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1.
Fish Shellfish Immunol ; 89: 170-178, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30928663

RESUMO

Peroxiredoxin 6 (Prx6) is an important member of the peroxiredoxin family that plays critical roles in protecting host against the toxicity of oxidative stress and participates in cell signaling. Herein, we report Prx6 gene from red swamp crayfish, Procambarus clarkii. The cDNA fragment of PcPrx6 was 660 bp, encoding a 219 amino acid residues protein. The quantitative real time PCR analysis showed ubiquitous expression of PcPrx6 mRNA in the tested tissues. The challenge with peptidoglycan and Poly I:C remarkably suppressed the mRNA level of PcPrx6 in hepatopancreas at 3, 12, 48 h compared with the PBS control. However, the expression level significantly increased after 36 h of their treatment. The knockdown of PcPrx6 by small interference RNA significantly enhanced the transcript levels of Toll pathway-responsive genes at 24 h. Recombinant PcPrx6 protein was purified using affinity chromatography and analyzed for its biological role. The results revealed that the recombinant PcPrx6 protein manifested the ability to protect supercoiled DNA damage from oxidative stress elicited by mixed function oxidative assay. Altogether, PcPrx6 may have multiple functional roles in the physiology of P. clarkii, since it negatively regulates the Toll signaling transduction and protects supercoiled DNA damage from oxidative stress.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Peroxirredoxina VI/genética , Peroxirredoxina VI/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Cromatografia de Afinidade , Dano ao DNA , DNA Super-Helicoidal/fisiologia , Perfilação da Expressão Gênica , Estresse Oxidativo , Peptidoglicano/farmacologia , Peroxirredoxina VI/química , Filogenia , Poli I-C/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência
2.
Fish Shellfish Immunol ; 87: 178-183, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30639478

RESUMO

Ras-related C3 botulinum toxin substrate 1 (Rac1) participates in many biological processes. In this study, a Rac1 gene was identified in the crayfish Procambarus clarkii with an open reading frame of 579 bp that encoded 192 amino acids. This predicted 21.4 kDa protein was highly homologous to those in other invertebrates. Real-time PCR analysis revealed that Pc-Rac1 was expressed in all examined tissues with the highest expression level in hemocytes. The transcriptional expression level of Pc-Rac1 was significantly upregulated in hemocytes and hepatopancreas after lipopolysaccharide (LPS) or polyinosinic: polycytidylic acid (poly I: C) induction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis suggested that a recombinant Pc-Rac1 protein was successfully expressed in E. coli. Far-western blot analysis demonstrated that Rac1 can interact with the PBD domain of p21-activated kinase 1 (PAK1). RNA interference of Pc-Rac1 affected the mRNA expression levels of immune-related genes lectin, Toll, crustin, TNF, ALF and cactus. These results suggest that Pc-Rac1 is involved in the innate immune responses in P. clarkii.


Assuntos
Astacoidea/imunologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/genética , Animais , Astacoidea/genética , Astacoidea/metabolismo , Escherichia coli , Expressão Gênica , Hemócitos/metabolismo , Hepatopâncreas/metabolismo , Imunidade Inata/genética , Lipopolissacarídeos/farmacologia , Proteínas Monoméricas de Ligação ao GTP/química , Poli I-C/farmacologia , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Quinases Ativadas por p21/metabolismo
3.
Fish Shellfish Immunol ; 86: 882-891, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30553892

