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2.
Parasit Vectors ; 13(1): 20, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931867

RESUMO

BACKGROUND: MF6p/host defense molecules (HDMs) are a broad family of small proteins secreted by helminth parasites. Although the physiological role of MF6p/HDMs in trematode parasites is not fully understood, their potential biological function in maintaining heme homeostasis and modulating host immune response has been proposed. METHODS: A gene encoding the MF6p/HDM of Clonorchis sinensis (CsMF6p/HDM) was cloned. Recombinant CsMF6p/HDM (rCsMF6p/HDM) was expressed in Escherichia coli. The biochemical and immunological properties of rCsMF6/HDM were analyzed. CsMF6p/HDM induced pro-inflammatory response in RAW 264.7 cells was analyzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. The structural feature of CsMF6p/HDM was analyzed by three-dimensional modeling and molecular docking simulations. RESULTS: The CsMF6p/HDM shares a high level of amino acid sequence similarity with orthologs from other trematodes and is expressed in diverse developmental stages of the parasite. The rCsMF6p/HDM bound to bacteria-derived lipopolysaccharide (LPS), without effectively neutralizing LPS-induced inflammatory response in RAW 264.7 macrophage cells. Rather, the rCsMF6p/HDM induced pro-inflammatory immune response, which is characterized by the expression of TNF-α and IL-6, in RAW 264.7 cells. The rCsMF6p/HDM-induced pro-inflammatory immune response was regulated by JNK and p38 MAPKs, and was effectively down-regulated via inhibition of NF-κB. The structural analysis of CsMF6p/HDM and the docking simulation with LPS suggested insufficient capture of LPS by CsMF6p/HDM, which suggested that rCsMF6p/HDM could not effectively neutralize LPS-induced inflammatory response in RAW 264.7 cells. CONCLUSIONS: Although rCsMF6p/HDM binds to LPS, the binding affinity may not be sufficient to maintain a stable complex of rCsMF6p/HDM and LPS. Moreover, the rCsMF6p/HDM-induced pro-inflammatory response is characterized by the release of IL-6 and TNF-α in RAW 264.7 macrophage cells. The pro-inflammatory response induced by rCsMF6p/HDM is mediated via NF-κB-dependent MAPK signaling pathway. These results collectively suggest that CsMF6p/HDM mediates C. sinensis-induced inflammation cascades that eventually lead to hepatobiliary diseases.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Clonorchis sinensis/metabolismo , Macrófagos/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Clonagem Molecular , Clonorquíase/etiologia , Citocinas/metabolismo , Doenças do Sistema Digestório/etiologia , Doenças do Sistema Digestório/parasitologia , Escherichia coli , Imunidade Celular , Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/parasitologia , Camundongos , Simulação de Acoplamento Molecular/métodos , NF-kappa B/metabolismo , Células RAW 264.7 , Trematódeos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-31707088

RESUMO

LEAP-2, a multifunctional peptide, not only exhibits a regulatory role in pathogenic infection, but also participates in the regulation of teleostean immunity. In this study, ORF sequence of WR-LEAP-2 was 240 bp and encoded 79 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-LEAP-2 was observed in liver. Aeromonas hydrophila challenge can sharply increase WR-LEAP-2 mRNA expression in liver, kidney and spleen. The purified WR-LEAP-2 peptide can directly bind to A. hydrophila and S. agalactiae, reduce the relative bacterial activity and limit bacterial growth in vitro. In addition, the treatment of WR-LEAP-2 can restrict bacterial dissemination in vivo and reduce production of pro-inflammatory cytokines. These results indicated that WR-LEAP-2 can confer protection against A. hydrophila- or S. agalactiae-stimulated MyD88-dependent pro-inflammatory cytokines activation.


Assuntos
Aeromonas hydrophila , Peptídeos Catiônicos Antimicrobianos/imunologia , Quimera/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/metabolismo , Carpa Dourada/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Quimera/genética , Citocinas/metabolismo , Diploide , Doenças dos Peixes/genética , Carpa Dourada/genética , Infecções por Bactérias Gram-Negativas/genética , Fígado/imunologia
4.
Nat Commun ; 10(1): 5731, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844052

