Genomic sequences comparison and differential expression of miiuy croaker MHC class I gene, after infection by Vibrio anguillarum

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. MHC gene family contains two main subgroups of immunologically active molecules. In order to study the molecular function and genomic characteristic of class I gene in teleost, the full lengths of MHC class Iα cDNA and genomic sequence were cloned from miiuy croaker (Miichthys miiuy). Seven exons and six introns were identified in miiuy croaker class Iα gene. This genomic structural feature of miiuy croaker is similar to that present in some fishes such as Japanese flounder and Atlantic salmon, but different from that present in some other fishes such as half-smooth tongue sole and channel catfish. The deduced amino acid sequence of class Iα gene had 25.9-54.1% identity with those of mammal and teleost. Real-time quantitative RT-PCR demonstrated that the MHC class Iα gene was ubiquitously expressed in 10 normal tissues; expression levels of MHC Iα gene were found first upregulated and then downregulated throughout the pathogenic bacteria infection process in spleen and kidney.     

Identification of 21 novel immune-type receptors in miiuy croaker and expression pattern of three typical inhibitory members

Novel immune-type receptor (NITR) genes belong to the immunoglobulin superfamily and are encoded by clusters of multigene families. NITRs encode type I transmembrane proteins and are only found in teleosts. In the current study, total 21 NITR genes are identified from miiuy croaker (Miichthys miiuy) and named as MmNITR1 to MmNITR21. Miiuy croaker NITR genes that encoded one or two extracellular immunoglobulin (Ig) domains, a transmembrane (TM) region, an immunoreceptor tyrosine-based inhibitor motif (ITIM) in the cytoplasmic (Cyt) region. The majority of MmNITRs possess cytoplasmic ITIM that can be classified as inhibitory receptors. However, a smaller number of NITRs (MmNITR8, MmNITR15 and MmNITR16) can be classified as activating receptors by the lack of cytoplasmic ITIMs and presence of a positively charged residue within their transmembrane domain. As typical inhibitory receptors, MmNITR1, MmNITR2 and MmNITR3 have different characteristics of the structure. In MmNITR1 gene, variable (V) and intermediate (I) domains are encoded by two separate exons. In contrast to MmNITR1, MmNITR3 gene encode V and I domains in a single exon. And MmNITR2 gene is characterized by the presence of only one Ig-like (V-type) extracellular domain and lack of J or J-like motifs. Also MmNITR2 gene displays an additional exon which is 48 bp long between the V domain and the TM region. Two and four potential N-link giycosylation sites (N-X-S/T) are present in the extracellular Ig domains. Real-time RT-PCR results showed that upon induction with Vibrio anguillarum, NITR gene expressions were induced by bacteria in kidney, liver and spleen. Meanwhile, NITRs are also primarily detected in different tissues. Phylogenetic analyses of NITR V domains indicate that MmNITR1 and MmNITR2 are more similar than MmNITR3.     

Characterization and expression of the CXCR1 and CXCR4 in miiuy croaker and evolutionary analysis shows the strong positive selection pressures imposed in mammal CXCR1

The innate immune system can recognize non-self, danger signals, and pathogen associated molecular patterns and provides a first line of antimicrobial host defense. Therefore, it plays an instructive role and is pretty important in vertebrates. In innate immune responses, CXCRs act as the main receptors of CXC chemokines and play a vital role in host defense and inflammation. In present study, we cloned two cDNA molecules of CXCR1 and CXCR4 in Miichthys miiuy (miiuy croaker). In these two genes, we found the most highly conserved DRY motif in the second intracellular loop adjacent to the third transmembrane domain. The expressions of CXCR1 and CXCR4 showed that they were ubiquitously expressed in ten normal tissues. After infection with Vibrio anguillarum and Vibrio harveyi, the expressions of CXCRs in the immune tissues were significantly regulated in most of tissues except that of CXCR1 in the kidney after V. harveyi injection. Evolutionary analysis showed that only the ancestral lineages of CXCR4 in amphibians underwent positive selection, indicating that the ancestors of amphibians boarded the land and had to further evolve to adapt to terrestrial environments. Multiple ML methods were implemented to detect the robust positively selected candidates for sites. In total, we detected 12 and 3 positively selected sites in the subsets of current mammal and fish CXCR1 genes, and only one site under positive selection was found in mammalian CXCR4 subsets. These positively selected sites were mainly located in the extracellular domains of CXCRs. The sliding window analysis and evolution test tended to favor positive selection acting on the N-terminal domain of CXCR1, which was the critical region for ligand/receptor signaling for neutrophils and receptor–ligand interaction, indicating that the N-terminal of CXCR1 in mammals underwent more positive selection than that of fish.     

