Analysis of the immune-inducible genes of Plutella xylostella using expressed sequence tags and cDNA microarray

Eum, J.H.,Seo, Y.R.,Yoe, S.M.,Kang, S.W.,Han, S.S. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2007 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.31 No.11

In the present study, the complex gene expression responses of Plutella xylostella to microbial challenges and injury were surveyed using a newly constructed expressed sequence tag (EST) clone collection and cDNA microarray analysis. A total of 1132 P. xylostella ESTs were cloned, annotated and categorized by their putative functions; these included proteases, protease inhibitors, recognition molecules and anti-microbial peptides. GeneOntology revealed that 4% of the P. xylostella ESTs corresponded to immunity-related genes potentially involved in innate immunity. We then used microarray analysis to identify 44 genes that were differentially expressed with at least a two-fold expression difference in P. xylostella before and after pathogen challenge. Together, our EST categorization and microarray profiling analyses allowed us to identify 70 genes that should be considered candidate immune response genes, providing important new insights into the molecular events that occur during the innate immune response in P. xylostella.

Characterization and antiviral function of a cytosolic sensor gene, MDA5, in Japanese flounder, Paralichthys olivaceus

Ohtani, M.,Hikima, J.i.,Kondo, H.,Hirono, I.,Jung, T.S.,Aoki, T. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 Developmental and comparative immunology Vol.35 No.5

Cytosolic pattern recognition receptors such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) play an important role in sensing viral RNAs. The receptor encoded by melanoma differentiation-associated gene 5 (MDA5), an RLR, recognizes viral RNA in the cytoplasm and enhances antiviral response in host cells. The full-length MDA5 gene in Japanese flounder, Paralichthys olivaceus was cloned and found to have 11,251 nucleotides. MDA5 transcript abundance was significantly increased in whole kidney infected with viral hemorrhagic septicemia virus (VHSV) as well as whole kidney and peripheral blood leukocytes stimulated with poly I:C in vitro. Hirame natural embryo (HINAE) cells overexpressing MDA5 showed a lower cytopathic effect (CPE) against VHSV, hirame rhabdovirus (HIRRV) and infectious pancreatic necrosis virus (IPNV) infection. When infected with VHSV, MDA5-overexpressing HINAE cells had 24-75 fold lower virus titer than normal HINAE cells. These results suggest that Japanese flounder MDA5 is involved in the induction of antiviral response.

Innate immune response in the hemolymph of an ascidian, Halocynthia roretzi, showing soft tunic syndrome, using label-free quantitative proteomics

Cha, I.S.,Castillo, C.S.d.,Nho, S.W.,Hikima, J.i.,Aoki, T.,Jung, T.S. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 Developmental and comparative immunology Vol.35 No.8

Soft tunic syndrome of Halocynthia roretzi manifests as soft, weak, and rupturable tunics, causing mass mortality. Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS), innate immune response was established by comparing hemolymph protein profiles of ascidians with healthy or softened tunics. Of 100 proteins in each individual ascidian, 59 proteins from healthy and 56 proteins from diseased ascidians were functionally classified. Proteins found only in diseased individuals included trypsin inhibitor and Hr-29, and with high exponentially modified protein abundance index (emPAI) values. From 41 proteins identified to be common to both healthy and diseased ascidians, 15 were associated with innate immune response. Ficolin 3, a component of the lectin-complement system, was significantly decreased in diseased ascidians, but a cell surface protein, type II transmembrane serine protease-1 (TTSP), was considerably elevated. These results suggest that trypsin inhibitor, ficolin 3, and TTSP are probably involved in the innate immune response related to this tunic disease. Beside, Hr-29 could be suggested as a biomarker for soft tunic syndrome.