RESUMO

Antimicrobial peptides (AMPs) are key effectors of the innate immune system and promising therapeutic agents. Yet, knowledge on how to design AMPs with minimal cross-resistance to human host-defense peptides remains limited. Here, we systematically assess the resistance determinants of Escherichia coli against 15 different AMPs using chemical-genetics and compare to the cross-resistance spectra of laboratory-evolved AMP-resistant strains. Although generalizations about AMP resistance are common in the literature, we find that AMPs with different physicochemical properties and cellular targets vary considerably in their resistance determinants. As a consequence, cross-resistance is prevalent only between AMPs with similar modes of action. Finally, our screen reveals several genes that shape susceptibility to membrane- and intracellular-targeting AMPs in an antagonistic manner. We anticipate that chemical-genetic approaches could inform future efforts to minimize cross-resistance between therapeutic and human host AMPs.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Membrana Externa Bacteriana/efeitos dos fármacos , Membrana Externa Bacteriana/imunologia , Evolução Molecular Direcionada , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/imunologia , Genes Bacterianos/genética , Genes Bacterianos/imunologia , Testes de Sensibilidade Microbiana , Mutação
5.
PLoS One ; 14(12): e0227080, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31877198

RESUMO

Recurrent and chronic otitis media (OM) are often refractory to antibiotics due to bacterial persistence in biofilm within the middle ear. In vitro and in vivo studies have demonstrated that antimicrobial proteins and peptides (AMPs) are bactericidal against otopathogens, indicating potential therapeutic value for recalcitrant OM. We measured concentrations of 6 AMPs and 14 cytokines in middle ear effusion (MEE) from 67 children undergoing ventilation tube insertion for recurrent acute OM. Sixty one percent of children had bacterial otopathogens detected in their MEE, 39% by PCR and 22% by PCR and culture. Groups were defined as: PCR-negative/culture-negative (absence of bacterial otopathogen), n = 26; PCR-positive/culture-negative (presence of nonculturable bacterial otopathogen), n = 26; PCR-positive/culture-positive (presence of culturable bacterial otopathogen), n = 15. Age, antibiotic usage, day-care attendance, presence of respiratory viruses in MEE and number of AOM episodes were similar between groups. AMP and cytokine concentrations were higher in children with bacterial otopathogens in their MEE compared to those with no bacterial otopathogens. Median concentrations of AMPs (except HBD2) were 3 to 56-fold higher in MEE from children with bacterial otopathogens detected in their MEE (P ≤ 0.01). Similarly, median cytokine concentrations (except TGFß) were >16-fold higher in MEE with bacterial otopathogens detected (P ≤ 0.001). This is the first study to measure AMPs in MEE and together with the cytokine data, results suggest that elevated AMPs and cytokines in MEE are a marker of inflammation and bacterial persistence. AMPs may play an important role in OM pathogenesis.


Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Bactérias/imunologia , Citocinas/imunologia , Orelha Média/imunologia , Otite Média com Derrame/imunologia , Otite Média com Derrame/microbiologia , Bactérias/isolamento & purificação , Infecções Bacterianas/complicações , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Doença Crônica , Estudos de Coortes , Orelha Média/microbiologia , Feminino , Humanos , Lactente , Masculino , Otite Média com Derrame/complicações
6.
J Immunol Res ; 2019: 1016567, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31871952

RESUMO

Sublancin is a glycosylated antimicrobial peptide produced by Bacillus subtilis 168 possessing antibacterial and immunomodulatory activities. This study was aimed at investigating the effects of sublancin on immune functions and serum antibody titer in specific pathogen-free (SPF) broiler chickens vaccinated with Newcastle disease (ND) vaccine. For this purpose, 3 experiments were performed. Experiment 1: SPF broiler chicks (14 days old) were randomly allotted to 1 of 7 groups including a blank control (BC), vaccine control (VC), and 5 (3-7) vaccinated and sublancin supplemented at 5, 15, 30, 45, and 60 mg activity/L of water, respectively. Vaccinated groups (2-7) were vaccinated with ND vaccine by intranasal and intraocular routes at the 14th day. On 7, 14, 21, and 28 days post vaccination (dpv), the blood samples were collected for the determination of serum hemagglutination inhibition (HI) antibody titer. Experiment 2: SPF broiler chicks were divided into 1 of 3 groups, i.e., blank control (BC), vaccine control (VC), and sublancin treatment (ST). On 7, 14, and 21 dpv, the blood samples were collected for measuring HI antibody titer by micromethod. Experiment 3: the design of this experiment was the same as that of experiment 2. On 7 and 21 dpv, pinocytosis of peritoneal macrophages, B lymphocyte proliferation assay, measurement of CD4+ and CD8+ T cells, and serum cytokine quantitation were carried out. It was noted that sublancin promoted B lymphocyte proliferation, increased the proportion of CD8+ T lymphocyte subpopulations, and enhanced the antibody titer in broiler chickens. In addition, it was also observed that sublancin has the potential to induce the secretion of IFN-γ, IL-10, and IL-4. In conclusion, these findings suggested that sublancin could promote both humoral and cellular immune responses and has the potential to be a promising vaccine adjuvant.


