AUTHOR=Liu Zixu , Yuan Biao , Zhao Lingmin , Huang Lixing , Qin Yingxue , Zhang Jiaonan , Zhang Jiaolin , Hu Bing , Yan Qingpi 

TITLE=Function of the fliK Gene in Pseudomonas plecoglossicida Pathogenicity and Epinephelus coioides’ Immune Response

JOURNAL=Frontiers in Marine Science

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/marine-science/articles/10.3389/fmars.2022.879333

DOI=10.3389/fmars.2022.879333

ISSN=2296-7745

ABSTRACT=<p><italic>Pseudomonas plecoglossicida</italic> is a gram-negative pathogenic bacterium that causes visceral white spot disease in several marine and aquaculture fish species, resulting in high mortality and severe financial loss. Based on previous RNA sequencing (RNA-seq), <italic>fliK</italic> gene expression is significantly up-regulated in <italic>P. plecoglossicida</italic> during infection, indicating that <italic>fliK</italic> may contribute to its bacterial pathogenicity. To investigate the role of <italic>fliK</italic>, four specific short hairpin RNAs (shRNAs) were designed and synthesized according to the <italic>fliK</italic> gene sequence, with three of the four mutants exhibiting a significant decrease in <italic>fliK</italic> gene expression in <italic>P. plecoglossicida</italic>. The shRNA-406 mutant with the maximum silencing efficiency (97.5%) was chosen for further study. Compared with the wild-type (WT) <italic>P. plecoglossicida</italic> strain, silencing <italic>fliK</italic> in the <italic>fliK</italic>-RNA interference (RNAi) strain resulted in a significant decrease in growth, motility, chemotaxis, adhesion, and biofilm formation in <italic>P. plecoglossicida</italic>. Silencing of <italic>fliK</italic> also resulted in a 95% increase in the survival rate, a 2-day delay in the onset of death, and a significant decrease in the number of white spots on the spleen surface in infected orange-spotted groupers (<italic>Epinephelus coioides</italic>). In addition, <italic>fliK</italic> gene expression and pathogen load were significantly lower in the spleens of <italic>E. coioides</italic> infected with the <italic>fliK</italic>-RNAi strain than in those infected with the WT strain of <italic>P. plecoglossicida</italic>. RNA-seq of the spleens further revealed that <italic>fliK</italic> silencing significantly regulated the immune response of <italic>E. coioides</italic> during the pathogenic process. Compared with the WT-infected group, the differentially expressed genes (DEGs) in the <italic>fliK</italic>-RNAi-infected group were enriched in 344 and 345 KEGG pathways at 3 and 5 days post infection (dpi), respectively. Among these pathways, 21 immune system-related pathways were enriched, including the natural killer (NK) cell-mediated cytotoxicity, platelet activation, and Th17 cell differentiation signaling pathways. The NK cell-mediated cytotoxicity pathway was the most significantly enriched, which may enhance the host’s ability to remove pathogens and reduce inflammation. This study revealed the effects of the <italic>fliK</italic> gene in <italic>P. plecoglossicida</italic> pathogenicity and identified the main pathways involved in the immune response of <italic>E. coioides.</italic></p>