RESUMO
OBJECTIVES: This study evaluated the effect of milk supplemented with Lactobacillus rhamnosus SP1 on the occurrence of caries and the salivary concentration of human ß-defensin-3 (hßD-3) in preschool children with high caries risk. MATERIALS AND METHODS: A sample of 42 children was randomly assigned to two groups; children in the intervention group were given 150 mL of milk supplemented with 107 CFU/mL of Lactobacillus rhamnosus SP1, while children in the control group were given standard milk, for 10 months. The occurrence of dental caries was assessed using the International Caries Detection and Assessment System (ICDAS), and the concentration of hßD-3 was measured in unstimulated saliva using an ELISA test at baseline and after the intervention. RESULTS: There was an increase in the number of teeth with carious lesions (dICDAS2-6 mft) in the control group, and this increase was statistically significant (p = 0.0489). The concentration of hßD-3 in saliva from the intervention group decreased from 597.91 to 126.29 pg/mL (p = 0.0061), unlike in the control group, where no change in hßD-3 salivary concentration was found. CONCLUSIONS: These findings showed that regular intake of probiotic-supplemented milk in preschool children with high caries risk decreased the occurrence of caries and the salivary levels of hßD-3. CLINICAL RELEVANCE: Our results suggest the need for developing and implementing probiotic supplementation, as adjuvants to the conventional treatments for caries and allow to considerate the salivary levels of hßD-3 as markers of oral tissue homeostasis.
Assuntos
Cárie Dentária , Probióticos , beta-Defensinas , Animais , Pré-Escolar , Cárie Dentária/prevenção & controle , Suscetibilidade à Cárie Dentária , Suplementos Nutricionais , Humanos , Leite , Saliva , Streptococcus mutansRESUMO
Vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC) are two forms of a disease caused by Candida spp. ß-defensin (BD) is one of the most important families of antimicrobial peptides in the female genital tract and includes molecules that exert essential local functions as antimicrobial and PMN chemoattractant peptides. However, the information on their role during murine and human VVC and RVVC is limited. Thus, we analyzed the behavior and contribution of BD1 to the local response in a VVC mice model and the local cytokine profile and human BD1 and BD3 expression in cervicovaginal lavage from patients with VVC and RVVC. We demonstrated that, in patients with RVVC BD1, mRNA and protein expression were severely diminished and that the aspartate proteinase and lipase secreted by C. albicans are involved in that decrease. This study provides novel information about the pathogenesis of VVC and describes a highly efficient C. albicans escape strategy for perpetuating the infection; these results may contribute to the development of new or combined treatment approaches.
RESUMO
Abstract Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and lung as well as involvement of kidney, gastrointestinal system and heart. Aetiology and exact mechanism of disease is poorly understood. The association between antimicrobial peptides (AMPs) and other diseases such as idiopathic pulmonary fibrosis, diffuse panbronchiolitis, pulmoner alveolar proteinosis and psoriasis have been reported. A small number of studies have examined the role of AMPs on autoimmune diseases which has not been studied in scleroderma yet. We aimed to investigate AMP serum levels and their association with disease characteristics of SSc. Methods: Forty-two patients (40 female, mean age 42 years) and 38 healthy subjects (32 female, mean age 38 years) were enrolled. For SSc patients, the following data were recorded: disease subset (limited/diffuse), autoantibodies (antinuclear, anti-centromere (ACA), and anti-SCL-70), blood tests, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP), modified Rodnan skin score, presence and history of digital ulcers, kidney, gastrointestinal disease and lung involvement assessed by computed tomography and pulmonary function tests. Association between serum AMPs and disease characteristics were analysed. Results: Twenty-nine of the patients had diffuse (69%) and 13 of the patients had limited (31%) systemic sclerosis. Average disease duration was 5.5 years. Pulmonary involvement was detected in 20 patients (47.6%). Serum concentration of alpha defensin was higher than healthy subjects (563 ± 415 vs 377 ± 269 ng/mL, p = 0.02). However, no difference was observed for beta-1 and beta-2 defensins in SSc patients and healthy controls. In sub-group analysis patients with interstitial lung disease had higher levels of alpha defensin than those without lung involvement (684 ± 473 vs 430 ± 299 ng/ml, p = 0.04). There was also correlation between alfa defensin serum concentrations and CRP (r = 0.34). Conclusions: Alpha defensin levels are increased in scleroderma patients and correlated with lung involvement indicating a role in the pathogenesis of disease. Trial registration: This study is not a clinical trial study.(AU)
