多鳞白甲鱼X型凝集素(OmITLN)基因克隆及在抗菌免疫中的功能研究

为探究秦巴山溪半洞穴鱼类——多鳞白甲鱼的先天性免疫因子X型凝集素的免疫功能,研究从多鳞白甲鱼(Onychostoma macrolepis)肝脏转录组数据库筛选出一种Intelectin基因片段,通过克隆鉴定获得Intelectin基因cDNA序列全长,命名为OmITLN;采用qRT-PCR分析OmITLN基因在健康多鳞白甲鱼8个组织和嗜水气单胞菌感染后两个免疫组织的表达情况;构建OmITLN基因的原核表达载体并在大肠杆菌BL21中诱导表达,获得Om ITLN重组蛋白,采用ELISA法以及荧光标记法检测Om ITLN对病原菌的凝集效果及糖结合情况。结果显示, OmITLN是一种X型凝集素基因,其cDNA全长960 bp,编码315个氨基酸。Om ITLN蛋白含有1个N端的纤维蛋白原相关结构域(Fibrinogen-Related Domain, FReD)和1个C端Intelectin特异性区域,不具有信号肽和跨膜区。系统进化树分析发现, Om ITLN与鲫、团头鲂、鳙、草鱼等鲤科鱼类的Intelectin聚为一支,亲缘关系最近。OmITLN基因在所检测组织均有表达,且在脾脏和肝脏中的...     

抗菌肽HBβ-C在全雄和杂交黄颡鱼对多子小瓜虫抗性差异中的作用

为研究全雄黄颡鱼(Pelteobagrus fulvidraco)、瓦式黄颡鱼(Pelteobagrus vachelli)和杂交黄颡鱼(黄颡鱼P.fulvidraco♀×瓦氏黄颡鱼P. vachelli♂)对多子小瓜虫(Ichthyophthirius multifiliis)的抗性差异,通过生物信息学分析黄颡鱼皮肤黏液蛋白质组,发现其血红蛋白源抗菌肽(HBβ-C)位于血红蛋白β链HBβ的碳端,共33个氨基酸。利用化学合成的不同浓度的HBβ-C肽段进行体外抗虫实验,研究发现其能有效杀死滋养体、包囊体和掠食体阶段的多子小瓜虫,其中15μg/mL的HBβ-C能在3min内杀死所有滋养体。基因表达量分析显示,在杂交黄颡鱼的鳃和皮肤组织中, HBβ的mRNA表达量高于全雄黄颡鱼;但在应对小瓜虫感染的过程中,全雄黄颡鱼的HBβmRNA转录水平快速提升,其表达水平和上升倍率显著高于杂交黄颡鱼。蛋白表达量分析显示,HBβ在全雄黄颡鱼鳃组织中的蛋白表达量明显高于杂交黄颡鱼。免疫荧光定位结果显示,抗菌肽HBβ-C特异地在红细胞中表达,可以分泌并附着在滋养体上。综上所述,相对于杂交黄颡鱼,全雄黄颡鱼中H...     

黄颡鱼HSC70基因及其组织表达分析

热休克蛋白70（HSP70）与生物体的抗胁迫能力密切相关。本文采用RACE (Rapid amplification of cDNA ends) 技术,从黄颡鱼Pelteobagrus fulvidraco克隆到一种组成型热休克蛋白（HSC70）基因及其cDNA。该cDNA全长2245bp,包括5′非编码区82bp,3′非编码区225bp,开放阅读框(ORF) 1938bp,编码645个氨基酸组成的蛋白质。黄颡鱼HSC70基因含有8个内含子,与人、鼠、虹鳟和花斑溪鳉的HSC70基因内含子数目相同,位置相似。其中,最长内含子（873bp）位于5′端非编码区,其余内含子（长度在80－251bp之间不等）均在编码区以内。黄颡鱼HSC70基因编码的氨基酸序列与南方鲶的相似度最高,达96.13%,与欧洲银鲫和团头鲂的相似度分别为94.45%和94.14%。RT-PCR检测显示,正常情况下黄颡鱼HSC70在血细胞、心脏、肝、头肾、脾、鳃、肌肉和脑中均有表达,但表达量在鳃中最高,肌肉中最低;统计结果显示,热激后HSC70在血细胞、肝、头肾和脑中的表达量显著上升(p<0.05),而在其余组织中热激前后的表达差异不显著(p>0.05)。     

Decreased expression of intelectin 1 in the human airway epithelium of smokers compared to nonsmokers