A genome-wide survey of expansive NLR-C subfamily in miiuy croaker and characterization of the NLR-B30.2 genes

NOD-like receptors (NLRs) are essential intracellular pattern-recognition receptors that respond to pathogens and regulate innate immunity. NLRs include three distinct subfamilies: NLR-A, NLR-B and NLR-C, thereinto, NLR-C as a large subfamily is unique to bony fish and little research about it has been done. In the current study, we identified the members of NLR-B and NLR-C subfamilies containing 2 and 48 genes respectively in miiuy croaker. Compared with other teleosts except for zebrafish, NLR-C subfamily genes occurred expansion in miiuy croaker. The gene expansions of NLR-C subfamily may illustrate adaptive genome evolution in response to specific aquatic environments. Structural analysis showed that the N-terminus of NLR-C subfamily receptors has different characteristics of the domains including RING domain, FISNA domain or PYRIN domain. Interestingly, the C-terminus of 18 NLR-C subfamily members contains an extra B30.2 domain (named NLR-B30.2 genes) which plays an important role in antiviral immune recognition. Simultaneously, molecular evolutionary analysis indicated that the positively sites in miiuy croaker are mainly located in NACHT domain which was the vital region for signal transduction in immune response. Significantly, pathogens challenge in spleen and macrophages demonstrated that NLR-B30.2 genes exhibited more sensitive response to virus than bacteria, suggesting these genes play enhanced roles in innate antiviral immunity, which may represent a new family used for antiviral infection.     

Identification and characterization of Cynoglossus semilaevis microRNA response to Vibrio anguillarum infection through high-throughput sequencing

MicroRNAs (miRNA) play key regulatory roles in diverse biological processes. Cynoglossus semilaevis is an important commercial mariculture fish species in China. To identify miRNAs and investigate immune-related miRNAs of C. semilaevis, we performed high-throughput sequencing on three small RNA libraries prepared from C. semilaevis immune tissues (liver, head kidney, spleen, and intestine). One library was prepared under normal conditions (control, CG); two were prepared during Vibrio anguillarum infection, where vibriosis symptoms were obvious and non-obvious (HOSG and NOSG, respectively). We obtained 11,216,875, 12,313,404, and 11,398,695 clean reads per library, respectively. Bioinformatic analysis identified 452 miRNAs, including 24 putative novel miRNAs. We analyzed differentially expressed miRNAs between two libraries using pairwise comparison. For NOSG–CG, there was significant differential expression of 175 (38.72%) miRNAs. There was significant differential expression of 215 (47.57%) miRNAs between HOSG and CG. Compared with CG, The HOSG–NOSG comparison revealed significantly different expression of 122 (26.99%) miRNAs respectively. Real-time quantitative PCR (RT-qPCR) experiments were performed for 10 miRNAs of the three samples, and agreement was found between the sequencing and RT-qPCR data. For miRNAs that were significantly differentially expressed, functional annotation of target genes by Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that a set of miRNAs that were expressed highly abundantly and significantly differentially were might involved in immune system development and immune response. To our understanding, this is the first report of comprehensive identification of C. semilaevis miRNAs being differentially regulated in immune tissues (liver, head kidney, spleen, and intestine) in normal conditions relating to V. anguillarum infection. Many miRNAs were differentially regulated upon pathogen exposure. This work provides an opportunity for further understanding of the molecular mechanisms of miRNA regulation in C. semilaevis host–pathogen interactions.     

