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1.
Dev Comp Immunol ; : 103960, 2020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-33301793

RESUMO

The versatile fish pathogen Edwardsiella tarda is an intracellular pathogen with the ability to invade and replicate in host phagocytes. However, the mechanism mediating the uptake of E. tarda in fish monocytes/macrophages (MO/MΦ) is not yet understood. Generating mudskipper kidney-derived MO/MФ transcriptomic resources from mudskipper challenged by E. tarda is crucial for understanding the molecular mechanisms underlying the mudskipper invasion process. In the present study, a total of 1,185 up-regulated and 885 down-regulated differentially expressed genes (DEGs) were identified using RNA-seq. Enrichment and pathway analysis of DEGs revealed the centrality of the phagosome and regulation of actin cytoskeleton pathways in pathogen entry. The progress of phagosome formation was observed by transmission electron microscopy. Eight conserved integrin (ITG) subunit genes, belonging to the phagocytic receptors, were found in the transcriptomic sequence data. Additionally, quantitative real-time PCR showed that the mRNA expressions of most ITG subunit genes were related to the different infection times of E. tarda and the different bacterial pathogens. Further assays demonstrated that phagocytosis of FITC-labeled E. tarda by mudskipper MO/MФ was significantly reduced by the tetrapeptide Asp-Gly-Arg-Ser (RGDS). In summary, phagocytosis is one of the entry pathways into mudskipper MO/MΦ, and RGD-binding ITGs are involved in the phagosome formation process.

2.
J Fish Dis ; 2020 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-33340375

RESUMO

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

3.
Zool Res ; 41(2): 123-137, 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32150792

RESUMO

Interleukin-34 (IL-34) is a novel cytokine that plays an important role in innate immunity and inflammatory processes by binding to the colony-stimulating factor-1 receptor (CSF-1R). However, information on the function of IL-34 in fish remains limited. In the present study, we identified an IL-34 homolog from mudskippers ( Boleophthalmus pectinirostris). In silico analysis showed that the mudskipper IL-34 (BpIL-34) was similar to other known IL-34 variants in sequence and structure and was most closely related to an orange-spotted grouper ( Epinephelus coioides) homolog. BpIL-34 transcripts were constitutively expressed in various tissues, with the highest level of expression found in the brain. Edwardsiella tarda infection significantly up-regulated the mRNA expression of BpIL-34 in the mudskipper tissues. The recombinant mature BpIL-34 peptide (rBpIL-34) was purified and used to produce anti-rBpIL-34 IgG. Western blot analysis combined with PNGase F digestion revealed that native BpIL-34 in monocytes/macrophages (MOs/MФs) was N-glycosylated. In vitro, rBpIL-34 treatment enhanced the phagocytotic and bactericidal activity of mudskipper MOs/MФs, as well as the mRNA expression of pro-inflammatory cytokines like tumor necrosis factor α ( BpTNF-α) and BpIL-1ß in these cells. Furthermore, the knockdown of mudskipper CSF-1R1 ( BpCSF-1R1), but not mudskipper BpCSF-1R2, significantly inhibited the rBpIL-34-mediated enhanced effect on MO/MФ function. In conclusion, our results indicate that mudskipper BpIL-34 modulates the functions of MOs/MФs via BpCSF-1R1.


Assuntos
Edwardsiella tarda/fisiologia , Proteínas de Peixes/genética , Peixes/genética , Interleucinas/genética , Fator Estimulador de Colônias de Macrófagos/genética , Macrófagos/imunologia , Monócitos/imunologia , Animais , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Peixes/imunologia , Imunidade Inata , Interleucinas/imunologia , Fator Estimulador de Colônias de Macrófagos/imunologia
4.
Dev Comp Immunol ; 103: 103511, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31580833