Lectins are innate immune defense proteins that recognize bacterial cell wall components. Based on the knowledge that cigarette smoking is associated with an increased risk of infections, we hypothesized that cigarette smoking may modulate the expression of lectin genes in airway epithelium. Affymetrix microarrays were used to survey the expression of lectin genes in large airway epithelium from nine nonsmokers and 20 healthy smokers and in small airway epithelium from 13 nonsmokers and 20 healthy smokers. There were no changes (>2-fold change; p < 0.05) in lectin gene expression among healthy smokers compared with nonsmokers except for down-regulation of intelectin 1, a lectin that binds to galactofuranosyl residues in bacterial cell walls (large airway epithelium, p < 0.01; small airway epithelium, p < 0.01). This was confirmed by TaqMan RT-PCR in both large (p < 0.05) and small airway epithelium (p < 0.02). Immunohistochemistry assessment of airway biopsies demonstrated that intelectin 1 was expressed in secretory cells, while Western analysis confirmed the decreased expression of intelectin 1 in airway epithelium of healthy smokers compared with healthy nonsmokers (p < 0.02). Finally, compared with healthy nonsmokers, intelectin 1 expression was also decreased in small airway epithelium of smokers with lone emphysema and normal spirometry (n = 13, p < 0.01) and smokers with established chronic obstructive pulmonary disease (n = 14, p < 0.01). In the context that intelectin 1 plays a role in defense against bacteria, its down-regulation in response to cigarette smoking is another example of the immunomodulatory effects of smoking on the immune system and may contribute to the increase in susceptibility to infections observed in smokers.     

Tandem repeat L-rhamnose-binding lectin from the skin mucus of ponyfish, Leiognathus nuchalis

Two lactose-binding lectins (PFL-1 and -2) were identified in the skin mucus of ponyfish, Leiognathus nuchalis. The molecular masses of PFL-1 and -2 were estimated to be 24 and 30kDa, respectively. Cloning of the PFL-1 cDNA demonstrated its unique tandem repeat structure composed of two homologous domains with 41.7% internal identity. Furthermore, PFL-1 exhibited homology with L-rhamnose-binding lectins previously purified from the eggs of fish and sea urchins. The N-terminal amino acid sequence of PFL-2 was similar to that of PFL-1, suggesting that this protein is an isotype of PFL-1. The PFL-1 gene was expressed in the skin, an important line of defense against pathogens in fish, but was not expressed in any of the other tissues tested here. PFL-1 is the fourth type of fish skin mucus lectin to be identified, suggesting that different species of fish express different types of lectin in their skin mucus.     

Flavobacterium columnare chemotaxis to channel catfish mucus

Flavobacterium columnare is a Gram-negative pathogen of many species of wild and cultured fish. Isolates from diseased channel catfish belong to either genomovar I or II. Genomovar II isolates were found to be more virulent than genomovar I isolates. The objective of the present study was to determine whether differences exist in the chemotactic response of these genomovars to mucus obtained from the skin, gills and intestines of healthy channel catfish using the capillary chemotaxis assay. Mucus from the skin and gill induced a greater chemotactic response by F. columnare than mucus from the intestine. Sixty percent of mucus from the skin of individual catfish yielded a positive chemotactic response from F. columnare. Finally, skin mucus induced a greater chemotactic response in genomovar II F. columnare than in genomovar I F. columnare isolates. The data indicate that mucus from channel catfish results in a chemotactic response by F. columnare. This positive chemotactic response may be an important first step for F. columnare colonization of channel catfish skin or gills. Although the role that chemotaxis plays in the virulence of F. columnare is not fully defined, the chemotactic response of genomovar ll isolates suggests that chemotaxis is associated with virulence.     

Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge

The adhesion dynamics of Flavobacterium columnare to fish tissues were evaluated in vivo by immersion challenge followed by bacterial plate count and confirmatory observations of gill-adhered bacterial cells using scanning electron microscopy. Adhesion of F. columnare genomovar I (ARS-1) and II (BGFS-27) strains to skin and gill of channel catfish Ictalurus punctactus and gill of zebrafish Danio rerio was compared. At 0.5 h post-challenge, both strains adhered to gill of channel catfish at comparable levels (10(6) colony forming units [CFU] g(-1)), but significant differences in adhesion were found later in the time course. Channel catfish was able to effectively reduce ARS-1 cells on gill, whereas BGFS-27 persisted in gill beyond the first 24 h post-challenge. No significant difference was found between both strains when adhered to skin, but adhered cell numbers were lower (10(3) CFU g(-1)) than those found in gill and were not detectable at 6 h post-challenge. Adhesion of BGFS-27 cells to gill of zebrafish also occurred at high numbers (> 10(6) CFU g(-1)), while only < 10(2) CFU g(-1) of ARS-1 cells were detected in this fish. The results of the present study show that particular strains of F. columnare exhibit different levels of specificity to their fish hosts and that adhesion to fish tissues is not sufficient to cause columnaris disease.     

Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall

Galactofuranosyl residues are present in various microorganisms but not in mammals. In this study, we identified a human lectin binding to galactofuranosyl residues and named this protein human intelectin (hIntL). The mature hIntL was a secretory glycoprotein consisting of 295 amino acids and N-linked oligosaccharides, and its basic structural unit was a 120-kDa homotrimer in which 40-kDa polypeptides were bridged by disulfide bonds. The hIntL gene was split into 8 exons on chromosome 1q21.3, and hIntL mRNA was expressed in the heart, small intestine, colon, and thymus. hIntL showed high levels of homology with mouse intelectin, Xenopus laevis cortical granule lectin/oocyte lectin, lamprey serum lectin, and ascidian galactose-specific lectin. These homologues commonly contained no carbohydrate recognition domain, which is a characteristic of C-type lectins, although some of them have been reported as Ca(2+)-dependent lectins. Recombinant hIntL revealed affinities to d-pentoses and a d-galactofuranosyl residue in the presence of Ca(2+), and recognized the bacterial arabinogalactan of Nocardia containing d-galactofuranosyl residues. These results suggested that hIntL is a new type lectin recognizing galactofuranose, and that hIntL plays a role in the recognition of bacteria-specific components in the host.     

Characterization of a galactose binding serum lectin from the Indian catfish, Clarias batrachus: possible involvement of fish lectins in differential recognition of pathogens

A lectin with molecular mass around 200 kDa was isolated from the serum of the Indian catfish Clarias batrachus. The bioactivity of this serum lectin was Ca2+ and pH dependent. The lectin appeared to be specific for alpha-methyl galactose and sialoglycoproteins like porcine and bovine submaxillary mucin and could agglutinate human, rabbit, mice, rat and chicken erythrocytes. This fish lectin was able to specifically agglutinate different gram negative bacteria. When it was checked against different strains of the fish pathogen Aeromonas sp., it significantly altered the viability and pathogenicity of the bacteria. Binding of the lectin to Aeromonas sp., resulted in a dose dependent increase in the bactericidal activity of fish macrophages. However, when the lectin was checked against different gram positive bacteria it could not agglutinate or affect the viability of those strains and also failed to bring about any significant change in the bactericidal potential of fish macrophages. The lectin was able to induce the proliferation of head kidney lymphocytes of Clarias and helped in the release of 'IL-1' like cytokines from head kidney macrophages.     

Identification, cloning and tissue localization of a rainbow trout (Oncorhynchus mykiss) intelectin-like protein that binds bacteria and chitin

Intelectins are a recently identified group of animal lectins involved in innate immune surveillance. This paper describes the primary structure, expression and immunohistochemical localization of a rainbow trout plasma intelectin (RTInt). RTInt exhibited calcium-dependent binding to N-acetylglucosamine (GlcNAc) and mannose conjugated Toyopearl Amino 650 M matrices. When GlcNAc eluates from chromatography matrices were analyzed by reducing 1D PAGE and Western blots, the lectin appeared as approximately 37 kDa and approximately 72 kDa bands. Similar analysis of plasma revealed a single 72 kDa band under reducing conditions. MALDI-TOF MS demonstrated five, approximately 37 kDa isoforms (pI 5.3-6.1) separated by 2D-PAGE. A 975 bp cDNA sequence obtained by RT-PCR from liver and spleen tissue encoded a 325 amino acid secretory protein with homology to human and murine intelectins, which bind bacterial components and are induced during parasitic infections. Gene expression and immunohistochemistry detected RTInt in gill, spleen, hepatic sinusoid, renal interstitium, intestine, skin, swim bladder and within leukocytes. Direct binding assays demonstrated the ability of RTInt to bind relevant bacterial and chitinous targets. These findings suggest that RTInt plays a role in innate immune defense against bacterial and chitinous microbial organisms.     

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3' RACE, 5' RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H(2)O(2)-generation assay and substrate specificity for only l-Lys with a K(m) of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H(2)O(2) is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.     

Intelectin: a novel lipid raft-associated protein in the enterocyte brush border

Intelectin is a mammalian Ca2+-dependent, D-galactosyl-specific lectin expressed in Paneth and goblet cells of the small intestine and proposed to serve a protective role in the innate immune response to parasite infection. In addition, it is structurally identical to the intestinal lactoferrin receptor known to reside in the enterocyte brush border. To clarify this apparent discrepancy with regard to localization, the aim of this work was to study the cellular and subcellular distribution of small intestinal intelectin by immunofluorescence and immunogold electron microscopy. Secretory granules of lysozyme-positive Paneth cells in the bottom of the crypts as well as goblet cells along the crypt-villus axis were intensively labeled with intelectin antibodies, but quantitatively, the major site of intelectin deposition was the enterocyte brush border. This membrane is organized in stable glycolipid-based lipid raft microdomains, and like the divalent lectin galectin-4, intelectin was enriched in microvillar "superrafts", i.e., membranes that resist solubilization with Triton X-100 at 37 degrees C. This strategic localization suggests that the trimeric intelectin, like galectin-4, serves as an organizer and stabilizer of the brush border membrane, preventing loss of digestive enzymes to the gut lumen and protecting the glycolipid microdomains from pathogens.     

Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections

Bass hepcidin was purified from the gill of hybrid striped bass (Morone chrysops x Morone saxatilis) based on antimicrobial activity against Escherichia coli. This 21-amino acid peptide has 8 cysteines engaged in 4 disulfide bonds and is very similar to human hepcidin, an antimicrobial peptide with iron regulatory properties. To gain insight into potential role(s) of bass hepcidin in innate immunity in fish, we synthesized the peptide, characterized its antimicrobial activities in vitro, determined its solution structure by NMR, and quantified hepatic gene expression in vivo following infection of bass with the fish pathogens, Streptococcus iniae or Aeromonas salmonicida. Its structure is very similar to that of human hepcidin, including the presence of an antiparallel beta-sheet, a conserved disulfide-bonding pattern, and a rare vicinal disulfide bond. Synthetic bass hepcidin was active in vitro against Gram-negative pathogens and fungi but showed no activity against key Gram-positive pathogens and a single yeast strain tested. Hepcidin was non-hemolytic at microbicidal concentrations and had lower specific activity than moronecidin, a broad spectrum, amphipathic, alpha-helical, antimicrobial peptide constitutively expressed in bass gill tissue. Good synergism between the bacterial killing activities of hepcidin and moronecidin was observed in vitro. Hepcidin gene expression in bass liver increased significantly within hours of infection with Gram-positive (S. iniae) or Gram-negative (A. salmonicida) pathogens and was 4-5 orders of magnitude above base-line 24-48 h post-infection. Our results suggest that hepcidin plays a key role in the antimicrobial defenses of bass and that its functions are potentially conserved between fish and human.     

Effects of 2-methylisoborneol (MIB), and ethanol on the expression and activity of cytochrome P450s from the channel catfish

Injection of 1 mg kg⁻¹ of 2-methylisoborneol (MIB) caused an increase in a CYP1A-like protein of approximately 56 kDa from the kidneys of juvenile channel catfish as determined by western blot analysis using antibodies raised against trout CYP1A1. Ethoxyresorufin O-deethylase activity from kidneys of MIB-injected fish was significantly elevated as compared to ethanol-injected controls. Static exposure of juvenile channel catfish to 10 ppm MIB caused a statistically significant induction of a CYP2K-like protein of approximately 53 kDa from the livers of treated catfish as determined by western blot analysis using antibodies raised against trout CYP2K1. Treatment of juvenile channel catfish with ethanol (1 ml kg⁻¹) reduced the expression of one kidney and two constitutive liver P450s, while increasing another kidney form. There was no difference in carbon tetrachloride-induced lipid peroxidation in livers after ethanol treatment. Thus, MIB and ethanol affect the expression of at least three P450 isoforms in channel catfish tissues.     

Channel catfish (Ictalurus punctatus) protein disulphide isomerase, PDIA6: molecular characterization and expression regulated by bacteria and virus inoculation

Protein disulfide isomerases (PDIs) are thought to aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Currently, increasing evidence suggests PDIs play an important role in host cell invasion and they are relevant targets for the host immune response. However the roles of specific PDIs in teleosts are little known. Here, we characterized the Protein disulfide isomerase family A, member 6 (PDIA6) from channel catfish, Ictalurus punctatus (named as ccPDIA6). The catfish ccPDIA6 gene was homologous to those of other vertebrate species with 13 exons and 12 introns. The consensus full-length ccPDIA6 cDNA contained an ORF of 1320 bp encoding a putative protein of 439 amino acids. It had a 19 amino acid signal peptide and two active thioredoxin-like domains. Sequence of phylogenic analysis and multiple alignments showed that ccPDIA6 was conserved throughout vertebrate evolution. Southern blot analysis suggested the presence of one copy of the ccPDIA6 gene in the catfish genome. Tissue distribution shows that ccPDIA6 was expressed in all examined tissues at the mRNA level. When using the aquatic zoonotic pathogens such as Edwardsiella tara, Streptococcus iniae, and channel catfish reovirus （CCRV） to challenge channel catfish, ccPDIA6 expression was significant changed in immune-related tissues such as head kidney, intestine, liver and spleen. The results suggested that ccPDIA6 might play an important role in the immunity of channel catfish. This is the first report that the PDI gene may be involved in fish host defense against pathogen infection.