Phosvitin plays a critical role in the immunity of zebrafish embryos via acting as a pattern recognition receptor and an antimicrobial effector

How fish embryos that develop externally survive microbial attacks is poorly understood. Here, we clearly demonstrated that the embryo extract of zebrafish and its early embryo both displayed antimicrobial activity against microbes, including pathogenic Aeromonas hydrophila, and phosvitin (Pv), a nutritional protein abundant in eggs, was related to this antimicrobial activity. We also showed that recombinant Pv (rPv) acted as a pattern recognition receptor capable of recognizing the microbial signature molecules LPS, lipoteichoic acid, and peptidoglycan, as well as binding the Gram-negative and -positive microbes Escherichia coli, A. hydrophila, and Staphylococcus aureus and functioned as an antimicrobial agent capable of killing the microbes. Furthermore, we revealed that its C-terminal 55 residues (Pt5) with the functional sites Arg(242) and Ala(201)/Ile(203) were indispensable for Pv antimicrobial activity. Importantly, microinjection of rPv or Pt5 into early embryos significantly enhanced their resistance to A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-Pv antibody plus rPv (or Pt5) but not by injection of anti-actin antibody plus rPv. Moreover, the generated mutants with in vitro antimicrobial activity, when injected into the embryos, could also promote their resistance to A. hydrophila, but those without in vitro antimicrobial activity could not. It is thus proposed that Pv participates in the protection of early embryos against pathogenic attacks via binding and disrupting potential pathogens. This work also opens a new way for the study of the immunological roles of yolk proteins in oviparous animals that rely on yolk proteins for embryonic development.     

Complete Genome Sequence of Aeromonas hydrophila Phage CC2

Aeromonas hydrophila is one of the major pathogenic bacteria for fish and people. To develop an effective antimicrobial agent, we isolated a bacteriophage from sewage, named CC2, and sequenced its genome. Comparative genome analysis of phage CC2 with its relatives revealed that phage CC2 has higher sequence homology to A. salmonicida phage 65 than to A. hydrophila phage Aeh1. Here, we announce the complete genome sequence of CC2 and report major findings from the genomic analysis.     

Immune response of zebrafish (Danio rerio) against a newly isolated bacterial pathogen Aeromonas hydrophila

A strain of Aeromonas hydrophila associated with unusual mortalities in zebrafish (Danio rerio) culture facilities was isolated, identified and characterized. In challenge experiments, adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection with viable bacteria and its extracellular products (ECPs) reaching very high mortalities in a few hours. The infection, by the viable bacteria or the ECPs, caused cell death in kidney, due to the cytotoxic and haemolytic activities of the bacterial ECPs. Moreover, the infection affected the release of oxygen (ROS) and nitrogen (NO) reactive free radicals. To determine if this A. hydrophila infection induces an inflammatory response, mRNA expression levels of tumour necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), interferon-gamma (IFNgamma) and inducible nitric oxide (iNOS) were assessed by real time PCR. The expression levels of TNFalpha, IL-1beta and IFNgamma were upregulated in the kidneys of infected zebrafish with viable bacteria, heat-killed bacteria and ECPs. Expression levels of iNOS were upregulated by ECPs. Mortality rate (LD(50)) and histopathology were also determined.     

Identification, cloning and characterization of interleukin-17 and its family from zebrafish

Cytokines are one of the major signaling molecules involved in immunity. Many of these cytokines have been isolated in vertebrates and found to play a significant role in host defense mechanism. Interleukin-17 (IL-17) family of genes are known to have pro-inflammatory actions and associated with specific disease conditions, these genes are conserved across vertebrate evolution. In this study, computational screening for the zebrafish (Danio rerio) genome resulted in identification of five contigs harboring IL-17 genes. Zebrafish cDNA encoding five IL-17 genes exhibited percentage identities of 19.3%-61.9% with that of human homologs. The molecules show conservation of cysteines, important for disulphide bonds for IL-17 molecules. The structural composition of these genes shows two introns and three exons except for IL-17D gene that has only one intron and two exons. Phylogenetic analysis using maximum parsimony algorithm showed that zebrafish IL-17 genes clustered well with other IL-17 homologs further proving the structural similarity with IL-17 genes from other organisms. Expression analysis by RT-PCR revealed expression of IL-17 genes in normal and stimulated tissues of kidney, spleen, gills and intestine. The expression of IL-17 in un-stimulated tissues indicates that these genes may play important roles in normal conditions as well.     