RESUMO

ß-defensin is a cationic host defense peptide actively participating in host innate immune response against pathogens. In teleost fish, ß-defensin exhibits a diversity in genotypes and functions. Herein, a ß-defensin homolog (PaBD) was identified from ayu, Plecoglossus altivelis, showing multiple tissues' upregulation against Vibrio anguillarum challenge. In vivo experiments revealed that intraperitoneal injection of chemically synthesized mature PaBD (mPaBD) increased the survival rate of V. anguillarum-infected ayu, accompanied by reduced bacterial load and decreased tissue mRNA levels of tumor necrosis factor α (PaTNF-α) and interleukin 1ß (PaIL-1ß). However, in vitro, mPaBD showed weak bactericidal activity against V. anguillarum. Interestingly, mPaBD enhanced phagocytosis, intracellular bacterial killing, and respiratory burst of ayu monocytes/macrophages (MO/MΦ). Moreover, it inhibited mRNA levels of PaIL-1ß and PaTNF-α in MO/MФ upon V. anguillarum infection. In conclusion, PaBD protects ayu against V. anguillarum challenge not only through its direct antibacterial ability, but also through its immunomodulation in MO/MΦ.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Osmeriformes/imunologia , Vibrioses/veterinária , Vibrio/fisiologia , beta-Defensinas/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Carga Bacteriana/efeitos dos fármacos , Citocinas/genética , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Proteínas de Peixes/administração & dosagem , Proteínas de Peixes/genética , Imunomodulação , Macrófagos/imunologia , Macrófagos/microbiologia , Monócitos/imunologia , Monócitos/microbiologia , Osmeriformes/classificação , Osmeriformes/genética , Fagocitose , Filogenia , Explosão Respiratória , Alinhamento de Sequência , Taxa de Sobrevida , Distribuição Tecidual , Vibrio/efeitos dos fármacos , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrioses/prevenção & controle , beta-Defensinas/administração & dosagem , beta-Defensinas/genética
5.
Zool Res ; 41(1): 39-50, 2020 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-31709785

RESUMO

D-dopachrome tautomerase (DDT), a member of the macrophage migration inhibitory factor (MIF) protein superfamily, is a newly described cytokine with chemokine-like characteristics. However, research on fish DDT remains limited. In this study, we identified a DDT homolog (LjDDT) from the Japanese sea bass, Lateolabrax japonicus. Sequence analysis showed that LjDDT had typical sequence features of known DDT and MIF homologs and was most closely related to DDT of rock bream ( Oplegnathus fasciatus). LjDDT transcripts were detected in all tested tissues of healthy Japanese sea bass, with the highest expression found in the liver. Upon infection with Vibrio harveyi, LjDDT transcripts were significantly down-regulated in the three tested tissues, including the liver, spleen, and head kidney. Recombinant LjDDT (rLjDDT) and the corresponding antibody (anti-rLjDDT) were subsequently prepared. The administration of 100 µg/g anti-rLjDDT had a statistically significant protective effect on the survival of V. harveyi-infected fish. Moreover, rLjDDT was able to induce the migration of monocytes/macrophages (MO/MФ) and lymphocytes both in vitro and in vivo, but without significant influence on the migration of neutrophils. rLjDDT exhibited chemotactic activity for lipopolysaccharide (LPS) -stimulated M1-type MO/ MΦ in vitro, but not for cAMP-stimulated M2-type MO/MΦ. Furthermore, the knockdown of LjCD74, but not LjCXCR4, significantly down-regulated the rLjDDT-enhanced migration of MO/MΦ and relieved the rLjMIF-inhibited migration of MO/MΦ. These results indicate that LjCD74 may be the major chemotactic receptor of LjDDT and LjMIF in Japanese sea bass MO/MΦ. Combined rLjDDT+ rLjMIF treatment had no significant effect on the migration of MsiRNA, LjCD74si-, or LjCXCR4sitreated MO/MΦ compared to the control group, suggesting that the roles of LjDDT and LjMIF may be antagonistic. In conclusion, our study demonstrates for the first time that DDT may play a role in the immune responses of fish against bacterial infection through chemotactic recruitment of MO/MΦ via mediation of CD74 as an antagonist of MIF.


Assuntos
Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Sequência de Aminoácidos , Animais , Movimento Celular , Relação Dose-Resposta a Droga , Peixes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/fisiologia , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/farmacologia , RNA Mensageiro , Vibrio , Vibrioses/enzimologia , Vibrioses/microbiologia , Vibrioses/veterinária
6.
Zool Res ; 40(5): 404-415, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31343855

RESUMO

Classical Fc receptors (FcRs) mediate the binding to and recognition of the Fc portion of antibodies and play an important role during immune responses in mammals. Although proteins similar to soluble FcRs have been identified in fish, little is known about the role of such proteins in fish immunity. Here, we cloned a cDNA sequence encoding a soluble Fc receptor for an immunoglobulin G (FcγR) homolog from ayu (Plecoglossus altivelis) (PaFcγRl). The predicted protein was composed of two immunoglobulin C2-like domains but lacked a transmembrane segment and a cytoplasmic tail. The PaFcγRl transcripts were distributed at low levels in all tested tissues, but significantly increased after Vibrio anguillarum infection. The PaFcγRl protein was expressed in the head kidney, trunk kidney, and neutrophils. Recombinant PaFcγRl (rPaFcγRl) was secreted when transfected into mammalian cells and the native protein was also detected in serum upon infection. rPaFcγRl was also demonstrated to bind to ayu IgM, as assessed by cell transfection. Suppressive activity of the recombinant mature protein of PaFcγRl (rPaFcγRlm) on in vitro anti-sheep red blood cell (SRBC) responses was detected by a modified hemolytic plaque forming cell assay. In conclusion, our study revealed that PaFcγRl is closely involved in the negative regulation of IgM production in the ayu spleen.