Comparison of 16SrDNA and toxR genes as targets for detection of Vibrio anguillarum in Dicentrarchus labrax kidney and liver

Vibrio anguillarum is a pathogen that causes high mortality in marine and freshwater fish. The aim of this study was to develop a real-time PCR assay for identification and quantification of V. anguillarum in fish tissue. The assay was carried out using two target genes, 16SrDNA and toxR, to evaluate the influence of differences in the operon copy number in quantitative assessment, both in pure cultures of V. anguillarum serovar O1 (strain 975/I), as a reference, and in the liver and kidney of a sea bass (Dicentrarchus labrax) specimen. Real-time PCR analysis showed high specificity for both target genes, with a detection limit of approximately 1-10 bacterial cells per reaction in pure culture and 10/100 V. anguillarum cells per reaction in fish tissue, which corresponds to 2 × 10²/2 × 10³ cells g⁻¹ fish tissue. Moreover, both genes showed high specificity but differing sensitivity due to the different operon copy number; as a result, it is possible to target the high copy number gene to improve sensitivity. Our results suggest that the protocol we tested can be used as a sensitive and specific molecular method for the detection of the fish pathogen V. anguillarum in fish tissue.     

Transcriptomic profiling revealed the signatures of intestinal barrier alteration and pathogen entry in turbot (Scophthalmus maximus) following Vibrio anguillarum challenge

The mucosal immune system serves as the frontline barriers of host defense against pathogen infection, especially for the fishes, which are living in the pathogen rich aquatic environment. The intestine constitutes the largest surface body area in constantly contact with the external pathogens, and plays a vital role in the immune defense against inflammation and pathogen infection. Previous studies have revealed that fish intestine might serves as the portal of entry for Vibrio anguillarum. To characterize the immune actors and their associated immune activities in turbot intestine barrier during bacterial infection, here we examined the gene expression profiles of turbot intestine at three time points following experimental infection with V. anguillarum utilizing RNA-seq technology. A total of 122 million reads were assembled into 183,101 contigs with an average length of 1151 bp and the N50 size of 2302 bp. Analysis of differential gene expression between control and infected samples at 1 h, 4 h, and 12 h revealed 2079 significantly expressed genes. Enrichment and pathway analysis of the differentially expressed genes showed the centrality of the pathogen attachment and recognition, antioxidant/apoptosis, mucus barrier modification and immune activation/inflammation in the pathogen entry and host immune responses. The present study reported the novel gene expression patterns in turbot mucosal immunity, which were overlooked in previous studies. Our results can help to understand the mechanisms of turbot host defense, and may also provide foundation to identify the biomarkers for future selection of disease-resistant broodstock and evaluation of disease prevention and treatment options.     

Differential expression of microRNAs in shrimp Marsupenaeus japonicus in response to Vibrio alginolyticus infection

Till date numerous microRNAs (miRNAs) have been discovered from various organisms, including mammals, plants, insects, nematodes and viruses. They are known to have antiviral functions in crustaceans such as shrimp Marsupenaeus japonicas. However, little is known about the role of miRNAs against bacterial infection in this shrimp caused by Vibrio alginolyticus. We performed small RNA sequencing to characterize the differentially expressed microRNAs in V. alginolyticus challenged shrimp, in comparison to that in control uninfected shrimp, at 24 h and 48 h. In total, 55 host miRNAs were differentially expressed in response to the infection and most of these were downregulated at both the time-points. TargetScan and miRanda algorithms showed that the target genes of these down-regulated miRNAs were related to innate immune functions such as production of phenoloxidase enzyme, apoptosis and phagocytosis. Further, gene ontology analysis revealed that many immune signaling pathways were mediated by these miRNAs. This study is one of the earliest attempts at characterizing shrimp miRNAs that respond to V. alginolyticus infection, and will help unravel the miRNA pathways involved in antibacterial action in shrimp.     