Assuntos
Humanos , Escleroderma Sistêmico/patologia , Peptídeos Catiônicos Antimicrobianos/sangue , alfa-Defensinas/sangue , beta-Defensinas/sangue , Pneumopatias/etiologiaRESUMO
Human ß-defensin 1 (hBD-1) is a multifaceted antimicrobial peptide being a tumour suppressor and, depending on call of duty, capable of inducing self-nets and neutrophil extracellular traps (NETs) to capture and/or kill bacteria, participates in inflammatory responses in chronic diseases including hBD-3 upregulation and also capable of up/downregulation in the presence of certain species of Lactobacillus sp. Thus, is regulated by host microbiota. Alleles, genotypes and/or altered gene expression of its coding gene, DEFB1, have been associated with several human diseases/conditions ranging from metabolic/chronic (e.g. cancer), infectious (e.g. tuberculosis, HIV/AIDS), inflammatory (gastrointestinal diseases), male infertility and more recently, neurologic (e.g. depression and Alzheimer) and autoimmune diseases (e.g. vitiligo and systemic lupus erythematosus). The present update focuses on novel DEFB1/hBD-1 properties and biomarker features, its biological function and the pharmaceutical potential uses of antimicrobial peptide elicitors (APEs) or the engineered peptide in the treatment of hBD-1-related human diseases.
Assuntos
beta-Defensinas/metabolismo , Regulação da Expressão Gênica , Humanos , Indústrias , beta-Defensinas/química , beta-Defensinas/genéticaRESUMO
Evidências indicam um relacionamento bidirecional entre a periodontite crônica (PC) e o Diabetes Mellitus tipo 2 (DM2). Estudos recentes demonstraram que componentes da resposta imune inata, como as beta-defensinas humanas (hBD), podem apresentar-se alterados em indivíduos com DM2 ou PC. Tendo em vista que a resposta imune inata é a primeira barreira de defesa do organismo, alterações nas hBDs poderiam ser parcialmente responsáveis por um aumento na susceptibilidade dos indivíduos à PC. O presente estudo teve 2 propostas de investigação com objetivos específicos: (1) avaliar a influência da periodontite e da condição inflamatória do sítio nos níveis das hDBs-2 e -3 no fluido crevicular gengival (FCG) de indivíduos com e sem periodontite; e (2) avaliar a influência do DM2 e do controle glicêmico nos níveis das hBDs-1, -2 e -3 no FCG de indivíduos com e sem DM2. Para a proposta 1, foram selecionados indivíduos saudáveis (S) e com periodontite (P) entre os pacientes das clínicas da Faculdade de Odontologia da Universidade Federal de Minas Gerais. Para a proposta 2 foram selecionados indivíduos com DM2 compensados (DM2c) e descompensados (DM2d) entre os usuários do sistema público de saúde da região metropolitana de Belo Horizonte, compreendendo 20 indivíduos em cada grupo. Todos os indivíduos foram submetidos a exame periodontal completo e dados médicos e sociodemográficos foram registrados. Amostras de FCG foram coletadas em sítios saudáveis (Ss) nos indivíduos sem periodontite; amostras de sítios saudáveis (Ps), com gengivite (Pg) e com periodontite (Pp) foram coletadas de indivíduos com periodontite. A quantificação das hBDs no FCG foi realizada pela técnica Enzyme Linked ImmunonoSorbent Assay (ELISA), método de sanduíche. O presente estudo foi aprovado pelo Comitê de Ética em Pesquisa da Universidade Federal de Minas Gerais COEP UFMG (CAAE #0529.0.203.0001-11). Os níveis de hBDs foram comparados entre sítios saudáveis, sítios com gengivite e sítios com periodontite, aninhados em indivíduos com e sem periodontite, através de modelagem hierárquica linear. Os níveis de hBDs foram comparados entre indivíduos saudáveis, DM2c e DM2d por teste de Kruskal-Wallis seguido do teste post-hoc de Dunn para comparações entre os pares. Os resultados mostraram que a periodontite foi associada a elevados níveis de hBD-2 (coeficiente 14,68; p = 0,023) e hBD-3 (coeficiente 1091.86, p < 0.001) no nível indivíduo, no entanto não houve efeito da condição do sítio sobre estes níveis. Foi ainda demonstrado que o DM2, independente do controle glicêmico, foi associado a níveis reduzidos de hBD-1 (p < 0,001), que o DM2 descompensado foi associado com níveis reduzidos de hBD-2 (p < 0,01); e que o DM2 não resulta em alteração nos valores de hBD-3. Uma maior expressão de hBD2 e -3 no FCG de indivíduos com periodontite pode sugerir uma resposta normal do organismo perante à infecção dos tecidos gengivais ou ser considerado um fator parcialmente responsável pela resposta imunoinflamatória exacerbada comumente encontrada nestes indivíduos. O DM2 parecer afetar o padrão de expressão das hBD1 e -2 de maneira distinta.(AU)