Assuntos
Peixes/fisiologia , Imunoglobulina M/metabolismo , Receptores de IgG/metabolismo , Baço/citologia , Animais , Receptores de IgG/genética
7.
Int J Oral Sci ; 9(12): e7, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32987970

RESUMO

Dental caries are the most prevalent chronic infections in the oral cavity, and Streptococcus mutans acts as the main cariogenic bacterial species. Antibacterial quaternary ammonium compounds (QAs) have been developed to preveFnt or treat dental caries. However, there is no report on the tolerance of S. mutans to QAs. In this study, we investigated the development of S. mutans persistence induced by a novel dental caries defensive agent, dimethylaminododecyl methacrylate (DMADDM), for the first time. Typical biphasic killing kinetics for persisters were observed in both S. mutans planktonic and biofilm cultures challenged by DMADDM at concentrations of 20 and 200 µg·mL-1, respectively. The persisters tolerated six other antibiotics with different antibacterial mechanisms, while only daptomycin and vancomycin could slightly reduce the persister numbers in planktonic cultures. The distribution of persisters in DMADDM-treated biofilms was similar to that in the untreated control, except that the total biomass and biofilm height were significantly reduced. A higher exopolysaccharides (EPS):bacteria ratio was observed in DMADDM-treated biofilms. Persisters in biofilms significantly upregulated gtf gene expression, indicating an increase in the bacteria's ability to produce EPS and an elevated capability of cariogenic virulence. Carbon source metabolism was significantly reduced, as related metabolic genes were all downregulated in persisters. Concentrations of 0.1 mM, 1 mM and 10 mM of extra glucose significantly reduced the number of persisters both in planktonic and biofilm conditions. The formation of non-inheritable and multidrug tolerant persisters induced by DMADDM suggested that drug tolerance and new persistent eradication strategies should be considered for oral antibacterial agents.

8.
Nan Fang Yi Ke Da Xue Xue Bao ; 36(10): 1328-1333, 2016 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-27777193

RESUMO

OBJECTIVE: To analyze the whole microbial structure in a case of rampant caries to provide evidence for its prevention and treatment. METHODS: Clinical samples including blood, supragingival plaque, plaque in the caries cavity, saliva, and mucosal swabs were collected with the patient's consent. The blood sample was sent for routine immune test, and the others samples were stained using Gram method and cultured for identifying colonies and 16S rRNA sequencing. DNA was extracted from the samples and tested for the main cariogenic bacterium (Streptococcus mutans) with qPCR, and the whole microbial structure was analyzed using DGGE. RESULTS: The patient had a high levels of IgE and segmented neutrophils in his blood. Streptococci with extremely long chains were found in the saliva samples under microscope. Culture of the samples revealed the highest bacterial concentration in the saliva. The relative content of hemolytic bacterium was detected in the samples, the highest in the caries cavity; C. albicans was the highest in the dental plaque. In addition, 33 bacterial colonies were identified by VITEK system and 16S rDNA sequence phylogenetic analysis, and among them streptococci and Leptotrichia wade were enriched in the dental plaque sample, Streptococcus mutans, Fusobacterium nucleatum, and Streptococcus tigurinus in the caries cavity, and Lactobacillus in the saliva. S. mutans was significantly abundant in the mucosal swabs, saliva and plaque samples of the caries cavity as shown by qPCR. Compared to samples collected from a healthy individual and another two patients with rampant caries, the samples from this case showed a decreased bacterial diversity and increased bacterial abundance shown by PCR-DGGE profiling, and multiple Leptotrichia sp. were detected by gel sequencing. CONCLUSION: The outgrowth of such pathogenic microorganisms as S. mutans and Leptotrichia sp., and dysbiosis of oral microbial community might contribute to the pathogenesis of rampant caries in this case.