Molecular cloning and expression analysis of two distinct F-type lectins from the rock bream, Oplegnathus fasciatus

Park, H.J.,Kim, J.W.,Kim, E.G.,Kim, H.N.,Chae, Y.S.,Jeong, J.M.,Kim, D.H.,Park, C.I. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 Developmental and comparative immunology Vol.36 No.1

Several lectin families characterized by distinct signature sequence motifs and structural folds, such as C-type, peptidoglycan recognition protein, ficolin, pentraxins, and most recently galectins, have been implicated in immune surveillance. In this study, two distinct F-type lectins RbFTL-1 and RbFTL-2, from the rock bream (Oplegnathus fasciatus), were identified and their expression was analyzed. The full-length cDNA of RbFTL-1 was composed of 1204bp with a 945-bp open reading frame (ORF) that encoded a 314 amino-acid protein, while that of RbFTL-2 consisted of 1614bp with a 951-bp ORF encoding a 316 amino-acid protein. RbFTL-1 and RbFTL-2 mRNAs were predominately expressed in the head-kidney and in the liver, respectively. Levels of the RbFTL-1 mRNA transcript increased up to 5.0- and 2.8-fold in the head-kidney and trunk-kidney compared to the muscle, respectively, while those of the RbFTL-2 mRNA transcript increased up to 12.0-fold in liver. The expression of RbFTL-1 and RbFTL-2 were differentially up-regulated in rock bream challenged with Edwardsiella tarda, Streptococcus iniae, and RSIV, with significant increases at 1 and 3h post-challenge compared to the controls.

Rock bream (Oplegnathus fasciatus) serpin, protease nexin-1: Transcriptional analysis and characterization of its antiprotease and anticoagulant activities

Umasuthan, N.,Whang, I.,Kim, J.O.,Oh, M.J.,Jung, S.J.,Choi, C.Y.,Yeo, S.Y.,Lee, J.H.,Noh, J.K.,Lee, J. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 Developmental and comparative immunology Vol.35 No.7

Protease nexin-1 (PN-1) is a serine protease inhibitor (SERPIN) protein with functional roles in growth, development, patho-physiology and injury. Here, we report our work to clone, analyze the expression profile and characterize the properties of the PN-1 gene in rock bream (Rb), Oplegnathus fasciatus. RbPN-1 encodes a peptide of 397 amino acids (AA) with a predicted molecular mass of 44kDa and a 23 AA signal peptide. RbPN-1 protein was found to harbor a characteristic SERPIN domain comprised of a SERPIN signature and having sequence homology to vertebrate PN-1s. The greatest identity (85%) was observed with PN-1 from the three-spined stickleback fish, Gasterosteus aculeatus. The functional domains, including a heparin binding site and reactive centre loop were conserved between RbPN-1 and other fish PN-1s; in particular, they were found to correspond to components of the human plasminogen activator inhibitor 1, PAI-1. Phylogenetic analysis indicated that RbPN-1 was closer to homologues of green spotted pufferfish and Japanese pufferfish. Recombinant RbPN-1 demonstrated antiprotease activity against trypsin (48%) and thrombin (89%) in a dose-dependent manner, and its antithrombotic activity was potentiated by heparin. The anticoagulant function prolonged clotting time by 3.7-fold, as compared to the control in an activated partial thromboplastin time assay. Quantitative real-time PCR results indicated that RbPN-1 is transcribed in many endogenous tissues at different levels. Lipopolysaccharide (LPS) stimulated a prolonged transcriptional response in hematic cells, and Rb iridovirus up-regulated the RbPN-1 mRNA level in hematic cells to a maximum of 3.4-fold at 12h post-infection. Interestingly, LPS and Edwardsiella tarda significantly induced the RbPN-1 transcription at the late phase of infection. In vivo studies indicated that injury response caused a temporal suppression in RbPN-1 transcription, in conjunction with that of another SERPIN, rock bream heparin cofactor II, RbHCII. Taken together, our findings suggest that PN-1 functions as an antiprotease and anticoagulant and that SERPINs (PN-1 and HCII) are likely to contribute to immunity and post-injury responses.

