12 results
Search Results
Now showing 1 - 10 of 12
Publication Metadata only Identification of immune-related genes in hemocytes of black tiger shrimp (Penaeus monodon)(2002-01-01) Premruethai Supungul; Sirawut Klinbunga; Rath Pichyangkura; Sarawut Jitrapakdee; Ikuo Hirono; Takashi Aoki; Anchalee Tassanakajon; Chulalongkorn University; Thailand National Center for Genetic Engineering and Biotechnology; Mahidol University; National University Corporation Tokyo University of Marine Science and TechnologyAn expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 × 105. Of these, 615 clones having inserts larger than 500 bp were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the clotting system and the prophenoloxidase-activating system, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported. © Springer-Verlag New York Inc.Publication Metadata only Gene silencing of a prophenoloxidase activating enzyme in the shrimp, Penaeus monodon, increases susceptibility to Vibrio harveyi infection(2009-07-01) Walaiporn Charoensapsri; Piti Amparyup; Ikuo Hirono; Takashi Aoki; Anchalee Tassanakajon; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; National University Corporation Tokyo University of Marine Science and TechnologyThe prophenoloxidase (proPO) activating system is an important innate immune response against microbial infections in invertebrates. The major enzyme, phenoloxidase (PO), is synthesized as an inactive precursor and its activation to an active enzyme is mediated by a cascade of clip domain serine proteinases (clip-SPs). In this study, a cDNA encoding a proPO activating enzyme (PPAE) from the black tiger shrimp, Penaeus monodon, designated as PmPPAE1, was cloned and characterized. The full-length cDNA contains an open reading frame (ORF) of 1392 bp encoding a predicted protein of 463 amino acids including an 18 amino acid signal peptide. The PmPPAE1 protein exhibits a characteristic sequence structure of clip-SPs consisting of the clip domain at the N-terminus and a SP domain at the C-terminus. Sequence analysis showed that PmPPAE1 exhibited the highest amino acid sequence similarity (70%) to a PPAE of the crayfish, Pacifastacus leniusculus. PmPPAE1 mRNA is abundantly expressed in hemocytes, and this is regulated after systemic Vibrio harveyi infection supporting that it is an immune-responsive gene. RNA interference-mediated suppression of PmPPAE1, performed by injection of double-stranded RNA (dsRNA) corresponding to the PmPPAE1 gene into shrimp, resulted in a significant reduction of PmPPAE1 but not other clip-SP and related gene transcript levels of P. monodon, suggesting gene-specific knockdown. RNAi-mediated silencing of PmPPAE1 gene significantly decreased the total PO activity (36.7%) in shrimp and additionally increased the mortality of V. harveyi infected shrimp, the latter of which correlated with an increase in the number of viable bacteria in the hemolymph. These results indicate that PmPPAE1 functions in the proPO system and is an important component in the shrimp immune system. © 2009 Elsevier Ltd. All rights reserved.Publication Metadata only WSV399, a viral tegument protein, interacts with the shrimp protein PmVRP15 to facilitate viral trafficking and assembly(2016-06-01) Phattarunda Jaree; Saengchan Senapin; Ikuo Hirono; Chu Fang Lo; Anchalee Tassanakajon; Kunlaya Somboonwiwat; Chulalongkorn University; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; National University Corporation Tokyo University of Marine Science and Technology; National Cheng Kung University© 2016 Elsevier Ltd. Viral responsive protein 15 (PmVRP15) has been identified as a highly up-regulated gene in the hemocyte of white spot syndrome virus (WSSV)-infected shrimp Penaeus monodon. However, the function of PmVRP15 in host-viral interaction was still unclear. To elucidate PmVRP15 function, the interacting partner of PmVRP15 from WSSV was screened by yeast two-hybrid assay and then confirmed by co-immunoprecipitation (Co-IP). Only WSV399 protein was identified as a PmVRP15 binding protein; however, the function of WSV399 has not been characterized. Localization of WSV399 on the WSSV virion was revealed by immunoblotting analysis (in vitro) and immunoelectron microscopy (in vivo). The results showed that WSV399 is a structural protein of the WSSV virion and is particularly located on the tegument. Gene silencing of wsv399 in WSSV-infected shrimp reduced the percentage of cumulative mortality by 74%, although the expression level of a viral replication marker gene, vp28, was not changed suggesting that WSV399 might not involved in viral replication but viral assembly. Because it has