Assuntos
Cárie Dentária/microbiologia , Microbiota , Anormalidades Múltiplas , Placa Dentária/microbiologia , Fusobacterium/isolamento & purificação , Humanos , Imunoglobulina E/sangue , Lactobacillus/isolamento & purificação , Leptotrichia/isolamento & purificação , Deformidades Congênitas dos Membros , Mucosa Bucal/microbiologia , Neutrófilos/citologia , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Saliva/microbiologia , Streptococcus/isolamento & purificação , Anormalidades Dentárias
9.
Int J Oral Sci ; 8(4): 231-238, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27740612

RESUMO

D-alanine (D-Ala) is an essential amino acid that has a key role in bacterial cell wall synthesis. Alanine racemase (Alr) is a unique enzyme that interconverts L-alanine and D-alanine in most bacteria, making this enzyme a potential target for antimicrobial drug development. Streptococcus mutans is a major causative factor of dental caries. The factors involved in the survival, virulence and interspecies interactions of S. mutans could be exploited as potential targets for caries control. The current study aimed to investigate the physiological role of Alr in S. mutans. We constructed alr mutant strain of S. mutans and evaluated its phenotypic traits and interspecies competitiveness compared with the wild-type strain. We found that alr deletion was lethal to S. mutans. A minimal supplement of D-Ala (150 µg·mL-1) was required for the optimal growth of the alr mutant. The depletion of D-alanine in the growth medium resulted in cell wall perforation and cell lysis in the alr mutant strain. We also determined the compromised competitiveness of the alr mutant strain relative to the wild-type S. mutans against other oral streptococci (S. sanguinis or S. gordonii), demonstrated using either conditioned medium assays or dual-species fluorescent in situ hybridization analysis. Given the importance and necessity of alr to the growth and competitiveness of S. mutans, Alr may represent a promising target to modulate the cariogenicity of oral biofilms and to benefit the management of dental caries.


Assuntos
Alanina Racemase/metabolismo , Cárie Dentária/microbiologia , Streptococcus mutans/crescimento & desenvolvimento , Biofilmes , Humanos , Hibridização in Situ Fluorescente , Streptococcus mutans/metabolismo
10.
Fish Shellfish Immunol ; 57: 136-147, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27546554

RESUMO

Mammalian interleukin 4 (IL-4) and interleukin 13 (IL-13) molecules are anti-inflammatory cytokines mediating the alternative activation of macrophages. However, the role of fish IL-4/13 homologs in monocytes/macrophages (MO/MФ) polarization remains unclear. In this study, we have functionally identified an IL-4/13B homolog in grass carp (Ctenopharyngodon idella), which is termed as CiIL-4/13B. Multiple alignment showed that CiIL-4/13B shared the typical characteristics and structure with other known fish IL-4/13. Phylogenetic analysis showed that CiIL-4/13B is evolutionarily closely related to zebrafish (Danio rerio) and common carp (Cyprinus carpio) IL-4/13B. CiIL-4/13B mRNA was constitutively expressed in tissues and peripheral blood lymphocytes (PBLs) examined, with its highest expression seen in PBLs. Following Aeromonas hydrophila infection, CiIL-4/13B mRNA expression was upregulated. Recombinant CiIL-4/13B (rCiIL-4/13B) was overexpressed in Escherichia coli and purified for a functional study. Using prepared anti-rCiIL-4/13B antiserum, Western blot analysis showed that native CiIL-4/13B in grass carp plasma is N-glycosylated. Intraperitoneal injection of bioactive rCiIL-4/13B significantly increased the survival rate of grass carp against A. hydrophila, and decreased the tissue bacterial load, with a higher dose having better effects. Bioactive rCiIL-4/13B treatment decreased nitrite production and mRNA expression of proinflammatory cytokines (IL-1ß and TNF-α), while it increased arginase activity and mRNA expression of anti-inflammatory cytokines (TGF-ß and IL-10). The phagocytosis by grass carp MO/MФ had no significant changes by the 8 h treatment of bioactive rCiIL-4/13B compared to that of the negative control, while it was significantly inhibited by the 24 h treatment of bioactive rCiIL-4/13B. The inhibitory effect of rCiIL-4/13B on MO/MФ phagocytosis may be a consequence of MO/MФ proliferation. In summary, our results suggest that CiIL-4/13B plays a protective effect in grass carp against A. hydrophila by inducing alternatively activated MO/MФ.