Crevicular fluid levels of beta-defensins in individuals with chronic periodontitis and type 2 diabetes Scientific evidence indicates a bidirectional relationship between chronic periodontitis and Type 2 Diabetes Mellitus (DM2). Recent studies have shown that components of the innate immune response, such as human beta-defensins (hBDs), may be altered in individuals with CP or DM2. Given that the innate immune response is the body's first defense barrier against microbial invasion, changes in hBDs could partially account for an increase in the susceptibility CP. The present study had 2 research proposals with specific objectives: (1) to evaluate the influence of periodontitis and inflammatory site condition on hDBs-2 and -3 levels in the gingival crevicular fluid (GCF) of individuals with and without periodontitis; and (2) to assess the influence of DM2 and glycemic control on the levels of hBDs-1, -2 and -3 in the GCF of individuals with and without DM2. For the proposal 1, healthy subjects (H) and subjects with periodontitis (P) were selected among the patients from the School of Dentistry of the Federal University of Minas Gerais. For the proposal 2, individuals with DM2 with good (DM2g) and poor (DM2p) glycemic control were selected among the users of the public health system of the metropolitan area of Belo Horizonte, comprising 20 individuals in each group. All subjects underwent complete periodontal examination and medical and sociodemographic data were recorded. GCF samples were collected in healthy sites (Hh) from subjects without periodontitis; samples of healthy sites (Ph), gingivitis (Pg) and periodontitis (Pp) were collected from individuals with periodontitis. Quantification of hBDs in the GCF was performed by the Enzyme Linked ImmunonoSorbent Assay (ELISA) sandwich method. The present study was approved by the Research Ethics Committee of the Federal University of Minas Gerais - COEP UFMG (CAAE # 0529.0.203.0001-11). Levels of hBDs were compared among healthy sites, sites with gingivitis and sites with periodontitis, nested in individuals with and without periodontitis, through linear hierarchical modeling. Levels of hBDs were compared between healthy subjects, with DM2 with good and poor glycemic control through the Kruskal-Wallis test followed by Dunn's post-hoc test for pair-wise comparisons. Results showed that periodontitis was associated with high levels of hBD-2 (coefficient 14.68, p = 0.023) and hBD-3 (coefficient 1091.86, p <0.001) at the individual level. However, there was no effect of the site condition on these levels. It was also demonstrated that DM2, regardless of the glycemic control, was associated with reduced levels of hBD-1 (p <0.001), that DM2p was associated with reduced levels of hBD-2 (p <0.01); and that DM2 did not result in changes in hBD-3 values. Increased expression of hBD-2 and -3 in the GCF of individuals with periodontitis may suggest either a normal response of the organism to the infection of the gingival tissues or can be considered a partially responsible factor for the exacerbated immunoinflammatory response commonly found in individuals with periodontitis. DM2 appears to affect the expression pattern of hBD-1 and -2 differently.(AU)
Assuntos
Humanos , beta-Defensinas , Periodontite Crônica , Diabetes Mellitus Tipo 2 , Líquido do Sulco Gengival , Medição de Níveis de Água , Estudo Comparativo , Estudos Transversais , HiperglicemiaRESUMO
Candida albicans is the prevalent etiological agent in acute vulvovaginal infection and the most severe chronic condition known as recurrent vulvovaginal candidiasis (VVC). A critical role of local innate immunity in defense and pathogenesis of vaginal infection by Candida is proposed. The fungal recognition by the innate immune receptor is an essential step for the induction of local responses including cytokines and antimicrobial peptides (AMPs) production for host protection. Using TLR2-deficient mice, we characterized the early innate immune response during VVC. Intravaginal challenge of TLR2-/- mice with C. albicans demonstrated that in response to the initial massive penetration, a strong local inflammatory reaction with recruitment of polymorphonuclear neutrophils was developed. Both interleukin 1ß (IL1ß)-regarded as the hallmark of VVC immunopathogenesis-and IL6 were increased in vaginal lavage. Murine beta defensin 1 (mBD1), a constitutive AMP with fungicidal and chemotactic activity, was significantly upregulated in wild type (WT) animals in response to infection. Interestingly, in the absence of TLR2 recognition, levels of mBD1 RNA more than twice higher than those in WT infected animals were observed. Interestingly, our results demonstrate that TLR2 signaling is important to control the fungal burden in the vaginal tract. These finding provide new evidence about the role of this innate receptor during VVC.
