Asunto(s)
Bacillus anthracis/metabolismo , Técnicas de Química Analítica/métodos , Animales , Carbunco/metabolismo , Antígenos Bacterianos/química , Medios de Cultivo/farmacología , Fluoresceína-5-Isotiocianato/química , Liofilización , Caballos , Control de Calidad , Reproducibilidad de los Resultados , Esporas Bacterianas/metabolismoAsunto(s)
Bacillus anthracis/metabolismo , Técnicas de Química Analítica/métodos , Cromatografía de Gases/métodos , Ésteres/química , Ácidos Grasos/química , Agar/química , Calibración , Medios de Cultivo/farmacología , Metilación , Modelos Estadísticos , Control de Calidad , Glycine max/química , Esporas Bacterianas/metabolismo , Factores de TiempoRESUMEN
Anthrax protective antigen (PA, 83 kDa), a pore-forming protein, upon protease activation to 63 kDa (PA(63)), translocates lethal factor (LF) and edema factor (EF) from endosomes into the cytosol of the cell. The relatively small size of the heptameric PA(63) pore (approximately 12 angstroms) raises questions as to how large molecules such as LF and EF can move through the pore. In addition, the reported high binding affinity between PA and EF/LF suggests that EF/LF may not dissociate but remain complexed with activated PA(63). In this study, we found that purified (PA(63))(7)-LF complex exhibited biological and functional activities similar to the free LF. Purified LF complexed with PA(63) heptamer was able to cleave both a synthetic peptide substrate and endogenous mitogen-activated protein kinase kinase substrates and kill susceptible macrophage cells. Electrophysiological studies of the complex showed strong rectification of the ionic current at positive voltages, an effect similar to that observed if LF is added to the channels formed by heptameric PA(63) pore. Complexes of (PA(63))(7)-LF found in the plasma of infected animals showed functional activity. Identifying active complex in the blood of infected animals has important implications for therapeutic design, especially those directed against PA and LF. Our studies suggest that the individual toxin components and the complex must be considered as critical targets for anthrax therapeutics.
Asunto(s)
Carbunco/metabolismo , Antígenos Bacterianos/química , Antígenos Bacterianos/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Animales , Western Blotting , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Electrofisiología , Cobayas , Técnicas In Vitro , Cinética , Membrana Dobles de Lípidos , Quinasas de Proteína Quinasa Activadas por Mitógenos/química , Péptidos/química , Unión Proteica , Conejos , Especificidad por Sustrato , Temperatura , Factores de TiempoRESUMEN
A two-component direct fluorescent-antibody (DFA) assay, using fluorescein-labeled monoclonal antibodies specific to the Bacillus anthracis cell wall (CW-DFA) and capsule (CAP-DFA) antigens, was evaluated and validated for rapid identification of B. anthracis. We analyzed 230 B. anthracis isolates; 228 and 229 were positive by CW-DFA and CAP-DFA assays, respectively. We also tested 56 non-B. anthracis strains; 10 B. cereus and 2 B. thuringiensis were positive by the CW-DFA assay, and 1 B. megaterium strain was positive by CAP-DFA. Analysis of the combined DFA results identified 227 of 230 B. anthracis isolates; all 56 strains of the other Bacillus spp. were negative. Both DFA assays tested positive on 14 of 26 aging clinical specimens from the 2001 anthrax outbreak investigation. The two-component DFA assay is a sensitive, specific, and rapid confirmatory test for B. anthracis in cultures and may be useful directly on clinical specimens.