Assuntos
Carga Bacteriana , Carpas , Proteínas de Peixes/genética , Infecções por Bactérias Gram-Negativas/veterinária , Imunidade Inata , Interleucina-4/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Interleucina-4/química , Interleucina-4/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/veterinária
11.
Dongwuxue Yanjiu ; 37(3): 126-35, 2016 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-27265650

RESUMO

Ayu (Plecoglossus altivelis) fish, which are an amphidromous species distributed in East Asia, live in brackish water (BW) during their larval stage and in fresh water (FW) during their adult stage. In this study, we found that FW-acclimated ayu larvae exhibited a slower growth ratio compared with that of BW-acclimated larvae. However, the mechanism underlying FW acclimation on growth suppression is poorly known. We employed transcriptome analysis to investigate the differential gene expression of FW acclimation by RNA sequencing. We identified 158 upregulated and 139 downregulated transcripts in FW-acclimated ayu larvae compared with that in BW-acclimated larvae. As determined by Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway mapping, functional annotation of the genes covered diverse biological functions and processes, and included neuroendocrinology, osmotic regulation, energy metabolism, and the cytoskeleton. Transcriptional expression of several differentially expressed genes in response to FW acclimation was further confirmed by real-time quantitative PCR. In accordance with transcriptome analysis, iodothyronine deiodinase (ID), pro-opiomelanocortin (POMC), betaine-homocysteine S-methyltransferase 1(BHMT), fructose-bisphosphate aldolase B (aldolase B), tyrosine aminotransferase (TAT), and Na(+)-K(+) ATPase (NKA) were upregulated after FW acclimation. Furthermore, the mRNA expressions of b-type natriuretic peptide (BNP) and transgelin were downregulated after FW acclimation. Our data indicate that FW acclimation reduced the growth rate of ayu larvae, which might result from the expression alteration of genes related to endocrine hormones, energy metabolism, and direct osmoregulation.


Assuntos
Perfilação da Expressão Gênica , Larva/efeitos dos fármacos , Larva/genética , Osmeriformes/genética , Salinidade , Aclimatação/efeitos dos fármacos , Aclimatação/genética , Animais , Tamanho Corporal/efeitos dos fármacos , Tamanho Corporal/genética , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Água Doce/química , Larva/crescimento & desenvolvimento , Larva/fisiologia , Osmeriformes/crescimento & desenvolvimento , Osmeriformes/fisiologia
12.
Fish Shellfish Immunol ; 55: 140-8, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27235369

RESUMO

Recognizing the presence of invading pathogens by pattern recognition receptors (PRRs) is key to mounting an effective innate immune response. Mammalian CD302 is an unconventional C-type lectin like receptor (CTLR) involved in the functional regulation of immune cells. However, the role of CD302 in fish remains unclear. In this study, we characterized a novel CD302 gene from ayu (Plecoglossus altivelis), which was tentatively named PaCD302. The cDNA sequence of PaCD302 is 1893 nucleotides in length, and encodes a polypeptide of 241 amino acids with molecular weight 27.1 kDa and pI 4.69. Sequence comparison and phylogenetic tree analysis showed that PaCD302 is a type I transmembrane CTLR devoid of the known amino acid residues essential for Ca(2+)-dependent sugar binding. PaCD302 mRNA expression was detected in all tissues and cells tested, with the highest level in the liver. Following Vibrio anguillarum infection, PaCD302 mRNA expression was significantly upregulated in all tissues tested. For further functional analysis, we generated a recombinant protein for PaCD302 (rPaCD302) by prokaryotic expression and raised a specific antibody against rPaCD302. Western blot analysis revealed that the native PaCD302 is glycosylated. Refolded rPaCD302 was unable to bind to five monosaccharides (l-fucose, d-galactose, d-glucose, d-mannose and N-acetyl glucosamine) or two other polysaccharides (lipopolysaccharide and peptidoglycan). It was able to bind to three Gram-positive and seven Gram-negative bacteria, but show no bacterial agglutinating activity. PaCD302 function blocking using anti-PaCD302 IgG resulted in inhibition of phagocytosis and bactericidal activity of ayu monocytes/macrophages (MO/MΦ), suggesting that PaCD302 regulates the function of ayu MO/MΦ. In summary, our study demonstrates that PaCD302 may participate in the immune response of ayu against bacterial infection via modulation of MO/MΦ function.