RESUMO

Ferritin is a protein related to the storage of iron and widely distributed in animals. It participates in many biological process, including antioxidation, cell activation, angiogenesis, regulation of iron metabolic balance and immune defense. In the present study, a novel ferritin gene was identified from red swamp crayfish Procambarus clarkii, with a cDNA sequence encoding a predicted 221 amino-acid residues. The ferritin protein contains a 19-residue signal peptide and 145-residue classic ferritin domain. The NJ phylogenetic analysis showed PcFer clustered with other crustacean peptides. The recombinant PcFer protein was produced and purified in E. coli, and the anti-rabbit polyclonal antibody was obtained. The rPcFer exhibited iron binding activity at a dose-dependent effect. The qPCR and western blot analysis revealed that PcFer was highly expressed in hemocytes, hepatopancreas, and gills. After challenged with WSSV and Aeromonas hydrophila, the mRNA and protein expression patterns of PcFer were significantly up-regulated in hemocytes and hepatopancreas. dsRNA interfering technique was utilized to silence the expression of PcFer gene. The WSSV copy number in PcFer silenced shrimp was much higher than that in the control group. The present study indicated that PcFer was involved in the immune defense against WSSV and Aeromonas hydrophila, and might inhibit WSSV replication in P. clarkii. These results will provide important data support for further study of the functional role of the ferritin gene.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Ferritinas/genética , Ferritinas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Ferritinas/química , Perfilação da Expressão Gênica , Brânquias/metabolismo , Hemócitos/metabolismo , Hepatopâncreas/metabolismo , Filogenia , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/fisiologia
4.
Dev Comp Immunol ; 91: 101-107, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385317

RESUMO

Caspase, an aspartate specific proteinase mediating apoptosis, plays a key role in immune response. In our previous study, the expression of a caspase gene was up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To further reveal the effect of caspase on WSSV infection, we cloned this caspase gene (denominated as CqCaspase) with an open reading frame of 1062 bp, which encoded 353 amino acids with a caspase domain (CASc) containing a p20 subunit and a p10 subunit. Tissue distribution analysis indicated that the mRNA transcript of CqCaspase was widely expressed in all tested tissues with the highest expression in Hpt, while the lowest expression in muscle. To further explore the effect of CqCaspase on WSSV replication, recombinant protein of CqCaspase (rCqCaspase) was delivered into Hpt cells followed by WSSV infection, which resulted in a significantly decreased expression of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV, suggesting that CqCaspase, possibly by the enhanced apoptotic activity, had a strong negative effect on the WSSV replication. These data together indicated that CqCaspase was likely to play a vital role in immune defense against WSSV infection in a crustacean C. quadricarinatus, which shed a new light on the mechanism study of WSSV infection in crustaceans.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/imunologia , Caspases/genética , Infecções por Vírus de DNA/imunologia , Hemócitos/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/metabolismo , Astacoidea/virologia , Caspases/metabolismo , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Imunidade Inata/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral
5.
Fish Shellfish Immunol ; 80: 155-164, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29870827

RESUMO

Prophenoloxidase (proPO) activating system is an important immune response for arthropods. ß-1, 3-glucanase related protein (previously named as lipopolysaccharide and ß-1, 3-glucan binding protein (LGBP) in crustaceans) is a typical pattern recognition receptor family involved in the proPO activation by recognizing the invading microbes. In this study, we pay special attention to a bacteria-induced ß-1,3-glucanase related protein from red swamp crayfish Procambarus clarkii, an important aquaculture specie in China. This protein, designated PcBGRP, was found a typical member of crustacean BGRP family with the glucanase-related domain and the characteristic motifs. PcBGRP was expressed in hemcoyes and hepatopancreas, and its expression could be induced by the carbohydrate and bacteria stimulants. The induction by lipopolysaccharide (LPS) and ß-1,3-glucan (ßG) was more significant than by peptidoglycan (PG). The response of PcBGRP to the native Gram-negative bacterial pathogen Aeromonas hydrophila was more obvious than to Gram-positive bacteria. Using RNA interference and recombinant protein, PcBGRP was found to protect crayfish from A. hydrophila infection revealed by the survival test and morphological analysis. A mechanism study found PcBGRP could bind LPS and ßG in a dose-dependent manner, and the LPS recognizing ability determined the Gram-negative bacterium binding activity of PcBGRP. PcBGRP was found to enhance the PO activation both in vitro and in vivo, and the protective role was related to the PO activating ability of PcBGRP. This study emphasized the role of BGRP family in crustacean immune response, and provided new insight to the immunity of red swamp crayfish which suffered serious disease during the aquaculture in China.


