Major histocompatibility complex class I (MHC Iα) of Japanese flounder (Paralichthys olivaceus) plays a critical role in defense against intracellular pathogen infection.

The major histocompatibility complex (MHC) is a highly polymorphic region of the vertebrate genome that plays a critical role in initiating immune responses towards invading pathogens. It is well known that MHC I molecules play a central role in the immune response to viruses. However, rare literatures were reported the role of MHC I in the resistance to intracellular bacteria. Sequences of MHC Iα were identified in multiple teleost species, including Japanese flounder (Paralichthys olivaceus), however, the immunological function of MHC Iα remain largely unknown. In this study, we examined the expression profile and biological activity of an MHC Iα homologue, PoMHC Iα, from P. olivaceus. Structural analysis showed that PoMHC Iα possesses conserved structural characteristics of MHC Iα proteins, including MHC_I domain, IGc1 domain, transmembrane region. Expression of PoMHC Iα was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen infection. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoMHC Iα increased the capability of host cells to defense against intracellular pathogen Edwardsiella tarda infection and enhanced the expression of immune related genes. The knockdown of PoMHC Iα attenuated the ability of cells to eliminate E. tarda, which was sustained by the in vivo results that overexpression of PoMHC Iα promoted the host defense against invading E. tarda. Antigen uptake assay indicated PoMHC Iα participated in cells antigen presentation. Collectively, this study is the first report that MHC Iα plays an important role in immune defense against intracellular bacterial pathogen in teleost. Taken together, these findings add new insights into the biological function of teleost MHC Iα and emphasize the importance of MHC I gene products for the control of E. tarda infection.

Granulocyte colony stimulating factor (GCSF) of Japanese flounder (Paralichthys olivaceus): Immunoregulatory property and anti-infectious function.

Granulocyte colony stimulating factor (GCSF) is a key regulator of neutrophil production, and plays a vital role in immune response of mammals and teleost against pathogen. Sequences of GCSF were identified in several teleost species, however, the function and activity of GCSF in teleost remain largely unknown. In this study, we examined the biological activity and the immunomodulatory property of a GCSF homologue, PoGCSF, from Japanese flounder (Paralichthys olivaceus). Structural analysis showed that PoGCSF possesses conserved structural characteristics of GCSF proteins, including a signal peptide and a typical IL-6 domain. The expression of PoGCSF was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoGCSF increased the capability of host cells to defense against pathogen infection and enhanced the expression of immune related genes. The knockdown of PoGCSF attenuated the ability of host cells to eliminate pathogenic bacteria. In vivo results showed that overexpression of PoGCSF promoted the host defense against invading pathogenic microorganism. Collectively, this study is the first report about the immunoregulatory property and anti-infectious immunity of GCSF in teleost. These findings suggested that PoGCSF serves as an immune-related cytokine and plays an important role in the immune defense system of Japanese flounder.

High mobility group box 2 of black rockfish Sebastes schlegelii: Gene cloning, immunoregulatory properties and antibacterial effect.

High-mobility group box 2 (HMGB2) is a non-histone chromosomal protein that involved diverse functions such as transcriptional regulation and innate immune responses in mammalian. In teleost, very limited studies on HMGB2 proteins have been documented. Black rockfish (Sebastes schlegelii) is an economic fish species and cultured worldwide. However, the study of black rockfish about immunology is very scarce. In the present study, a HMGB2 homologue gene (SsHMGB2) was identified and characterized in black rockfish. The open reading frame of SsHMGB2 is 648 bp, and the deduced amino acid sequence of SsHMGB2 shares 74.4%-91.2% overall sequence identities with the HMGB2 proteins of several fish species. In silico analysis identified several conserved features, including two basic HMG boxes and an acidic C-terminal tail composed of 24 Asp/Glu residues. Expression of SsHMGB2 occurred in multiple tissues and was upregulated during pathogens infection. Recombinant SsHMGB2 (rSsHMGB2) exhibited apparent binding activities against DNA. In vivo studies showed that the expressions of multiple immune-related genes in head kidney were significantly enhanced when black rockfish were treated with rSsHMGB2. Furthermore, rSsHMGB2 reduced pathogen dissemination and replication in fish kidney and spleen. Taken together, these results suggest that SsHMGB2 possesses apparent immunoregulatory properties and played a role in fighting bacterial infection.

Global discovery of small RNAs in the fish pathogen Edwardsiella piscicida: key regulator of adversity and pathogenicity.

Recently, bacterial small RNA (sRNA) has been shown to be involved as a key regulator in stress responses. sRNAs of Edwardsiella piscicida, an important aquatic pathogen, are not well characterized to date. In this study, using RNA-seq technology, we globally found and identified sRNA candidates expressed from E. piscicida grown in normal LB medium, acid pressure, iron deficiency stress, and oxidation pressure. A total of 148 sRNAs were found, including 19 previously annotated sRNAs and 129 novel sRNA candidates by searching against the Rfam database. Compared in normal condition, the expression of 103 sRNAs (DEsRNA, differentially expressed sRNA) and 1615 mRNAs (DEmRNAs, differentially expressed mRNA) showed significant differences in three stress sample. Based on the prediction by IntaRNA and relational analysis between DEsRNAs and DEmRNAs, 103 DEsRNAs were predicted to regulate 769 target mRNAs. Pleiotropic function of target DEmRNAs indicated that sRNAs extensively participated in a variety of physiological processes, including response to adversity and pathogenicity, the latter was further confirmed by infection experiment. A large number transcription factors appeared in target genes of sRNAs, which suggested that sRNAs likely deeply interlaced within complex gene regulatory networks of E. piscicida. Moreover, 49 Hfq-associated sRNAs were also identified in this study. In summary, we globally discovered sRNAs for the first time in pathogenic bacteria of fish, and our findings indicated that sRNAs in E. piscicida have important roles in adaptation to environmental stress and pathogenicity. These results also provide clues for deciphering regulation mechanism of gene expression related to physiological response and pathogenicity.