RESUMEN
Proteins which are secreted or associated with the cell envelope of Mycobacterium tuberculosis may contain protective T-cell epitopes. Prior to this study, a recombinant clone bank of enzymatically active M. tuberculosis-alkaline phosphatase fusions, were screened for immunogenicity in a murine T-cell model. Five of these were selected for further study, and the IFN-gamma secretion and proliferation of human PBMC from purified protein derivative- (PPD)-positive and PPD-negative donors were measured in response to oligopeptides, Mtb-PhoA fusions and one full-length protein. Epitopes from four of the five selected antigens were immunoreactive in the human model and corresponded to cytochrome d ubiquinol oxidase, cytochrome c oxidase subunit II, MTV005.02 and MTV033.08. Thus, this strategy identified novel human immunogenic peptides as possible candidates for a subunit vaccine.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Activación de Linfocitos , Linfocitos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Fosfatasa Alcalina/química , Fosfatasa Alcalina/inmunología , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Proteínas Bacterianas/química , Clonación Molecular , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Epítopos/inmunología , Escherichia coli , Humanos , Interferón gamma/biosíntesis , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism analysis demonstrated that these upstream flanking sequences and the 231-bp, 5' coding region are highly conserved among both drug-sensitive and multiply-drug-resistant isolates of M. tuberculosis. The efpA gene was present in the slow-growing human pathogens M. tuberculosis, Mycobacterium leprae, and Mycobacterium bovis and in the opportunistic human pathogens Mycobacterium avium and Mycobacterium intracellular. However, efpA was not present in 17 other opportunistically pathogenic or nonpathogenic mycobacterial species.
Asunto(s)
Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Transporte de Membrana , Mycobacterium tuberculosis/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Transporte Biológico/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Mycobacterium bovis/genética , Mycobacterium leprae/genética , Reacción en Cadena de la PolimerasaRESUMEN
Phagemid vectors were constructed to allow fusions of alkaline phosphatase to proteins encoded by G+C-rich DNA, by engineering a BstBI site (TT/CGAA) in front of a phoA gene that lacks an encoded signal peptide. Three vectors (pJDT1, pJDT2 and pJDT3), each with phoA in a different reading frame with respect to the BstBI site, were produced; a lacP region is present in each plasmid upstream of the BstBI site. The presence of the BstBI site allows the random cloning of G+C-rich DNA digested with a number of restriction enzymes that generate cohesive ends.