Zebrafish as a useful model for zoonotic Vibrio parahaemolyticus pathogenicity in fish and human

Vibrio parahaemolyticus is an important aquatic zoonotic pathogen worldwide that causes vibriosis in many marine fish, and sepsis, gastroenteritis and wound infection in humans. However, the pathogenesis of different sources of V. parahaemolyticus is not fully understood. Here, we examined the pathogenicity and histopathology of fish (V. parahaemolyticus 1.2164) and human (V. parahaemolyticus 17) strains in a zebrafish (Danio rerio). We found that different infection routes resulted in different mortality in zebrafish. Moreover, death due to V. parahaemolyticus 1.2164 infection occurred quicker than that caused by V. parahaemolyticus 17 infection. Hematoxylin-eosin staining of liver, kidney and intestine sections showed histological lesions in all three organs after infection with either strain. V. parahaemolyticus 1.2164 caused more severe damage than V. parahaemolyticus 17. In particular, V. parahaemolyticus 1.2164 treatment induced more serious hydropic degeneration and venous sinus necrosis in the liver than V. parahaemolyticus 17 treatment. The expression levels of three proinflammatory cytokines, interleukin 1β (il1β), interferon phi 1 (ifnϕ1) and tumor necrosis factor α (tnfα), as determined by quantitative real-time PCR, were upregulated in all examined tissues of infected fish. Notably, the peak levels of tnfα were significantly higher than those of il1β and ifnϕ1, suggesting, together with pathological results, that tnfα and il1β play an important role in acute sepsis. High amounts of tnfα may be related to acute liver necrosis, while ifnϕ1 may respond to V. parahaemolyticus and play an antibacterial role for chronically infected adult zebrafish. Taken together, our results suggest that the zebrafish model of V. parahaemolyticus infection is useful for studying strain differences in V. parahaemolyticus pathogenesis.     

Expression of IL-1Ra,IL-6, IL-8, IL-18, TNF-α and IFN-γ genes in peripheral blood leukocytes of rabbits infected with RHDV (Rabbit Haemorrhagic Disease Virus)

Rabbit haemorrhagic disease virus (RHDV) induces a highly contagious and extremely lethal disease that fulfils many requirements of an animal model of fulminant hepatic failure (FHF); however, the pathogenesis of RHD has still not been fully elucidated. Cytokines play an important role in regulation of the immune response and pathogenesis of many diseases, including those caused by viral infections. Furthermore, recent studies indicate a role of the immune response, especially peripheral blood leukocytes (PBL), in the pathogenesis of RHD. Thus, in the present study we investigated the expression of IL-1Ra, IL-6, IL-8, IL-18, TNF-α and IFN-γ genes in PBL of RHDV-infected rabbits. We also compared the expression of genes encoding these cytokines in rabbits with different course of RHDV infection (in animals that died 36 h post infection or survived even over 60 h after infection). The study revealed increased expression of genes encoding pro-inflammatory cytokines: IL-6, IL-8, TNF-α, IFN-γ in PBL of RHDV-infected rabbits. Moreover, the level of cytokine gene expression depended on the course of RHD. Hence, the results obtained indicate the potential role of these cytokines in RHDV infection and their influence on the survival time of infected rabbits.     

Identification and characteristic analysis of TLR28: A novel member of the TLR1 family in teleost

Toll-like receptors (TLRs) play a critical role in the innate immune response of fish to recognize microorganisms. Fish TLRs have significant variety and distinct features. This study focuses on a novel TLR member that belongs to the TLR1 family and was first discovered in miiuy croaker (designated as TLR28, mmiTLR28). In phylogenetic analysis, the mmiTLR28 clustered in the TLR1 family. Further characteristic analysis showed a high homology with TLR2 despite some differences between them. The predicted tertiary structure of mmiTLR28 possesses a hydrophobic pocket in the ectodomain region. Expression analysis showed the high expression level in the liver of miiuy croaker. Further functional experiments on the liver after Vibrio anguillarum, Staphylococcus aureus, lipopolysaccharides (LPS), and poly (I:C) stimulation showed significant upregulation; these results indicate the potential role of mmiTLR28 in immune response. For LPS stimulation in miiuy croaker leukocytes, mmiTLR28 also displayed significant upregulation. The discovery of mmiTLR28 will enrich the information on TLR family; the functional experiments have shown the role of mmiTLR28 in immunity. The results of this study lay the foundation for future research on fish immune systems.     

