RESUMEN
Bacterial biofilms cause numerous problems in health care and industry; notably, biofilms are associated with a large number of infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics, making it hard to eradicate biofilm-associated infections. Bacteria rely on efflux pumps to get rid of toxic substances. We discovered that efflux pumps are highly active in bacterial biofilms, thus making efflux pumps attractive targets for antibiofilm measures. A number of efflux pump inhibitors (EPIs) are known. EPIs were shown to reduce biofilm formation, and in combination they could abolish biofilm formation completely. Also, EPIs were able to block the antibiotic tolerance of biofilms. The results of this feasibility study might pave the way for new treatments for biofilm-related infections and may be exploited for prevention of biofilms in general.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Transporte Biológico Activo/efectos de los fármacos , Dipéptidos/farmacología , Humanos , Piperazinas/farmacología , Tioridazina/farmacologíaRESUMEN
In order to investigate the ability of the Vitreoscilla hemoglobin (VHb) to act as a peroxidase, the protein was overexpressed in Escerichia coli and purified using a 6xHis-tag. The peroxidase activity of VHb was studied using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferrocene carboxylic acid (FcCOOH) dopamine and L-dopa as substrates. The effects of external agents such as pH, salt concentration/ionic strength, and the thermal stability of VHb on the catalytic activity were assessed. The optimum pH for VHb using ABTS as a substrate was estimated to be 6-7. The VHb protein proved to be stable up to 80 degrees C, as judged by its peroxidase activity. Furthermore, NaCl concentrations up to 100 mM did not exert any significant effect on the activity. The catalytic activity against ABTS and FcCOOH was similar to that measured for horseradish peroxidase, whereas in the case of the phenolic substrates dopamine and L-dopa the activity was several orders of magnitude lower. The Michaelis constants, KmH2O2, were in good agreement with the data for human and bovine hemoglobin. No activity could be detected for the negative controls lacking VHb. These results demonstrate that VHb exhibits peroxidase activity, a finding in line with the hypothesis that VHb has cellular functions beyond the role as an oxygen carrier.
Asunto(s)
Proteínas Bacterianas/química , Hemoglobinas/química , Peroxidasas/química , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Benzotiazoles/química , Catálisis , Dopamina/química , Escherichia coli/genética , Compuestos Ferrosos/química , Hemoglobinas/biosíntesis , Hemoglobinas/aislamiento & purificación , Cinética , Levodopa/química , Metalocenos , Peroxidasas/biosíntesis , Peroxidasas/aislamiento & purificación , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Espectrofotometría Ultravioleta , Relación Estructura-Actividad , Especificidad por Sustrato , Ácidos Sulfónicos/química , Hemoglobinas TruncadasRESUMEN
Management of bacterial infections is becoming increasingly difficult due to the rising frequency of strains that are resistant to many current antibiotics. New types of antibiotics are, therefore, urgently needed. Virulence factors or virulence-associated phenotypes such as adhesins and biofilm formation are highly attractive targets for new drugs. Specific adhesion provides bacteria with target selection and prevents removal by hydrodynamic flow forces. Bacterial adhesion is of paramount importance for bacterial pathogenesis. Adhesion is also the first step in biofilm formation. Biofilm formation is particularly problematic in medical contexts because biofilm-associated bacteria are particularly hard to eradicate. Several promising candidate drugs that target bacterial adhesion and biofilm formation are being developed. Some of these might be valuable weapons for fighting infectious diseases in the future. Here we use illustrative examples, mainly from the enterics, to demonstrate the principles.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Antibacterianos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Infecciones Bacterianas/inmunología , Biopelículas/crecimiento & desarrollo , Adhesión Bacteriana/fisiología , Biopelículas/efectos de los fármacosRESUMEN
Experimentation was initiated to explore insight into the redox-catalysis reaction derived from the heme prosthetic group of chimeric Vitreoscilla hemoglobin (VHb). Two chimeric genes encoding chimeric VHbs harboring one and two consecutive sequences of Fc-binding motif (Z-domain) were successfully constructed and expressed in E. coli strain TG1. The chimeric ZVHb and ZZVHb were purified to a high purity of more than 95% using IgG-Sepharose affinity chromatography. From surface plasmon resonance, binding affinity constants of the chimeric ZVHb and ZZVHb to human IgG were 9.7 x 10(7) and 49.1 x 10(7) per molar, respectively. More importantly, the chimeric VHbs exhibited a peroxidase-like activity determined by activity staining on native PAGE and dot blotting. Effects of pH, salt, buffer system, level of peroxidase substrate and chromogen substrate were determined in order to maximize the catalytic reaction. From our findings, the chimeric VHbs displayed their maximum peroxidase-like activity at the neutral pH (approximately 7.0) in the presence of high concentration (20-40 mM) of hydrogen peroxide. Under such conditions, the detection limit derived from the calibration curve was at 250 ng for the chimeric VHbs, which was approximately 5-fold higher than that of the horseradish peroxidase. These findings reveal the novel functional role of Vitreoscilla hemoglobin indicating a high trend of feasibility for further biotechnological and medical applications.