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J Proteomics ; 80: 34-42, 2013 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-23159400


Trypanosoma cruzi proteins with molecular weight between 30 and 34 kDa have shown high reactivity in western blot assays with serum samples from chagasic individuals. However, in-depth analysis of the constituents of these protein fractions has not been performed. This is the first report of an immunoaffinity proteomic approach to identify the immunodominant 30-34 kDa proteins of T. cruzi that could eventually be used for the diagnosis of Chagas disease. We used two different sample preparation protocols for protein digestion coupled to mass spectrometry to identify proteins in the protein fraction. The immunodominant proteins and their respective epitopes were then identified by co-immunoprecipitation and excision-epitope mapping/mass spectrometry, using human sera followed by the prediction and three-dimensional structural modeling of reactive epitopes. The use of different sample preparation methods allowed the identification of a relatively high number of proteins, some of which were only identified after one or multiple sample preparation and digestion protocols. Seven immunodominant proteins were identified by co-immunoprecipitation with purified IgGs from chagasic serum samples. Moreover, six reactive peptide epitopes were detected in four of these proteins by excision-epitope mapping/mass spectrometry. Three-dimensional structural models were obtained for the immunoreactive peptides, which correlated well with the linear B-cell epitope prediction tools.

Anticuerpos/química , Enfermedad de Chagas/sangre , Enfermedad de Chagas/parasitología , Epítopos/química , Trypanosoma cruzi/metabolismo , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Electroforesis en Gel de Poliacrilamida , Mapeo Epitopo , Epítopos de Linfocito B/química , Humanos , Inmunoglobulina G/química , Inmunoprecipitación , Espectrometría de Masas , Peso Molecular , Péptidos/química , Conformación Proteica , Proteoma , Proteómica/métodos
Microbiology ; 156(Pt 10): 3011-20, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20688821


The putative phosphoporin encoded by vca1008 of Vibrio cholerae O1 is expressed in vivo during infection and is essential for the intestinal colonization of infant mice. In vitro, its expression is induced under inorganic phosphate (P(i)) limitation in a PhoB/R-dependent manner. In this work we demonstrated that VCA1008 has a strain-specific role in the physiology and pathogenicity of V. cholerae O1. Disruption of vca1008 led to a growth defect, an inability to colonize and a high susceptibility to sodium deoxycholate (DOC; the major bile compound) in the El Tor biotype strain N16961, but did not affect the classical strain O395 in the same way. Furthermore, vca1008 promoter activity was higher in N16961 cells grown under a low P(i) supply in the presence of DOC than in the absence of the detergent. In the P(i)-limited cells, vca1008 was positively regulated by PhoB, but when DOC was added to the medium, it negatively affected the PhoB-mediated activation of the gene, and enhanced vca1008 expression in a ToxR-dependent manner. These findings reveal for the first time a complex strain-specific interplay between ToxR and PhoB/R systems to control porin genes, as well as the influence of DOC on the expression of PhoB- and ToxR-regulated genes and pathogenesis in pandemic strains of V. cholerae.

Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácido Desoxicólico/farmacología , Porinas/metabolismo , Factores de Transcripción/metabolismo , Vibrio cholerae/patogenicidad , Animales , Proteínas Bacterianas/genética , Clonación Molecular , Proteínas de Unión al ADN/genética , Prueba de Complementación Genética , Ratones , Mutación , Fosfatos/metabolismo , Porinas/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Vibrio cholerae/genética , Vibrio cholerae/crecimiento & desarrollo , Vibrio cholerae/metabolismo , Virulencia
Proteomics ; 6(5): 1495-511, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16447160


A proteomic analysis of a wild-type and of a phoB mutant showed that Vibrio cholerae expresses genes of two major regulons in response to phosphate starvation. The Pho regulon, expressed by the wild-type, allowed the cells to adapt to the new environment. Induction of the general stress regulon was mainly observed in the phoB mutant as a strategy to resist stress and survive. Some functions of the adaptative and survival responses play roles in the pathogenicity of the bacteria. Among the members of the Pho regulon, we found a porin described as an important factor for the intestinal colonisation. Other functions not obviously related to phosphate metabolism, expressed preferentially by the wild-type cells, have also been implicated in virulence. These findings might explain the lack of virulence of the phoB mutant. The Pho regulon picture of V. cholerae, however, will not be complete until minor members and membrane proteins are identified. Among the phosphate-starvation induced genes we have found 13 hypothetical ones and for some of them functions have been assigned. The majority of the genes identified here have not been described before, thus they could be used to expand the proteomic reference map of V. cholerae El Tor.

Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Fosfatos/metabolismo , Proteoma/análisis , Regulón , Vibrio cholerae O1/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Electroforesis en Gel Bidimensional , Datos de Secuencia Molecular , Operón , Vibrio cholerae O1/genética , Vibrio cholerae O1/patogenicidad
Proteomics ; 4(5): 1491-504, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15188416


A proteome reference map has been constructed for Vibrio cholerae El Tor, in the pI range of 4.0 to 7.0. The map is based on two-dimensional gels (2-D) and the identification, by peptide mass fingerprint, of proteins in 94 spots, corresponding to 80 abundant proteins. Two strains are compared, strain N16961 and a Latin American El Tor strain C3294. The consensus map contains 340 spots consistently seen with both strains grown in Luria-Bertani broth (LB) or minimal M9 medium. The results were obtained from nine gels run with 18 cm immobilized pH gradient strips and precast gels. The 2-D gels were anchored to real N16961 proteins identified by mass spectrometry. Various energy metabolism components and periplasmic ATP-binding cassette (ABC) transporter proteins were identified among the abundant proteins. Two isoforms of OmpU were found. Five operons are proposed and seven hypothetical proteins were experimentally confirmed. Comparisons are made with protein 2-D gels for a classical strain and to microarray analysis available for the N16961 El Tor strain. New results were obtained from the proteome analysis, indicating an abundance of periplasmic ABC transporter proteins not found in microarray studies.

Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteoma/normas , Vibrio cholerae/clasificación , Vibrio cholerae/metabolismo , Proteínas Bacterianas/química , Medios de Cultivo , Electroforesis en Gel Bidimensional , Focalización Isoeléctrica , Espectrometría de Masas , Peso Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Operón , Mapeo Peptídico , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estándares de Referencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vibrio cholerae/genética , Vibrio cholerae/crecimiento & desarrollo