Assuntos
Doenças dos Peixes/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Imunidade Inata , Lectinas Tipo C/genética , Osmeriformes , Vibrioses/veterinária , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Monócitos/imunologia , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Alinhamento de Sequência/veterinária , Vibrio/fisiologia , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologia
13.
Dongwuxue Yanjiu ; 37(2): 96-102, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-27029867

RESUMO

Colony-stimulating factor 1 receptor (CSF-1R) is an important regulator of monocytes/macrophages (MO/MΦ). Although several CSF-1R genes have been identified in teleosts, the precise role of CSF- 1R in ayu (Plecoglossus altivelis) remains unclear. In this study, we characterized the CSF-1R homologue from P. altivelis, and named it PaCSF-1R. Multiple sequence alignment and phylogenetic tree analysis showed that PaCSF-1R was most closely related to that of Japanese ricefish (Oryzias latipes). Tissue distribution and expression analysis showed that the PaCSF-1R transcript was mainly expressed in the head kidney-derived MO/MΦ, spleen, and head kidney, and its expression was significantly altered in various tissues upon Vibrio anguillarum infection. After PaCSF-1R neutralization for 48 h, the phagocytic activity of MO/MΦ was significantly decreased, suggesting that PaCSF-1R plays a role in regulating the phagocytic function of ayu MO/MΦ.


Assuntos
Proteínas de Peixes/genética , Regulação da Expressão Gênica , Osmeriformes/genética , Osmeriformes/microbiologia , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Vibrio/fisiologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Fagocitose , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo
14.
Fish Shellfish Immunol ; 51: 70-76, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26876329

RESUMO

Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine involved in many diseases in which immune dysfunction is present. Ayu LECT2 (PaLECT2), which interacts with a C-type lectin receptor (PaCLR), was shown to activate ayu head kidney-derived monocytes/macrophages (MO/MΦ) to improve the outcomes of fish upon bacterial infections. However, it is not known if PaCLR mediates PaLECT2 effects on ayu MO/MΦ. In this study, we determined the role of PaCLR in signal transduction of PaLECT2 on ayu MO/MΦ. We expressed the PaCLR ectodomain in Escherichia coli and produced a refolded recombinant protein (rPaCLR) that was then used to produce the anti-PaCLR IgG (anti-PaCLR) for neutralization. Addition of the refolded PaLECT2 mature peptide (rPaLECT2m) to ayu MO/MΦ cultures, increased cytokine expression, induced chemotaxis, and enhanced phagocytosis and bactericidal activity of these cells were observed. When we added anti-PaCLR to block the ectodomain of PaCLR, these effects were significantly inhibited. Based on our previous works and the data presented here, we conclude that PaCLR mediates the immunomodulatory effects of PaLECT2 on ayu MO/MΦ, thus defining a mechanism by which LECT2 protects fish against pathogens.


Assuntos
Proteínas de Peixes/genética , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intercelular/genética , Lectinas Tipo C/genética , Lectinas/genética , Osmeriformes/genética , Animais , Quimiotaxia , Escherichia coli/genética , Proteínas de Peixes/metabolismo , Rim Cefálico/metabolismo , Imunomodulação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lectinas/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Organismos Geneticamente Modificados/genética , Osmeriformes/imunologia , Osmeriformes/metabolismo
15.
Fish Shellfish Immunol ; 47(2): 878-85, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26525517

RESUMO

Cathelicidins (CATHs) are a family of endogenous antimicrobial peptides that are capable of both direct bacteria-killing and immunomodulatory effects. P2X7 receptor (P2X7R) is a mediator of CATH in mammalian immune cells. Here, we studied the function and regulation of CATH in head kidney-derived monocytes/macrophages (MO/MФ) from ayu, Plecoglossus altivelis. We investigated the chemotaxis of MO/MФ in response to ayu CATH (PaCATH), and found that PaCATH had a dose-dependent effect on MO/MФ chemotaxis with the optimal concentration of 10.0 µg/ml. The qPCR and Western blot analysis revealed that PaCATH inhibited the expression of ayu P2X7R (PaP2X7R) at both mRNA and protein levels. Knockdown of the PaP2X7R expression in ayu MO/MФ by RNA interference not only significantly inhibited the chemotactic effect of PaCATH on MO/MФ, but also obviously reduced the effect of PaCATH on the phagocytosis, bacteria-killing, respiratory burst, and cytokine expression of ayu MO/MФ. Our study revealed that the immunomodulatory effect of fish CATH is mediated by P2X7R.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Peixes/genética , Macrófagos/imunologia , Monócitos/imunologia , Osmeriformes/imunologia , Receptores Purinérgicos P2X7/genética , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Quimiotaxia , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Osmeriformes/genética , Receptores Purinérgicos P2X7/metabolismo , Análise de Sequência de DNA/veterinária
16.
Dongwuxue Yanjiu ; 36(3): 133-41, 2015 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-26018856