Assuntos
Proteínas de Artrópodes/imunologia , Astacoidea/imunologia , Proteínas de Transporte/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções Estafilocócicas/imunologia , Aeromonas hydrophila , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Astacoidea/genética , Proteínas de Transporte/genética , Glucana 1,3-beta-Glucosidase/farmacologia , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/veterinária , Hemócitos/imunologia , Hepatopâncreas/imunologia , Lipopolissacarídeos/farmacologia , Fases de Leitura Aberta , Peptidoglicano/farmacologia , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/veterinária , Staphylococcus aureus
6.
Dev Comp Immunol ; 87: 109-115, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29909090

RESUMO

Antimicrobial peptides (AMPs) play important roles in innate immunity against pathogens and lysozymes are a particularly type of AMP. Lysozymes are hydrolytic enzymes that are characterized by their ability to cleave the beta-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, which is the major bacterial cell wall polymer. In this work, a lysozyme was identified from Procambarus clarkii and designated PcLys-i3. Quantitative RT-PCR was used to analyze the tissue distribution and expression profiles of PcLys-i3. PcLys-i3 was present in all tested tissues and had high expression levels in gills, stomach and intestine. The expression levels of PcLys-i3 were up-regulated in gills and intestine after challenge with Vibrio parahaemolyticus, Staphylococcus aureus and Aeromonas hydrophila. PcLys-i3 and PcFer proteins can enhance the bacterial elimination in crayfish, whereas the bacterial elimination was weakened when the expression level of PcLys-i3 or PcFer RNAs was suppressed by RNAi. Recombinant PcLys-i3 and PcFer significantly reduced the mortality of crayfish with bacterial infections. Further study found that PcLys-i3 could interact with PcFer in vitro. Finally, the PcLys-i3 and PcFer proteins could bind to bacteria and inhibit bacterial replication. These results suggest that both PcLys-i3 and PcFer play important roles in the antibacterial immunity of red swamp crayfish.


Assuntos
Antibacterianos/imunologia , Proteínas de Artrópodes/imunologia , Astacoidea/imunologia , Muramidase/imunologia , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/fisiologia , Animais , Antibacterianos/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Astacoidea/genética , Astacoidea/microbiologia , Perfilação da Expressão Gênica/métodos , Brânquias/imunologia , Brânquias/metabolismo , Brânquias/microbiologia , Interações Hospedeiro-Patógeno , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Intestinos/microbiologia , Muramidase/genética , Muramidase/metabolismo , Interferência de RNA , Staphylococcus aureus/imunologia , Staphylococcus aureus/fisiologia , Regulação para Cima , Vibrio/imunologia , Vibrio/fisiologia
7.
Dev Comp Immunol ; 85: 134-141, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29680689

RESUMO

Lysozymes possess antibacterial activities, making them crucial defense proteins in innate immunity. In this study, a chicken-type (c-type) lysozyme (designated PcLyzc) was cloned and characterized from red swamp crayfish Procambarus clarkii. The full-length cDNA had an open reading frame of 435 base pairs encoding a polypeptide of 144 amino acid residues. Multiple alignments and phylogenetic analysis revealed that PcLyzc shared high similarity to the other known invertebrate c-type lysozymes. PcLyzc transcripts were steadily expressed in a wide range of tissues in healthy crayfish, and were prominently up-regulated in the hepatopancreas and gills after Vibrio anguillarum or Aeromonas hydrophila challenge. Recombinant PcLyzc showed inhibitory activity in vitro against both Gram-positive bacteria, including Staphylococcus aureus, Micrococcus luteus and Bacillus thuringiensis, and Gram-negative bacteria, including A. hydrophila, V. anguillarum and Escherichia coli. By overexpressing PcLyzc through introducing exogenous recombinant protein, or silencing PcLyzc expression through injecting double strand RNA, it was found that PcLyzc could help eliminate the invading bacteria in crayfish hemolymph and could protect crayfish from death, possibly by promoting the hemocytic phagocytosis. These results indicated that PcLyzc played a role in the antibacterial immunity of crustaceans, and laid a foundation of developing new therapeutic agents in aquaculture.


