RESUMEN
The identification of factors involved in the interaction of Mycobacterium bovis with the hosts will lead to new strategies to control bovine tuberculosis. In this study we compared the transcriptional profile of an attenuated M. bovis strain and a virulent M. bovis strain as a means to elucidate the molecular basis for their differential phenotype. Microarray and RT-qPCR results demonstrated that the expression of mce4D, Mb2607/Mb2608 and Mb3706c were up-regulated in the virulent strain whereas alkB, Mb3277c and Mb1077c were expressed at higher levels in the attenuated strain. These differential expression profiles were confirmed for Mb2607/Mb2608, mce4D, Mb1077c, alkB and Mb3277c during the replication of bacteria inside macrophages.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Mycobacterium bovis/genética , Mycobacterium bovis/patogenicidad , Animales , Bovinos , Células Cultivadas , Macrófagos/microbiología , Ratones , Mycobacterium bovis/aislamiento & purificación , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sus scrofaRESUMEN
The mce operons constitute four homologous regions in the Mycobacterium tuberculosis genome, each of which has 8-13 ORFs. Although the function of the Mce protein family has not been clearly established, its members are believed to be membrane lipid transporters. Based on functional experiments, we found that the regulator of the mce3 locus, Mce3R, negatively regulates the expression of the Rv1933c-Rv1935c and Rv1936-Rv1941 transcriptional units. These operons are adjacent to one another and divergently transcribed. The predicted functions of most of these genes are related to either lipid metabolism or redox reactions. Bioinformatic analysis of the 5' UTR sequences of the differentially expressed genes allowed us to define a putative Mce3R motif. Importantly, the Mce3R motif was present six and three times in the mce3R-yrbE3A and Rv1935c-Rv1936 intergenic regions, respectively. Two occurrences of this motif mapped within the two regions of the mce3 operon that were protected by Mce3R in a footprinting analysis, thus indicating that this motif is likely to serve as an operator site for the Mce3R regulator in the promoter. In addition, alterations in the lipid content of M. tuberculosis were detected in the absence of Mce3R. Taken together, these results suggest that Mce3R controls the expression of both the putative transport system encoded in the mce3 operon and the enzymes implicated in the modification of the Mce3-transported substrates.
Asunto(s)
Proteínas Bacterianas/fisiología , Metabolismo de los Lípidos , Mycobacterium tuberculosis/metabolismo , Regulón , Proteínas Represoras/fisiología , Animales , Secuencia de Bases , Línea Celular , Secuencia Conservada , ADN Bacteriano/análisis , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Lípidos/análisis , Ratones , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidación-Reducción , Regiones Promotoras GenéticasRESUMEN
Tuberculosis in seals is caused by Mycobacterium pinnipedii, a member of the Mycobacterium tuberculosis complex. In this study, we evaluated the extent of genetic variability among Mycobacterium bovis and M. pinnipedii by microarray-based comparative genomics. We identified two deletions that are exclusive to M. pinnipedii: PiD1 that removes the orthologues of the M. tuberculosis genes Rv3530c and Rv3531c, and PiD2 that encompasses genes Rv1977 and Rv1978. Interestingly, a deletion overlapping the previously described RD2 region was identified in some isolates of Mycobacterium microti and further characterised.