already been known that tegument proteins function in capsid transport during viral trafficking and assembly, interaction between PmVRP15 on hemocyte nuclear membrane and the WSV399 viral tegument protein suggests that PmVRP15 might be required for trafficking and assembly of WSSV during infection.Publication Metadata only Molecular cloning, genomic organization and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp Penaeus monodon(2008-02-01) Piti Amparyup; Hidehiro Kondo; Ikuo Hirono; Takashi Aoki; Anchalee Tassanakajon; Thailand National Center for Genetic Engineering and Biotechnology; Mahidol University; National University Corporation Tokyo University of Marine Science and TechnologyA novel crustin-like antimicrobial peptide (Crus-likePm) was identified from haemocytes of Penaeus monodon. The deduced amino acid sequence of a Crus-likePm consists of 124 amino acid residues of the mature peptide and a signal peptide of 17 amino acid residues. The mature peptide contains a glycine-rich domain at the N-terminus and 12 conserved cysteine residues containing a single WAP domain at the C-terminus. Phylogenetic tree and sequence comparison clearly confirmed a distinct between a Crus-likePm and other shrimp crustins. Genomic organization and upstream region of a Crus-likePm gene was investigated. The gene consisted of two exons and one intron. The 5′-flanking regions of a Crus-likePm gene contain multiple putative transcription factor binding sites. mRNA transcript of a Crus-likePm was found to be abundantly expressed in haemocyte and highly up-regulated after Vibrio harveyi injection. The mature Crus-likePm was cloned into the pET28b with an N-terminal hexa-histidine tag fused in-frame, and expressed in E. coli. The purified recombinant Crus-likePm showed strong antimicrobial activity against both Gram-positive and Gram-negative bacteria including V. harveyi, a major pathogenic bacteria in shrimp aquaculture. © 2007 Elsevier Ltd. All rights reserved.Publication Metadata only Cloning, expression and antimicrobial activity of crustinPm1, a major isoform of crustin, from the black tiger shrimp Penaeus monodon(2008-01-01) Premruethai Supungul; Sureerat Tang; Cherdsak Maneeruttanarungroj; Vichien Rimphanitchayakit; Ikuo Hirono; Takashi Aoki; Anchalee Tassanakajon; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; National University Corporation Tokyo University of Marine Science and TechnologyCrustin antibacterial homologues, containing a whey acidic protein (WAP) domain, have been identified from the haemocyte library of the black tiger shrimp, Penaeus monodon. Sequence analysis of these cDNAs indicates the presence of several isoforms of crustin in P. monodon. CrustinPm1, the most abundant isoform, contains an open reading frame of 435 bp encoding a precursor of 145 amino acids that comprises 17 amino acid signal peptides and 128 amino acid mature peptides. The peptides contain a Gly-Pro rich region at the amino-terminus and a single whey acidic protein (WAP) domain at the carboxyl-terminus. In order to characterize the properties and biological activities of this peptide, crustinPm1 was overexpressed in Escherichia coli. The recombinant crustinPm1 has a molecular mass of 14.7 kDa with a predicted pI of 8.3. Antimicrobial assays demonstrated that recombinant crustinPm1 exhibited antimicrobial activity against only Gram-positive bacteria with strong inhibition against Staphylococcus aureus and Streptococcus iniae. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of crustinPm1 was a result of bactericidal effect. In situ hybridization with crustinPm1 antisense probes showed strong hybridization signals in a certain haemocyte population of unchallenged shrimp, indicating that crustinPm1 transcript is differentially expressed in different subsets of haemocyte cells. © 2007 Elsevier Ltd. All rights reserved.Publication Metadata only Molecular characterization and expression analysis of a c-type and two novel muramidase-deficient i-type lysozymes from Penaeus monodon(2010-03-01) Premruethai Supungul; Vichien Rimphanitchayakit; Takashi Aoki; Ikuo Hirono; Anchalee Tassanakajon; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; National University Corporation Tokyo University of Marine Science and TechnologyLysozyme is a widely distributed hydrolase possessing a hydrolytic activity against peptidoglycan in the bacterial cell wall and, hence, causing lysis of the bacteria. Two types of lysozymes; the c-type (PmLyzc) and the two catalytic residue ablated i-type lysozymes (PmLyzi1 and 2), were identified from the Penaeus monodon EST database (http://pmonodon.biotec.or.th). By RT-PCR, PmLyzc transcript