ABSTRACT
The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-A resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.
Subject(s)
Humans , Animals , Bacterial Outer Membrane Proteins/immunology , Brucella abortus/immunology , Bacterial Outer Membrane Proteins/analysis , Brucella abortus/chemistry , Brucella abortus/enzymology , Brucella Vaccine , Brucellosis/diagnosis , Chromatography, Affinity , Crystallography , Enzyme-Linked Immunosorbent Assay , Protein Structure, Quaternary , Protein Structure, Tertiary , Pteridines/chemical synthesisABSTRACT
Se presentan resultados que muestan que el análisis por immunoblotting de la respuesta inmune humoral de pacientes brucelosos permite la caracterización de componentes antigénicso de posible utilidad para el diagnóstico de la enfermedad. Se analizó el suero de 90 pacientes: 20 brucelosos agudos, 23 crónicos y 47 individuos serológicamente positivos sin evidencias clínicas de infección activa al momento del examen (SPI); se utilizó además el suero de 35 personas sanas como control. Todos los sueros fueron ensayados frente a tres fracciones antigéneticas: citoplasmáticas (CYT), membrana externa (OM) y membrana interna (IM). Dichas fracciones, que incluyen virtualmente todas las proteínas bacterianas, fueron preparadas a partir de Brucella abortus 1119/3 por solubilización con detergentes, digestión enzimática y ultracentrifugación. Los resultados obtenidos revelan la existencia de antígenos que permiten detectar a pacientes brucelosos con alta sensibilidad, y diferenciar a éstos del grupo SPI