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1.
Lett Appl Microbiol ; 77(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38719546

RESUMO

Aeromonas dhakensis is reported as an emerging pathogenic species within the genus Aeromonas and is widely distributed in tropical coastal areas. This study provided a detailed description and characterization of a strain of A. dhakensis (202108B1) isolated from diseased Ancherythroculter nigrocauda in an inland region of China. Biochemical tests identified the isolate at the genus level, and the further molecular analysis of concatenated housekeeping gene sequences revealed that the strain belonged to the species A. dhakensis. The isolated A. dhakensis strain was resistant to five antibiotics, namely, penicillin, ampicillin, clindamycin, cephalexin, and imipenem, while it was susceptible to or showed intermediate resistance to most of the other 15 tested antibiotics. The isolated strain of A. dhakensis caused acute hemorrhagic septicemia and tissue damage in artificially infected A. nigrocauda, with a median lethal dose of 7.76 × 104 CFU/fish. The genome size of strain 202108B1 was 5 043 286 bp, including 1 chromosome and 4 plasmids. This is the first detailed report of the occurrence of infection caused by an A. dhakensis strain causing infection in an aquaculture system in inland China, providing important epidemiological data on this potential pathogenic species.


Assuntos
Aeromonas , Antibacterianos , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , China , Aeromonas/genética , Aeromonas/isolamento & purificação , Aeromonas/classificação , Aeromonas/efeitos dos fármacos , Aeromonas/patogenicidade , Animais , Antibacterianos/farmacologia , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Peixes/microbiologia , Filogenia , Testes de Sensibilidade Microbiana , Aquicultura , Genoma Bacteriano , RNA Ribossômico 16S/genética , Plasmídeos/genética
2.
Fish Shellfish Immunol ; 134: 108548, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36690268

RESUMO

Cristaria plicata is one of the more important freshwater pearl bivalves in China, which is susceptible to pathogen infection, and greatly impacts the ability of breeding pearls. Nrf2/ARE signaling pathway and its downstream target gene Prx5 have endogenous antioxidant functions to protect cells from oxidative damage. The full-length cDNA of Prx5 was cloned from C. Plicata, which was 1420 bp, encoding a total of 189 amino acids and had two conserved cysteine residues (Cys78 and Cys179). The amino acid sequence of CpPrx5 was highly similar to Prx5 of other species. Real-time fluorescence quantitative PCR showed that CpPrx5 was distributed in various tissues of mussels, and the highest expression was in hepatopancreas. The expression of CpPrx5 up-regulated in hepatopancreas and gills after LPS, PGN and Poly:I:C stimulation. The recombinant plasmid DE3-PGEX-4T-1-CpPrx5 was expressed in Escherichia coli BL21 and showed antioxidant activity. With the increase of CpPrx5 protein concentration, the superhelical form of DNA was protected. The expression of CpPrx5 was up-regulated after interference CpKeap1 and down-regulated after interference CpNrf2. Gel block assay showed that CpNrf2 and CpMafK proteins blocked CpPrx5 promoter. Subcellular localization showed that CpPrx5 was located in 293T nucleus and cytoplasm and CpMafK was located in 293T nucleus. GST-Pull down verified that CpMafK and CpPrx5 could bind in vitro. These results indicated that Prx5 had antioxidant function and could protects DNA from oxidative damage, and participated in transcriptional regulation by combining with the transcription factor MafK. In addition, MafK could combine with Nrf2 to regulate the downstream target gene Prx5.


Assuntos
Bivalves , Unionidae , Animais , Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/genética , Clonagem Molecular , Sequência de Bases , Unionidae/genética , Bivalves/genética , DNA Complementar/genética , Transdução de Sinais
3.
Fish Shellfish Immunol ; 143: 109184, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37884104

RESUMO

Caspase-3 is generally considered to be the most important terminal shear enzyme in the process of apoptosis, as well as an important part of cytotoxic T lymphocytes (CTL) killing mechanism, which is confirmed to play an important role in vertebrate cell apoptosis and immune system, and is poorly reported in invertebrates. In this paper, we used bioinformatics to perform amino acid multiple sequence alignment and protein structural domain analysis, and constructed a phylogenetic tree to identify the full-length cDNA of the cloned caspase-3 of Cristaria plicata (Named CpCaspase-3). The expression of caspase-1, caspase-7, caspase-8, and caspase-9 was found to be down-regulated by double-stranded RNA interference of CpCaspase-3 in C. plicata. Some degree of disruption of the caspase signaling pathway occurs. The expression of CpCaspase-3 was affected after injection of Lipopolysaccharide (LPS), Peptidoglycan (PGN), polyinosinic-polycytidylic acid (poly(I:C)), and Aeromonas hydrophila. These results were suggested that CpCaspase-3 was involved in the immune response of C. plicata. The wound recovery process of C. plicata was simulated and CpCaspase-3 was found to promote wound recovery. An autophagy inhibition and autophagy activation model of mussels was constructed, where apoptosis and autophagy undergo crosstalk, and inhibition of autophagy induces the onset of apoptosis, and similarly autophagy activation inhibits the process of apoptosis instead. In addition, a recombinant CpCaspase-3-pEGFP-C1 plasmid was constructed for subcellular localization experiments and found that CpCaspase-3 was distributed in both the nucleus and the cytoplasm. This paper aims to unveil the immune mechanism of C. plicata and provide a theoretical basis for the healthy culture of shellfish.