Assuntos
Antibacterianos/imunologia , Astacoidea/imunologia , Galinhas/metabolismo , Imunidade Inata/imunologia , Muramidase/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Brânquias/imunologia , Brânquias/microbiologia , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Positivas/imunologia , Hemócitos/imunologia , Hemócitos/microbiologia , Hepatopâncreas/imunologia , Hepatopâncreas/microbiologia , Peptídeos/imunologia , Filogenia , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Vibrio/imunologia
8.
Fish Shellfish Immunol ; 77: 438-444, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29625245

RESUMO

As a new-found aquaculture pathogen, Spiroplasma eriocheiris, has resulted in inconceivable economic losses in aquaculture. In the infection of S. eriocheiris, the Procambarus clakii hemocytes have indicated to be major target cells. What was designed to examine in our study is the hemocytes' immune response at the protein levels. Before the pathogen was injected and after 192 h of post-injection, the differential proteomes of the crayfish hemocytes were analyzed immediately by isobaric tags for relative and absolute quantization (iTRAQ) labeling, followed by liquid chromatogramphytandem mass spectrometry (LC-MS/MS). This research had identified a total of 285 differentially expressed proteins. Eighty-three and 202 proteins were up-regulated and down-regulated, respectively, caused by the S. eriocheiris infection. Up-regulated proteins included alpha-2-macroglobulin (α2M), vitellogenin, ferritin, etc. Down-regulated proteins, involved with serine protease, peroxiredoxin 6, 14-3-3-like protein, C-type lectin, cdc42 homolog precursor, etc. The prophenoloxidase-activating system, antimicrobial action involved in the immune responses of P. clarkii is considered to be damaged due to S. eriocheiris infection. The present work could lay the foundation for future research on the proteins related to the susceptibility/resistance of P. clarkii to S. eriocheiris. In addition, it is helpful for our understanding molecular mechanism of disease processes in crayfishes.


Assuntos
Astacoidea/genética , Hemócitos/imunologia , Imunidade Inata/genética , Proteoma/imunologia , Spiroplasma/fisiologia , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Astacoidea/imunologia , Astacoidea/microbiologia , Proteômica
9.
Fish Shellfish Immunol ; 77: 131-138, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29605503

RESUMO

In crustaceans, lectins and hemocytes of the innate immune system provide the first line of defense. Although evidence points to the potential role of lectins in regulating hemocyte activity, the processes underlying the lectin activation have not been evaluated. In the present study, the receptor for CqL, a humoral lectin from Cherax quadricarinatus specific for galactose/sialic acid, was identified in a granular subset of hemocytes. The CqL receptor (CqLR) is a 490-kDa glycoprotein, composed of four identical 120-kDa subunits. As shown by immunohistochemistry, CqL at 7.5 µg/mL as optimal dose, after 2 min, induced, specifically on granular hemocytes, increased phosphorylation of serine (152%), threonine (192%), and tyrosine (242%) as compared with non-treated hemocytes; moreover, CqL induced increased generation of reactive oxygen species (ROS). Specific kinase inhibitors showed inhibition (P < 0.001) of ROS production induced by CqL. These results strongly suggest that CqL actively participated in the generation of ROS through kinases induced by a CqLR in a subset of granular hemocytes of the crayfish C. quadricarinatus. The results provide strong evidence that CqL activates, through specific granular hemocytes, receptors that modulate cellular functions in C. quadricarinatus.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Imunidade Inata , Lectinas/genética , Lectinas/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/sangue , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Hemócitos/imunologia , Lectinas/sangue , Alinhamento de Sequência , Transdução de Sinais
10.
Fish Shellfish Immunol ; 77: 112-119, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29578050

RESUMO

Crustacean hemocytes are known to remove invading pathogens by phagocytosis. In this study, we investigated how the semigranular cells (SGCs) and granular cells (GCs) of crayfish Cherax quadricarinatus participated in this process. By injecting the animals with excessive amounts of fluorescent microspheres (FMs), we showed that only a small portion of the circulating hemocytes were phagocytic cells, and they took up FMs in a size-dependent manner. The 0.2 µm FMs were internalized almost entirely by SGCs, while GCs and SGCs both contributed to the uptake of 2 µm FMs. Further analysis of the hemocytes from the animals injected with a mixture of FMs suggested that there were a subpopulation of SGCs specifically ingesting 0.2 µm FMs. The size-dependent manner was also applied to biological particles. Escherichia coli was internalized by both SGCs and GCs, whereas white spot syndrome virus (WSSV) was mostly ingested by SGCs. However, the bacterial cells were rapidly taken and cleared from the circulation by the hemocytes, while the WSSV virions were gradually internalized and remained in the cells for a relatively longer period of time. These findings provide basic information of the phagocytic hemocytes of crayfish and how they respond to different foreign particles.


