Nuclear factor 45 of tongue sole (Cynoglossus semilaevis): Evidence for functional differentiation between two isoforms in immune defense against viral and bacterial pathogens

Nuclear factor 45 (NF45) is known to play an important role in regulating interleukin-2 expression in mammals. The function of fish NF45 is largely unknown. In a previous study, we reported the identification of a NF45 (named CsNF45) from half smooth tongue sole (Cynoglossus semilaevis). In the present study, we identified an isoform of CsNF45 (named CsNF45i) from half smooth tongue sole and examined its biological properties in comparison with CsNF45. We found that CsNF45i is a truncated version of CsNF45 and lacks the N-terminal 38 residues of CsNF45. Genetic analysis showed that the CsNF45 gene consists of 14 exons and 13 introns, and that CsNF45 and CsNF45i are the products of alternative splicing. Constitutive expression of CsNF45 and CsNF45i occurred in multiple tissues but differed in patterns. Experimental infection with viral and bacterial pathogens upregulated the expression of both isoforms but to different degrees, with potent induction of CsNF45 being induced by bacterial pathogen, while dramatic induction of CsNF45i being induced by viral pathogen. Transient transfection analysis showed that both isoforms were localized in the nucleus and able to stimulate the activity of IL-2 promoter to comparable extents. To examine their in vivo effects, the two isoforms were overexpressed in tongue sole. Subsequent analysis showed that following viral and bacterial infection, the viral loads in CsNF45i-overexpressing fish were significantly lower than those in CsNF45-overexpressing fish, whereas the bacterial loads in CsNF45-overexpressing fish were significantly lower than those in CsNF45i-overexpressing fish. These results indicate that both CsNF45 and CsNF45i possess immunoregulatory properties, however, the two isoforms most likely participate in different aspects of host immune defense that target different pathogens.     

Construction and characterization of an auxotrophic ctxA mutant of O139 Vibrio cholerae

Cholera caused by the O139 serogroup still remains a public health concern in certain regions of the world and the existing O1 vaccines do not cross-protect cholera caused by this serogroup. An aminolevulinic acid (ALA) auxotroph vaccine candidate against the O139 serogroup, designated as VCUSM2, was recently developed. It was found to be immunogenic in animal model studies but showed mild reactogenic effects due to the presence of two intact copies of Vibrio cholerae toxin (CTX) genetic element. In the present study we have modified the ctx operon by systematic allelic replacement methodology to produce a mutant strain, designated as VCUSM14. This strain has two copies of chromosomally integrated and mutated ctxA gene, encoding immunogenic but not toxic cholera toxin A subunit (CT-A). The amino acids arginine and glutamic acid at position 7th and 112th, respectively, in CT-A of VCUSM14 were substituted with lysine (R7K) and glutamine (E112Q), respectively. Two copies of the ace and zot genes present in the ctx operon were also deleted. Cholera toxin-ELISA using GM1 ganglioside showed that the both wild type CT and mutated CT were recognized by anti-CT polyclonal antibodies. VCUSM14 produced comparatively less amount of antigenic cholera toxin when compared to the VCUSM2 and Bengal wild type strain. VCUSM14 did not elicit fluid accumulation when inoculated into rabbit ileal loops at doses of 10⁶ and 10⁸ CFU. The colonization efficiency of VCUSM14 was one log lower than the parent strain, VCUSM2, which can be attributed to the ALA auxotrophy and less invasive properties of VCUSM14. VCUSM14, thus a non-reactogenic auxotrophic vaccine candidate against infection by O139 V. cholerae.     

Proteomic analysis of Vibrio vulnificus M2799 grown under iron-repleted and iron-depleted conditions

