Two catalase homologs are involved in host protection against bacterial infection and oxidative stress in Crassostrea hongkongensis

Catalase is one of the key antioxidant enzymes and it appears to be involved in protection against immune infection and oxidative stress. Here, two catalase cDNAs (ChCat-1 and ChCat-2) were isolated from hemocytes of Crassostrea hongkongensis using SSH and RACE. The full-length cDNAs of ChCat-1 and ChCat-2 are 1913 and 2466 bp in length, encoding proteins of 515 and 511 amino acids, respectively. Multiple alignments of amino acid sequences revealed that both ChCat-1 and ChCat-2 possess several characteristic features of the catalase family of enzymes, including one proximal active site signature, one heme-ligand signature, and three catalytic amino acid residues (His(72), Asn(145) and Tyr(355)). Phylogenetic analysis indicates that these two catalases may share a common ancestral gene and result from a gene duplication event following the divergence of bivalves and gastropods. Constitutive expression of ChCat-1 and ChCat-2 was observed in all tissues studied, with highest levels of expression in gill and muscle, respectively. The expression of both genes was inducible by bacterial infection, and reached the maximum at 8 h (9.0-fold) and 12 h (2.3-fold) post-infection, respectively. Furthermore, both the purified ChCat-1 and ChCat-2 protein displayed a strong catalase activity, and S2 cells carrying ChCat-1 or ChCat-2 showed a higher degree of resistance to H(2)O(2) than that of control cells. In a word, this is the first report of the presence of two catalase genes in a single marine bivalve, and our results highlight the involvement of both ChCat-1 and ChCat-2 in host protection against pathogen infection and oxidative stress in C. hongkongensis.     

Cloning and expression of a heat shock protein (HSP) 90 gene in the haemocytes of Crassostrea hongkongensis under osmotic stress and bacterial challenge

Heat shock protein 90 (HSP90) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Here, we report the cloning of the HSP90 homologue in Crassostrea hongkongensis (ChHSP90) through SSH in combination with RACE from cDNA of haemocytes. The full-length cDNA of ChHSP90 is 2459 bp in length, consisting of a 3', 5'-untranslated region (UTR) and an open reading frame of 2169 bp encoding 722 amino acids. The identity analysis of the amino acid sequence of HSP90 revealed that ChHSP90 is highly conserved. Distribution of ChHSP90 mRNA in gonad, heart, adductor muscle, mantle, gill, digestive gland, and haemocytes suggested that ChHSP90 is ubiquitously expressed. The mRNA levels of ChHSP90 under salinity and bacterial challenges were analyzed by real-time PCR. Under hypo-osmotic treatment, ChHSP90 mRNA in gonad, heart and haemocytes were significantly up-regulated on day 2 and onwards; while in gill, digestive gland and adductor muscle it was significantly down-regulated; the expression in mantle was decreased significantly on day 2 and 3 (P < 0.01), and then up-regulated on day 4 (P < 0.05). Under hyper-osmotic treatment, the mRNA level in gonad, heart, adductor muscle was increased on day 2 and onwards; in gill, it was firstly increased, and then gradually decreased, reaching a minimum on day 3. On day 4, the expression level in gill recovered to pre-treatment level; in mantle and digestive gland, the expression levels were decreased, reaching to the minimum on day 3. During Vibrio alginolyticus challenge, the mRNA level of ChHSP90 increased 3-fold at 4 h post-infection, returned to its pre-challenge level at 6 h post-infection, then was further up-regulated from 8 to 36 h post-infection. These experiments demonstrate that ChHSP90 mRNA is constitutively expressed in various tissues and apparently inducible in haemocytes under salinity and bacterial challenges, suggesting its important role in response to both osmotic stress and bacterial invasion.     