Assuntos
Astacoidea/citologia , Escherichia coli/fisiologia , Hemócitos/fisiologia , Imunidade Inata , Fagocitose , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Astacoidea/imunologia , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Hemócitos/ultraestrutura , Hemócitos/virologia , Masculino , Microscopia Eletrônica de Transmissão , Internalização do Vírus
11.
Fish Shellfish Immunol ; 76: 279-286, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29496475

RESUMO

The PI3K/AKT signaling pathway is commonly exploited to regulate viral replication and affect the fate of infected cells. In the present study, a PI3K-specific inhibitor (LY294002) was employed to pretreat crayfish to evaluate the effects of PI3K/AKT signaling pathway in WSSV replication. The results showed that the WSSV copy numbers in crayfish pretreated with LY294002 were significantly lower than those in Tris-HCl pretreatment crayfish on the sixth and tenth day after WSSV infection. In semigranular cells, the apoptosis rates were up-regulated on the third day post-WSSV infection, and a significantly lower proportion of apoptosis cells were observed in LY294002-pretreatment group. The expression level of Bax, Bax inhibitor-1 and lectin mRNA in haemocytes of crayfish were increased after WSSV infection. After the secondary stimulation with Tris-HCl, the Bax expression level in LY294002-pretreatment crayfish was significantly higher than that of crayfish pretreated with Tris-HCl on the third or sixth day, but the Toll and lectin mRNA expression decreased significantly on the third, sixth and tenth day. The Bax mRNA expression levels in LY294002-WSSV group were significantly higher than those in Tris-HCl-WSSV group on the third and tenth day. The Bax inhibitor-1 mRNA expression levels in LY294002-WSSV group were significantly lower than those in Tris-HCl-WSSV crayfish on the third day. These results together indicated that the hosts PI3K/AKT signaling pathway play positive roles in WSSV replication through the balance between host cell apoptois and innate immune responses. This information is helpful to further understand the role of PI3K/AKT signaling pathway on WSSV replication in Decapoda crustaceans.


Assuntos
Proteínas de Artrópodes/antagonistas & inibidores , Astacoidea/imunologia , Cromonas/farmacologia , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Astacoidea/genética , Astacoidea/virologia , Vírus da Síndrome da Mancha Branca 1/efeitos dos fármacos
12.
Dev Comp Immunol ; 84: 264-272, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29510164

RESUMO

Influenza A virus non-structural-1A binding protein (named as Ns1abp) was originally identified as a host protein from human that bound to the viral NS-1 protein. In our previous study, the expression of an Ns1abp-like gene (denoted as CqNs1abp-like gene) was found to be up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To elucidate the role of CqNs1abp-like gene involved in WSSV infection, we cloned the CqNs1abp-like gene in which the open reading frame was 2232 bp, encoding 743 amino acids with two typical domains of one BTB (Broad-Complex, Tramtrack and Bric a brac) domain at N-terminal and six Kelch domains at C-terminal. The gene expression profile showed that the mRNA transcript of CqNs1abp-like gene was widely expressed in all the tested tissues with highest expression in nerve, relatively high expression in Hpt and lowest expression in eyestalk. Importantly, both the WSSV entry and the viral replication were significantly reduced in Hpt cells after gene silencing of CqNs1abp-like gene. By using protein pull-down assay, we found that the recombinant BTB domain, six Kelch domains and CqNs1abp-like intact protein were all bound to the WSSV envelope protein VP28, respectively, in which the BTB domain showed slightly less binding affinity than that of the six Kelch domains or the recombinant intact protein. Besides, the WSSV entry into Hpt cells was clearly decreased when the virus was pre-incubated with the recombinant BTB domain, six Kelch domains, or the recombinant CqNs1abp-like intact protein, respectively, suggesting that the CqNs1abp-like gene was likely to function as a putative recognition molecular towards WSSV infection in a crustacean C. quadricarinatus. Taken together, these data shed new light on the mechanism of WSSV infection and a putatively novel target on anti-WSSV infection in crustacean farming.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/imunologia , Infecções por Vírus de DNA/imunologia , Hemócitos/fisiologia , Tecido Nervoso/fisiologia , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/metabolismo , Células Cultivadas , Clonagem Molecular , Humanos , Vírus da Influenza A/fisiologia , Proteínas Nucleares/metabolismo , Domínios Proteicos/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Proteínas do Envelope Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
13.
Fish Shellfish Immunol ; 75: 391-399, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29427719

