RESUMO
Attenuated strains of mycobacteria can be exploited to determine genes essential for their pathogenesis and persistence. To this goal, we sequenced the genome of H37Ra, an attenuated variant of Mycobacterium tuberculosis H37Rv strain. Comparison with H37Rv revealed three unique coding region polymorphisms. One polymorphism was located in the DNA-binding domain of the transcriptional regulator PhoP, causing the protein's diminished DNA-binding capacity. Temporal gene expression profiles showed that several genes with reduced expression in H37Ra were also repressed in an H37Rv phoP knockout strain. At later time points, genes of the dormancy regulon, typically expressed in a state of nonreplicating persistence, were upregulated in H37Ra. Complementation of H37Ra with H37Rv phoP partially restored its persistence in a murine macrophage infection model. Our approach demonstrates the feasibility of identifying minute but distinct differences between isogenic strains and illustrates the consequences of single point mutations on the survival stratagem of M. tuberculosis.
Assuntos
Proteínas de Bactérias/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/patogenicidade , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Células Cultivadas , Teste de Complementação Genética , Macrófagos/microbiologia , Camundongos , Dados de Sequência Molecular , Mutação Puntual , Polimorfismo Genético , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Fatores de Transcrição/química , VirulênciaRESUMO
DNA microarray technology has been increasingly applied for studies of clinical samples. Frequently, RNA probes from clinical samples are available in limited amounts. We describe a reliable amplification method for bacterial RNA. We verified this method on mycobacterial RNA applying mycobacterial genome-directed primers (mtGDPs). Glass slide-based oligoarrays were employed to assess the quality of the amplification method. We observed a relatively small bias in amplified RNA pool when compared to the unamplified one. Up to 1000-fold linear RNA amplification in a single amplification round was obtained. To our knowledge, this study describes the first amplification method for mycobacterial RNA.
Assuntos
Mycobacterium tuberculosis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Bacteriano/análise , Vírus da Leucemia Murina de Moloney/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , DNA Polimerase Dirigida por RNA/farmacologiaRESUMO
A 9.5-kb section of DNA called region of deletion 1 (RD1) is present in virulent Mycobacterium tuberculosis strains but is deleted in all attenuated Mycobacterium bovis BCG vaccine strains. This region codes for at least nine genes. Some or all RD1 gene products may be involved in virulence and pathogenesis, and at least two, ESAT-6 and CFP-10, represent potent T- and B-cell antigens. In order to produce the entire set of RD1 proteins with their natural posttranslational modifications, a robust expression system for M. tuberculosis proteins in the fast-growing saprophytic strain Mycobacterium smegmatis was developed. Our system employs the inducible acetamidase promoter and allows translational fusion of recombinant M. tuberculosis proteins with polyhistidine or influenza hemagglutinin epitope tags for affinity purification. Using eGFP as reporter gene, we showed that the acetamidase promoter is tightly regulated in M. smegmatis and that this promoter is much stronger than the widely used constitutive groEL2 promoter. We then cloned 11 open reading frames (ORFs) found within RD1 and successfully expressed and purified the respective proteins. Sera from tuberculosis patients and M. tuberculosis-infected mice reacted with 10 purified RD1 proteins, thus demonstrating that Rv3871, Rv3872, Rv3873, CFP-10, ESAT-6, Rv3876, Rv3878, Rv3879c and ORF-14 are expressed in vivo. Finally, glycosylation of the RD1 proteins was analyzed. We present preliminary evidence that the PPE protein Rv3873 is glycosylated at its C terminus, thus highlighting the ability of M. smegmatis to produce M. tuberculosis proteins bearing posttranslational modifications.