Assuntos
Unionidae , Animais , Sequência de Bases , Caspase 3/genética , Filogenia , Clonagem Molecular , Unionidae/genética , Imunidade
4.
Fish Shellfish Immunol ; 141: 108977, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37579811

RESUMO

Nitazoxanide (NTZ) is a broad-spectrum immunomodulatory drug, and little information is about the immunotoxicity of aquatic organisms induced by NTZ. In the present study, reduced body length and decreased yolk sac absorption in the NTZ-treated group were observed. Meanwhile, the number of innate immune cells and adaptive immune cells was substantially reduced upon NTZ exposure, and the migration and retention of macrophages and neutrophils in the injured area were inhibited. Following NTZ stimulation, oxidative stress levels in the zebrafish increased obviously. Mechanistically, RNA-seq, a high-throughput method, was performed to analyze the global expression of differentially expressed genes (DEGs) in zebrafish embryos treated with NTZ. 531 DEGs were identified by comparative transcriptome analysis, including 121 up-regulated and 420 down-regulated genes in zebrafish embryos after NTZ exposure. The transcriptome sequences were further subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) and analysis, showing phototransduction and metabolic pathway, respectively, and were most enriched. In addition, some immune-related genes were inhibited after NTZ exposure. RNA-seq results confirmed by qRT-PCR were used to verify the expression of the 6 selected genes. The other immune-related genes such as two pro-inflammatory cytokines (IL-1ß, tnfα) and two chemokines (CXCL8b.3, CXCL-c1c) were further confirmed and were differentially regulated after NTZ exposure. In summary, NTZ exposure could lead to immunotoxicity and increased ROS in zebrafish embryos, this study provides valuable information for future elucidating the molecular mechanism of exogenous stimuli-induced immunotoxicity in aquatic ecosystems.


Assuntos
Ecossistema , Peixe-Zebra , Animais , Perfilação da Expressão Gênica , Macrófagos , Transcriptoma
5.
Fish Shellfish Immunol ; 127: 65-73, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35705131

RESUMO

As members of arrestins family, ß-arrestins are widely expressed in monocytes, macrophages, neutrophils and other immune cells. They can regulate the immune response of bodies through various ways. In the present study, a ß-arrestin homolog named Hcß-arrestin was cloned and identified from Hyriopsis cumingii. Predicted Hcß-arrestin protein contained a conserved arrestin domain, which could be further divided into arrestin-N (39-192aa) and arrestin-C (211-365aa). Amino acid sequence alignment showed that it had the highest identity with Mytilus galloprovincialis and Mytilus edulis counterpart, which was 89.02% and 87.68%, respectively. Furthermore, real-time quantitative PCR analysis showed that the Hcß-arrestin gene was widely expressed in the detected tissues and with the highest expression in hepatopancreas. The transcription of Hcß-arrestin in hepatopancreas and gill of mussels was significantly up-regulated after stimulation with peptidoglycan, lipopolysaccharide (LPS) and polyinosinic polycytidylic acid. Knockdown of Hcß-arrestin gene significantly increased the expression of some antibacterial effector genes, such as lysozyme, LPS-binding protein/bactericidal permeability increasing protein and theromacin in hepatopancreas and gills of LPS stimulated mussels, but only had little effect on TLR pathway genes. In addition, GST pull-down assay confirmed that Hcß-arrestin can bind to HcTRAF6 protein in vitro. Dual luciferase reporter assay showed that the co-expression of HcTRAF6 and Hcß-arrestin inhibited the activation of NF-κB reporter by HcTRAF6. These findings indicated that Hcß-arrestins could interact with HcTRAF6 to negatively regulate the NF-κB pathway in H. cumingii.