Assuntos
Adjuvantes Imunológicos , Galinhas/imunologia , Galinhas/virologia , Imunidade , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Citocinas/metabolismo , Imunização , Imunomodulação , Linfócitos/imunologia , Linfócitos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Pinocitose/imunologia , Fatores de Tempo , Vacinas Virais/administração & dosagem
7.
Int J Mol Sci ; 21(1)2019 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-31877866

RESUMO

There is a growing interest in the complex role of host defense peptides (HDPs) in the pathophysiology of several immune-mediated inflammatory diseases. The physicochemical properties and selective interaction of HDPs with various receptors define their immunomodulatory effects. However, it is quite challenging to understand their function because some HDPs play opposing pro-inflammatory and anti-inflammatory roles, depending on their expression level within the site of inflammation. While it is known that HDPs maintain constitutive host protection against invading microorganisms, the inducible nature of HDPs in various cells and tissues is an important aspect of the molecular events of inflammation. This review outlines the biological functions and emerging roles of HDPs in different inflammatory conditions. We further discuss the current data on the clinical relevance of impaired HDPs expression in inflammation and selected diseases.


Assuntos
Imunidade Adaptativa/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Bactérias/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/classificação , Peptídeos Catiônicos Antimicrobianos/genética , Bactérias/classificação , Catelicidinas/genética , Catelicidinas/imunologia , Catelicidinas/metabolismo , Defensinas/genética , Defensinas/imunologia , Defensinas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Inflamação/genética , Inflamação/microbiologia
8.
Fish Shellfish Immunol ; 95: 624-634, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31698072

RESUMO

Two lipopolysaccharides (LPS) and ß-1, 3-glucan binding protein (LGBP), designated as PcLGBP isoform1 and PcLGBP isoform2, respectively, were identified from Procambarus clarkii in this study. The full-length cDNA of PcLGBP isoform1 was 1308 bp containing an open reading frame (ORF) of 1113 bp encoding a protein of 370 amino acids. The full-length cDNA of PcLGBP isoform2 was 1440 bp containing an ORF of 1245 bp encoding a protein of 414 amino acids. Predicted PcLGBP isoform1 and PcLGBP isoform 2 proteins contained a signal peptide, a glycoside hydrolase domain, and a low-complexity region. The difference between the two LGBP isoforms was that PcLGBP isoform2 had 44 more amino acids behind the signal peptide than the PcLGBP isoform1. The PcLGBP isoform1 and PcLGBP isoform2 transcripts mainly expressed in the hepatopancreas in female and male crayfish. Moreover, the expression levels of the two genes in the hepatopancreas were higher in male than that in female crayfish. Upon being challenged with Vibrio parahaemolyticus or LPS, the expression levels of PcLGBP isoform1 and PcLGBP isoform2 in the hepatopancreas of female and male crayfish were most significantly up-regulated at different time points. The transcripts of anti-lipopolysaccharide factors (ALF5, ALF6, ALF8, and ALF9) and crustins (CRU1, CRU2, CRU3, and CRU4) were evidently down-regulated in the hepatopancreas of V. parahaemolyticus-challenged total PcLGBP (including PcLGBP isoform1 and PcLGBP isoform2)-silenced male crayfish. In addition, the phenoloxidase (PO) activity in the hepatopancreas of male crayfish was evidently higher than that of female crayfish. PcLGBP knock down could significantly decrease the PO activity in the hepatopancreas lysate (HLS) in male crayfish. The PO activity of male crayfish HLS was significantly increased when incubated with a mixture of recombinant LGBP protein and LPS or ß-1, 3 glucan. We conclude that LGBP isoforms from P. clarkii function as a pattern recognition protein for recognizing and binding LPS and ß-1, 3 glucan, and thus regulate the synthesis of antimicrobial peptides and activate the prophenoloxidase system.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Artrópodes/genética , Astacoidea/genética , Astacoidea/imunologia , Proteínas de Transporte/genética , Hepatopâncreas/imunologia , Lectinas/genética , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Proteínas de Transporte/imunologia , Feminino , Imunidade Inata , Lectinas/imunologia , Lipopolissacarídeos , Masculino , Isoformas de Proteínas , Vibrio parahaemolyticus/imunologia
9.
Fish Shellfish Immunol ; 95: 606-616, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31682999