RESUMO

Hemocyanins (HMC): the copper-containing respiratory proteins present in invertebrate hemolymph, which plays many essential roles in the immune system. Currently, little is known about the HMC domains of Procambarus clarkii (P. clarkii) and their function in antimicrobial immune response. In this present study, we comparatively studied the expression pattern of native PcHMC with the three recombinant proteins of variable domains of crayfish hemocyanin (PcHMC-N, N-terminal domain of hemocyanin; PcHMC-T, tyrosinase domain of hemocyanin; PcHMC-C, C-terminal domain of hemocyanin). The results showed that three purified recombinant proteins had a strong binding to various bacteria and lipopolysaccharides that further highly agglutinated. The HMCs recombinant proteins showed strong antibacterial activity against V. parahaemolyticus and S. aureus by bacterial growth inhibition, phenoloxidase (PO) and phagocytosis assays. Specifically, rPcHMC1-T and rPcHMC1-C inhibited both the bacteria efficiently, rPcHMC1-T was highly upregulated the PO activity than the other recombinant proteins. Whereas, recombinant proteins pretreated crayfish hemocytes participated in phagocytosis activity, rPcHMC1-N and rPcHMC1-C proteins had a profound effect than the rPcHMC1-T on S. aureus and V. parahaemolyticus phagocytosis. The crayfish hemocyanin domains clearly exhibited antibacterial and phagocytic activities against both the bacteria, suggesting that its variable domains of hemocyanin have the different function on specific pathogen during the assault of pathogens.


Assuntos
Antibacterianos/farmacologia , Proteínas de Artrópodes/farmacologia , Astacoidea/imunologia , Astacoidea/microbiologia , Fenômenos Fisiológicos Bacterianos , Hemocianinas/farmacologia , Animais , Antibacterianos/química , Proteínas de Artrópodes/química , Hemocianinas/química , Lipopolissacarídeos/farmacologia , Peptidoglicano/farmacologia , Domínios Proteicos , Ácidos Teicoicos/farmacologia
14.
Dev Comp Immunol ; 84: 109-116, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29428488

RESUMO

The ß-thymosins are a group of structurally related, highly conserved intracellular small peptides in vertebrates with various biological functions, including cytoskeletal remodeling, neuronal development, cell migration, cell survival, tissue repair and inhibition of inflammation. In contrast to vertebrates, the function of ß-thymosin is not fully understood in crustaceans. Previously, we found that a thymosin-repeated protein1 (CqTRP1) gene was up-regulated after white spot syndrome virus (WSSV) challenge in hematopoietic tissue (Hpt) cells from the red claw crayfish Cherax quadricarinatus. To further identify the effect of CqTRP1 on WSSV infection, a full length cDNA sequence of ß-thymosin homologue was cloned and analyzed from red claw crayfish followed by functional study. The CqTRP1 cDNA contains an open reading frame of 387 nucleotides encoding a protein of 129 amino acids with a putative molecular mass of 14.3 kDa. The amino acid sequence showed high identity with other ß-thymosins and contained three characteristic thymosin ß actin-binding motifs, suggesting that CqTRP1 was a member of the ß-thymosin family. Tissue distribution analysis revealed a ubiquitous presence of CqTRP1 in all the examined tissues with the highest expression in hemocytes, Hpt and gonad at the transcriptional level. Interestingly, the gene silencing of endogenous CqTRP1 by RNAi enhanced the WSSV replication in Hpt cells. Meanwhile, the WSSV replication was significantly reduced in the Hpt cell cultures if overloaded with a recombinant CqTRP1. Taken together, these data clearly indicated that CqTRP1 was likely to be associated with the anti-WSSV response in a crustacean C. quadricarinatus, which provides new strategy against white spot disease in crustacean aquaculture.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/imunologia , Infecções por Vírus de DNA/imunologia , Gônadas/metabolismo , Hemócitos/metabolismo , Timosina/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Aquicultura , Proteínas de Artrópodes/metabolismo , Astacoidea/virologia , Clonagem Molecular , Gônadas/imunologia , Gônadas/virologia , Hemócitos/imunologia , Hemócitos/virologia , Proteínas dos Microfilamentos/genética , RNA Interferente Pequeno/genética , Frutos do Mar , Timosina/metabolismo , Replicação Viral
15.
Dev Comp Immunol ; 82: 104-112, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29341872

