Xu-Jie Zhang

Distinctive Structural Hallmarks and Biological Activities of the Multiple Cathelicidin Antimicrobial Peptides in a Primitive Teleost Fish

Cathelicidin antimicrobial peptides (CAMPs) represent a crucial component of the innate immune system in vertebrates. Although widely studied in mammals, little is known about the structure and function of fish CAMPs. Further to the previous findings, two more cathelicidin genes and multiple transcripts from rainbow trout were identified in the present study. Interestingly, we found that trout have evolved energy-saving forms of cathelicidins with the total deletion of the characteristic cathelin-like domain. Sequence analysis revealed that salmonid CAMPs have formed a special class of antimicrobial peptides in vertebrates with three distinctive hallmarks: the N terminus is intensified by positive charges, the central region consists of repetitive motifs based on RPGGGS, and the C terminus is lowly charged. Immunofluorescence localization of trout CAMPs demonstrated that these peptides expressed mainly at the mucosal layer of gut. Meanwhile, signals around sinusoids were also detected in head kidney. Moreover, the biological activities of trout CAMPs were proved to be mediated by the N terminus. Additionally, the repetitive motifs characteristically existing in Salmonidae increased the structural flexibilities of peptides and further increased the antibacterial and IL-8–stimulating activities. Unlike most α helical and cytotoxic mammalian CAMPs, trout CAMPs, mainly consisting of β-sheet and random coil, exhibited no cytotoxic activities. The distinctive structural features of trout CAMPs provide new insights into the understanding of the evolution of CAMPs in vertebrates. Moreover, the high bacterial membrane selectivity of trout CAMPs will help to design excellent peptide antibiotics.

Spring Viremia of Carp Virus N Protein Suppresses Fish IFNφ1 Production by Targeting the Mitochondrial Antiviral Signaling Protein

For a virus to replicate efficiently, it must try and inhibit host IFN expression because IFN is an important host defense at early stages after viral infection. For aquatic viruses, the mechanisms used to escape the hosts IFN system are still unclear. In this study, we show that the N protein of spring viremia of carp virus (SVCV) inhibits zebrafish IFNφ1 production by degrading the mitochondrial antiviral signaling protein (MAVS). First, the upregulation of IFNφ1 promoter activity stimulated by polyinosinic:polycytidylic acid, retinoic acid–inducible gene I (RIG-I) or MAVS was suppressed by the SVCV infection. However, the upregulation by the downstream factor of the RIG-I–like receptor signaling pathway, TANK-binding kinase 1, was not affected. Notably, at the protein level, MAVS decreased remarkably when cells were infected with SVCV. Second, consistent with the result of the SVCV infection, overexpression of the N protein of SVCV blocked the IFNφ1 transcription activated by MAVS and downregulated MAVS expression at the protein level but not at the mRNA level. Further analysis demonstrated that the N protein targeted MAVS for K48-linked ubiquitination, which promoted the degradation of MAVS. These data indicated that fish MAVS could be degraded by the N protein of SVCV through the ubiquitin-proteasome pathway. To our knowledge, this is the first article of a fish RIG-I–like receptor pathway interfered by an aquatic virus in an ubiquitin-proteasome manner, suggesting that immune evasion of a virus also exists in lower vertebrates.

Fish gut-liver immunity during homeostasis or inflammation revealed by integrative transcriptome and proteome studies

The gut-associated lymphoid tissue, connected with liver via bile and blood, constructs a local immune environment of both defense and tolerance. The gut-liver immunity has been well-studied in mammals, yet in fish remains largely unknown, even though enteritis as well as liver and gallbladder syndrome emerged as a limitation in aquaculture. In this study, we performed integrative bioinformatic analysis for both transcriptomic (gut and liver) and proteomic (intestinal mucus and bile) data, in both healthy and infected tilapias. We found more categories of immune transcripts in gut than liver, as well as more adaptive immune in gut meanwhile more innate in liver. Interestingly reduced differential immune transcripts between gut and liver upon inflammation were also revealed. In addition, more immune proteins in bile than intestinal mucus were identified. And bile probably providing immune effectors to intestinal mucus upon inflammation was deduced. Specifically, many key immune transcripts in gut or liver as well as key immune proteins in mucus or bile were demonstrated. Accordingly, we proposed a hypothesized profile of fish gut-liver immunity, during either homeostasis or inflammation. Current data suggested that fish gut and liver may collaborate immunologically while keep homeostasis using own strategies, including potential unique mechanisms.

Alterations of the gut microbiome of largemouth bronze gudgeon (Coreius guichenoti) suffering from furunculosis.