Assuntos
Candidíase Vulvovaginal/genética , Receptor 2 Toll-Like/metabolismo , Animais , Candida albicans , Candidíase Vulvovaginal/microbiologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Predisposição Genética para Doença , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 2 Toll-Like/genéticaRESUMO
PROBLEM: A wide variety of mediators are involved in inflammatory processes. However, the identity of those participating in vaginal immune responses has not been established. We correlated extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase-8 (MMP-8), hyaluronan (HA), hyaluronidase-1 (Hyal-1), human ß-defensin-2 (hBD2), and neutrophil gelatinase-associated lipocalin (NGAL) concentrations with the extent of leukocyte infiltration into the vagina and suggest their participation in vaginal inflammation. METHODS OF STUDY: Vaginal fluid was obtained from 233 women seen at the outpatient clinic in the Department of Obstetrics and Gynecology at Campinas University, Brazil. The magnitude of vaginal inflammation was determined by the leukocyte count on vaginal smears and categorized as no inflammation (0 leukocytes/field), moderate inflammation (1-4 leukocytes/field), and intense inflammation (>4 leukocytes/field). Concentrations of EMMPRIN, MMP-8, HA, Hyal-1, hBD2, and NGAL were determined in vaginal fluid by ELISA. RESULTS: EMMPRIN, MMP-8, HA, hBD2, and NGAL concentration increased with elevated leukocyte numbers (P < 0.05), while Hyal-1 did not. EMMPRIN concentrations were correlated with HA and MMP-8 levels. CONCLUSION: EMMPRIN, MMP-8, HA, ß-defensin, and NGAL are elevated in women with vaginal inflammation.
Assuntos
Proteínas de Fase Aguda/imunologia , Basigina/imunologia , Ácido Hialurônico/imunologia , Lipocalinas/imunologia , Metaloproteinase 8 da Matriz/imunologia , Proteínas Proto-Oncogênicas/imunologia , Vaginite/imunologia , beta-Defensinas/imunologia , Adulto , Feminino , Humanos , Hialuronoglucosaminidase/imunologia , Leucócitos/imunologia , Lipocalina-2 , Adulto JovemRESUMO
BACKGROUND AND OBJECTIVE: Antimicrobial peptides, such as beta-defensins, secreted by gingival epithelial cells, are thought to play a major role in preventing periodontal diseases. In the present study, we investigated the ability of green tea polyphenols to induce human beta-defensin (hBD) secretion in gingival epithelial cells and to protect hBDs from proteolytic degradation by Porphyromonas gingivalis. MATERIAL AND METHODS: Gingival epithelial cells were treated with various amounts (25-200 µg/mL) of green tea extract or epigallocatechin-3-gallate (EGCG). The secretion of hBD1 and hBD2 was measured using ELISAs, and gene expression was quantified by real-time PCR. The treatments were also carried out in the presence of specific kinase inhibitors to identify the signaling pathways involved in hBD secretion. The ability of green tea extract and EGCG to prevent hBD degradation by proteases of P. gingivalis present in a bacterial culture supernatant was evaluated by ELISA. RESULTS: The secretion of hBD1 and hBD2 was up-regulated, in a dose-dependent manner, following the stimulation of gingival epithelial cells with a green tea extract or EGCG. Expression of the hBD gene in gingival epithelial cells treated with green tea polyphenols was also increased. EGCG-induced secretion of hBD1 and hBD2 appeared to involve extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. Lastly, green tea extract and EGCG prevented the degradation of recombinant hBD1 and hBD2 by a culture supernatant of P. gingivalis. CONCLUSION: Green tea extract and EGCG, through their ability to induce hBD secretion by epithelial cells and to protect hBDs from proteolytic degradation by P. gingivalis, have the potential to strengthen the epithelial antimicrobial barrier. Future clinical studies will indicate whether these polyphenols represent a valuable therapeutic agent for treating/preventing periodontal diseases.