Assuntos
Bivalves , Unionidae , Animais , Arrestina/metabolismo , Arrestinas/metabolismo , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , beta-Arrestinas/metabolismo
6.
Fish Shellfish Immunol ; 119: 533-541, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34737132

RESUMO

Metalloproteinase tissue inhibitors (TIMPs) have the activity of inhibiting matrix metalloproteinases (MMPs), which can promote cell growth, bind to the matrix, inhibit angiogenesis, and play a key role in extracellular matrix (ECM) metabolism regulation. In this study, TIMP-1, 2 from Hyriopsis cumingii (designated as HcTIMP-1, 2) were cloned and identified. Full-length cDNA of HcTIMP-1, 2 was 1160 bp and 729 bp, encoding 235 and 150 amino acid residues, respectively. The predicted molecular weight of HcTIMP-1 and 2 protein was 27.26 and 16.58 kDa, with isoelectric points of 8.89 and 8.72, respectively. HcTIMP-2 contained only one netrin (NTR) domain at the N-terminal but lacked a C-terminal domain. The mRNA of HcTIMP-1, 2 was expressed in hepatopancreas, gills, muscles, hemocytes, and mantles, which had the highest expression in hemocytes and muscles. The expression of HcTIMP-1, 2 had increased remarkably in hemocytes after bacterial challenge. After trauma, HcTIMP-1, 2 genes had the highest expression level in the first day. This indicated that HcTIMP-1 and 2 were involved in the immune response of H. cumingii. The soluble recombinant proteins HcTIMP-1, 2 were expressed efficiently in Escherichia coli BL21 (DE3) by constructing pET32a-TIMP1, 2 recombinant plasmids. The concentration of the recombinant was 0.14 and 0.31 mg/mL, respectively. The recombinant HcTIMP-1, 2 proteins were shown to inhibit human MMP2 activity and promoted the growth of NBL-7 and HUVE cells.


Assuntos
Unionidae , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Alinhamento de Sequência , Unionidae/genética
7.
Fish Shellfish Immunol ; 97: 455-464, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31870970

RESUMO

As a specific pearl mussel in China, Hyriopsis cumingii has enormous economic value. However, the organism damage caused by pearl insertion is immeasurable. TGF-ß/Smad signal transduction pathways are involved in all phases of wound healing. We have previously reported on two cytoplasmic signal transduction factors, Smad3 and Smad5 in mussel H. cumingii (named HcSmads), suggesting their involvements in wound healing. Here, Smad4 was cloned and described. The full length cDNA of HcSmad4 was 2543 bp encoded 515 amino acids. Deduced HcSmad4 protein possessed conserved MH1 and MH2 domains, nuclear location signals (NLS), nuclear exput signals (NES) and Smad activation domain (SAD). Transcripts of Smad3, 4 and 5 were constitutively expressed in all detected tissues, at highest levels in muscles. Furthermore, HcSmad4 mRNA levels were significantly increased at incision site post wounding, and expression of downstream target genes of Smad4, such as HcMMP1, HcMMP19, HcTIMP1 and HcTIMP2 were upregulated to a certain extent. Whatever knocked down HcSmad3/4 or treated by specific inhibitors of Smad 3 (SIS3), expression levels of these genes displayed a significantly downregulated tendency compared with the wound group. In addition, histological evaluation suggested that Smad3 knockdown or SIS3 treatment was accelerated wound healing, and then Smad4 knockdown delayed the process of wound healing in mussels. These data implicate that Smad3/4 play an important role in tissue repair in mollusks.


Assuntos
Proteína Smad4/antagonistas & inibidores , Proteína Smad4/genética , Unionidae/genética , Cicatrização/genética , Animais , China , Técnicas de Silenciamento de Genes , RNA Mensageiro , Transdução de Sinais , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/genética , Unionidae/fisiologia
8.
Fish Shellfish Immunol ; 91: 180-187, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31078645

RESUMO

Manganese superoxide dismutase (MnSOD) is a sort of important metalloenzyme that can catalyze ROS in the organisms. In this study, MnSOD cDNA of C. plicata, designated as CpMnSOD (accession no. MK465057), was cloned from hemocytes. The full-length cDNA of MnSOD was 1096 bp with a 672 bp open reading frame encoding 223 amino acids. The deduced amino acid sequence contained a mitochondrial-targeting sequence (MTS) of 18 amino acids in the N-terminus, and four conserved amino acids for manganese binding (H49, H97, D182, H186). CpMnSOD showed a high level (65-73%) of sequence similarity to MnSODs from other species. The results of Real-time quantitative PCR revealed that CpMnSOD mRNA constitutively expressed in tissues. The highest expression level was in hepatopancreas, followed by muscle, mantle and gill, and the lowest expression level was in hemocytes. After microcystin challenge, the expression levels of CpMnSOD mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpMnSOD was cloned into the plasmid pColdI-ZZ, and the recombinant protein was expressed in Escherichia coli BL21 (DE3). The enzyme stability assay showed that the purified CpMnSOD protein maintained more than 80% enzyme activity at temperature up to 70 °C, at pH 2.0-10.0, and resistant to 8 mol/L urea or 8% SDS.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Superóxido Dismutase/genética , Superóxido Dismutase/imunologia , Unionidae/genética , Unionidae/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Escherichia coli , Perfilação da Expressão Gênica , Filogenia , Proteínas Recombinantes , Alinhamento de Sequência , Superóxido Dismutase/química , Unionidae/enzimologia
9.
Fish Shellfish Immunol ; 60: 436-446, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27979780