近江牡蛎HSP70基因对溶藻弧菌感染的反应

采用实时荧光定量RT-PCR方法,检测了注射溶藻弧菌(Vibiro alginolyticus)后近江牡蛎鳃,闭壳肌,消化腺,外套膜,心脏以及血细胞中HSP70基因的表达变化。结果显示近江牡蛎这五种器官组织中的HSP70基因表达量均出现显著性高表达,且在鳃、外套膜和血细胞中的HSP70基因表达变化规律表现为典型的时间依赖性。血细胞中,显著高表达的峰值出现在24h,至72h恢复到对照水平,高表达持续时间最长:鳃中表达峰值出现时间较早,在第3h,随后在第12h便恢复到对照水平;外套膜,消化腺以及心脏中的峰值分别出现在6h,6h和3h,而在闭壳肌组织中,没出现显著性高表达。由此可见,近江牡蛎HSP70s可能在机体抗菌免疫过程中起了重要作用。     

The expression analysis of inflammatory and antimicrobial genes in the goldfish (Carassius auratus L.) infected with Trypanosoma carassii

We report results of a comprehensive analysis of inflammatory gene expression during the course of infection of Trypanosoma carassii in the goldfish. We observed significant increases in mRNA levels of genes encoding pro-inflammatory cytokines IFN-γ, TNFα1 and TNFα2; IL-1β-1 and IL-1β-2; IL-12-p35 and IL-12-p40; CCL1; CXCL8, anti-inflammatory cytokines IL-10 and TGFβ and iNOS A and iNOS B, using quantitative PCR. Expression levels and profiles of these cytokines and iNOS isoforms varied in the different tissues (kidney, spleen, liver) of goldfish during the course of T.?carassii infection. The expression of majority of genes that encode pro- and anti-inflammatory cytokines were up-regulated during the acute phase of infection (days 7-21 post-infection). The mRNA levels of these cytokines returned to normal levels or were down-regulated during the elimination phase of infection (days 28-56), with exception of IL-10 in the spleen and liver of infected fish. A parallel up-regulation of IFN-γ and IL-10 mRNA levels were observed in all tissues of infected fish during the acute phase of the infection. The expression of iNOS genes (iNOS A and B) was significantly delayed (day 14?pi) in the kidney, liver and spleen of infected fish. These results provide insights into the interaction between T.?carassii and goldfish, and suggest that Th1/Th2-like responses may be important for controlling T.?carassii infection in the goldfish.     

Molecular cloning and expression analysis of two lipopolysaccharide-induced TNF-α factors (LITAFs) from rock bream,Oplegnathus fasciatus

Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α factor (LITAF) plays an important role controlling the expression of TNF-α and the other cytokine genes in the presence of LPS. However, two LITAF homologues have not been characterized in fish. In this study, we cloned two distinct LITAF (RbLITAF1 and RbLITAF2) cDNAs from rock bream (Oplegnathus fasciatus) and characterized their expression profiles after infection with Edwardsiella tarda, Streptococcus iniae or red seabream iridovirus (RSIV). The coding regions of RbLITAF1 and RbLITAF2 cDNAs were 492 bp and 417 bp, encoding 153 and 138 amino acid residues, respectively. The genes consisted of a LITAF domain. RbLITAF1 was highly expressed in the spleen and heart of healthy rock bream, whereas RbLITAF2 was highly expressed in the gill, intestine and stomach. In spleen, the gene expression of RbLITAF1 and RbLITAF2 were increased until 5 days post-infection (dpi), and then decreased at 7 dpi. In kidney, E. tarda and RSIV infection led to induction of the RbLITAF1 gene at 1 dpi, RbLITAF2 gene was down-regulated after pathogen infection. These results suggest that RbLITAFs may be involved in the LITAF-mediated immune response and regulate systemic immune responses against pathogen infection.     

Identification and characterisation of the Mpeg1 homologue in the South African abalone, Haliotis midae