RESUMO

To develop an alternative to conventional antibiotics used in the aquaculture and livestock industries, we employed Bacillus subtilis, considered a biosafe microorganism, to express the degradable antimicrobial peptide lactoferricin. An expression plasmid pP43-6LFBII-GFP, in which reporter GFP cDNA was fused downstream of lactoferricin cDNA driven by an endogenous constitutive P43 promoter was electroporated into B. subtilis, followed by regeneration and cultivation. The putative colonies harboring plasmids were primarily screened by PCR-amplification of lactoferricin cDNA. Four transformants which were stable inheritance of plasmid containing lactoferricin cDNA included strains T1, T4, T7 and T13. Based on Western blot and Southern blot analyses, we found that transgenic strains T1 and T13 not only highly expressed exogenous recombinant lactoferricin, but also exhibited more stable inheritance of plasmids with 931 and 647 copies per cell, respectively. In the antibacterial in vitro experiment, the bactericidal activity of each microliter of cell lysate from transgenic strains T1 and T13 (5 × 108 CFU) for Escherichia coli was equivalent to 56 and 53 ng of Ampicillin dosage, respectively, while for Staphylococcus epidermidis, the equivalency T1 and T13 was 154 and 130 ng of Ampicillin dosage, respectively. Equivalencies of bacterial activity for Vibrio parahaemolyticus and Edwardsiella tarda followed suit. In the antibacterial in vivo experiment, we oral-in-tube fed tilapia fry (Oreochromis mossambicus X O. niloticus) with cell lysate from transgenic strain T1 and T13 individually. After 1-h of incubation, we immersed these treated fish fry in a water tank containing E. tarda (5 × 1011 CFU) for a 5-hr bacterial challenge. After one month cultivation, an average survival rate of 63 and 67% was observed after having fed the fish fry with transgenic strains T1 and T13, respectively. However, the average survival rate of fish fry fed with B. subtilis WT strain and transgenic strain T19 without expressing recombinant lactoferricin reached only 5 and 9%, respectively. These data indicate that the survival of fish fry infected by the intestinal pathogen tested could be significantly enhanced by feeding transgenic B. subtilis containing antibacterial peptide. Therefore, we suggest that this strategy could be applied to both aquaculture and livestock industries to (i) reduce the dependency on conventional antibiotics during seasonal outbreaks and (ii) eliminate the problem of antibiotic resistance.


Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Bacillus subtilis/genética , Resistência à Doença/imunologia , Doenças dos Peixes/imunologia , Organismos Geneticamente Modificados/imunologia , Probióticos/administração & dosagem , Tilápia/microbiologia , Administração Oral , Animais , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Aquicultura/métodos , Bactérias/patogenicidade , Doenças dos Peixes/microbiologia
10.
Int J Mol Sci ; 20(23)2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31766730

RESUMO

The alarming escalation of infectious diseases resistant to conventional antibiotics requires urgent global actions, including the development of new therapeutics. Antimicrobial peptides (AMPs) represent potential alternatives in the treatment of multi-drug resistant (MDR) infections. Here, we focus on Cecropins (Cecs), a group of naturally occurring AMPs in insects, and on synthetic Cec-analogs. We describe their action mechanisms and antimicrobial activity against MDR bacteria and other pathogens. We report several data suggesting that Cec and Cec-analog peptides are promising antibacterial therapeutic candidates, including their low toxicity against mammalian cells, and anti-inflammatory activity. We highlight limitations linked to the use of peptides as therapeutics and discuss methods overcoming these constraints, particularly regarding the introduction of nanotechnologies. New formulations based on natural Cecs would allow the development of drugs active against Gram-negative bacteria, and those based on Cec-analogs would give rise to therapeutics effective against both Gram-positive and Gram-negative pathogens. Cecs and Cec-analogs might be also employed to coat biomaterials for medical devices as an approach to prevent biomaterial-associated infections. The cost of large-scale production is discussed in comparison with the economic and social burden resulting from the progressive diffusion of MDR infectious diseases.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas , Proteínas de Insetos , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/imunologia , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/imunologia , Humanos , Proteínas de Insetos/uso terapêutico
11.
Fish Shellfish Immunol ; 94: 861-870, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31585246