RESUMO

Lipopolysaccharide-binding protein and bactericidal permeability-increasing protein (LBP/BPI) play crucial role in modulating cellular signals in response to Gram-negative bacteria infection. In the present study, two isoforms of LBP/BPI genes, designated as HcLBP/BPI1 and HcLBP/BPI2, respectively, were cloned from the mussel Hyriopsis cumingii by RACE approach. The full-length cDNA sequences of HcLBP/BPI1 and HcLBP/BPI2 were 1887 and 2227 bp and encoded two secreted proteins of 501 and 518 amino acid residues, respectively. The deduced amino acid of HcLBP/BPI1 and HcLBP/BPI2 contained several conserved domains, such as signal peptide, two BPI/LBP and one central domain. Phylogentic analysis further supported that HcLBP/BPI1 and HcLBP/BPI2 belonged to new members of invertebrate LBP/BPI family. The mRNA transcripts of HcLBP/BPI1 and HcLBP/BPI2 were ubiquitously expressed in all examined tissues, and the expression level of HcLBP/BPI1 was higher than that of HcLBP/BPI2. The mRNA expression of HcLBP/BPI1 in hepatopancreas and hemocytes was significantly up-regulate after Aeromonas hydrophila and LPS challenge, and HcLBP/BPI2 in hepatopancreas was only up-regulated at 6 and 12 h after LPS challenge and at 12 h after A. hydrophila challenge. In addition, the recombinant HcLBP/BPIs displayed antibacterial activity against Gram-negative bacteria, and the antibacterial index of HcLBP/BPI1 was higher than that of HcLBP/BPI2. These results indicated that HcLBP/BPI1 and HcLBP/BPI2 probably played distinct roles in bacterial mediating immune response in Mollusca.


Assuntos
Proteínas de Fase Aguda/genética , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Sanguíneas/genética , Proteínas de Transporte/genética , Imunidade Inata/genética , Glicoproteínas de Membrana/genética , Unionidae/genética , Unionidae/imunologia , Proteínas de Fase Aguda/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Sequência de Bases , Proteínas Sanguíneas/imunologia , Proteínas de Transporte/imunologia , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/imunologia , Filogenia , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Unionidae/classificação , Unionidae/microbiologia
10.
Fish Shellfish Immunol ; 66: 254-263, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28499967

RESUMO

Peroxiredoxins (Prxs) play an important role against various oxidative stresses by catalyzing the reduction of hydrogen peroxide (H2O2) and organic hydroperoxides to less harmful form. A 2-cys peroxiredoxin, designated as CpPrx, was cloned from hemocytes of freshwater mussel Cristaria plicata. The full length cDNA of CpPrx is 1247 bp, which includes an open reading frame (ORF) of 591bp, encoding 196 amino acids. CpPrx possesses two conserved cysteine residues (Cys49, Cys170). The deduced amino acid sequence of CpPrx showed a high level (67-74%) of sequence similarity to 2-Cys Prxs from other species. The results of real-time quantitative PCR revealed that CpPrx mRNA was constitutively expressed in tissues, and the highest expression levels were in hepatopancreas and gills. After peptidoglycan (PGN) and Aeromonas hydrophila challenge, the expression levels of CpPrx mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpPrx was cloned into the plasmid pET-32, and the recombinant protein was expressed in Escherichia coli BL21(DE3). Comparison with DE3-pET-32 and DE3 strain, the cells of DE3-pET-32-CpPrx exhibited resistance to the concentration of 0.4, 0.8 and 1.2 mmoL/L H2O2 in vivo.


Assuntos
Antioxidantes/metabolismo , Expressão Gênica , Peroxirredoxinas/genética , Peroxirredoxinas/imunologia , Unionidae/genética , Unionidae/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Hemócitos/metabolismo , Hemócitos/microbiologia , Hepatopâncreas/metabolismo , Hepatopâncreas/microbiologia , Peptidoglicano/farmacologia , Peroxirredoxinas/química , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Unionidae/microbiologia
11.
Fish Shellfish Immunol ; 67: 129-140, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28546027

RESUMO

The proteins of Smad family are critical components of the TGF-ß superfamily signal pathway. In this paper, we cloned two intracellular mediators of TGF-ß signaling, Smad3 and Smad5, from the pearl mussel Hyriopsis cumingii. The full length cDNA of HcSmad3 and HcSmad5 were 2052 bp and 1908 bp and encoded two polypeptides of 418 and 461amino acid residues, respectively. The deduced amino acid of HcSmad3 and HcSmad5 possessed two putative conserved domains, MH1 and MH2, a conserved phosphorylation motif SSXS at the carboxyl-terminal. The two Smad genes were detected muscle, mantle, hepatopancreas and gill, but with a very low level in heamocytes. The transcripts of Smad3 and Smad5 were up-regulated in hemocytes and hepatopancreas after A. hydrophila and PGN stimulation. However, the expression of Smad3 and Smad5 were only up-regulated in hepatopancreas after A. hydrophila stimulation. The transcripts of Smad3 and Smad5 had a slight change in hepatopancreas after PGN stimulation. The transcripts of HcSmad3 showed very little increase and HcSmad5 mRNA significantly up-regulated after wounding.