Vibrio vulnificus is an opportunistic marine bacterium that causes a serious, often fatal, infection in human. An important factor that determines the survival of V. vulnificus in the human body is the ability to acquire iron. The differential expression of proteins in whole-cell lysates of V. vulnificus M2799, a clinical isolate, was evaluated under iron-repleted and iron-depleted conditions during the early, mid and late logarithmic growth phases. A total of 32, 53 and 42 iron-regulated spots were detected by two-dimensional differential gel electrophoresis (2D-DIGE) in the early, mid and late logarithmic growth phases, respectively. Of these, 18 (early logarithmic growth phase), 31 (mid logarithmic growth phase) and 26 (late logarithmic growth phase) proteins were subsequently identified by matrix-assisted laser desorption/ionization-time of flight analysis. These proteins were classified into 10 functional categories, including inorganic ion transport and metabolism, carbohydrate transport and metabolism, and amino acid transport and metabolism. Based on this classification, the expression of proteins involved in the iron acquisition system increased from the early to the mid logarithmic growth phases, while that of proteins involved in other metabolic pathways increased from the mid to the late logarithmic growth phases. Furthermore, when the protein expression profile of the wild type bacterium was compared with that of the fur mutant grown under the iron-repleted condition, the expression of 18 proteins was found to be regulated by iron and Fur.     

Evaluation of molecular methods to discriminate the closely related species Vibrio fluvialis and Vibrio furnissii

Vibrio furnissii and Vibrio fluvialis are two closely related species which are regarded as emerging human pathogens. Human infections have been mainly associated with consumption of seafood or drinking of contaminated water. V. furnissii strains can be distinguished from V. fluvialis by their ability to produce gas from fermentation of carbohydrates. In this study, we compare two phenotypic (biochemical testing and matrix-assisted laser desorption/ionisation time of flight mass spectrometry, MALDI–TOF MS) and three genotypic techniques (rpoB sequencing, conventional PCR and real-time PCR) for determination of the two species. The methods were evaluated on a collection of 42 V. furnissii and 32 V. fluvialis strains, which were isolated from marine environments and from animals intended for food production. Four of the applied methods allowed the unambiguous discrimination of the two species, while the biochemical testing was the least reliable technique, due to a high variation in the phenotype of gas production from carbohydrates. In view of the One Health concept reliable diagnostic techniques are a prerequisite for preventive public health measurements, as pathogens isolated from animals can cross species borders and methods for detection of sources, reservoirs and ways of transmission of pathogenic bacteria are indispensable for the prevention of infectious diseases in humans and animals.     

Construction and characterization of rtxA and rtxC mutants of auxotrophic O139 Vibrio cholerae

Vibrio cholerae is a Gram-negative bacterium that causes diarrheal disease. V. cholerae O1 and O139 serogroups are toxigenic and are known to cause epidemic cholera. These serogroups produce cholera toxin and other accessory toxins such as accessory cholera enterotoxin, zonula occludens toxin, and multifunctional, autoprocessing repeat in toxin (MARTX). In the present study, we incorporated mutated rtxA and rtxC genes that encode MARTX toxin into the existing aminolevulinic acid (ALA) auxotrophic vaccine candidate VCUSM2 of V. cholerae O139 serogroup. The rtxC mutant was named VCUSM9 and the rtxC/rtxA mutant was named VCUSM10. VCUSM9 and VCUSM10 were able to colonize intestinal cells well, compared with the parent vaccine strain, and produced no fluid accumulation in a rabbit ileal loop model. Cell rounding and western blotting assays indicated that mutation of the rtxC gene alone (VCUSM9 strain) did not abolish MARTX toxicity. However mutation of both the rtxA and rtxC genes (VCUSM10) completely abolished MARTX toxicity. Thus we have produced a new, less reactogenic, auxotrophic rtxC/rtxA mutated vaccine candidate against O139 V. cholerae.     

Biology and host response to Cyprinid herpesvirus 3 infection in common carp

Viruses from the family Alloherpesviridae form an aquatic clade of herpesviruses infecting fish and amphibia. Diseases caused by these herpesviruses are of increasing importance because of the high morbidity and mortality associated with the infection, and the difficulties in diagnosing latently infected carriers. Cyprinid herpesvirus 3 (CyHV-3) induces a severe disease and mortality in common carp and thus greatly affects carp aquaculture and trade. This review summarises advancements in the understanding of the infection process and the current knowledge on immune responses of carp to CyHV-3. A focus is laid on host genetics and immunity responsible for resistance/survival from the disease and on the viral mechanisms accountable for evasion of carp immune responses. As current knowledge of immune responses to CyHV-3 is still limited, perspectives for future studies are outlined. Analysing CyHV-3 fish-host interactions will be useful and thought-provoking for a basic understanding of fish immune responses.     