中华鳖致病病原菌的分离及药物试验研究

嗜水气单胞菌为该病的条件致病菌。在适宜的条件下 ,该菌可通过体表伤口进入鳖体引起疾病。此病以体表腐皮与赤斑 ,有的中华鳖背部有明显的乳白色小点和出血性败血症为主要病理学表现 ,以各器官功能衰竭为致病原因。解剖后 ,肝脏病变呈污泥色 ,腐烂液化 ,肾呈青紫色、肿大 ,肠内无食物 ,呈灰白色 ,部分肠壁有出血点 ,脾脏明显肿大。经分离鉴定 ,生理生化特性分析 ,结果为典型的嗜水气单胞菌嗜水亚种 (Aeromonashydrophilasubsp .hydrophila)。     

湖北地区暴发病池塘中嗜水气单胞菌的遗传多样性和毒力特征研究

为了调查引起鱼类运动型气单胞菌败血症(俗称暴发病)的嗜水气单胞菌的遗传多样性和毒力特征, 阐明其流行规律, 研究于2006-2009年度从湖北省内3个不同地区的6个发病鱼塘中分离了30株嗜水气单胞菌, 其中20株为临床株(分离自血液、肝脏、肾脏或腹水), 6株为肠道株, 4株为池水株。基于所有菌株gyrB基因序列, 构建了系统发育树; 通过ERIC (Enterobacterial repetitive intergenic consensus, 肠道细菌基因间重复序列)指纹图谱进行菌株的遗传分型; 用PCR方法检测了7个毒力基因在菌株中的分布模式。这7个基因包括气溶素(aerA)、溶血素(hlyA)、热不稳定性细胞兴奋性肠毒素(alt)、热稳定性细胞兴奋性肠毒素(ast)、弹性蛋白酶(ahpB)、脂酶(lip)和鞭毛基因(fla)。此外, 以斑马鱼为感染对象, 通过腹腔注射测定了15株代表菌株的毒力。结果表明: 不同来源的20株临床株、1株肠道株和3株池水株具有相同的遗传特性, 体现为在系统树上聚为一枝, 序列相似性为100%, 具有相同的ERIC指纹图谱, 毒力基因分布模式为: aerA+hlyA+alt+ast+ahpB+lip+fla+, 且均为强毒株(LD50 9.74104cfu/尾)。与临床株相比, 其余5株肠道株和1株池水株或具有不同的ERIC指纹图谱或具有不同的毒力基因分布模式, 显示出了遗传多样性, 且毒力均弱于临床株(LD501.01106cfu/尾)。这说明在一定时间、一定区域内, 作为暴发病病原的嗜水气单胞菌为同一克隆系在流行, 不存在明显的变异或遗传多样性。此结果有助于阐明嗜水气单胞菌引起的暴发病的流行规律, 制定相应的防御措施。多种毒力基因在致病性菌株中的联合流行为发病机理的解析奠定了基础。此外, 鉴于毒力基因谱与致病性之间的相关性, 表明毒力基因可作为标记基因, 用于致病性菌株的检测。       

IPNV modulation of pro and anti-inflammatory cytokine expression in Atlantic salmon might help the establishment of infection and persistence

IPNV is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species and persistent infection in surviving fish after outbreaks. Because modulation of the host expression of pro and anti-inflammatory cytokines can help establish persistence, in this study, we examined the expression of IL-1β, IL-8, IFNα1 and IL-10 during acute and persistent IPNV infection of Atlantic salmon. Results showed that IPNV infection induces an increase of the IFNα1 and IL-10 mRNA levels in the spleen and head kidney (HK) of fish after acute experimental infection. Levels of the pro-inflammatory cytokines IL-1β and IL-8 did not rise in the spleen although an increase of IL-1β, but not of IL-8, was observed in head kidney. In carrier asymptomatic salmon, cytokine gene expression of IFNα1 in the spleen and IL-10 in head kidney were also significantly higher than expression in non-carrier fish. Interestingly, a decrease of IL-8 expression was also observed. IPNV infection of SHK-1, which is a macrophage-like cell line of salmon, also induced an increase of expression of the anti-inflammatory cytokine IL-10 with no effects on the expression of IL-1β and IL-8. The effects are induced by an unknown mechanism during viral infection because poly I:C and the viral genomic dsRNA showed the opposite effects on cytokine expression in SHK-1 cells. In summary, IPNV always induces up-regulation of the anti-inflammatory cytokine IL-10 in Atlantic salmon. As this is accompanied by a lack of induction of the pro-inflammatory cytokines IL-1β and IL-8, the anti-inflammatory milieu may explain the high frequency, prevalence and persistence of IPNV in salmon. Effects might be part of the viral mechanisms of immune evasion.     