Assuntos
Imunidade Inata/genética , Proteínas Smad/genética , Proteínas Smad/imunologia , Unionidae/genética , Unionidae/imunologia , Cicatrização/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Filogenia , Alinhamento de Sequência , Proteínas Smad/química
12.
Microbiol Immunol ; 59(12): 744-55, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26589461

RESUMO

The freshwater bivalve Cristaria plicata, which is widely distributed in Eastern Asia, is a key species in the pearl culture industry. In this study, a novel invertebrate-type lysozyme, designated as CpLYZ2, was cloned from hemocytes of C. plicata. This lysozyme shares high sequence identity and is homologous to a previously identified lysozyme CpLYZ1 isolated from C. plicata and with HcLyso3 isolated from Hyriopsis cumingii. The full-length cDNA of CpLYZ2 is 913 bp long, which includes an open reading frame (ORF) of 486 bp, a 3' untranslated region (UTR) of 389 bp and a 5' UTR of 38 bp. The ORF encodes a putative polypeptide of 161 amino acids with a predicted molecular mass of 18.2 kDa and a theoretical isoelectric point of 6.56. CpLYZ2 mRNA transcripts can be detected in hemocytes, hepatopancreas, muscle, gills and mantle tissues, the greatest expression being observed in the gills. CpLYZ2 expression in hemocytes, hepatopancreas and gills increased significantly after the mussel was challenged with Aeromonas hydrophila. Furthermore, the optimal pH and temperature for enzyme activity of the recombinant CpLYZ2 were 5.5 and 50°C, respectively. The recombinant lysozyme protein exhibited bacteriolytic activity against Escherichia coli, A. hydrophila, Staphylococcus aureus, Bacillus subtilis, Streptococcus sp. and Staphylococcus epidermidis. The findings of this study help to elucidate immune responses in molluscs and will thus expedite disease management of these key freshwater species, in turn boosting pearl culture in eastern Asia.


Assuntos
Muramidase/genética , Muramidase/metabolismo , Unionidae/enzimologia , Unionidae/genética , Aeromonas hydrophila , Sequência de Aminoácidos , Animais , Anti-Infecciosos/farmacologia , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Água Doce , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Ponto Isoelétrico , Dados de Sequência Molecular , Muramidase/química , Muramidase/farmacologia , Fases de Leitura Aberta , Filogenia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Análise de Sequência , Unionidae/imunologia
13.
Fish Shellfish Immunol ; 40(2): 446-54, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25038281

RESUMO

Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. The Cathepsin L cDNA and genome of Cristaria plicata(CpCL) was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends (RACE) PCR. The genomic DNA was 9353 bp long and had a total of six introns and seven exons. The full-length cDNA of CpCL was 1144 bp, the cDNA contained a 5' untranslated region (UTR) of 34 nucleotides, the 3' UTR of 108 bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1002 bp, encoding 333 amino acid residues with 37.65 kDa predicted molecular weight. The theoretical isoelectric point was 8.61. The prepro-cathepsin L was consisted of a typical signal peptide (Met1-Gly20), a pro-region peptide (Leu21-Glu116) and a mature peptide (Tyr117-Val333). Many members of the papain family possessed of a proline residue at position 2 in the mature enzymem, this was also observed in CpCL. The preproprotein included an oxyanion hole (Gln 135), the active center formed by Cys141, His280 and Asn 300, the potential N-glycosylation site (Asn38, Asn 113 and Asn 272) and the conserved GCXGG motifs, which was characteristic of cathepsin, the conserved ERWNIN and GNFD motifs, which were characteristic for cathepsin L. Homology analysis revealed that the CpCL shared 49-87% identity to other known cathepsin L sequences. The phylogenetic tree showed that the CpCL clustered with the invertebrate cathepsin L cysteine proteases, and was closely related to the cathepsin L of Hyriopsis cumingii. The expression of CpCL mRNA was detected in hepatopancreas, hemocytes, mantle, gills and adductor muscle, and the higher expression level was in hepatopancreas. After A. hydrophila stimulation, the expression of the CpCL mRNA was up-regulated in hemocytes and hepatopancreas, and the expression level was significantly lower in gill than one after PBS challenge group.