was detected in all tissues: gill, antennal gland, epipodite, heart, hemocyte, hepatopancreas, eyestalk, lymphoid organ and intestine, and highly expressed in hemocyte. The expression of PmLyzi2 mRNA was highest in heart while undetected in gill, lymphoid organ and intestine. The PmLyzi1 transcript was expressed only in hepatopancreas. The up-regulation of mRNA transcription after bacterial challenge was observed only with PmLyzc. To investigate their biological activities, the three mature recombinant proteins were expressed in an Escherichia coli system. Although the turbidimetric assay revealed that only recombinant PmLyzc possessed the muramidase activity, all of them variably exhibited antimicrobial activity against both Gram-positive and -negative bacteria especially the shrimp pathogens, Vibrio species. The antimicrobial activities of recombinant PmLyzc was the most effective one. These results demonstrated that the ability of lysozyme to inhibit the growth of bacteria did not depend only on the muramidase activity. Differences in tissue expression pattern of these gene transcripts and their antimicrobial activities indicated the multifunction of lysozyme as immune defense and digestive enzymes in P. monodon. © 2010 Elsevier Ltd. All rights reserved.Publication Metadata only Anti-lipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) exhibits antiviral activity by interacting with WSSV structural proteins(2014-01-01) Sivalee Suraprasit; Thanachai Methatham; Phattarunda Jaree; Kornsunee Phiwsaiya; Saengchan Senapin; Ikuo Hirono; Chu Fang Lo; Anchalee Tassanakajon; Kunlaya Somboonwiwat; Chulalongkorn University; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; National University Corporation Tokyo University of Marine Science and Technology; National Taiwan University; National Cheng Kung UniversityIn innate immunity, antimicrobial peptides (AMPs) play a vital role in combating microbial pathogens. Among the AMPs identified in Penaeus monodon, only anti-lipopolysaccharide factor isoform 3 (ALFPm3) has been reported to exhibit activity against white spot syndrome virus (WSSV). However, the mechanism(s) involved are still not clear. In the present study, ALFPm3-interacting proteins were screened for from a WSSV library using the yeast two-hybrid screening system, revealing the five potential ALFPm3-interacting proteins of WSSV186, WSSV189, WSSV395, WSSV458 and WSSV471. Temporal transcriptional analysis in WSSV-infected P. monodon revealed that all five of these WSSV gene transcripts were expressed in the late phase of infection (24 h and 48 h post-infection). Of these, WSSV189 that was previously identified as a structural protein, was selected for further analysis and was shown to be an enveloped protein by Western blot and immunoelectron microscopy analyses. The in vitro pull-down assay using recombinant WSSV189 (rWSSV189) protein as bait confirmed the interaction between ALFPm3 and WSSV189 proteins. Moreover, pre-incubation of rWSSV189 protein with rALFPm3 protein interfered with the latter's neutralization effect on WSSV in vivo, as shown by the increased cumulative mortality of shrimp injected with WSSV following prior treatment with pre-incubated rWSSV189 and rALFPm3 proteins compared to that in shrimp pre-treated with rALFPm3 protein. Thus, ALFPm3 likely performs its anti-WSSV action by binding to the envelope protein WSSV189 and possibly other WSSV structural proteins. © 2014 Elsevier B.V. All rights reserved.Publication Metadata only A novel white spot syndrome virus protein WSSV164 controls prophenoloxidases, PmproPOs in shrimp melanization cascade(2018-09-01) Pakkakul Sangsuriya; Walaiporn Charoensapsri; Jantiwan Sutthangkul; Saengchan Senapin; Ikuo Hirono; Anchalee Tassanakajon; Piti Amparyup; Chulalongkorn University; National University Corporation Tokyo University of Marine Science and Technology; Mahidol University; Thailand National Science and Technology Development Agency© 2018 Elsevier Ltd Melanization, mediated by the prophenoloxidase (proPO)-activating system, is an important innate immune response in invertebrates. The implication of the proPO system in antiviral response and the suppression of host proPO activation by the viral protein have previously been demonstrated in shrimp. However, the molecular mechanism of viral-host interactions in the proPO cascade remains largely unexplored. Here, we characterized the viral protein, namely, WSSV164, which was initially identified from the forward suppression subtractive hybridization (SSH) cDNA library of the PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon that was challenged with white spot syndrome