RESUMO

Interleukin 1ß (IL-1ß), the first interleukin to be characterized, plays a key role in regulating the immune response. In this study, we determined the cDNA and genomic DNA sequences of the IL-1ß gene from the large yellow croaker, Larimichthys crocea. Phylogenetic analysis indicated that the IL-1ß (LcIL-1ß) gene was most closely related to that of European seabass (Dicentrarchus labrax), sharing 67.8% amino acid identity. In healthy large yellow croaker, LcIL-1ß transcription was detected in all tested tissues, with the highest level found in the head kidney. Upon Vibrio alginolyticus infection, LcIL-1ß transcription in all tested tissues was significantly upregulated. Intraperitoneal injection of recombinant LcIL-1ß (rLcIL-1ß) improved the survival rate and reduced the tissue bacterial load after V. alginolyticus infection. In addition, rLcIL-1ß induced monocytes/macrophages (MO/MΦ) chemotaxis and increased phagocytosis and bactericidal activity in vitro. These results suggest that LcIL-1ß plays an important role in the large yellow croaker immune response against V. alginolyticus.


Assuntos
Doenças dos Peixes/genética , Proteínas de Peixes/genética , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Vibrioses/veterinária , Vibrio alginolyticus/fisiologia , Animais , Sequência de Bases , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/imunologia , Dados de Sequência Molecular , Perciformes/classificação , Perciformes/genética , Perciformes/imunologia , Filogenia , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrio alginolyticus/genética , Vibrio alginolyticus/isolamento & purificação
17.
Gene ; 556(2): 98-105, 2015 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-25447921

RESUMO

Neuregulin receptor degradation protein-1 (Nrdp1) was recently identified in humans as an important immune factor responding to the challenge of virus, LPS or cytokine. Its role in fish immune defense and whether it is involved in anti-parasite immunity have not been proven yet. In this report, the full-length cDNA sequence and genomic structure of Nrdp1 in the large yellow croaker Larimichthys crocea (LcNrdp1) were identified and characterized. The full-length cDNA of LcNrdp1 was 1248bp, including a 5' untranslated region (UTR) of 32bp, a 3' UTR of 259bp and an open reading frame (ORF) of 937bp, encoding a polypeptide of 318 amino acid residues. The full-length genomic DNA sequence of LcNrdp1 was composed of 2635 nucleotides, including four exons and three introns. The putative LcNrdp1 protein had no signal peptide sequence and contained a characteristic Nrdp1 consensus motif C3HC3D ring finger and a Coiled-coil domain. Phylogenetic analysis showed that Nrdp1 in fish was closer with that in other vertebrates (79%-90% amino acid identity) than in invertebrates and bacteria (27%-65%). In fishes, Nrdp1 in large yellow croaker was closer with that in Takifugu rubripes. The expression profile showed that LcNrdp1 was constitutively expressed in all tested tissues, especially highly expressed in brain, muscle and kidney. Post-infection (PI) with Cryptocaryon irritans, an increased expression of LcNrdp1 was induced in infection sites (skin and gill), whereas in immune organs, the expression of LcNrdp1 was up-regulated in spleen (except the 1st d and 10th d PI) but suppressed in head kidney. These results suggested that LcNrdp1 might play an important immune role in the finfish L. crocea in the defense against the parasite C. irritans.


Assuntos
Cilióforos/imunologia , Proteínas de Peixes/metabolismo , Perciformes/imunologia , Perciformes/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sequência de Bases , Infecções por Cilióforos/imunologia , Infecções por Cilióforos/parasitologia , Infecções por Cilióforos/veterinária , Clonagem Molecular , Evolução Molecular , Doenças dos Peixes/imunologia , Doenças dos Peixes/parasitologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Perciformes/parasitologia , Filogenia , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética
18.
Dongwuxue Yanjiu ; 35(2): 99-107, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24668652

RESUMO

The Dmrt family of genes are involved in sex differentiation in different species of invertebrates, and some vertebrates including human. In this study, we cloned the full-length cDNA of ayu (Plecoglossus altivelis) Dmrt1 and DmrtA2. Sequence and phylogenetic tree analyses showed ayu Dmrt1 showed highest similarity to that of Oncorhynchus mykiss while ayu DmrtA2 is most similar to that of Oryzias latipes. Fluorescence-based quantitative reverse transcription PCR (qRT-PCR) revealed the Dmrt1 was predominantly expressed in the testis. At the larval stages, Dmrt1 mRNA expression level was highest during 52-64 days post hatching (dph) and at the gastrula stage during embryonic development. DmrtA2, meanwhile, was specifically expressed in the ovary and was highly expressed in the female brain tissue, but not male brain tissue. During the larval stages, DmrtA2 expression remained high before day 34, and then fluctuated while generally decreasing. During embryonic development, DmrtA2 expression increased gradually and peaked at the hatching stage. Our data suggest that ayu Dmrt1 might participate in the differentiation and maintenance of testis while DmrtA2 may play a role in ovary-differentiation and mature-ovary maintenance. DmrtA2 might also participate in brain development.


