RESUMO
We have previously shown that the small metal-binding proteins CusF3H+ and SmbP can be used as fusion proteins for the expression and purification of recombinant proteins in Escherichia coli. Because of their small size, both around 10 kDa, they are suitable for the production of peptides to avoid meager yields after the final purification step of tag removal. Bin1b is a beta-defensin found in the epididymis of rats that has shown to have antimicrobial activity. Previous methodologies used to express this antimicrobial peptide in E. coli involve the expression of the peptide as inclusion bodies followed by in vitro refolding or the supplementation of the proteins necessary for proper folding of the peptide in the cytoplasm via a second plasmid. Here, we developed a methodology that forgoes these approaches and instead uses the fusion proteins CusF3H+ or SmbP and the E. coli strain SHuffle to obtain a soluble recombinant protein that contains the mature Bin1b peptide. The recombinant protein is purified using IMAC chromatography and is subsequently cleaved with enterokinase to separate the fusion protein from Bin1b. The purified peptide displays antimicrobial activity against E. coli, as previously shown. Furthermore, we also tested its antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus and found that Bin1b is also capable of inhibiting the growth of this bacterium. In conclusion, we developed a practical methodology for the expression and purification of the bioactive Bin1b peptide in E. coli using the fusion proteins CusF3H+ and SmbP. This approach could be further applied for the production of more biologically active peptides.
Assuntos
Escherichia coli , Proteínas Citotóxicas Formadoras de Poros , Proteínas Recombinantes de Fusão , Staphylococcus aureus/crescimento & desenvolvimento , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Citotóxicas Formadoras de Poros/biossíntese , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Hemoglobin is one of the most important molecules of the human body. Beyond its physiological activity, hemoglobins are able to inhibit the growth of several microorganisms. Since 1999, studies have reported that antimicrobial peptides can be produced by blood-feeding insects through hemoglobin digestion, and it has been reported that Triatoma infestans can generate an antimicrobial fragment from human fibrinopeptide. Thus T. infestans intestinal content was analyzed through Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), the eluted fractions were tested against Micrococcus luteus, Escherichia coli and Staphylococcus aureus, and the active fractions submitted to mass spectrometry. The data obtained were compared to hemoglobin databases to verify the presence of hemoglobin-derived fragments. Ten fractions eluted from chromatography presented antimicrobial activity, and when analyzed through mass spectrometry revealed the presence of 8 murine hemoglobin α-chain fragments and 24 fragments from murine hemoglobin ß fragments. Through the compilation of the fragments is possible to obtain over 67% coverage of both sequences. Part of the amino acid sequences corresponds to the sequences already identified on other intestinal contents of arthropods, and are highly conserved between the blood of other wild animals that are the most common intermediate hosts of Chagas' disease in Brazil and some of the main natural blood source for triatomines.
Assuntos
Hemoglobinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/biossíntese , Triatoma/metabolismo , Sequência de Aminoácidos/genética , Animais , Antibacterianos/biossíntese , Antibacterianos/metabolismo , Anti-Infecciosos , Doença de Chagas , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Camundongos , Proteínas Citotóxicas Formadoras de Poros/sangue , Trypanosoma cruziRESUMO
UNLABELLED: Exposure to high pressure is an efficient method of bacterial inactivation that is particularly important for reducing the microbial load present in foods. In this study, we examined the high pressure inactivation of Aeromonas hydrophila AH 191, a virulent strain that produces aerolysin, a cytotoxic, enterotoxic, and hemolytic toxin. High pressure treatment (250 MPa for 30 min at 25 °C in 0.1 M PBS, pH 7.4) of A. hydrophila grown in milk reduced bacterial viability by at least 9 orders of magnitude. Under these conditions, the enterotoxic, hemolytic, and cytotoxic activities of A. hydrophila culture supernatants were unaltered. These results indicate the need for caution in the use of high pressure for food processing since although truly toxigenic bacteria may be inactivated, their toxins may not be, thus posing a risk to human health. At higher pressure (350 MPa) the inactivation of bacteria was much more effective. Scanning electron microscopy showed a significant decrease in the number of bacteria after higher pressurization (350 MPa for 1 h) and transmission electron microscopy showed irregular shaped bacteria, suggestive of important cell wall and membrane damage, and cytoplasm condensation. PRACTICAL APPLICATION: High pressure inactivates Aeromonas hydrophila efficiently but is enhanced when combined with moderate temperature (40 °C). The biological activities of toxins from this bacterium are unaltered under these conditions.