High-throughput sequencing was applied to compare the intestinal microbiota in largemouth bronze gudgeon either healthy or affected by furunculosis. Proteobacteria, Actinobacteria, Tenericutes, Firmicutes and Bacteroidetes were detected as the predominant bacterial phyla in the gut of both diseased and healthy fish. The abundance of Proteobacteria differed significantly between the two groups of fish, mainly due to the overwhelming prevalence of Aeromonas in the diseased fish (81% ± 17%), while the genus was unevenly spread among the apparently healthy fish (33% ± 33%). The bacterial diversity in the intestine of diseased fish was markedly lower than in healthy fish. Analysis revealed the significant dissimilarity between the gut microbiota of diseased and healthy fish. The bacterial profiles in the gut were further characterized with the 28 phylotypes that were shared by the two groups. In diseased fish, two shared OTUs (OTU0001 and OTU0013) were closely related to Aeromonas salmonicida, their total proportion exceeding 70% of the sequences in diseased fish, while averaging 5.2% ± 4.6% in the healthy fish. This result suggested the presence of healthy carriers of pathogenic A. salmonicida among the farmed fish, and the gut appeared as a probable infection source for furunculosis in largemouth bronze gudgeon.

Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed

Bacillus subtilis is widely used as probiotic species in aquaculture for water quality control, growth promoting, or immunity enhancing. The aim of this study is to find novel B. subtilis strains from fish as potential probiotics for aquaculture. Eleven B. subtilis isolates derived from the intestinal tract of grass carp were identified by gene sequencing and biochemical tests. These isolates were classified into 4 groups, and the representatives (GC-5, GC-6, GC-21 and GC-22) of each group were further investigated for antibiotic susceptibility, sporulation rate, biofilm formation, activity against pathogenic bacteria, resistance to stress conditions of intestinal tract (high percentage of bile and low pH) and high temperature, which are important for probiotics to be used as feed additives. Additionally, the adhesion properties of the 4 characterized strains were assessed using Caco-2 cell and gut mucus models. The results showed that the 4 strains differed in their capacities to adhere to intestinal epithelial cells and mucus. Furthermore, the strains GC-21 and GC-22 up-regulated the expression levels of IL-10 and TGF-β but down-regulated IL-1β, suggesting their potential anti-inflammatory abilities. Based on physiological properties of the 4 characterized B. subtilis strains, one or more strains may have potential to be used as probiotics in aquaculture.

B Cell Functions Can Be Modulated by Antimicrobial Peptides in Rainbow Trout Oncorhynchus mykiss: Novel Insights into the Innate Nature of B Cells in Fish

B cells in fish were recently proven to have potent innate immune activities like macrophages. This inspired us to further explore the innate nature of B cells in fish. Moreover, antimicrobial peptides (AMPs) are representative molecules of innate immunity, and they can modulate the functions of macrophages. These make fish an appropriate model to study the interactions between B cells and AMPs. Interestingly, the results in this study revealed that the IgM+ and IgT+ B cells of rainbow trout could express multiple AMP genes, including four cathelicidin genes and one β-defensin gene. The expression levels of the cathelicidin genes were obviously higher than that of the β-defensin gene. Further studies revealed that intracellular, extracellular, in vitro, and in vivo stimulations could significantly increase the expression of the cathelicidin genes in trout IgM+ and IgT+ B cells but not the expression of the β-defensin gene, indicating that cathelicidin peptides are the main innate immune effectors of trout B cells. More interestingly, we found that cathelicidin peptides could significantly enhance the phagocytic, intracellular bactericidal, and reactive oxygen species activities of trout IgM+ and IgT+ B cells, a phenomenon previously reported only in macrophages, and these activities might also be mediated by the P2X7 receptor. These results collectively suggest that B cells play multiple roles in the innate immunity of fish, and they provide new evidence for understanding the close relationship between B cells and macrophages in vertebrates.

Molecular characterization and expression analysis of three subclasses of IgT in rainbow trout (Oncorhynchus mykiss).

ABSTRACT As the teleost specific immunoglobulin, IgT plays important roles in systemic and mucosal immunity. In the current study, in rainbow trout, we have cloned the heavy chain (Ig&tgr;) genes of a secretory form of IgT2 as well as the membrane and secretory forms of a third IgT subclass, termed IgT3. Conserved cysteine and tryptophan residues that are crucial for the folding of the immunoglobulin domain as well as hydrophobic and hydrophilic residues within CART motif were identified in all IgT subclasses. Through analysis of the rainbow trout genome assembly, Ig&tgr;3 gene was found localized upstream of Ig&tgr;1 gene, while Ig&tgr;2 gene situated on another scaffold. At the transcriptional level, Ig&tgr;1 was mainly expressed in both systemic and mucosal lymphoid tissues, while Ig&tgr;2 was largely expressed in systemic lymphoid organs. After LPS and poly (I:C) treatment, Ig&tgr;1 and Ig&tgr;2 genes exhibited different expression profiles. Interestingly the transcriptional level of Ig&tgr;3 was negligible, although its protein product could be identified in trout serum. Importantly, a previously reported monoclonal antibody directed against trout IgT1 was able to recognize IgT2 and IgT3. These data demonstrate that there exist three subclasses of IgT in rainbow trout, and that their heavy chain genes display different expression patterns during stimulation. Overall, our data reflect the diversity and complexity of immunoglobulin in trout, thus provide a better understanding of the IgT system in the immune response of teleost fish. HIGHLIGHTSThe heavy chain gene of a third IgT subclass (Ig&tgr;3) is characterized in rainbow trout.Trout Ig&tgr; genes are located probably at two Ig loci.Trout Ig&tgr; genes respond differentially to stimulants.The anti‐trout IgT1 mAb recognizes all of the three IgT subclasses.