Assuntos
Camellia sinensis , Catequina/análogos & derivados , Extratos Vegetais/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , beta-Defensinas/efeitos dos fármacos , Butadienos/farmacologia , Catequina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Gengiva/efeitos dos fármacos , Gengiva/metabolismo , Humanos , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Porphyromonas gingivalis/metabolismo , Proteólise/efeitos dos fármacos , Piridinas/farmacologia , Regulação para Cima , beta-Defensinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
To evaluate KGF and human beta defensin-4 (HBD-4) levels produced by dermic fibroblasts and keratinocytes cultivated from burned patients' skin samples. Keratinocytes and fibroblasts of 10 patients (four major burns, four minor burns and two controls) were primarily cultivated according to standard methods. HBD-4 and KGF genes were analyzed by quantitative PCR. In fibroblasts, KGF gene expression was 220±80 and 33.33±6.67 (M±SD; N=4), respectively for major and minor burn groups. In keratinocytes, KGF gene expression was 11.2±1.9 and 3.45±0.37 (M±SD; N=4), respectively for major and minor burn groups. In fibroblasts, HBD-4 gene expression was 15.0±4.0 and 11.5±0.5 (M±SD; N=4), respectively for major and minor burn. In keratinocyte, HBD-4 gene expression was 0.0±0.0 and 13.4±4.8 (M±SD; N=4), respectively for major and minor burn. KGF expression was increased in burn patient fibroblasts compared to control group. In keratinocytes culture, KGF suppression is inversely proportional to burn extension; it is active and increased in major burn but decreased in minor burn. HBD-4 expression was increased in fibroblasts and decreased in keratinocytes from all burned patients.(AU)
Assuntos
Animais , Queimaduras/complicações , Queratinócitos/citologia , beta-Defensinas , Fibroblastos/citologiaRESUMO
PURPOSE: To evaluate KGF and human beta defensin-4 (HBD-4) levels produced by dermic fibroblasts and keratinocytes cultivated from burned patients' skin samples. METHODS: Keratinocytes and fibroblasts of 10 patients (four major burns, four minor burns and two controls) were primarily cultivated according to standard methods. HBD-4 and KGF genes were analyzed by quantitative PCR. RESULTS: In fibroblasts, KGF gene expression was 220±80 and 33.33±6.67 (M±SD; N=4), respectively for major and minor burn groups. In keratinocytes, KGF gene expression was 11.2±1.9 and 3.45±0.37 (M±SD; N=4), respectively for major and minor burn groups. In fibroblasts, HBD-4 gene expression was 15.0±4.0 and 11.5±0.5 (M±SD; N=4), respectively for major and minor burn. In keratinocyte, HBD-4 gene expression was 0.0±0.0 and 13.4±4.8 (M±SD; N=4), respectively for major and minor burn. CONCLUSIONS: KGF expression was increased in burn patient fibroblasts compared to control group. In keratinocytes culture, KGF suppression is inversely proportional to burn extension; it is active and increased in major burn but decreased in minor burn. HBD-4 expression was increased in fibroblasts and decreased in keratinocytes from all burned patients. .
Assuntos
Feminino , Humanos , Masculino , Adulto Jovem , Queimaduras/genética , /análise , Fibroblastos/metabolismo , Queratinócitos/metabolismo , beta-Defensinas/genética , Células Cultivadas , /genética , Expressão Gênica , Reação em Cadeia da Polimerase , RNA , Pele/citologia , Pele/lesões , beta-Defensinas/metabolismoRESUMO
Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95%). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86% at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.
RESUMO
Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95 percent). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86 percent at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.
Assuntos
Escherichia coli/genética , Isopropiltiogalactosídeo , Peptídeos Catiônicos Antimicrobianos/análise , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Recombinantes de Fusão/análise , beta-Defensinas/análise , beta-Defensinas/genética , Fenômenos Fisiológicos Bacterianos , Métodos , MétodosRESUMO
Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34°C in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95%). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86% at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.
RESUMO
Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95%). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86% at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.