Although Haliotis midae is the most economically important cultured abalone species in South Africa, infectious diseases have the potential to severely limit the production of this shellfish. Consequently, it is becoming increasingly important to characterise the abalone immune system in order to better understand their ability to combat infection. This study reports the identification and characterisation of a perforin-like protein, designated hmMpeg1, which is believed to be involved in the H. midae immune system. hmMpeg1 encodes for a 78 kDa protein that has significant sequence similarity to Mpeg proteins from other abalone species and includes the conserved cytolytic membrane attack complex/perforin (MACPF) domain of perforin. Real-time quantitative PCR (qPCR) analysis demonstrated expression of hmMpeg1 mRNA in haemocytes and epipodia samples from H. midae exposed to a heat-killed, Gram-negative bacterial pathogen, Vibrio anguillarum 5676. hmMpeg1 mRNA in haemocytes increased significantly 48 h post-infection (h.p.i) (8.2 fold; P < 0.05), coinciding with a decrease in the total number of circulating haemocytes, and reached a maximum at 96 h.p.i (17.2 fold; P < 0.05). Similarly, a significant increase in the level of hmMpeg1 mRNA occurred at 24 h.p.i in epipodia samples (3.8 fold; P < 0.05), reaching a maximum at 48 h.p.i (4.5 fold; P < 0.05). In addition, western blot analysis detected a significant increase in hmMpeg1 between 24 h.p.i (4.2 fold; P < 0.05) and 48 h.p.i (3.1 fold; P < 0.05) in the epipodia, and between 48 h.p.i (1.7 fold; P < 0.05) and 96 h.p.i (1.9 fold; P < 0.05) in haemocytes, sampled from abalone exposed to the abalone pathogen V. anguillarum 5676. The importance of hmMpeg1, in terms of its function and importance in the H. midae immune response, is discussed.     

Primary culture of hemocytes from Eriocheir sinensis and their immune effects to the novel crustacean pathogen Spiroplasma eriocheiris

To investigate the interaction between Chinese mitten crab Eriocheir sinensis hemocytes and the pathogen Spiroplasma eriocheiris, a system for in vitro culture of E. sinensis hemocytes with high viability was developed. Following optimization of conditions, hemocytes survived for?>35 days. After challenge with the novel crustacean pathogen S.?eriocheiris, E. sinensis hemocytes began to develop vacuoles, and then they began to die (within 60?h). Real-time RT-PCR analysis of S. eriocheiris infected hemocytes identified increased expression levels of anti-lipopolysaccharide factor (ALF), peroxinectin (Pox) and clip domain serine protease (cSP) genes. The expression levels of ALF, Pox, and cSP genes in hemocytes of E. sinensis demonstrated that all three immune genes were significantly induced by challenge with S. eriocheiris. Increases in Pox mRNA levels were highest (up to 36-fold) and peaked at 24-48?h post-challenge (pc) (P?相似文献   

Cloning and characterization of a hsp70 gene from Asiatic hard clam Meretrix meretrix which is involved in the immune response against bacterial infection

In the present study, a 71.43 kDa heat shock protein cDNA was cloned from Asiatic hard clam Meretrix meretrix. The cDNA was 2292 bp, containing an open reading frame (ORF) of 1959 bp, which encodes a protein of 652 amino acids with a theoretical molecular weight of 71.43 kDa and an isoelectric point of 5.32. Based on the amino acid sequence analysis and phylogenetic analysis, this hsp70 cDNA is a member of cytoplasmic hsc70 (constitutive genes) subfamily in the hsp70 family, and is designated as MmeHsc71. Quantitative RT-PCR was carried out to compare the spatial and temporal expression patterns of MmeHsc71 in the mRNA level between control clams and Vibrio parahaemolyticus-infected clams. Spatially, MmeHsc71 mRNA was found in all tested tissues, including foot, hepatopancreas, mantle and gill. MmeHsc71 mRNA expression level in hepatopancreas and gill displayed a significant increase in vibrio-challenged clams at 24h post-infection compared to control clams (P < 0.05). Temporally, there was a significant increase of MmeHsc71 mRNA level in hepathopancreas of vibrio-challenged clams compared to control clams at 6, 12, and 24h post-challenge, respectively. The result of quantitative immunofluorescence also indicated that there was obvious increase of MmeHsc71 in hepatopancreas of vibrio-challenged clams compared to control clams in protein level at 24h post-infection. The results suggested that MmeHsc71 may play an important role in mediating the immune responses of M. meretrix to bacterial challenge.     