Chicken IL-17F: Identification and comparative expression analysis in Eimeria-infected chickens

Kim, W.H.,Jeong, J.,Park, A.R.,Yim, D.,Kim, Y.H.,Kim, K.D.,Chang, H.H.,Lillehoj, H.S.,Lee, B.H.,Min, W. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 Developmental and comparative immunology Vol.38 No.3

Interleukin-17F (IL-17F) is a proinflammatory cytokine, which plays an important role in gut homeostasis. A full-length chicken IL-17F (chIL-17F) cDNA with a 510-bp coding region was identified from ConA-activated chicken splenic lymphocytes. ChIL-17F shares 53% amino acid sequence identity with the previously described chicken IL-17 (chIL-17A) and 38-43% with mammalian homologues. The locus harboring chIL-17 and chIL-17F displayed inverted order compared to those of mammals. ChIL-17F transcript expression was high in lymphoblast cell line CU205 and at moderate levels in small and large intestines and liver. ChIL-17F and chIL-17 expression profiles were examined by quantitative real-time RT-PCR in mitogen-stimulated splenic lymphocytes and intestinal areas affected by Eimeria maxima and Eimeria tenella infections. Expression levels of chIL-17F, like chIL-17, were elevated in mitogen-activated splenic lymphocytes. ChIL-17F, but not chIL-17, expression was upregulated in intestinal tissues affected by E. maxima and E. tenella infections. Recombinant chIL-17F biological activities were similar to that of chIL-17 in primary chicken embryonic fibroblasts. These results suggest that chIL-17F is a unique member of the IL-17 family of cytokines.

Cathepsins in the kidney of olive flounder, Paralichthys olivaceus, and their responses to bacterial infection

Cha, I.S.,Kwon, J.,Mun, J.Y.,Park, S.B.,Jang, H.B.,Nho, S.W.,del Castillo, C.S.,Hikima, J.i.,Aoki, T.,Jung, T.S. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 Developmental and comparative immunology Vol.38 No.4

Cathepsin activities are responsible for mediating various pathways involved in immune response, including the apoptosis pathway, toll-like receptor (TLR) signaling, cytokine induction and activation of granule serine proteases. In the present study, we investigated cathepsin responses in the kidneys of olive flounder infected with Streptococcus parauberis, analyzing cathepsin expression using a label-free, quantitative proteomic approach in conjunction with quantitative real-time polymerase chain reaction (qRT-PCR). In proteomic analyses, we detected cathepsin B, D, L and S proteins, noting significant decreases and increases in cathepsins B and L, respectively, with infection. Taken together with an evaluation of cathepsin B, D, F, K, L, S and X gene expression in normal and infected kidneys by qRT-PCR, our results indicate that cathepsins B, D, L and S are the dominant lysosomal proteases in the immune system of the teleostei, olive flounder. Cathepsins F, K and X were regarded as minor cathepsins.

Bombyx mori transferrin: Genomic structure, expression and antimicrobial activity of recombinant protein

Yun, E.Y.,Lee, J.K.,Kwon, O.Y.,Hwang, J.S.,Kim, I.,Kang, S.W.,Lee, W.J.,Ding, J.L.,You, K.H.,Goo, T.W. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2009 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.33 No.10

Transferrin (Tf) is a multifunctional, iron binding protein found in both vertebrates and invertebrates. Although transferrin has been suggested to play a role in innate immunity, its immunological function during infection has not been characterized. In this study, we identified and characterized Bombyx mori transferrin (BmTf). The promoter region of BmTf has numerous putative NF-κB binding sites, suggesting its possible function in innate immunity. Analysis of BmTf gene expression shows that it is highly inducible in response to a wide variety of pathogens including bacteria, fungus, and viruses. Recombinant BmTf protein produced in a baculovirus system exhibits iron binding capacity and antibacterial activity against various Gram-positive and -negative bacteria. Taken together, our results indicate that BmTf is an inducible immune effector molecule that may play an important role in pathogen clearance of insect innate immunity.

Protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus: Sequence analysis, expression profile, and a possible biological role in host immunosuppression

Ibrahim, A.M.A.,Choi, J.Y.,Je, Y.H.,Kim, Y. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2007 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.31 No.10

A genome project has been launched and aims to sequence total genome of Cotesia plutellae bracovirus (CpBV). On this process, several putative open reading frames have been proposed, among which there was a large gene family coding for protein tyrosine phosphatases (PTPs). This study analyzed the deduced amino acid sequences of 14 CpBV-PTPs in terms of conserved domains with other known polydnaviral PTPs and determined their expression patterns in diamondback moth, Plutella xylostella, parasitized by C. plutellae. The analyzed CpBV-PTPs share the common 10 motifs with classical type of PTPs. However, there are variations among CpBV-PTPs in active site sequence and phosphorylation sites. Quantitative real-time polymerase chain reaction (PCR) indicated that most PTPs in the parasitized P. xylostella were expressed from the first day of parasitization and increased the expression levels during parasitization. All 14 PTPs were expressed in both immune-associated tissues of fat body and hemocytes in the parasitized host. During last instar, the PTP enzyme activity of the parasitized P. xylostella was significantly lower than that of the nonparasitized. The reduction of the PTP activity was observed in cytosolic fraction, but not in membrane fraction. The hemocytes of parasitized P. xylostella markedly lost their spreading ability in response to a cytokine (PSP1: plasmatocyte-spreading peptide 1). The functional link between the reduced PTP activity and the suppressed hemocytic behavior was evidenced by the inhibitory effect of sodium orthovanadate (a specific PTP inhibitor) on hemocyte-spreading behavior of nonparasitized P. xylostella. These results suggest that CpBV-PTPs are expressed in the parasitized P. xylostella and affect cellular PTP activity, which may be associated with host immunosuppression.

Genomic characterization and expression profiles upon bacterial infection of a novel cystatin B homologue from disk abalone (Haliotis discus discus)

Premachandra, H.K.A.,Wan, Q.,Elvitigala, D.A.S.,De Zoysa, M.,Choi, C.Y.,Whang, I.,Lee, J. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.38 No.4

Cystatins are a large family of cysteine proteinase inhibitors which are involved in diverse biological and pathological processes. In the present study, we identified a gene related to cystatin superfamily, AbCyt B, from disk abalone Haliotis discus discus by expressed sequence tag (EST) analysis and BAC library screening. The complete cDNA sequence of AbCyt B is comprised of 1967 nucleotides with a 306bp open reading frame (ORF) encoding for 101 amino acids. The amino acid sequence consists of a single cystatin-like domain, which has a cysteine proteinase inhibitor signature, a conserved Gly in N-terminal region, QVVAG motif and a variant of PW motif. No signal peptide, disulfide bonds or carbohydrate side chains were identified. Analysis of deduced amino acid sequence revealed that AbCyt B shares up to 44.7% identity and 65.7% similarity with the cystatin B genes from other organisms. The genomic sequence of AbCyt B is approximately 8.4Kb, consisting of three exons and two introns. Phylogenetic tree analysis showed that AbCyt B was closely related to the cystatin B from pacific oyster (Crassostrea gigas) under the family 1.Functional analysis of recombinant AbCyt B protein exhibited inhibitory activity against the papain, with almost 84% inhibition at a concentration of 3.5μmol/L. In tissue expression analysis, AbCyt B transcripts were expressed abundantly in the hemocyte, gill, mantle, and digestive tract, while weakly in muscle, testis, and hepatopancreas. After the immune challenge with Vibrio parahemolyticus, the AbCyt B showed significant (P<0.05) up-regulation of relative mRNA expression in gill and hemocytes at 24 and 6h of post infection, respectively. These results collectively suggest that AbCyst B is a potent inhibitor of cysteine proteinases and is also potentially involved in immune responses against invading bacterial pathogens in abalone.