Multi-Omics Analysis Reveals a Correlation between the Host Phylogeny, Gut Microbiota and Metabolite Profiles in Cyprinid Fishes

Gut microbiota play key roles in host nutrition and metabolism. However, little is known about the relationship between host genetics, gut microbiota and metabolic profiles. Here, we used high-throughput sequencing and gas chromatography/mass spectrometry approaches to characterize the microbiota composition and the metabolite profiles in the gut of five cyprinid fish species with three different feeding habits raised under identical husbandry conditions. Our results showed that host species and feeding habits significantly affect not only gut microbiota composition but also metabolite profiles (ANOSIM, p ≤ 0.05). Mantel test demonstrated that host phylogeny, gut microbiota, and metabolite profiles were significantly related to each other (p ≤ 0.05). Additionally, the carps with the same feeding habits had more similarity in gut microbiota composition and metabolite profiles. Various metabolites were correlated positively with bacterial taxa involved in food degradation. Our results shed new light on the microbiome and metabolite profiles in the gut content of cyprinid fishes, and highlighted the correlations between host genotype, fish gut microbiome and putative functions, and gut metabolite profiles.

Preferential combination between the light and heavy chain isotypes of fish immunoglobulins

Immunoglobulin light chain (IgL) is necessary for the assembly of an Ig molecule, which plays important roles in the immune response. IgL genes were identified in various teleost species, but the basic functions of different IgL isotypes and the preferential combination between IgL and IgH (Ig heavy chain) isotypes remain unclear. In the current study, by EST database searching and cDNA cloning in rainbow trout, 8 IgL sequences were obtained, which could be classified into the IgLκF, IgLκG, IgLσ and IgLλ isotypes, respectively. Trout IgL isotypes were highly expressed in the immune-related tissues, and participated in the immune responses in spleen and gut by stimulation with LPS and poly (I:C). The results of FACS and LC-MS/MS indicated that the IgLκG and IgLσ isotypes preferentially bonded with the heavy chains of IgM and IgT, respectively, in trout B cells and serum. In addition, the genomic organization of trout IgL isotypes and the utilization of recombination signal sequences were studied.

Identification of another primordial CD80/86 molecule in rainbow trout: Insights into the origin and evolution of CD80 and CD86 in vertebrates

Abstract In mammals, the binding of distinct costimulatory ligands CD80 and CD86 to their receptors is essential for optimal T cell activation. Previous studies have shown that only a single CD80/86 gene exists in rainbow trout (termed rtCD80/86A) and other teleost fish, suggesting that CD80 and CD86 arose by gene duplication in the tetrapod branch, after the separation of fish and tetrapods. However, in this study, another CD80/86 gene has been cloned from rainbow trout, termed rtCD80/86B. The sequence identity between trout CD80/86 is significantly higher than that between CD80 and CD86 in tetrapods, indicating that CD80 and CD86 underwent divergent evolution in vertebrates, especially in tetrapods. Gene synteny analyses showed that the CD80 and CD86 genes are closely located in the same chromosome in tetrapods. However, CD80/86 genes are located in two distinct chromosomes in rainbow trout and Atlantic salmon, suggesting that salmonid CD80/86 genes arose by the salmonid‐specific whole‐genome duplication (WGD) event. Expression analysis showed that rtCD80/86A was more abundant and inducible than rtCD80/86B in various tissues, indicating the important role of rtCD80/86A in trout immunity. Interestingly, we found that head kidney B cells showed higher expression level of rtCD80/86A and rtCD80/86B when compared with the other leukocytes, suggesting a potential role for trout B cells as antigen‐presenting cells (APCs). HighlightsA second CD80/86 gene was identified in rainbow trout.The origin and evolution relationships of trout CD80/86 genes were analyzed.Trout CD80/86 genes were highly expressed in B cells.Two CD80/86 genes also exist in Atlantic salmon.