RESUMO

It is well known that iron is an essential element for all living organism. The intracellular iron availability is also important for the host's innate immune response to various pathogens, in which the iron homeostasis can be regulated by ferritin due to its iron storage property. In this study, a full-length cDNA sequence of ferritin (named as CqFerritin) was identified with 1410 bp from red claw crayfish Cherax quadricarinatus, which contained an open reading frame of 513 bp, encoding 170 amino acids with a conserved ferritin domain. Tissue distribution analysis demonstrated that CqFerritin was widely expressed in various tissues with high presence in haemocyte, haematopoietic tissue (Hpt) and heart, while lowest expression in hepatopancreas. In addition, loss-of-function of CqFerritin by gene silencing resulted in significantly higher expression of an envelope protein VP28 of white spot syndrome virus (WSSV) in red claw crayfish Hpt cell cultures, indicating the potential antiviral response of CqFerritin. To further explore the effect on WSSV replication by CqFerritin, recombinant CqFerritin protein (rCqFerritin) was transfected into Hpt cells followed by WSSV infection. Importantly, the replication of WSSV was obviously decreased in Hpt cells if transfected with rCqFerritin protein, suggesting that CqFerritin had clearly negative effect on WSSV infection. Furthermore, intracellular accumulation of iron ions was found to promote the WSSV replication in a dose-dependent manner, illustrating that the iron level regulated by CqFerritin was likely to be vital for WSSV infection in red claw crayfish. Taken together, these data suggest that CqFerritin plays an important role in immune defense against WSSV infection in a crustacean C. quadricarinatus.


Assuntos
Proteínas de Artrópodes/metabolismo , Astacoidea/imunologia , Infecções por Vírus de DNA/imunologia , Ferritinas/metabolismo , Sistema Hematopoético/metabolismo , Ferro/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/genética , Astacoidea/virologia , Células Cultivadas , Clonagem Molecular , DNA Complementar/genética , Ferritinas/genética , Imunidade Inata , Transporte de Íons , Miocárdio/metabolismo , Replicação Viral
16.
Fish Shellfish Immunol ; 72: 383-388, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29097323

RESUMO

Cyclophilin A (Cyp A) is the main intracellular receptor of cyclosporin A (CsA) belonging to the immunophilin family, which is known as an effective immunosuppressive drug. This study aimed to gain insights into the structure and biological function of cyclophilin A in the red swamp crayfish, Procambarus clarkii (PcCypA). We cloned PcCypA by homology cloning and anchored polymerase chain reaction (PCR), and assessed its mRNA and protein expression levels in different tissues using quantitative real-time PCR and western blot analysis, respectively. The full-length DNA contained a 5' untranslated region (UTR) comprising 108 base pairs (bp), an open reading frame of 495 bp encoding a polypeptide of 164 amino acids with an estimated molecular mass of 17.3 kDa, and a 3' UTR of 281 bp including a significant poly(A) plus tail sequence. The predicted amino acid sequence of PcCypA shared high identity with CypA in other organisms. PcCypA transcripts were detected in the hepatopancreas, gill, heart, muscle, testis, and ovary of crayfish, with the highest expression levels in the heart. Western blot analysis found one 17-kDa band in all of the tissues examined, except for the ovary. Molecular identification and expression analysis of PcCypA will facilitate further studies of the immune defense mechanisms in red swamp crayfish, and provide new insights into freshwater invertebrate immunology.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Ciclofilina A/genética , Ciclofilina A/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Ciclofilina A/química , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência
18.
Dev Comp Immunol ; 80: 94-98, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28502650