The immune response of white shrimp Litopenaeus vannamei following Vibrio alginolyticus injection

White shrimp Litopenaeus vannamei injected with saline, and injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus at 1.0 x 10(5) and 1.8 x 10(5) colony-forming units (cfu) shrimp(-1) were examined for hyaline cell (HC) counts, granular cell (GC) counts, total haemocyte counts (THCs), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after 1-168 h. Shrimp that received no injection served as the control. The shrimps which received V. alginolyticus at both doses showed significant decreases in these parameters after 6-96 h. The values for HC and SOD activity decreased earlier and then RB. The time to cause maximum depletion of haemocytes (haemocytopenia), PO activity, RB, and SOD activity were 12, 72, 48, and 24 h post-injection, respectively. The HC, GC, and RB returned to the original values earlier at 72 h, followed by SOD activity at 96 h, and then PO activity at 168 h post-infection. It was concluded that an injection of V. alginolyticus rapidly reduced the shrimp's immunity by decreasing HC, GC, SOD activity, RB, and PO activity within 3-24 h, followed by a slow recovery during 72-168 h post-injection. Furthermore, white shrimp L. vannamei which received V. alginolyticus showed a 6-9 h later response in PO activity, and a 72-96 h later recovery of PO activity, compared to the responses in RB and SOD activity indicating their roles in shrimp defence and immunity.     

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa

Background

The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na⁺-K⁺-ATPase.

Methods

Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na⁺-K⁺-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.

Results

The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α₁Na⁺-K⁺-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.

Conclusions

These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.     

An Mpeg (macrophage expressed gene) from the Pacific oyster Crassostrea gigas: molecular characterization and gene expression

Mpegs (macrophage expressed genes) encode members of the MACPF (membrane-attack complex/perforin) protein superfamily that play essential roles in innate immunity. In the present study, a homolog of Mpeg1 was identified in Crassostrea gigas and designed Cg-Mpeg1. The complete cDNA of Cg-Mpeg1 is 2781 bp in length, containing an ORF of 2226 bp, which encodes a putative protein of 742 amino acids with a predicted 19-aa hydrophobic signal peptide, an MACPF domain, and a transmembrane domain. Phylogenetic analysis shows that Cg-Mpeg1 is similar to other mollusk MACPF proteins and might originate in an ancient ancestor gene before the divergence of protostomes and deuterostomes. Localization study revealed that Cg-Mpeg1 protein is found primarily in late endosomes. The MACPF domain from Cg-Mpeg1 exhibits significant antibacterial activity to both Gram-negative and positive bacteria. Furthermore, Real-time Quantitative PCR analysis showed that Cg-Mpeg1 is expressed in all tissues detected with highest expression in gill and gonads. Moreover, Mpeg1 mRNA levels are significantly up-regulated following infection with Vibrio alginolyticus. These results highlight that Cg-Mpeg1 plays an essential role in host defense and elimination of pathogens in C. gigas.     

CD8alpha and CD8beta in Atlantic halibut, Hippoglossus hippoglossus: cloning, characterization and gene expression during viral and bacterial infection

CD8 is expressed on cytotoxic T-cells where it functions as a co-receptor for the TCR by binding to MHC class I proteins that present peptides on the cell surface. In this study we describe the cloning and sequencing of full length cDNAs encoding CD8alpha and CD8beta from Atlantic halibut (Hippoglossus hippoglossus L.) and subsequent isolation and characterization of the CD8alpha and CD8beta genes. The predicted halibut CD8alpha and CD8beta proteins are similar to those of mammals and other fish. Real time RT-PCR revealed that the highest levels of CD8 mRNA were found in the thymus, while some expression was also seen in the spleen, the gills, and the anterior and posterior kidney. In situ hybridization confirmed that the halibut thymus contained numerous CD8alpha and CD8beta expressing cells, while the anterior kidney had no CD8alpha positive cells but only a few CD8beta expressing cells. Only moderate changes in CD8 mRNA expression in other organs during either nodavirus or Vibrio anguillarum infection were observed. Both CD8alpha and CD8beta were significantly (P<0.05) down-regulated in spleen at 48h compared to their levels at 12h post-infection with nodavirus and V. anguillarum.     