Dual oxidases participate in the regulation of intestinal microbiotic homeostasis in the kuruma shrimp Marsupenaeus japonicus

The metazoan gut lumen harbors numerous microbial communities. Tolerance for high bacterial counts and maintenance of microbiota homeostasis remain insufficiently studied. In this study, we identified a novel dual oxidase (MjDUOX2) involved in reactive oxygen species (ROS) production in the kuruma shrimp Marsupenaeus japonicus. MjDUOX2 is a transmembrane protein with an N-signal peptide region (19 aa) and a peroxidase homology domain (PHD, 554 aa) in the extracellular region; seven transmembrane regions; and three EF (calcium-binding region) domains (110 aa), a FAD-binding domain (104 aa), and a NAD-binding domain (156 aa) in the intracellular region. The novel MjDUOX2 exhibits a relatively low similarity (26.84% identity) to a previously reported DUOX in the shrimp (designated as MjDUOX1). The mRNA of MjDUOXs was widely distributed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Oral infection of the shrimp with pathogenic bacteria upregulated the mRNA expression of MjDUOXs and increased the ROS level in the intestine. However, High ROS level could inhibit the expression of MjDUOXs in shrimp after Vibrio anguillarum infection. Knockdown of MjDUOXs by RNA interference (RNAi) decreased the ROS level, increased the bacterial count in the intestine, and decreased the survival rate of the MjDUOX-RNAi shrimp infected with V. anguillarum. These results suggest that MjDUOXs play an important role for microbiota homeostasis in intestine of shrimp.     

A novel C-type lectin (FcLec4) facilitates the clearance of Vibrio anguillarum in vivo in Chinese white shrimp

C-type lectins play important roles in innate immunity of invertebrates. In the present study, we report a novel C-type lectin, named FcLec4, from the Chinese white shrimp Fenneropenaeus chinensis. FcLec4 contains a single carbohydrate recognition domain (CRD) with a putative signal peptide. Phylogenetic analysis indicated that FcLec4 was distant from most reported C-type lectins from shrimps. The expression of FcLec4 increased at both mRNA and protein level after stimulation of Vibrio anguillarum. Recombinant FcLec4 could agglutinate both Gram-positive and -negative bacteria in the presence of calcium. The recombinant protein could bind to peptidoglycan and selectively bind to microorganisms. Interestingly, the tight binding of recombinant FcLec4 to V. anguillarum might facilitate the subsequent clearance of the bacteria in vivo. To the best of our knowledge, this might be the first report that a C-type lectin was found to be directly involved in the anti-V. anguillarum response in shrimps.     

The modulation of extracellular superoxide dismutase in the specifically enhanced cellular immune response against secondary challenge of Vibrio splendidus in Pacific oyster (Crassostrea gigas)

Extracellular superoxide dismutase (EcSOD) is a copper-containing glycoprotein playing an important role in antioxidant defense of living cells exposed to oxidative stress, and also participating in microorganism internalization and cell adhesion in invertebrates. EcSOD from oyster (designated CgEcSOD) had been previously reported to bind lipopolysaccharides (LPS) and act as a bridge molecule in Vibrio splendidus internalization. Its mRNA expression pattern, PAMP binding spectrum and microorganism binding capability were examined in the present study. The mRNA expression of CgEcSOD in hemocytes was significantly up-regulated at the initial phase and decreased sharply at 48 h post V. splendidus stimulation. The recombinant CgEcSOD protein (rCgEcSOD) could bind LPS, PGN and poly (I:C), as well as various microorganisms including Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Vibrio anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica at the presence of divalent metal ions Cu²⁺. After the secondary V. splendidus stimulation, the mRNA and protein of CgEcSOD were both down-regulated significantly. The results collectively indicated that CgEcSOD could not only function in the immune recognition, but also might contribute to the immune priming of oyster by inhibiting the foreign microbe invasion through a specific down-regulation.