virus (WSSV). Using the yeast two-hybrid system, WSSV164 was found to interact with the PmproPO2 protein. The subsequent validation assay by co-immunoprecipitation revealed that WSSV164 directly bound to both PmproPO1 and PmproPO2. The gene silencing experiment was carried out to explore the role of WSSV164 in the control of the proPO pathway in shrimp, and the results showed that suppression of WSSV164 can restore PO activity in WSSV-infected shrimp hemolymph. The recombinant proteins of PmproPO1 and PmproPO2 were produced in Sf-9 cells and were shown to be successfully activated by exogenous trypsin and endogenous serine proteinases from shrimp hemocyte lysate supernatant (HLS), yielding PO activity in vitro. Moreover, the activated PO activity in shrimp HLS was dose-dependently reduced by the recombinant WSSV164 protein, suggesting that WSSV164 may interfere with the activation of the proPO system in shrimp. Taken together, these results suggest an alternative infection route of WSSV through the encoded viral protein WSSV164 that binds to the PmproPO1 and PmproPO2 proteins, interfering with the activation of the melanization cascade in shrimp.Publication Metadata only Random amplified polymorphic DNA (RAPD) markers for determination of genetic variation in wild populations of the black tiger prawn (Penaeus monodon) in Thailand(1997-06-01) Anchalee Tassanakajon; Siriporn Pongsomboon; Vichien Rimphanitchayakit; Padermsak Jarayabhand; Vichai Boonsaeng; Chulalongkorn University; Mahidol UniversityRandom amplified polymorphic DNA (RAPD) analysis was used to amplify the genome of black tiger prawns (Penaeus monodon) to detect DNA markers and assess the utility of the RAPD method for investigating genetic variation in wild P. monodon in Thailand. A total of 200 ten-base primers were screened, and 84 primers yielded amplification products. Six positive primers that gave highly reproducible RAPD patterns were selected for the analysis of three geographically different samples of Thai P monodon. A total of 70 reproducible RAPD fragments ranging in size from 200 to 2000 bp were scored, and 40 fragments (57%) were polymorphic. The RAPD analysis of broodstocks from three different locales, Satun-Trang, Trat, and Angsila, revealed different levels of genetic variability among the samples. The percentages of polymorphic bands were 48% and 45% in Satun-Trang and Trat, respectively, suggesting a high genetic variability of the two samples to be used in selective breeding programs. Only 25% polymorphic bands were found in the Angsila sample, indicating the lowest polymorphic level among the three samples examined. Primer 428 detected a RAPD marker that was found only in P monodon originating from Satun-Trang, suggesting the potential use of this marker as a population-specific marker in this species.Publication Metadata only Isolation and characterization of microsatellite markers in the black tiger prawn Penaeus monodon(1998-03-01) Anchalee Tassanakajon; Amornrat Tiptawonnukul; Premruethai Supungul; Vichien Rimphanitchayakit; Doug Cook; Padermsak Jarayabhand; Sirawut Klinbunga; Vichai Boonsaeng; Chulalongkorn University; Dalhousie University; Thailand National Center for Genetic Engineering and Biotechnology; Mahidol UniversityIsolation and characterization of microsatellite sequences from the genome of the black tiger prawn Penaeus monodon are described. Ninety-seven (GT)(n) and 16 (CT)(n) microsatellites were isolated from partial genomic libraries composed of 18,000 and 5250 clones, respectively. The genomic library screening indicated that (GT)(n) microsatellites are more abundant than (CT)(n) in P. monodon genome. The microsatellite sequences were classified into three categories, perfect, imperfect, and compound. The predominant categories found in P. monodon microsatellites are imperfect repeats for both (GT)(n) and (CT)(n). Very long repeat arrays were found in P. monodon microsatellite clones, which resulted in difficulties in primer design. Two microsatellite loci, CUPmo 18 and CUPmo 386, were successfully amplified. The number of alleles of each locus was preliminarily determined. For the CUPmo 18 locus, Mendelian inheritance was tested by analysis of genotypic ratios in F1 offspring and their parents. The results of this study demonstrate the presence of highly polymorphic microsatellite markers in P. monodon. These markers will be useful in population studies and parental determination in P. monodon. However, the low abundance and difficulties in obtaining a large number of usable microsatellite loci indicated that these markers may not be appropriate for use in genome mapping of this species.