RESUMEN
Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.
Asunto(s)
Anticuerpos Monoclonales , Colorantes Fluorescentes , Genes Reporteros , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente/métodos , Técnicas de Sonda Molecular , Fragmentos de Inmunoglobulinas , Proteínas de la Membrana/ultraestructuraRESUMEN
Region of difference (RD1) genes are present in virulent Mycobacterium tuberculosis but not the vaccine strain M. bovis bacille Calmette-Guérin (BCG). The deletion of RD1 from M. tuberculosis produces an attenuation strikingly like that of BCG, which suggests the use of RD1 mutant strains for improvement of the tuberculosis (TB) vaccine. We performed long-term murine infection with M. tuberculosis H37Rv: Delta RD1 and BCG. Mice infected with H37Rv: Delta RD1 gained less weight than did BCG-infected control mice, and, after >1 year, their lungs harbored many more bacteria and displayed significant levels of inflammation. This difference in virulence has important implications for the pursuit of strains lacking RD1 in the development of the TB vaccine.
Asunto(s)
Eliminación de Gen , Genes Bacterianos , Mycobacterium bovis/patogenicidad , Mycobacterium tuberculosis/patogenicidad , Tuberculosis Pulmonar/patología , Animales , Vacuna BCG , Humanos , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/genética , Vacunas contra la Tuberculosis , Tuberculosis Pulmonar/microbiología , Vacunas Atenuadas , VirulenciaRESUMEN
The RD1 genomic region is present in virulent strains of Mycobacterium tuberculosis (MTB), missing from the vaccine strain M. bovis BCG, and its importance to virulence has been established experimentally. Based on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this region affects virulence is unknown. Here we examined mutants disrupted in five individual RD1 genes. Both in vitro and in vivo, each mutant displayed an attenuated phenotype very similar to a mutant missing the entire RD1 region. Genetic complementation of individual genes restored virulence. Attenuated mutants could multiply within THP-1 cells, but they were unable to spread to uninfected macrophages. We also examined export of two immunodominant RD1 proteins, CFP-10 and ESAT-6. Export of these proteins was greatly reduced or abolished in each attenuated mutant. Again, genetic complementation restored a wild-type phenotype. Our results indicate that RD1 genes work together to form a single virulence determinant, and argue that RD1 encodes a novel specialized secretion system that is required for pathogenesis of MTB.