RESUMO

The main advantage of antimicrobial peptides (AMPs) used as the effectors in the innate immunity system of invertebrates is that the high specificity is not indispensable. And they play important roles in the systemic defenses against microbial invasion. In this study, a new full-length cDNA of the crustins molecule was identified in red swamp crayfish, P. clarkii (named Pc-crustin 4). The ORF of Pc-crustin 4 contained 369 bp which encoded a protein of 122 amino acids, with a 20-amino-acid signal peptide sequence. On the base of the classification method established by Smith et al., Pc-crustin 4 belonged to Type Ⅰ crustin molecule. The Pc-crustin 4 transcripts were expressed in hemocytes at relatively high level, and relatively low level in hepatopancreas, gills, and intestine in normal crayfish. After respectively challenged with S. aureus or E. ictaluri, the expression levels of Pc-crustin 4 showed up-regulation trends at different degrees in the hemocytes, hepatopancreas, gills, and intestine tissues. Besides, the results of liquid antibacterial assay showed that rPc-crustin 4 inhibited obviously the growth of S. aureus and E. ictaluri. The results of bacteria binding assay showed that rPc-crustin 4 could bind strongly to S. aureus and E. ictaluri. Finally, RNAi assay was performed to study the immunity roles of Pc-crustin 4 in crayfish in vivo. Taken together, Pc-crustin 4 is an important immunity effector molecule, which plays crucial roles in defending against bacterial infection in crayfish.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Astacoidea/genética , Astacoidea/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Edwardsiella ictaluri/fisiologia , Perfilação da Expressão Gênica , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Alinhamento de Sequência , Staphylococcus aureus/fisiologia
12.
Expert Rev Gastroenterol Hepatol ; 13(10): 963-976, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31603356

RESUMO

Introduction: Inflammatory bowel diseases (IBD) are on the rise worldwide. This review covers the current concepts of the etiology of Crohn´s disease and ulcerative colitis by focusing on an unbalanced interaction between the intestinal microbiota and the mucosal barrier. Understanding these issues is of paramount importance for the development of targeted therapies aiming at the disease cause.Area covered: Gut microbiota alterations and a dysfunctional intestinal mucosa are associated with IBD. Here we focus on specific defense structures of the mucosal barrier, namely antimicrobial peptides and the mucus layer, which keep the gut microbiota at a distance under healthy conditions and are defective in IBD.Expert commentary: The microbiology of both forms of IBD is different but characterized by a reduced bacterial diversity and richness. Abundance of certain bacterial species is altered, and the compositional changes are related to disease activity. In IBD the mucus layer above the epithelium is contaminated by bacteria and the immune reaction is dominated by the antibacterial response. Human genetics suggest that many of the basic deficiencies in the mucosal response, due to Paneth cell, defensin and mucus defects, are primary. Nutrition may also be important but so far there is no therapy targeting the mucosal barrier.


Assuntos
Colite Ulcerativa/microbiologia , Doença de Crohn/microbiologia , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Animais , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/imunologia , Colite Ulcerativa/metabolismo , Doença de Crohn/tratamento farmacológico , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Disbiose , Microbioma Gastrointestinal/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Humanos , Imunidade nas Mucosas , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Muco/imunologia , Muco/metabolismo , Muco/microbiologia , Probióticos/uso terapêutico
13.
Fish Shellfish Immunol ; 95: 151-162, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31605765

RESUMO

G protein-coupled receptors (GPCRs) are important transmembrane receptors that participate in diverse physiological processes including metabolism, cell growth and immune processes by transmitting extracellular signals to intracellular effectors. In this study, a gene belonging to the GPCR family was cloned from Eriocheir sinensis and named EsGPCR89. The full-length gene includes an open reading frame (ORF) of 465 amino acid residues, and bioinformatic analysis confirmed the high conservation between species. EsGPCR89 was detected in various tissues of E. sinensis, and was up-regulated in brain following Staphylococcus aureus infection. Expression levels of cerebral antimicrobial peptides (AMPs) were also up-regulated following bacterial challenge, reflecting their function in cerebral immunity. Additionally, EsGPCR89 silencing in hemocytes by RNA interference, down-regulated AMPs in brain after S. aureus infection. Moreover, through Immunisation assay and Polyacrylamide gel electrophoresis (SDS-PAGE) experiments, we could infer that bacterially infected hemocytes released effectors under the regulation of EsGPCR89, thereby activating transcription of cerebral AMPs. These results demonstrate that EsGPCR89 plays important roles in cerebral antimicrobial function via hemocytes.


Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Proteínas de Artrópodes/imunologia , Braquiúros/imunologia , Encéfalo/imunologia , Hemócitos/imunologia , Receptores Acoplados a Proteínas-G/imunologia , Infecções Estafilocócicas/veterinária , Animais , Proteínas de Artrópodes/genética , Braquiúros/genética , Encéfalo/microbiologia , Clonagem Molecular , Regulação da Expressão Gênica , Imunidade Inata , Filogenia , Receptores Acoplados a Proteínas-G/genética , Infecções Estafilocócicas/imunologia , Staphylococcus aureus
14.
Fish Shellfish Immunol ; 94: 398-406, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31521782

RESUMO

Crustin is an antimicrobial peptide (AMP) that plays a key role in the innate immunity of crustaceans. This study cloned a new crustin from Pacific white shrimp Litopenaeus vannamei, which we designated as LvCrustinB, using rapid amplification of cDNA ends (RACE). The full-length cDNA of LvCrustinB is 751 bp with an open reading frame (ORF) of 591 bp encoding a peptide of 196 amino acids that includes a putative signal sequence. LvCrustinB is a type II crustin that has a glycine-rich region and a single whey acidic protein domain (WAP) domain. The mRNA transcript of LvCrustinB was detected in all examined tissues and was found to be most abundantly expressed in the epithelium and muscle. The expression of LvCrustinB in hemocytes was significantly upregulated after L. vannamei was challenged with LPS, Vibrio parahaemolyticus, and white spot syndrome virus (WSSV). When LvCrustinB was knocked down with RNAi, the mortality rate of L. vannamei significantly increased after V. parahaemolyticus or WSSV infection. Recombinant LvCrustinB was produced using Pichia pastoris GS115 and was shown to bind to 2 g-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and 2 g-negative bacteria (Escherichia coli and V. parahaemolyticus) via polysaccharides, which included PGN, LTA, and LPS. In vivo, the recombinant LvCrustinB remarkably protected L. vannamei from V. parahaemolyticus infection. These results suggest that LvCrustinB plays an important role in innate immunity and may be potentially utilized as antibacterial agents in shrimp.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Hemócitos/metabolismo , Lipopolissacarídeos/farmacologia , Filogenia , Interferência de RNA , Alinhamento de Sequência , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
15.
PLoS One ; 14(9): e0222878, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31550271

RESUMO

INTRODUCTION: Mucosal immune activation, in the context of sexual transmission of HIV-1 infection, is crucial, as the increased presence of activated T cells enhance susceptibility to infection. In this regard, it has been proposed that immunomodulatory compounds capable of modulating immune activation, such as Vitamin D (VitD) may reduce HIV-1 transmission and might be used as a safe and cost-effective strategy for prevention. Considering this, we examined the in vitro effect of the treatment of peripheral blood mononuclear cells (PBMCs) with the active form of VitD, calcitriol, on cellular activation, function and susceptibility of CD4+ T cells to HIV-1 infection. METHODS: We treated PBMCs from healthy HIV unexposed individuals (Co-HC) and frequently exposed, HIV-1 seronegative individuals (HESNs) from Colombia and from healthy non-exposed individuals from Canada (Ca-HC) with calcitriol and performed in vitro HIV-1 infection assays using X4- and R5-tropic HIV-1 strains respectively. In addition, we evaluated the activation and function of T cells and the expression of viral co-receptors, and select antiviral genes following calcitriol treatment. RESULTS: Calcitriol reduced the frequency of infected CD4+ T cells and the number of viral particles per cell, for both, X4- and R5-tropic viruses tested in the Co-HC and the Ca-HC, respectively, but not in HESNs. Furthermore, in the Co-HC, calcitriol reduced the frequency of polyclonally activated T cells expressing the activation markers HLA-DR and CD38, and those HLA-DR+CD38-, whereas increased the subpopulation HLA-DR-CD38+. Calcitriol treatment also decreased production of granzyme, IL-2 and MIP-1ß by T cells and increased the transcriptional expression of the inhibitor of NF-kB and the antiviral genes cathelicidin (CAMP) and APOBEC3G in PBMCs from Co-HC. CONCLUSION: Our in vitro findings suggest that VitD treatment could reduce HIV-1 transmission through a specific modulation of the activation levels and function of T cells, and the production of antiviral factors. In conclusion, VitD remains as an interesting potential strategy to prevent HIV-1 transmission that should be further explored.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Calcitriol/administração & dosagem , Infecções por HIV/prevenção & controle , Ativação Linfocitária/efeitos dos fármacos , Vitaminas/administração & dosagem , Desaminase APOBEC-3G/imunologia , Desaminase APOBEC-3G/metabolismo , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Feminino , Infecções por HIV/sangue , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Imunidade nas Mucosas/efeitos dos fármacos , Masculino , Cultura Primária de Células
16.
Fish Shellfish Immunol ; 93: 1007-1017, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31449978