Transgenic expression of tilapia hepcidin 1-5 and shrimp chelonianin in zebrafish and their resistance to bacterial pathogens

Recently, tilapia hepcidin (TH)1-5 was characterized, and its antimicrobial functions against several pathogens were reported. The antimicrobial functions of another shrimp antimicrobial peptide (AMP), chelonianin, were also characterized using a recombinant chelonianin protein (rcf) that was expressed by a stably transfected Chinese hamster ovary (CHO) cell line against pathogen infections in fish. The function of the overexpression of both AMPs in zebrafish muscles was not examined in previous studies. Herein, we investigated the antimicrobial functions of TH1-5 and chelonianin against Vibrio vulnificus (204) and Streptococcus agalactiae (SA48) in transgenic TH1-5 zebrafish and transgenic chelonianin zebrafish. The presence of TH1-5 and chelonianin enhanced the inhibitory ability in transgenic AMP zebrafish against the two different bacterial infections. The bacterial number of either V. vulnificus (204) or S. agalactiae (SA48) had decreased at 96?h after injection into transgenic AMP zebrafish muscle compared to non-transgenic zebrafish muscle. Additionally, immune-related gene expressions analyzed by real-time PCR studies showed the modulation of several genes including interleukin (IL)-10, IL-22, IL-26, MyD88, Toll-like receptor (TLR)-1, TLR-3, TLR-4, nuclear factor (NF)-κB, tumor necrosis factor (TNF)-α, and lysozyme, and significant differences were found between transgenic AMP zebrafish and wild-type zebrafish injected with PBS at 1-24?h. These results suggest that several immune-related gene expressions were induced in transgenic TH1-5 and chelonianin zebrafish which effectively inhibited bacterial growth. The survival rate dropped to 86.6% in transgenic chelonianin zebrafish after 28 days of infection compared of the 50% survival rate in transgenic TH1-5 zebrafish after 28 days of infection. Overall, these results indicate that TH1-5 and chelonianin possess the potential to be novel candidate genes for aquaculture applications to treat fish diseases.     

Genetic evidence for an evolutionarily conserved role of IL-7 signaling in T cell development of zebrafish

In mammals, the cytokine IL-7 is a key regulator of various aspects of lymphocyte differentiation and homeostasis. Because of the difficulty of identifying cytokine homologs in lower vertebrates and the paucity of assay systems and reagents, the degree of functional conservation of cytokine signaling pathways, particularly those pertaining to lymphocyte development, is unclear. In this article, we report on the analysis and characterization of three zebrafish mutants with severely impaired thymopoiesis. The identification of affected genes by positional cloning revealed components of the IL-7 signaling pathway. A presumptive null allele of the zebrafish homolog of the IL-7Rα-chain causes substantially reduced cellularity of the thymus but spares B cell development in the kidney. Likewise, nonsense mutations in the zebrafish homologs of janus kinases JAK1 and JAK3 preferentially affect T cell development. The functional interactions of the cytokine receptor components were examined in the three groups of fish hetero- or homozygous for either il7r and jak1, il7r and jak3, or jak1 and jak3 mutations. The differential effects on T cell development arising from the different genotypes could be explained on the basis of the known structure of the mammalian IL-7R complex. Because IL-7 signaling appears to be a universal requirement for T cell development in vertebrates, the mutants described in this article represent alternative animal models of human immunodeficiency syndromes amenable to large-scale genetic and chemical screens.     

Expression pattern and functions of autophagy-related gene atg5 in zebrafish organogenesis

The implications of autophagy-related genes in serious neural degenerative diseases have been well documented. However, the functions and regulation of the family genes in embryonic development remain to be rigorously studied. Here, we report on for the first time the important role of atg5 gene in zebrafish neurogenesis and organogenesis as evidenced by the spatiotemporal expression pattern and functional analysis. Using morpholino oligo knockdown and mRNA overexpression, we demonstrated that zebrafish atg5 is required for normal morphogenesis of brain regionalization and body plan as well as for expression regulation of neural gene markers: gli1, huC, nkx2.2, pink1, β-synuclein, xb51 and zic1. We further demonstrated that ATG5 protein is involved in autophagy by LC3-II/LC3I ratio and rapamycin-induction experiments, and that ATG5 is capable of regulating expression of itself gene in the manner of a feedback inhibition loop. In addition, we found that expression of another autophagy-related gene, atg12, is maintained at a higher constant level like a housekeeping gene. This indicates that the formation of the ATG12–ATG5 conjugate may be dependent on ATG5 protein generation and its splicing, rather than on ATG12 protein in zebrafish. Importantly, in the present study, we provide a mechanistic insight into the regulation and functional roles of atg5 in development of zebrafish nervous system.     