First characterization of a teleost Epstein-Barr virus-induced gene 3 (EBI3) reveals a regulatory effect of EBI3 on the innate immune response of peripheral blood leukocytes

Epstein-Barr virus-induced gene 3 (EBI3) encodes a protein that in mammals is known to be a subunit of interleukin (IL)-27 and IL-35, both which regulate cytokine production and inflammatory response. To date, no studies on fish EBI3 have been documented. In this work, we report the identification of an EBI3 homologue, CsEBI3, from tongue sole (Cynoglossus semilaevis) and analysis of its expression and biological effect. CsEBI3 is composed of 245 amino acid residues and possesses a Fibronectin type 3 (FN3) domain that is preserved in lower and higher vertebrates. Expression of CsEBI3 was detected in a wide range of tissues, in particular those of immune relevant organs, and upregulated in a time-dependent manner by experimental challenge with bacterial and viral pathogens. Bacterial infection of peripheral blood leukocytes (PBL) enhanced CsEBI3 expression and caused extracellular secretion of CsEBI3. Purified recombinant CsEBI3 (rCsEBI3) stimulated the respiratory burst activity of PBL and upregulated the expression of IL-1β, IL-8, Myd88, interferon-induced gene 15, CD28, and chemokines. In contrast, rCsEBI3M, a mutant CsEBI3 that lacks the FN3 domain failed to activate PBL and induced much weaker expression of the immune genes. Treatment of PBL with rCsEBI3, but not with the mutant rCsEBI3M, enhanced cellular resistance against bacterial invasion, whereas antibody blocking of CsEBI3 on PBL significantly reduced cellular resistance against bacterial infection. Taken together, these results indicate for the first time that a teleost EBI3 possesses immunoregulatory property in a manner that is dependent on the conserved FN3 domain, and that CsEBI3 is involved in the innate immune defense of PBL against microbial pathogens.     

Flagellin from Marinobacter algicola and Vibrio vulnificus activates the innate immune response of gilthead seabream

Adjuvants have emerged as the best tools to enhance the efficacy of vaccination. However, the traditional adjuvants used in aquaculture may cause adverse alterations in fish making necessary the development of new adjuvants able to stimulate the immune system and offer strong protection against infectious pathogens with minimal undesirable effects. In this respect, flagellin seems an attractive candidate due to its ability to strongly stimulate the immune response of fish. In the present study, we have evaluated the ability of recombinant flagellin from Marinobacter algicola (MA) and Vibrio vulnificus (Vvul), a non-pathogenic and a pathogenic bacteria, respectively, to stimulate the innate immune system of gilthead seabream (Sparus aurata L.) and compare the effect with that of the classical flagellin from Salmonella enterica serovar Typhimurium (Salmonella Typhimurium, STF). Intraperitoneal injection of MA and Vvul resulted in a strong inflammatory response characterized by increased reactive oxygen species production and the infiltration of acidophilic granulocytes at the injection site. Interestingly, however, only flagellin from MA consistently induced the expression of the gene encoding pro-inflammatory interleukin-1β. These effects were further confirmed in vitro, where a dose-dependent activation of macrophages and acidophilic granulocytes by MA and Vvul flagellins was observed. In contrast, STF flagellin was found to be less potent in both in vivo and in vitro experiments. Our results suggest the potential use of MA and Vvul flagellins as immunostimulants and adjuvants for fish vaccination.     

Regulation of the Vibrio vulnificus vvpE expression by cyclic AMP-receptor protein and quorum-sensing regulator SmcR

In Vibrio vulnificus, cAMP-receptor protein (CRP) and the quorum-sensing regulator SmcR are simultaneously and cooperatively required for the metalloprotease vvpE gene expression, rather than sequentially in a regulatory cascade. However, this study shows a new temporal and functional sequence between the two factors in regulating vvpE expression. A crp mutation inhibited vvpE expression with growth impairment from early stage. In contrast, a smcR mutation inhibited vvpE expression only at the late stage with no effect on growth. A crp–smcR double mutation severely inhibited vvpE expression with growth impairment from early stage. The inhibited vvpE expression was restored only at the early stage by a crp single complementation, but not at all by a smcR complementation. These results indicate that CRP functions as an essential activator, whereas SmcR functions in the presence of CRP for full vvpE expression.     