Assuntos
Aeromonas hydrophila/crescimento & desenvolvimento , Manipulação de Alimentos/métodos , Leite/microbiologia , Animais , Toxinas Bacterianas/biossíntese , Células CACO-2 , Chlorocebus aethiops , Enterotoxinas/biossíntese , Contaminação de Alimentos/análise , Contaminação de Alimentos/prevenção & controle , Microbiologia de Alimentos/métodos , Humanos , Concentração de Íons de Hidrogênio , Pressão Hidrostática , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Proteínas Citotóxicas Formadoras de Poros/biossíntese , Temperatura , Células VeroRESUMO
In Chagas disease, CD8(+) T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8(+) T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8(+) T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8(+) T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8(+) T-cells segregated into IFNγ(+) perforin (Pfn)(neg) or IFNγ(neg)Pfn(+) cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8(+)Pfn(+) cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8(-/-) recipients showed that the CD8(+) cells from infected ifnγ(-/-)pfn(+/+) donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8(+) cells from ifnγ(+/+)pfn(-/-) donors. Moreover, the reconstitution of naïve cd8(-/-) mice with CD8(+) cells from naïve ifnγ(+/+)pfn(-/-) donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ(+) cells accumulation, whereas reconstitution with CD8(+) cells from naïve ifnγ(-/-)pfn(+/+) donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn(+) cells in the cardiac tissue. Our data support a possible antagonist effect of CD8(+)Pfn(+) and CD8(+)IFNγ(+) cells during CCC. CD8(+)IFNγ(+) cells may exert a beneficial role, whereas CD8(+)Pfn(+) may play a detrimental role in T. cruzi-elicited heart injury.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Cardiomiopatia Chagásica/imunologia , Regulação da Expressão Gênica/imunologia , Interferon gama/imunologia , Miocárdio/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Trypanosoma cruzi/imunologia , Doença Aguda , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Movimento Celular/genética , Movimento Celular/imunologia , Cardiomiopatia Chagásica/genética , Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/patologia , Feminino , Regulação da Expressão Gênica/genética , Interferon gama/biossíntese , Interferon gama/genética , Masculino , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Citotóxicas Formadoras de Poros/biossíntese , Proteínas Citotóxicas Formadoras de Poros/genética , Trypanosoma cruzi/metabolismoRESUMO
BACKGROUND: Tim-3 was recently described as a Th1-specific molecule, participating in the regulation of immune responses and in the induction of allograft tolerance. Here, we studied Tim-3 mRNA expression together with molecular markers of T-cell activation and cytotoxicity, in rejected human kidney grafts. METHODS: Twenty human kidney grafts that had undergone nephrectomy due to an irreversible acute rejection episode were studied. We quantified intragraft expression of Tim-3, granzyme B, perforin, IFN-gamma and Fas-ligand mRNA by real-time RT-PCR, with probes and primers TaqMan. Protocol biopsies were studied as controls. Statistical analyses were performed to compare groups, and to investigate the potential association with gene transcripts measures and rejection. RESULTS: All molecules studied were up-regulated in the rejection group compared with controls (p<0.001). Acute rejection type III (Banff 97) profiles were associated with higher values, where granzyme B and perforin presented the highest (5672.51+/-9002.16 and 1866.59+/-2426.38, respectively) and Tim-3 had the lowest ones (166.62+/-174.94). Tim-3 had also a lower expression in those patients that did not respond to anti-rejection therapy. There was a positive correlation between Tim-3 and IFN-gamma mRNA expression levels (r(2)=0.73; p<0.001). CONCLUSIONS: Our results corroborate the concept that acute rejection is an active process, where inflammatory as well as regulatory factors have their roles. Severe episodes of acute rejection were associated with higher expression of cytotoxic molecules and lower expression of potential regulatory molecule.