Vibrio harveyi is commonly found in salt and brackish water, and is recognized as a serious bacterial pathogen in aquaculture worldwide. In this study, we cloned the ferric uptake regulator (Fur) gene from V. harveyi wild-type strain HA_1, which was isolated from diseased American eels (Anguilla rostrata) and has a length of 450 bp, encoding 149 amino acids. Then, a mutant strain, HA_1-ΔFur, was constructed through homologous recombination of a suicide plasmid (pCVD442). The HA_1-ΔFur mutant exhibited attenuated biofilm formation, intensified swarming motility, and 18-fold decrease (5.5%) in virulence to the American eels, but it showing no difference in growth and hemolysis with the wild-type strain. Transcriptome analysis revealed that 875 genes were differentially expressed in the ΔFur mutant, with 385 up-regulated and 490 down-regulated DEGs. GO and KEGG enrichment analysis revealed that, compared to the wild-type strain, the type II secretion systems (T2SS), type VI secretion systems (T6SS), amino acid synthesis and transport, and energy metabolism pathways were significantly down-regulated, but the ABC transporters and biosynthesis of siderophore group non-ribosomal peptides pathways were up-regulated in the ΔFur strain. The qRT-PCR results further confirmed that DEGs responsible for amino acid transport and energy metabolism were positively regulated, but DEGs involved in iron acquisition were negatively regulated in the ΔFur strain. These findings suggest that the gene Fur contribute to the virulence of V. harveyi through biofilm formation, energy metabolism, and transcript regulation.

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Edwardsiella anguillarum is a common bacterial pathogen mainly infected cultivated eel, and outer membrane protein A (OmpA) emulsified with Freund's adjuvant was evaluated as an effective fishery vaccine. However, the role of solo OmpA in the vaccine have not been explored on the molecular mechanism. In this study, we examined the RNA-seq in the liver of European eel (Anguilla anguilla) post the challenge of E. anguillarum in eels injected with the OmpA vaccine, Freund's adjuvant, or PBS to elucidate the alternative splicing (AS) and long noncoding RNA (lncRNA) in the process of E. anguillarum infection and host anti-infection using genome-wide transcriptome. After all eels were challenged by E. anguillarum at 28 d post the injection (dpi), compared to severe pathological changes in the liver of PBS injected eels (Con_inf group), the OmpA immunized eels (OmpA_inf group) showed infiltrated lymphocytes in the liver as well as the Freund's adjuvant inoculated eels (FCIA_inf group) showed slight edema of hepatocytes and blood coagulation. Compared to the Con_inf group, the relative percent survival (RPS) of eels was 77.7% and 44.4% in OmpA_inf and FCIA_inf group. The results of DEGs and DE-transcripts showed 6 samples in the OmpA_inf and FCIA_inf groups were clustered together, which different to 3 samples in the Con_inf group. We found only 35 DEGs between OmpA_inf and FCIA_inf compare to thousands of DEGs in two compares of OmpA_inf vs Con_inf and FCIA_inf Con_inf. GO and KEGG analysis of genes targeted by 37 DE-lncRNAs in co-expression and co-location showed at least 52 GO terms and 2 KEGG pathways were enrichment. The results of AS analysis showed 293 differentially alternative splicing (DAS) genes between OmpA_inf and FCIA_inf were mainly involved in GO terms of catalytic activity, membrane part and biological regulation, and KEGG pathways of signal transduction, immune system and infection disease. Finally, the interaction between proteins expressed by DAS genes and the interaction between DE-lncRNAs and target genes were explored using Cytoscape 3.9.1. The results indicated 66 DAS gene expressed proteins formed total of 50 degrees in 20 networks, and 33 DE-lncRNAs interacted with 194 target genes formed total of 246 and 41 networks in co-expression and co-location. Taken together, after the immunization of OmpA vaccine, compared to Freund's adjuvant inoculation, higher RPS was closely related to DAS genes and DE-lncRNAs in response to E. anguillarum infection in European eels. The OmpA role in vaccine was firstly revealed through AS genes and lncRNAs, and results of this study are of great significance to the development of fishery subunit vaccines.