Study on the immune response to recombinant Hsp70 protein from Megalobrama amblycephala

The expression of heat shock protein 70 (Hsp70) is induced in response to many factors including high temperature, infection, metal pollutants and toxic chemicals. In this study, Megalobrama amblycephala HSP70 promoter was cloned, and characteristic heat shock elements (HSEs) were identified in the promoter region. The recombinant M. amblycephala Hsp70 protein (rMaHsp70) was expressed and purified from Escherichia coli BL21 (DE3). To evaluate in vivo immune response of rMaHsp70, we administered intraperitoneal (IP) injection, and demonstrated that rMaHsp70 stimulated M. amblycephala immune activity by inducing the expression of HSP70, HIF-1α, HSC70, CXCR4b, TNF-α and IL-1β mRNAs in liver, headkidney, spleen and gill, as well as SOD, glutathione, lysozyme and interferon alpha proteins in serum and liver. The effect of rMaHsp70 as adjuvant against Aeromonas hydrophila was assessed by injecting a mixed vaccine of rMaHsp70 and A. hydrophila (A. hydrophila/Hsp70) into M. amblycephala, and the relative percent survival (RPS) in the A. hydrophila/Hsp70 group was 75% compared to 50% in the A. hydrophila/PBS group. Furthermore, rMaHsp70 also promoted the proliferation and suppressed apoptosis in M. amblycephala fin cells (MAF) in a dose-dependent manner. Taken together, these results suggest that rMaHsp70 can induce organic immune response and improve environmental tolerance.     

f57f4.4p::gfp as a fluorescent reporter for analysis of the C. elegans response to bacterial infection

Host defense mechanisms are multi-layered and involve constitutive as well as inducible components. The dissection of these complex processes can be greatly facilitated using a reporter gene strategy with a transparent animal. In this study, we use Caenorhabditis elegans as a model host and introduce a new pathogen-inducible fluorescent reporter involving the promoter of f57f4.4, a gene encoding a putative component of the glycocalyx. We show that this reporter construct does not respond to heavy metal or hypertonic environments, but is specifically and locally induced in the intestine upon Photorhabus luminescens and Pseudomonas aeruginosa infections. We further demonstrate that its upregulation requires live pathogens as well as elements of the nematode p38 MAP kinase and TGF-beta pathways. In addition to introducing a new tool for the study of the interactions between C. elegans and a pathogen, our results suggest a role for the glycocalyx in gut immunity.     

Intracellular Copper Zinc Superoxide dismutase (icCuZnSOD) from Asian seabass (Lates calcarifer): molecular cloning, characterization and gene expression with reference to Vibrio anguillarum infection

Copper Zinc Superoxide dismutase (CuZnSOD) is the family of most important antioxidant metalloenzymes that protects tissues from damage by reactive oxygen species (ROS). In the present study, the intracellular copper zinc SOD from the Asian seabass Lates calcarifer (Lc-icCuZnSOD) was identified by RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) technique. The full-length cDNA of Lc-icCuZnSOD consisted of 809 nucleotides with an open-reading frame of 465 bp encoding 154 amino acids and N-Glycosylation site (NVTA) within. The predicted molecular mass of the protein is 15.84 kDa with an estimated pI of 5.52. The deduced amino acid sequence of Lc-icCuZnSOD shared high degree of homology with known CuZnSODs from other species. CuZn binding sites (H47, H49, H64, and H121 for Cu²⁺ and H72, H81, and ASP84 for Zn²⁺), two cysteine residues (aa 58 and 147) that form a disulfide bond, and CuZnSOD family signature sequences (GFHVHAFGDNT, aa 45-55 and GNAGGRLACGVI, aa 139-150) were highly conserved among fish species. Temporal and tissue specific expression of Lc-icCuZnSOD was significantly differentially altered in Asian seabass challenged with Vibrio anguillarum indicating possible role in antioxidant activities involved in the innate immune defense mechanisms.     

