RESUMEN
The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.
Asunto(s)
Proteínas Bacterianas/metabolismo , Brucella abortus/genética , Proteómica , Factores de Virulencia/metabolismo , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Brucella abortus/metabolismo , Brucella abortus/patogenicidad , Línea Celular , Medios de Cultivo , Electroforesis en Gel Bidimensional , Genes Bacterianos , Proteínas HSP70 de Choque Térmico/metabolismo , Ratones , Datos de Secuencia Molecular , Isomerasa de Peptidilprolil/metabolismo , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Virulencia/genéticaRESUMEN
Brucella species are pathogenic agents that cause brucellosis, a debilitating zoonotic disease that affects a large variety of domesticated animals and humans. Brucella melitensis and Brucella abortus are considered major health threats because of their highly infectious nature and worldwide occurrence. The availability of the annotated genomes for these two species has allowed a comparative proteomics study of laboratory grown B. melitensis 16M and B. abortus 2308 by two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting. Computer-assisted analysis of the different 2-D gel images of strains 16M and 2308 revealed significant quantitative and qualitative differences in their protein expression patterns. Proteins involved in membrane transport, particularly the high affinity amino acids binding proteins, and those involved in Sec-dependent secretion systems related to type IV and type V secretion systems, were differentially expressed. Differential expression of these proteins may be responsible for conferring specific host preference in the two strains 2308 and 16M.
Asunto(s)
Proteínas Bacterianas/metabolismo , Brucella abortus/química , Brucella abortus/metabolismo , Brucella melitensis/química , Brucella melitensis/metabolismo , Proteoma/análisis , Animales , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Brucella abortus/genética , Brucella abortus/crecimiento & desarrollo , Brucella melitensis/genética , Brucella melitensis/crecimiento & desarrollo , Electroforesis en Gel Bidimensional , Regulación Bacteriana de la Expresión Génica , Mapeo PeptídicoRESUMEN
Bacillus anthracis, the causative agent of anthrax, secretes numerous proteins into the extracellular environment during infection. A comparative proteomic approach was employed to elucidate the differences among the extracellular proteomes (secretomes) of three isogenic strains of B. anthracis that differed solely in their plasmid contents. The strains utilized were the wild-type virulent B. anthracis RA3 (pXO1(+) pXO2(+)) and its two nonpathogenic derivative strains: the toxigenic, nonencapsulated RA3R (pXO1(+) pXO2(-)) and the totally cured, nontoxigenic, nonencapsulated RA3:00 (pXO1(-) pXO2(-)). Comparative proteomics using two-dimensional gel electrophoresis followed by computer-assisted gel image analysis was performed to reveal unique, up-regulated, or down-regulated secretome proteins among the strains. In total, 57 protein spots, representing 26 different proteins encoded on the chromosome or pXO1, were identified by peptide mass fingerprinting. S-layer-derived proteins, such as Sap and EA1, were most frequently observed. Many sporulation-associated enzymes were found to be overexpressed in strains containing pXO1(+). This study also provides evidence that pXO2 is necessary for the maximal expression of the pXO1-encoded toxins lethal factor (LF), edema factor (EF), and protective antigen (PA). Several newly identified putative virulence factors were observed; these include enolase, a high-affinity zinc uptake transporter, the peroxide stress-related alkyl hydroperoxide reductase, isocitrate lyase, and the cell surface protein A.
Asunto(s)
Bacillus anthracis/genética , Proteínas Bacterianas/análisis , Plásmidos , Proteoma , Electroforesis en Gel Bidimensional , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The proteomes of selected Brucella spp. have been extensively analyzed by utilizing current proteomic technology involving 2-DE and MALDI-MS. In Brucella melitensis, more than 500 proteins were identified. The rapid and large-scale identification of proteins in this organism was accomplished by using the annotated B. melitensis genome which is now available in the GenBank. Coupled with new and powerful tools for data analysis, differentially expressed proteins were identified and categorized into several classes. A global overview of protein expression patterns emerged, thereby facilitating the simultaneous analysis of different metabolic pathways in B. melitensis. Such a global characterization would not have been possible by using time consuming and traditional biochemical approaches. The era of post-genomic technology offers new and exciting opportunities to understand the complete biology of different Brucella species.
Asunto(s)
Proteínas Bacterianas/genética , Brucella melitensis/genética , Proteoma/genéticaRESUMEN
Brucella melitensis is a facultative, intracellular, gram-negative cocco-bacillus that causes Malta fever in humans and brucellosis in animals. There are at least six species in the genus, and the disease is classified as zoonotic because several species infect humans. Using 2-D gel electrophoresis and mass spectrometry, we have initiated (i) a comprehensive mapping and identification of all the expressed proteins of B. melitensis virulent strain 16M, and (ii) a comparative study of its proteome with the attentuated vaccinal strain Rev 1. Comprehensive proteome maps of all six Brucella species will be generated in order to obtain vital information for vaccine development, identification of pathogenicity islands, and establishment of host specificity and evolutionary relatedness.
Asunto(s)
Proteínas Bacterianas/metabolismo , Vacuna contra la Brucelosis , Brucella melitensis , Proteoma/análisis , Animales , Proteínas Bacterianas/genética , Vacunas Bacterianas , Brucella/genética , Brucella/metabolismo , Brucella/patogenicidad , Brucella melitensis/genética , Brucella melitensis/metabolismo , Brucella melitensis/patogenicidad , Brucelosis/prevención & control , Brucelosis/veterinaria , Mapeo Cromosómico/veterinaria , Electroforesis en Gel Bidimensional/veterinaria , Regulación Bacteriana de la Expresión Génica , Humanos , Especificidad de la Especie , Virulencia , ZoonosisRESUMEN
Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.
Asunto(s)
Proteínas Bacterianas/análisis , Brucella melitensis/química , Proteoma/análisis , Animales , Cromosomas Bacterianos , Electroforesis en Gel Bidimensional , Genes Bacterianos , Humanos , Focalización Isoeléctrica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The genus Brucella consists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the different Brucella species, Brucella melitensis is considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence in B. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the two B. melitensis proteomes revealed proteins expressed in either 16M or Rev 1, as well as up- or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1.