Assuntos
Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Osmeriformes/embriologia , Osmeriformes/metabolismo , Fatores de Transcrição/metabolismo , Animais , DNA Complementar/genética , Feminino , Larva/genética , Larva/metabolismo , Masculino , Osmeriformes/genética , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética
19.
Int J Oral Sci ; 5(4): 206-11, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24136674

RESUMO

Streptococcus mutans is a common Gram-positive bacterium and plays a significant role in dental caries. Tobacco and/or nicotine have documented effects on S. mutans growth and colonization. Sortase A is used by many Gram-positive bacteria, including S. mutans, to facilitate the insertion of certain cell surface proteins, containing an LPXTGX motif such as antigen I/II. This study examined the effect of nicotine on the function of sortase A to control the physiology and growth of S. mutans using wild-type S. mutans NG8, and its isogenic sortase-defective and -complemented strains. Briefly, the strains were treated with increasing amounts of nicotine in planktonic growth, biofilm metabolism, and sucrose-induced and saliva-induced antigen I/II-dependent biofilm formation assays. The strains exhibited no significant differences with different concentrations of nicotine in planktonic growth assays. However, they had significantly increased (P≤0.05) biofilm metabolic activity (2- to 3-fold increase) as the concentration of nicotine increased. Furthermore, the sortase-defective strain was more sensitive metabolically to nicotine than the wild-type or sortase-complemented strains. All strains had significantly increased sucrose-induced biofilm formation (2- to 3-fold increase) as a result of increasing concentrations of nicotine. However, the sortase-defective strain was not able to make as much sucrose- and saliva-induced biofilm as the wild-type NG8 did with increasing nicotine concentrations. These results indicated that nicotine increased metabolic activity and sucrose-induced biofilm formation. The saliva-induced biofilm formation assay and qPCR data suggested that antigen I/II was upregulated with nicotine but biofilm was not able to be formed as much as wild-type NG8 without functional sortase A.


Assuntos
Aminoaciltransferases/efeitos dos fármacos , Proteínas de Bactérias/efeitos dos fármacos , Cisteína Endopeptidases/efeitos dos fármacos , Nicotina/farmacologia , Peptidoglicano/efeitos dos fármacos , Streptococcus mutans/efeitos dos fármacos , Motivos de Aminoácidos , Aminoaciltransferases/genética , Antígenos de Bactérias/efeitos dos fármacos , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Cisteína Endopeptidases/genética , Relação Dose-Resposta a Droga , Humanos , Mutação/genética , Nicotina/administração & dosagem , Peptidoglicano/genética , Saliva/fisiologia , Streptococcus mutans/enzimologia , Streptococcus mutans/crescimento & desenvolvimento , Sacarose/farmacologia
20.
Dongwuxue Yanjiu ; 33(6): 603-8, 2012 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-23266980

RESUMO

Heat shock protein 60 is an essential chaperone that can maintain the natural structure and function of mitochondrial proteins. Here, we successfully cloned the full length cDNA of HSP60 from Neobenedenia melleni, designated as NmHSP60. Real-time quantitative PCR and Western blot were used to analyze the expression change of NmHSP60 under different temperature and salinity. Compared with the typical 25 Degrees Celsius, expressions decreased dramatically in eggs and adults at 18 Degrees Celsius. Conversely, at 32 Degrees Celsius, expression increase dramatically in adults. Compared with salinity 24, expressions were significantly down-regulated in adults at salinity 18, and up-regulated in eggs at salinity 30. Experimental results suggest that NmHSP60 may play an significant role in N. melleni's adaptation to adverse environmental conditions.


Assuntos
Chaperonina 60/genética , Clonagem Molecular , Proteínas de Helminto/genética , Platelmintos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Chaperonina 60/metabolismo , Feminino , Proteínas de Helminto/metabolismo , Masculino , Dados de Sequência Molecular , Óvulo/crescimento & desenvolvimento , Óvulo/metabolismo , Filogenia , Platelmintos/classificação , Platelmintos/crescimento & desenvolvimento , Platelmintos/metabolismo , Análise de Sequência
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