RESUMO

Pathogenic disease is a major factor affecting the aquaculture of the rockfish Sebastiscus marmoratus, an important commercial species inhabiting the nearshore waters of the Western Pacific Ocean. Antimicrobial peptides (AMPs), as critical components of innate immunity, have been considered as promising antibiotic substitutes. The aims of this study were 1) to identify major AMPs in the rockfish, 2) to assess their antimicrobial activity and 3) to evaluate their potential therapeutic application. Six AMPs were identified, Hepcidin 1, liver-expressed antimicrobial peptide 2 (LEAP-2), Piscidin, Moronecidin, NK-lysin and ß-defensin through analysis of the liver transcriptome of S. marmoratus. The transcriptional expression profiles of these AMPs were investigated by real-time quantitative PCR (RT-qPCR). These AMPs showed tissue-specific distribution patterns, and S. marmoratus displays a time-, dose- and tissue-dependent expression of AMPs in response to lipopolysaccharide (LPS) challenge. While the synthetic peptides of LEAP-2 and Moronecidin exerted broad-spectrum antimicrobial activity against important aquatic pathogens in vitro by directly disrupting microbial membrane, and no cytotoxicity against murine hepatic cells was observed at the effective concentrations from 5 µM to 40 µM. The existence of multiple AMPs and their distinct tissue distribution patterns and inducible expression patterns suggests a sophisticated, highly redundant, and multilevel network of antimicrobial defensive mechanisms of S. marmoratus. Therefore, S. marmoratus-derived AMPs appear to be potential therapeutic applications against pathogen infections in aquaculture.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Perciformes/genética , Perciformes/imunologia , Animais , Anti-Infecciosos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Linhagem Celular , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/veterinária , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Perciformes/metabolismo
17.
Fish Shellfish Immunol ; 93: 841-850, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31430558

RESUMO

Bactericidal permeability-increasing protein (BPI) is an antimicrobial protein with potent endotoxin-neutralising activity and plays a crucial role in innate immunity against bacterial infection. In the present study, a bpi (designed as rpbpi) was identified and characterized from manila clam Ruditapes philippinarum. Multiple alignments and phylogenetic analysis suggested that rpbpi was a new member of the bpis family. In non-stimulated clams, rpbpi transcripts were ubiquitously expressed in all tested tissues with the highest expression level in hemocytes. After Vibrio anguillarum challenge, the expression levels of rpbpi mRNA in hemocytes were up-regulated significantly at 3 h and 48 h compared with that in the control, which were 4.01- and 19.10-fold (P < 0.05), respectively. The recombinant RpBPI (rRpBPI) showed high antibacterial activities against Gram-negative bacteria Escherichia coli and V. anguillarum, but not Staphylococcus aureus. Moreover, membrane integrity analysis revealed that rRpBPI increased the membrane permeability of Gram-negative bacteria, and then resulted in cell death. Overall, our results suggested that RpBPI played an important role in the elimination of invaded bacteria through membrane-disruptive activity.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Bivalves/genética , Bivalves/imunologia , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Sequência de Bases , Proteínas Sanguíneas/química , Perfilação da Expressão Gênica , Bactérias Gram-Negativas/fisiologia , Filogenia , Alinhamento de Sequência
18.
Mol Immunol ; 114: 378-388, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31450183