两种沼虾溶菌酶基因ORF的克隆和罗氏沼虾溶菌酶基因的组织表达(英文)

分别提取罗氏沼虾和日本沼虾血细胞总RNA,RT-PCR扩增获得特异性cDNA片段,纯化后克隆到T载体上。序列测定表明所克隆的两种沼虾溶菌酶基因的开放阅读框(ORF)为477bp,共编码158个氨基酸,包括溶菌酶成熟肽140个氨基酸残基和信号肽18个氨基酸残基。同源性分析表明,罗氏沼虾和日本沼虾溶菌酶基因的碱基序列及推测氨基酸序列高度同源,分别为99.4%和98.1%。两种沼虾溶菌酶基因的碱基序列和推测氨基酸序列与Gen-Bank上其他对虾溶菌酶的同源性达83.0%和80.0%以上。两种沼虾溶菌酶都具有c-型溶菌酶典型的两个酶活性位点(Glu51)和(Asp68),以及8个保守结构氨基酸残基Cys,且在101、106和107位上缺少Asp,因而推测本实验所克隆的两种沼虾溶菌酶基因属c-型溶菌酶基因的非钙结合亚型。以PCR法制备罗氏沼虾溶菌酶基因的生物素标记探针,斑点杂交检测感染弧菌后溶菌酶基因mRNA在各组织中的转录水平,结果表明受感染6h后在眼、肌肉、鳃、肝胰腺、肠管中的表达量均有升高,其中在肝胰腺中的表达量最高,约为对照组的560%。在不同感染时间里,肝胰腺中该基因表达量有较大的变化:感染后3h表达量最低,24h后表达量升至最高,大约为对照组的430%,48h时的表达量又有所下降,但仍明显高于对照组(约为330%)。受弧菌感染后罗氏沼虾溶菌酶基因转录的上调证明溶菌酶基因在非特异性免疫中的直接作用,同时表明肝胰腺可能在沼虾的免疫防御过程起重要作用。       

cDNA cloning and mRNA expression of a tandem-repeat galectin (PoGal2) from the pearl oyster, Pinctada fucata

Galectins can recognize and specifically bind to β-galactoside residues, playing crucial roles in innate immune responses of vertebrates and invertebrates. We cloned the cDNA of a tandem-repeat galectin from the pearl oyster Pinctada fucata (designated as PoGal2). PoGal2 cDNA is 1347 bp long and consists of a 5'-untranslated region (UTR) of 3 bp, a 3'-UTR of 297 bp with one cytokine RNA instability motif (ATTTA), and an open reading frame of 1047 bp, encoding a polypeptide of 349 amino acids, with an estimated molecular mass of 38.1 kDa and a theoretical isoelectric point of 8.5. PoGal2 contains two carbohydrate recognition domains (CRDs); both have the conserved carbohydrate-binding motifs H-NPR and WG-EE. PoGal2 shares 50.6 and 50.9% identity with those of abalone (Haliotis discus) and the Manila clam (Venerupis philippinarum), respectively. Phylogenetic analysis revealed that the tandem-repeat galectins formed two clades for the different species. Molluscan tandem-repeat galectins were clustered into a single clade, and nematode tandem-repeat galectins were clustered into another single clade. In both clades, CRD-N and CRD-C were divided into different groups. PoGal2 mRNA was constitutively expressed in all tissues analyzed, and the expression level of PoGal2 mRNA was found to be significantly up-regulated in digestive glands, gills and hemocytes after Vibrio alginolyticus stimulation/infection. Expression profile analysis showed that the expression level of PoGal2 mRNA was significantly up-regulated at 8, 12 and 24 h after V. alginolyticus infection. These results suggest that PoGal2 is a constitutive and inducible acute-phase protein involved in the innate immune response of pearl oysters.     

Identification of rice genes induced in a rice blast-resistant mutant

To clarify mechanisms of rice blast resistance in rice plants we used suppression subtractive hybridization (SSH) to isolate genes induced upon rice blast inoculation in a rice blast-resistant mutant. A total of 26 rice cDNAs were isolated and found to have elevated expression upon rice blast infection in a rice blast-resistant derivative, SHM-11, of the rice cultivar, Sanghaehyanghyella. Sequencing of the cDNAs revealed that many of the proteins they encoded had been previously described as involved in plant responses against pathogen attack. Two interesting groups of the defense-related proteins consisted of three different PR5 homologues and four different protease inhibitors, all highly expressed in the rice blast mutant. Genes encoding proteins involved in signal transduction and regulation were also identified, including translation initiation factor eIF5A, C2 domain DNA binding protein, putative rice EDS and putative receptor like kinase. Most of the identified cDNAs were highly expressed 24 h after blast inoculation. Our results suggest that a pathway regulating defense gene expression may be altered in the mutant, resulting in early induction of the defense genes upon fungal infection.