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 (Δ phoP ), and complemented (C-Δ phoP ) strains. In artificial infection experiments, the Δ phoP strain exhibited significantly attenuated virulence, with an LD 50 value of 4.082 × 10 4 , 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 Δ phoP -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.