novel insights into the role of the PhoP/PhoQ system in host-pathogen interactions and lays a theoretical foundation for developing targeted control strategies against P. plecoglossicida infection in aquaculture.