Assuntos
Catepsina L/genética , Unionidae/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina L/química , Catepsina L/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Unionidae/metabolismo , Unionidae/microbiologia
14.
Fish Shellfish Immunol ; 34(5): 1033-41, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23333359

RESUMO

Lysozymes are important proteins to bivalve in the innate immune responses against bacterial infections, and provide nutrition as digestion enzymes. A new LYZ1 from the freshwater mussel Cristaria plicata was cloned by rapid amplification of cDNA ends (RACE) and nested PCR method. The full-length cDNA sequence of CpLYZ1 was 763 bp. The cDNA contained a 5'-terminal untranslated region (UTR) of 21 bp, a 3'- terminal UTR of 259 bp with a 29 bp poly(A) tail, a tailing signal (AATAAA) and the open reading frame of 483 bp. The CpLYZ1 cDNA encoded a polypeptide of 160 amino acids with a predicted molecular mass of 17.8 kDa, and a theoretical isoelectric point of 6.07. The comparison of the deduced amino acid sequences with LYZs from other species showed that the enzyme belonged to i-type lysozyme. The mRNA transcript of CpLYZ1 could be detected in all the examined tissues with the highest expression level in hepatopancreas. The expression levels of CpLYZ1 in hemocytes, hepatopancreas and gill significantly increased after Aeromonas hydrophila challenge. The expression level of CpLYZ1 in hemocytes sharply decreased from 6 h to 24 h and significantly increased at 48 h, and was the highest level in hepatopancreas at 24 h, and was the maximum level in gill at 48 h. Furthermore, the recombinant CpLYZ1 was induced to be expressed as an inclusion body form by IPTG at 37 °C for 4 h, and then was purified by using the Ni(2+) affinity chromatography. The relative enzyme activity of the recombinant CpLYZ1 was influenced on pH and temperature. The optimal pH and temperature was 5.5 and 50 °C, respectively. Against Escherichia coli, A. hydrophila, Staphyloccocus aureus, Bacillus subtilis, Streptococcus sp. and Staphylococcus epidermidis, the recombinant CpLYZ1 had bacteriolytic activity.


Assuntos
Muramidase/genética , Muramidase/imunologia , Unionidae/genética , Unionidae/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Escherichia coli/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Dados de Sequência Molecular , Muramidase/química , Muramidase/metabolismo , Especificidade de Órgãos , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Unionidae/química , Unionidae/enzimologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-37285927

RESUMO

Glutathione S-transferases (GSTs) are phase II metabolic detoxification enzymes, which are widely found in organisms, and play an important role in helping organisms to resist toxic compounds. In this study, the two Delta-class GSTs cDNA sequences were cloned from Procambarus clarkii (designated as PcGSTD1 and PcGSTD2). Tissue specific expression profile showed that PcGST1,2 were expressed in all 6 tissues, with the highest expression in hepatopancreas. Subcellular localization assay showed that PcGSTD1, 2 were mainly expressed in the cytoplasm of HEK-293 T cells. Recombinant PcGSTD1, 2 showed the highest catalytic activity to the GST model substrate 1-chloro-2,4-dinitrobenzene (CDNB) at 20 and 30 °C, pH 8 and 7, respectively. The mRNA expression of PcGSTD1, 2 and the GSTs activity varied with the time of imidacloprid challenge. The BL21(DE3) expressing PcGSTD1, 2 proteins could more resistant to H2O2. The dsRNA experiments showed that PcKeap1b, PcNrf1, and PcMafK affected the transcription levels of PcGSTD1, 2. The GST-Pulldown results revealed that PcbZIP and PcMafK recombinant proteins could bind to each other in vitro. The gel mobility shift assay demonstrated that PcMafK recombinant protein had affinity with the promoter of PcGSTD2. The Dual luciferase assays analyzed the activity of the promoters after different truncations, the core region of PcGSTD1 promoter was at -440 bp to +54 bp, and that of PcGSTD2 promoter was between -1609∼-1125 bp. These results suggested that PcGSTD1, 2 respond positively to imidacloprid stress in P. clarkii, and the transcriptional expressions of PcGSTD1, 2 were influenced by the factors of PcKeap1b/PcNrf1/PcMafK.


Assuntos
Antioxidantes , Astacoidea , Humanos , Animais , Astacoidea/genética , Células HEK293 , Peróxido de Hidrogênio , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Dev Comp Immunol ; 141: 104629, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36587710

