Transcriptional Regulation and Signaling of Type IV IFN with Identification of the ISG Repertoire in an Amphibian Model, Xenopus laevis.

The type IV IFN (IFN-υ) is reported in vertebrates from fish to primary mammals with IFN-υR1 and IL-10R2 as receptor subunits. In this study, the proximal promoter of IFN-υ was identified in the amphibian model, Xenopus laevis, with functional IFN-sensitive responsive element and NF-κB sites, which can be transcriptionally activated by transcription factors, such as IFN regulatory factor (IRF)1, IRF3, IRF7, and p65. It was further found that IFN-υ signals through the classical IFN-stimulated gene (ISG) factor 3 (ISGF3) to induce the expression of ISGs. It seems likely that the promoter elements of the IFN-υ gene in amphibians is similar to type III IFN genes, and that the mechanism involved in IFN-υ induction is very much similar to type I and III IFNs. Using recombinant IFN-υ protein and the X. laevis A6 cell line, >400 ISGs were identified in the transcriptome, including ISGs homologous to humans. However, as many as 268 genes were unrelated to human or zebrafish ISGs, and some of these ISGs were expanded families such as the amphibian novel TRIM protein (AMNTR) family. AMNTR50, a member in the family, was found to be induced by type I, III, and IV IFNs through IFN-sensitive responsive element sites of the proximal promoter, and this molecule has a negative role in regulating the expression of type I, III, and IV IFNs. It is considered that the current study contributes to the understanding of transcription, signaling, and functional aspects of type IV IFN at least in amphibians.

Molecular characterization of nineteen cytokine receptor family B (CRFB) members, CRFB1, CRFB2, CRFB4-17, with three CRFB9 and two CRFB14 in a cyprinid fish, the blunt snout bream Megalobrama amblycephala.

The class II cytokine receptor family members are receptors of class 2 helical cytokines in mammals, and are named cytokine receptor family B (CRFB) in fish. In zebrafish, sixteen members, including CRFB1, CRFB2 and CRFB4-17 were reported. With the availability of genome sequence, a total of nineteen CRFBs was identified in the blunt snout bream (Megalobrama amblycephala), including CRFB1, CRFB2, CRFB4-17 with the presence of three CRFB9 isoforms, and two CRFB14 isoforms. These CRFB molecules contain well conserved features, such as fibronectin type III (FNIII) domain, transmembrane and intracellular domains as other class II cytokine receptors, and are phylogenetically grouped into thirteen clades with their homologues from other species of fish. The CRFB genes were constitutively expressed in organs/tissues examined in the fish. The finding of more CRFB members in the bream may provide clues to understand possible receptor-ligand interaction and their diversity from an evolutionary point of view.

Unraveling and characterization of novel T3SS effectors in Edwardsiella piscicida.

Type III secretion system (T3SS) facilitates survival and replication of Edwardsiella piscicida in vivo. Identifying novel T3SS effectors and elucidating their functions are critical in understanding the pathogenesis of E. piscicida. E. piscicida T3SS effector EseG and EseJ was highly secreted when T3SS gatekeeper-containing protein complex EsaB-EsaL-EsaM was disrupted by EsaB deficiency. Based on this observation, concentrated secretomes of ΔesaB strain and ΔesaBΔesaN strain were purified by loading them into SDS-PAGE gel for a short electrophoresis to remove impurities prior to the in-the gel digestion and mass spectrometry. Four reported T3SS effectors and two novel T3SS effector candidates EseQ (ETAE_2009) and Trx2 (ETAE_0559) were unraveled by quantitative comparison of the identified peptides. EseQ and Trx2 were revealed to be secreted and translocated in a T3SS-dependent manner through CyaA-based translocation assay and immunofluorescent staining, demonstrating that EseQ and Trx2 are the novel T3SS effectors of E. piscicida. Trx2 was found to suppress macrophage apoptosis as revealed by TUNEL staining and cleaved caspase-3 of infected J774A.1 monolayers. Moreover, Trx2 has been shown to inhibit the p65 phosphorylation and p65 translocation into the nucleus, thus blocking the NF-κB pathway. Furthermore, depletion of Trx2 slightly but significantly attenuates E. piscicida virulence in a fish infection model. Taken together, an efficient method was established in unraveling T3SS effectors in E. piscicida, and Trx2, one of the novel T3SS effectors identified in this study, was demonstrated to suppress apoptosis and block NF- κB pathway during E. piscicida infection. IMPORTANCE Edwardsiella piscicida is an intracellular bacterial pathogen that causes intestinal inflammation and hemorrhagic sepsis in fish and human. Virulence depends on the Edwardsiella type III secretion system (T3SS). Identifying the bacterial effector proteins secreted by T3SS and defining their role is key to understanding Edwardsiella pathogenesis. EsaB depletion disrupts the T3SS gatekeeper-containing protein complex, resulting in increased secretion of T3SS effectors EseG and EseJ. EseQ and Trx2 were shown to be the novel T3SS effectors of E. piscicida by a secretome comparison between ∆esaB strain and ∆esaB∆esaN strain (T3SS mutant), together with CyaA-based translocation assay. In addition, Trx2 has been shown to suppress macrophage apoptosis and block the NF-κB pathway. Together, this work expands the known repertoire of T3SS effectors and sheds light on the pathogenic mechanism of E. piscicida.

Immunoexpression of MMP-8 and MMP-9 in chronic subdural hematoma.

To determine the possible role of matrix metallopeptidase (MMP)-8 and MMP-9 in the development of chronic subdural hematoma (CSDH), we investigated their expression in CSDH. In our previous study, we analyzed hematoma fluid and peripheral blood of 83 patients with CSDH, including 17 postoperative patients. Based on these results, we included 50 people in the normal group and analyzed 20 markers in the peripheral blood of each person. In order to identify representative markers, it was assessed by using overall differential gene expression. The concentration of MMP-8 was significantly higher in the normal group than that in the preoperative and postoperative groups. The concentration of MMP-9 was significantly lower in the normal group than in both preoperative and postoperative groups. Immunohistochemistry confirmed the expression of MMP-8 and MMP-9 in CSDH membranes. In conclusion, our results provide evidence of the expression of MMP-8 and MMP-9 in CSDH. In addition, the expression of MMP-8 and MMP-9 suggests angiogenesis in CSDH formation.