RESUMO

Freshwater crayfish is an important commodity as well as a successful model for studies on crustacean immunity. Due to the ease with which they are kept and the available methods for hemocyte separation and culture they have proven to be very useful. Here, recent progress regarding pattern recognition, immune effector production and antiviral mechanisms are discussed. Several cases of functional resemblance between vertebrate complement and the crayfish immune reactions are highlighted.


Assuntos
Proteínas de Artrópodes/imunologia , Astacoidea/imunologia , Hemócitos/fisiologia , Imunidade Inata , Receptores de Reconhecimento de Padrão/imunologia , Viroses/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Animais , Antivirais/imunologia , Técnicas de Cultura de Células , Especificidade da Espécie , Vertebrados/imunologia
19.
Dev Comp Immunol ; 80: 53-66, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28279805

RESUMO

The possibility of immunological memory in invertebrates is a topic that has recently attracted a lot of attention. Today, even vertebrates are known to exhibit innate immune responses that show memory-like properties, and since these responses are triggered by cells that are involved in the innate immune system, it seems that immune specificity and immune memory do not necessarily require the presence of B cells and T cells after all. This kind of immune response has been called "immune priming" or "trained immunity". In this report, we review recent observations and our current understanding of immunological memory within the innate immune system in cultured shrimp and crayfish after vaccination with live vaccine, killed vaccine and subunit vaccines. We also discuss the possible mechanisms involved in this immune response.


Assuntos
Antígenos de Bactérias/imunologia , Artemia/imunologia , Astacoidea/imunologia , Interferência de RNA/imunologia , Vacinas de Subunidades/imunologia , Vibrioses/imunologia , Vibrio/imunologia , Vacinas Virais/imunologia , Viroses/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Animais , Antígenos de Bactérias/genética , Aquicultura , Imunidade Inata , Memória Imunológica , Vacinação
20.
Dev Comp Immunol ; 79: 186-194, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29102705

RESUMO

White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, which has been causing huge economic losses in global aquaculture. Laminin receptor (LR) is a cell surface receptor which participates in the interactions between cells as well as cells and extracellular matrix. Previously, we found that a CqLR-like gene was responsive to WSSV infection in the hematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. To further reveal the role of CqLR-like gene involved in WSSV infection, the full-length cDNA of CqLR-like gene was cloned with 1000 bp, and the open reading frame encoded 308 amino acids with a conserved laminin-binding domain. Importantly, both the WSSV entry and viral replication were strongly reduced in Hpt cells after loss-of-function of CqLR-like gene by gene silencing. Protein interaction assay demonstrated that the recombinant CqLR-like protein could bind to WSSV virion in vitro by enzyme-linked immunosorbent assay and the binding affinity was in a dose-dependent manner. Furthermore, recombinant CqLR-like protein was found to bind to WSSV envelop protein VP28, but not other envelop proteins tested including VP19, VP24, and VP26, by pull down assay in HEK293T cells. In regarding to that LR is mainly localized on many types of cells' membrane, these data together suggested that CqLR-like protein was likely to function as a putative recognition molecule towards WSSV and act in the viral entry into a crustacean host cell, which may benefit the elucidation of the WSSV pathogenesis and further the pharmaceutical target for the possibly effective control of WSSV disease.


Assuntos
Astacoidea/imunologia , Infecções por Vírus de DNA/imunologia , Receptores de Laminina/genética , Proteínas do Envelope Viral/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Clonagem Molecular , Células HEK293 , Humanos , Terapia de Alvo Molecular , Ligação Proteica , RNA Interferente Pequeno/genética , Internalização do Vírus , Replicação Viral
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