Th2 Cytokines Augment IL-31/IL-31RA Interactions via STAT6-dependent IL-31RA Expression

Interleukin 31 receptor α (IL-31RA) is a novel Type I cytokine receptor that pairs with oncostatin M receptor to mediate IL-31 signaling. Binding of IL-31 to its receptor results in the phosphorylation and activation of STATs, MAPK, and JNK signaling pathways. IL-31 plays a pathogenic role in tissue inflammation, particularly in allergic diseases. Recent studies demonstrate IL-31RA expression and signaling in non-hematopoietic cells, but this receptor is poorly studied in immune cells. Macrophages are key immune-effector cells that play a critical role in Th2-cytokine-mediated allergic diseases. Here, we demonstrate that Th2 cytokines IL-4 and IL-13 are capable of up-regulating IL-31RA expression on both peritoneal and bone marrow-derived macrophages from mice. Our data also demonstrate that IL-4Rα-driven IL-31RA expression is STAT6 dependent in macrophages. Notably, the inflammation-associated genes Fizz1 and serum amyloid A (SAA) are significantly up-regulated in M2 macrophages stimulated with IL-31, but not in IL-4 receptor-deficient macrophages. Furthermore, the absence of Type II IL-4 receptor signaling is sufficient to attenuate the expression of IL-31RA in vivo during allergic asthma induced by soluble egg antigen, which may suggest a role for IL-31 signaling in Th2 cytokine-driven inflammation and allergic responses. Our study reveals an important counter-regulatory role between Th2 cytokine and IL-31 signaling involved in allergic diseases.     

Interleukin-22: A cytokine produced by T,NK and NKT cell subsets,with importance in the innate immune defense and tissue protection

Interleukin (IL)-22 is a member of the IL-10 cytokine family that is produced by special immune cell populations, including Th22, Th1, and Th17 cells, classical and non-classical (NK-22) NK cells, NKT cells, and lymphoid tissue inducer cells. This cytokine does not influence cells of the hematopoietic lineage. Instead, its target cells are certain tissue cells from the skin, liver and kidney, and from organs of the respiratory and gastrointestinal systems. The main biological role of IL-22 includes the increase of innate immunity, protection from damage, and enhancement of regeneration. IL-22 can play either a protective or a pathogenic role in chronic inflammatory diseases depending on the nature of the affected tissue and the local cytokine milieu. This review highlights the primary effects of IL-22 on its target cells, its role in the defense against infections, in tumorigenesis, in inflammatory diseases and allergy as well as the potential of the therapeutic modulation of IL-22 action.     

Apela Regulates Fluid Homeostasis by Binding to the APJ Receptor to Activate Gi Signaling

Apela (APJ early endogenous ligand, also known as elabela or toddler) is a recently discovered peptide hormone. Based on genetic studies in zebrafish, apela was found to be important for endoderm differentiation and heart development during embryogenesis. Although common phenotypes of apela and APJ-null zebrafish during embryonic development suggested that apela interacts with the APJ receptor, kinetics of apela binding to APJ and intracellular signaling pathways for apela remain unknown. The role of apela in adults is also uncertain. Using a chimeric apela ligand, we showed direct binding of apela to APJ with high affinity (K_d = 0.51 nm) and the ability of apelin, the known peptide ligand for APJ, to compete for apela binding. Apela, similar to apelin, acts through the inhibitory G protein pathway by inhibiting forskolin-stimulated cAMP production and by inducing ERK1/2 phosphorylation. In adult rats, apela is expressed exclusively in the kidney, unlike the wide tissue distribution of apelin. In vivo studies demonstrated the ability of apela to regulate fluid homeostasis by increasing diuresis and water intake. Dose-response studies further indicated that apela induces 2- and 5-fold higher maximal responses than apelin in ERK1/2 phosphorylation and diuresis/water intake, respectively. After designing an apela antagonist, we further demonstrated the role of endogenous ligand(s) in regulating APJ-mediated fluid homeostasis. Our results identified apela as a potent peptide hormone capable of regulating fluid homeostasis in adult kidney through coupling to the APJ-mediated G_i signaling pathway.