L. Ma

Krüppel-like factors (KLFs) are transcription factors that bind DNA via three tandem zinc fingers, critically regulating cellular processes like proliferation, differentiation, and apoptosis. Among KLFs, KLF9 is particularly notable for its immunological roles in mammalian immune systems, where it regulates tumor growth, modulates interferon-related genes and inflammatory cytokines. However, the potential role of klf9 in fish immune responses remains poorly understood. In this study, we characterized turbot (Scophthalmus maximus L.) klf9 (Sm_klf9) and explored its role against bacterial infection. In our results, the full length of Sm_klf9 was 3,372 bp, including an open reading frame (ORF) of 684 bp. In phylogenetic analyses, Sm_klf9 grouped closely with the klf9 genes of Japanese flounder and Atlantic halibut, consistent with the broader phylogenetic relationships among flatfish species. Sm_klf9 exhibited widespread expression across all tested tissues in turbot with the highest expression level in blood. The expression of Sm_klf9 was significantly induced in a time-dependent manner in turbot intestine after bacterial infection (dramatic upregulation with 2.78-fold at 2 h, followed by significant downregulations with 2.29- and 2.74-fold at 12 h and 24 h, respectively), and the urgently significant induction at 2h suggested rapid response measures against bacterial infection at the early stages. The overexpression of Sm_klf9 up-regulated the key immune-related genes in the NF-κB signaling pathway, such as NF-κB2 and Myd88. Meanwhile, the overexpression of Sm_klf9 was found to accelerate the invasion of Edwardsiella tarda and promote the cell apoptosis in turbot intestine cells. Overall, the findings in this study indicated the important roles of Sm_klf9 in turbot immune system during pathogenic infection, which could provide a theoretical basis for investigating the key roles of Sm_klf9 in bacteriostatic ability and inflammatory regulations in turbot during bacterial infection.

Growth arrest and DNA damage-inducible 45 alpha, b gene (gadd45ab) is a key homologue of gadd45a subfamily, which participates in several biological processes, such as inflammation and apoptosis. Black rockfish (Sebastes schlegelii) is an important commercial fish species in China. In current study, black rockfish gadd45ab (Ss_gadd45ab) was characterized as a target gene of miR-20b-5p. The results indicated that high evolutionary conservations were found in the sequence and structure of Ss_gadd45ab, which was also detected to locate on the cytomembrane in the black rockfish intestine. The expressions of Ss_gadd45ab and diverse cytokines related to immune response were significantly induced with different patterns by the infection of E. tarda. Moreover, all tested immune genes were dramatically up-regulated when Ss_gadd45ab was overexpressed in the intestine cells, and significant up-regulations and down-regulations of almost these immune genes were observed in the overexpressed cells during the LPS stimulation at 12 h and 24 h, respectively, which implied the potential regulations of Ss_gadd45ab in the immune system. Meanwhile, the overexpression of Ss_gadd45ab was found to prevent the invasion of E. tarda and promote the cell apoptosis in the intestine cells of black rockfish. Therefore, a profile of a novel regulatory mechanism of Ss_gadd45ab targeted by miR-20b-5p in the inflammatory and apoptotic responses of black rockfish during pathogenic infections was hypothesized. Even though the potential functions of Ss_gadd45ab on the inflammation and apoptosis in the black rockfish intestine during pathogenic infections have been described preliminarily in this study, more efforts are still urgent to be done for the further verification of this regulatory mechanism in black rockfish.

Recently, Vibrio anguillarum, a Gram-negative pathogenic bacterium, has been becoming a major constraint on the development of the turbot aquaculture industry because of its characteristics of worldwide distribution, broad host range and potentially devastating impacts. Although the functions of protein-coding mRNAs in the immune response against bacterial infection have been reported, as well as several non-coding RNAs (ncRNAs), such as circular RNAs (circRNAs) and microRNAs (miRNAs), the relationships between mRNAs and ncRNAs in the immune system of turbot liver are still limited during bacterial infection. In present study, the comprehensive analyses of whole-transcriptome sequencing were conducted in turbot liver infected by V. anguillarum. The differential expression was analyzed in the data of circRNAs, miRNAs, and mRNAs. The interactions of miRNA-circRNA pairs and miRNA-mRNA pairs were predicted basing on the negative regulatory relationships between miRNAs and their target circRNAs\mRNAs. The circRNA-related ceRNA regulatory networks were constructed for the analyses of regulated mechanism in turbot immune system. Subsequently, the RT-qPCR was carried out to verify the results of sequencing. Finally, we identified 31 circRNAs, 53 miRNAs and 948 mRNAs with differential expression. Gene set enrichment analyses using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that innate immunity was principally activated at the early stages of infection, while adaptive immunity was activated after 24 h. Finally, 65 circRNA-miRNA-mRNA pathways were constructed, based on the hypothesis of ceRNA regulatory networks. In conclusion, our findings provide new insights on the underlying immune response to bacterial infection and identify novel target genes for the prevention and control of disease in turbot.

In teleost, the immune roles of the gut-associated lymphoid tissue (GALT) have been well recognized. The antibacterial functions of the spleen have been also documented in various fish species. However, the mechanism of fish intestine-spleen immunity during bacterial infection still remains largely unknown. In this study, an integrative analysis was conducted in the black rockfish intestine and spleen after Aeromonas salmonicida and Edwardsiella tarda infection on the transcriptomic level. The immune gene libraries were constructed in the intestine and spleen. A total of 211 and 253 differentially expressed genes were observed in the immune gene library of spleen and intestine, respectively. One more category of immune genes was found in the intestine (9 categories) than in the spleen (8 categories). Potential evidence of the cooperation in the intestine-spleen axis was obtained based on the functional annotation and enrichment analyses of joint immune genes between intestine and spleen. In addition, bioinformatic and ceRNA analyses for transcriptomic data were performed. Finally, two key immune genes (CO3 and MRC1) and their related non-coding RNAs were observed in the functions of bacterial recognition and binding. According to the pathway “intestinal immune network for IgA production” predicted in the splenic transcriptome by functional enrichment, a novel regulatory pathway of the immunity of the intestine-spleen axis was hypothesized in black rockfish during bacterial infection. Moreover, the regulatory relationships between the immune transcripts in this pathway were verified preliminarily by RT-qPCR. Dual-luciferase reporter assays were performed to determine the regulations between the ceRNAs of MRC1/miR-23b-5p/LNC_00057980 and CO3/miR-214/LNC_00096415 axis. Overall, our findings demonstrate the key pathways of MRC1/miR-23b-5p/LNC_00057980 and CO3/miR-214/LNC_00096415 in the regulation mechanism of intestine-spleen axis during two different Gram-negative bacteria infection in black rockfish.