RESUMO

Antimicrobial peptides (AMPs) play an important role in the innate immune response of vertebrates by creating a hostile environment for any invading pathogens. Piscidins are potent teleost specific AMPs, which have a broad spectrum activity. We have identified a novel piscidin active peptide, in the greater amberjack, Seriola dumerili, that consists of 25 aa, which forms an amphipathic helix with distinct hydrophobic and positively charged regions. Following homology and phylogenetic analysis the greater amberjack piscidin was deemed to belong to the group 3 family of piscidins. Piscidin was expressed constitutively at immune sites, with transcript level highest in the spleen and gut, at an intermediate level in the gills and lowest in the head kidney. Following in vivo stimulation with PAMPs (poly I:C, LPS and flagellin) piscidin transcript level increased in gills in response to flagellin, in gut and spleen in response to poly I:C, and in head kidney in response to poly I:C, LPS and flagellin. Head kidney and spleen cells were then isolated from greater amberjack and incubated with each of the PAMPs for 4, 12 and 24 h. Piscidin expression was unchanged at 4 and 12 h post PAMP stimulation in head kidney cells but at 24 h transcript level was markedly upregulated compared to control (unstimulated) cells, especially with the bacterial PAMPs. In contrast, spleen cells upregulated piscidin expression by 4 h post stimulation with poly I:C and flagellin, and remained upregulated to 24 h with flagellin exposure, but had returned to baseline levels by 12 h using poly I:C. To determine if piscidin expression could be modulated by diet, greater amberjack were fed diets supplemented with MOS and cMOS for 30 days when transcript level was determined. It was found that MOS supplemented diets increased expression in the spleen, cMOS supplemented diets upregulated transcript levels in the gills and head kidney, whilst a diet containing both MOS and cMOS upregulated transcript in the gut, when compared to fish fed the control diet. Finally, a synthetic greater amberjack piscidin was produced and showed bacteriostatic activity against a number of bacterial strains, including both Gram positive and Gram negative fish pathogens.


Assuntos
Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perciformes/genética , Perciformes/imunologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Sequência de Bases , Brânquias/imunologia , Rim Cefálico/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Padrões Moleculares Associados a Patógenos/imunologia , Filogenia , Alinhamento de Sequência
19.
Fish Shellfish Immunol ; 94: 149-156, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31465873

RESUMO

Anti-lipopolysaccharide factors (ALFs) are important host-defense molecules of crustaceans. They all contain a lipopolysaccharide-binding domain (LBD) and some ALFs exhibit strong antimicrobial activity. In this research, a Group G ALF from Penaeus monodon (ALFPm11) was studied. It is an anionic peptide specifically having a cationic and highly amphipathic LBD, with five positively charged residues separated by aromatic residues. It was abundantly expressed in the hepatopancreas of P. monodon normally but the expression level in other tissues was relatively low or undetectable. However, in the shrimps challenged by Vibrio, expression of ALFPm11 could be detected in all tissues. Chemically synthesized ALFPm11-LBD displayed high inhibitory activity (minimum inhibition concentration≤ 4 µM) against various bacteria, e.g. Exiguobacterium sp. L33, Bacillus sp. T2, and Acinetobacter sp. L32. It also displayed apparent activity in the agar well diffusion assay. Furthermore, it could efficiently induce agglutination of both Gram-positive and Gram-negative bacteria and cause significant membrane permeabilization of the bacteria. As a comparative study, ALFPm11-LBD showed a better or equal antimicrobial function to ALFPm3-LBD which was reported to possess strong antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. Thus, this research found a new effective ALF in P. monodon and demonstrated its antimicrobial mechanism, suggesting its potential applications in the future.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Testes de Sensibilidade Microbiana , Alinhamento de Sequência
20.
Mar Drugs ; 17(9)2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31466296

RESUMO

Giant groupers, the largest grouper type in the world, are of economic importance in marine aquaculture for their rapid growth. At the same time, bacterial and viral diseases have become the main threats to the grouper industry. Here, we report a high-quality genome of a giant grouper sequenced by an Illumina HiSeq X-Ten and PacBio Bioscience Sequel platform. A total of 254 putative antimicrobial peptide (AMP) genes were identified, which can be divided into 34 classes according to the annotation of the Antimicrobial Peptides Database (APD3). Their locations in pseudochromosomes were also determined. Thrombin-, lectin-, and scolopendin-derived putative AMPs were the three largest parts. In addition, expressions of putative AMPs were measured by our transcriptome data. Two putative AMP genes (gapdh1 and gapdh2) were involved in glycolysis, which had extremely high expression levels in giant grouper muscle. As it has been reported that AMPs inhibit the growth of a broad spectrum of microbes and participate in regulating innate and adaptive immune responses, genome sequencing of this study provides a comprehensive cataloging of putative AMPs of groupers, supporting antimicrobial research and aquaculture therapy. These genomic resources will be beneficial to further molecular breeding of this economically important fish.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Bass/genética , Doenças dos Peixes/prevenção & controle , Proteínas de Peixes/genética , Pesqueiros , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Bass/imunologia , Bass/microbiologia , Cruzamento/métodos , Embaralhamento de DNA , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Sequenciamento Completo do Genoma
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