Identification and analysis of an intracellular Cu/Zn superoxide dismutase from Sepiella maindroni under stress of Vibrio harveyi and Cd

Superoxide dismutases (SODs) are ubiquitous family of metalloenzymes involved in protecting organisms from excess reactive oxygen species damage. In this paper, a novel intracellular Cu/ZnSOD from Sepiella maindroni (designated as SmSOD) was identified and characterized. The full-length cDNA sequence of SmSOD (GenBank accession No. KF908850) was 709 bp containing an open reading frame (ORF) of 459 bp, encoding 153 amino acid residues peptide with predicted pI/MW (6.02/15.75 kDa), a 131 bp-5′- and 116 bp-3′- untranslated region (UTR). BLASTn analysis and phylogenetic relationship strongly suggested that the sequence shared high similarity with known Cu/Zn SODs. Several highly conserved motifs, including two typical Cu/Zn SOD family domains, two conserved Cu-/Zn-binding sites (H-47, H-49, H-64, H-120 for Cu binding, and H-64, H-72, H-81, D-84 for Zn binding) and intracellular disulfide bond (C-58 and C-146), were also identified in SmSOD. Time-dependent mRNA expression of SmSOD in hepatopancreas was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd²⁺ exposure. The results indicated that SmSOD was an acute-phase protein involved in the immune responses against pathogens and biological indicator for metal contaminants in aquatic environment.     

Identification and functional characterization of an Rbx1 in an invertebrate Haliotis diversicolor supertexta

Rbx1 (RING box1) is an evolutionarily conserved RING-H2 finger protein and belongs to the RING-finger family of Ubiquitin ligase E3, which determines the substrate specificity of ubiquitination and regulates a variety of biological processes. We report here the identification and functional characterization of an Rbx1 homologue in abalone, which we named ab-Rbx1. Ab-Rbx1 contains conserved cysteine/histidine residues which are the characteristics of Rbx proteins. Phylogenetic tree analysis further demonstrated that ab-Rbx1 belongs to the Rbx1 family other than Rbx2 family. Real-time PCR analysis revealed that ab-Rbx1 was ubiquitously expressed in all examined tissues of abalone and the expression level of ab-Rbx1 was significantly induced by mitogenic situation. Immunohistochemical and immunofluorescent staining showed that the ab-Rbx1 was expressed predominantly in epithelial cells and localized both in the cytoplasmic and nuclear compartment. Ubiquitination assay demonstrated that ab-Rbx1 had ubiquitin ligase activity and could auto-ubiquitinated itself. These results suggest that ab-Rbx1 is an Rbx1 homologue and may be indirectly involved in the immune response of abalone through ubiquitination.     

Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.     

怀有先天性心脏病胎儿的孕妇与正常孕妇肠道菌群的高通量测序分析

目的:分析怀有先天性心脏病胎儿的孕妇(PW组)与正常孕妇(NW组)的肠道菌群特征。方法:对可能引起个体肠道菌群差异的因素加以控制,2013年6月18号~2013年12月17号收集15例正常孕妇和17例异常孕妇的粪便样本,提取细菌基因组DNA,用PCR方法扩增16S r DNA,进行二代Illumina测序。测序数据应用运算分类单位(operational taxonomic unit,OTU)聚类分析、多样性分析和分类学分析等生物信息技术分析2类孕妇肠道菌群的结构、多样性和丰度。结果:2类孕妇肠道菌群的高通量测序分析分别得到2 696 276(NW组)条和2 445 530(PW组)条优化序列,测序覆盖深度97%。经过97%相似度归并后分别得到77 243个(NW组)和75 600个OTUs(PW组);菌群多样性分析显示正常孕妇肠道菌群的Chao 1指数和Shannon指数均高于异常孕妇。在最佳分类水平上,正常孕妇肠道菌群由38个科的细菌构成,归属于14个门,以厚壁菌门(Firmicutes)为主,其次为变形菌门(Proteobacteria)、放线菌门(Actinobacteria)等。怀有先心胎儿孕妇肠道菌群构成与正常孕妇相似,最佳分类水平上由33个科的细菌构成,归属于9个门,其中Firmicutes为(75.7±15.8)%,Proteobacteria为(21.1±17.0)%,Actinobacteria为(2.0±2.1)%。在最佳分类水平上,方差分析显示两者的双歧杆菌科(Bifidobacteriaceae)和红蝽杆菌科(Coriobacteriaceae)菌种丰度有明显差异。结论:PW组孕妇与NW组孕妇相比,整体肠道菌群含量明显下降,提示PW组孕妇有更恶劣的肠道环境,其中部分菌群含量存在明显差异。先天性心脏病胎儿的发生可能与孕妇恶劣的肠道微环境有关,其中Bifidobacteriaceae和Coriobacteriaceae在先天性心脏病的预防方面可能有重要作用。