RESUMO

Microcystins (MC) are one of the most abundant and widely distributed cyanotoxins in aquatic systems. MC inhibits the functions of protein phosphatase 1 and 2A (PP1/2A), which can seriously affect ecosystem integrity. The NF-E2-related nuclear factor 2 (Nrf2)/Kelch-like epichlorohydrin-related protein-1 (Keap1) signaling pathway protects against oxidative damage by activating phase II detoxification/antioxidant enzymes. Our previous study revealed that MC upregulates the expression and enhances the activities of the antioxidant enzymes by stimulating the CpNrf2 signaling pathway. In the current study, to further clarify the regulatory role of Keap1 in response to MC-induced oxidative stress in shellfish, we cloned the full-length cDNA of Keap1a and Keap1b from Cristaria plicata (designated CpKeap1a and CpKeap1b), which are 2952 and 3710 bp peptides, respectively. The amino acid sequence of CpKeap1a and CpKeap1b contained Tram-track and Bric-a-brac (BTB), Intervening region (IVR), and Double glycine repeat (DGR) domain. Additionally, CpKeap1a contained two cysteine residues analogous to Cys-273 and -288 in zebrafish, but CpKeap1b did not. Moreover, CpKeap1a and -1b formed a homodimer and heterodimer, respectively, and also formed a heterodimer with CpNrf2. In the hepatopancreas, the expression levels of CpKeap1a and -1b were the highest, but MC treatment down-regulated the expression of these proteins. Moreover, the transcription of antioxidant enzymes with antioxidant response element (ARE-driven enzymes), including CpMnSOD, CpCu/ZnSOD, CpTRX, CpPrx, CpSe-GPx, and Cpsigma-GST was upregulated by CpNrf2 in the hepatopancreas. Compared with the MC-induced group, CpKeap1a-siRNA1117 injection significantly increased the transcription of mRNAs for ARE-driven enzymes and Nrf2. CpKeap1a-siRNA1117 also enhanced the activities of antioxidation enzymes. These findings demonstrated that Keap1a negatively regulated the expression of Nrf2 protein and MC-induced oxidative stress response in C. plicata. Therefore, we speculated that CpKeap1a promoted CpNrf2 by recognizing and binding MC. These events then protected molluscs from MC-induced oxidative damage.


Assuntos
Bivalves , Unionidae , Animais , Antioxidantes/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Epicloroidrina , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Peixe-Zebra/metabolismo , Microcistinas/metabolismo , Ecossistema , Bivalves/genética , Unionidae/genética , Estresse Oxidativo , Proteínas de Transporte/metabolismo , Proteínas de Peixe-Zebra/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-37689170

RESUMO

Nuclear factor E2-associated factor 2 (Nrf2)/Antioxidant Response Element (ARE) signaling pathway is an endogenous antioxidant pathway that protects cells from oxidative damage. This pathway is triggered when aquatic organisms are exposed to environmental toxicants. In this study, CpMafK (musculoaponeurotic fibrosarcoma K of Cristaria plicata) mRNA expression in hepatopancreas and gills were up regulated after Cristaria plicata (C. plicata) was exposed to microcystin (MC), which showed that CpMafK protected C. plicata from MC. After MC treatment and CpNrf2 (Nrf2 of Cristaria plicata) knockdown, the mRNA expression of CpMafK was down regulated. After MC treatment and CpMafK knockdown, the mRNA expression of CpNrf2 was down regulated. Indicating that the expression of CpNrf2 was positively correlated with CpMafK. CpGPx (GPx of Cristaria plicata) mRNA was also down regulated with the down regulation of CpMafK and CpNrf2. CpGPx promoter contains a variety of transcription factor binding sites, including Nrf2, ARE elements, etc. Gel blocking experiments showed that CpNrf2/CpMafK heterodimers were bound to CpGPx promoters in vitro. Dual luciferase reporter assay showed that CpNrf2/CpMafK heterodimer negatively regulated CpGPx promoter in cells. In conclusion, Nrf2 and MafK mediate regulation of GPx play a crucial role in protecting bivalves from MC.


Assuntos
Fibrossarcoma , Microcistinas , Animais , Microcistinas/toxicidade , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Glutationa Peroxidase/genética
18.
Aquat Toxicol ; 263: 106708, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37776712

RESUMO

Glutathione S-transferase is a crucial phase II metabolic enzyme involved in detoxification and metabolism in aquatic organisms. This study aimed to investigate the regulation of Nrf2/Keap1 pathway on microcystin-induced CpGST-Mu expression and CpGST-Mu resistance to hydrogen peroxide. A mu class GST from Cristaria plicata (CpGST-Mu) was identified. The full-length cDNA was 1026 bp, with an open reading frame of 558 bp. Subcellular localization revealed that CpGST-Mu was localized in cytoplasm. The optimum pH and temperature for the catalytic activity of CpGST-Mu protein was pH 6 and 40 °C, respectively. The results of Real-time quantitative PCR showed that CpGST-Mu mRNA was constitutively expressed in tissues, with the highest expression level in hepatopancreas and the lowest expression level in gill. The mRNA level of CpGST-Mu was significantly increased under the stress of microcystins and hydrogen peroxide. CpGST-Mu had an antagonistic effect on hydrogen peroxide. In the knockdown experiments, the mRNA levels of CpGST-Mu exhibited corresponding changes while Nrf2 and Keap1 genes were individually knocked down. These findings indicated that GST-Mu exhibited antioxidant properties and its expression was regulated by Nrf2/Keap1 signaling pathway. The study provided new information on the function of GST-Mu and could contribute to future studies on how to excrete microcystins in molluscs.


Assuntos
Unionidae , Poluentes Químicos da Água , Animais , Antioxidantes/metabolismo , Microcistinas/toxicidade , Microcistinas/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Peróxido de Hidrogênio/metabolismo , Poluentes Químicos da Água/toxicidade , Glutationa Transferase/metabolismo , RNA Mensageiro
19.
Aquat Toxicol ; 255: 106398, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36669434

RESUMO

Microcystins (MCs) are the most frequent and widely distributed type of cyanotoxin in aquatic systems, and they cause an imbalance of the body's oxidative system. In a previous experiment, we demonstrated that the mollusk Cristaria plicata can protect against MC-induced oxidative damage through the nuclear factor erythroid 2-related factor 2(Nrf2)/Kelch-like epichlorohydrin-related protein-1 (Keap1) pathway. Here, we evaluated whether selective autophagy affects the Nrf2/Keap1a anti-oxidative stress pathway in C. plicata. Full-length cDNA sequences of p62/SQSTM1 from C. plicata (Cpp62) were divided into 2484 bp fragments. From N-terminal to C-terminal, the amino acid sequence of Cpp62 contained PB1 (Phox and Bem1p domain), ZNF (zinc finger domain) chain, LIR (LC3 interacting region) and UBA (ubiquitin-associated domain) domains, but not the KIR (Keap1 interacting region) domain. We confirmed that Cpp62 did not bind to CpKeap1a in vitro, and the relative level of Cpp62 was the highest in the hepatopancreas. Moreover, MCs significantly upregulated the mRNA and protein levels of Cpp62 in the hepatopancreas after CpKeap1a knockdown, whereas Nrf2 upregulated the transcription levels of Cpp62, suggesting that MCs increased Cpp62 expression via the Nrf2/Keap1a signaling pathway. Moreover, Cpp62 and CpNrf2 proteins have a strong affinity for the NQO1 promoter, but MCs inhibited the ability of CpNrf2 and Cpp62 to upregulate luciferase activity. The results show that Nrf2 and the p62 protein induced p62 expression by binding to ARE (antioxidant response element) sequences in the p62 promoter of C. plicata, thereby promoting p62 to resist MC-induced oxidative stress. Therefore, we speculate that MCs induce p62-dependent autophagy in C. plicata, resulting in the inhibition of Nrf2 transcription and Cpp62 promoter activity. These findings help to reveal the mechanism by which the p62-Nrf2/Keap1 pathway mitigates MC-induced oxidative damage in mussels.


Assuntos
Unionidae , Poluentes Químicos da Água , Animais , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Microcistinas/toxicidade , Microcistinas/metabolismo , Poluentes Químicos da Água/toxicidade , Transdução de Sinais , Estresse Oxidativo
20.
Front Immunol ; 14: 1119902, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36793729

RESUMO

The phenomenon of intestinal dysfunction is widely observed in white shrimp (Litopenaeus vannamei) culture, and ß-1,3-glucan has been confirmed to be beneficial in intestinal health with a lack understanding of its underlying mechanism. Proteobacteria, Firmicutes, and Actinobacteria served as the predominant phyla inhabiting the intestine of white shrimp, whilst a significant variation in their proportion was recorded in shrimp fed with basal and ß-1,3-glucan supplementation diets in this study. Dietary supplementation of ß-1,3-glucan could dramatically increase the microbial diversity and affect microbial composition, concurrent with a notable reduction in the ratio of opportunistic pathogen Aeromonas, gram-negative microbes, from Gammaproteobacteria compared to the basal diet group. The benefits for microbial diversity and composition by ß-1,3-glucan improved the homeostasis of intestinal microbiota through the increase of specialists' number and inhibition of microbial competition caused by Aeromonas in ecological networks; afterward, the inhibition of Aeromonas by ß-1,3-glucan diet dramatically suppressed microbial metabolism related to lipopolysaccharide biosynthesis, followed by a conspicuous decrease in the intestinal inflammatory response. The improvement of intestinal health referred to the elevation in intestinal immune and antioxidant capacity, ultimately contributing to the growth of shrimp fed ß-1,3-glucan. These results suggested that ß-1,3-glucan supplementation improved the intestinal health of white shrimp through the modulation of intestinal microbiota homeostasis, the suppression of intestinal inflammatory response, and the elevation of immune and antioxidant capacity, and subsequently promoted the growth of white shrimp.


Assuntos
Microbioma Gastrointestinal , Penaeidae , Animais , Suplementos Nutricionais/análise , Antioxidantes , Glucanos , Intestinos/microbiologia
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