RESUMEN
Mycobacterium tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a 'non-culturable' state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the 'non-culturable' cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.
Asunto(s)
Proteínas Bacterianas/fisiología , Citocinas/fisiología , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/patogenicidad , Factores de Virulencia/fisiología , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Recuento de Colonia Microbiana , Medios de Cultivo/metabolismo , Citocinas/genética , Detergentes/farmacología , Femenino , Eliminación de Gen , Prueba de Complementación Genética , Humanos , Macrófagos/microbiología , Ratones , Viabilidad Microbiana/genética , Mutagénesis Insercional , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Virulencia , Factores de Virulencia/genéticaRESUMEN
Mycobacterium tuberculosis contains five genes, rpfA through rpfE, that bear significant homology to the resuscitation-promoting factor (rpf) gene of Micrococcus luteus, whose product is required to resuscitate the growth of dormant cultures of M. luteus and is essential for the growth of this organism. Previous studies have shown that deletion of any one of the five rpf-like genes did not affect the growth or survival of M. tuberculosis in vitro. In conjunction with the results of whole-genome expression profiling, this finding was indicative of their functional redundancy. In this study, we demonstrate that the single deletion mutants are phenotypically similar to wild-type M. tuberculosis H37Rv in vivo. The deletion of individual rpf-like genes had no discernible effect on the growth or long-term survival of M. tuberculosis in liquid culture, and the ability to resuscitate spontaneously from a nonculturable state in a most probable number assay was also unaffected for the three strains tested (the DeltarpfB, DeltarpfD, and DeltarpfE strains). In contrast, two multiple strains, KDT8 (DeltarpfA-mutation DeltarpfC DeltarpfB) and KDT9 (DeltarpfA DeltarpfC DeltarpfD), which lack three of the five rpf-like genes, were significantly yet differentially attenuated in a mouse infection model. These mutants were also unable to resuscitate spontaneously in vitro, demonstrating the importance of the Rpf-like proteins of M. tuberculosis in resuscitation from the nonculturable state. These results strongly suggest that the biological functions of the five rpf-like genes of M. tuberculosis are not wholly redundant and underscore the potential utility of these proteins as targets for therapeutic intervention.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citocinas/metabolismo , Regulación Bacteriana de la Expresión Génica , Mutación , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/patogenicidad , Animales , Proteínas Bacterianas/genética , Medios de Cultivo , Citocinas/genética , Eliminación de Gen , Genes Bacterianos , Humanos , Ratones , Ratones Endogámicos C57BL , Familia de Multigenes , Mycobacterium tuberculosis/genética , Tuberculosis Pulmonar/microbiología , VirulenciaRESUMEN
The function of the Mycobacterium tuberculosis eukaryotic-like protein serine/threonine kinase PknG was investigated by gene knock-out and by expression and biochemical analysis. The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase. An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C-terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif. Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane. The pknG gene was inactivated by allelic exchange in M. tuberculosis. The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice. The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient-depleted media. The PknG-deficient mutant accumulates glutamate and glutamine. The cellular levels of these two amino acids reached approximately threefold of their parental strain levels. Higher cellular levels of the amine sugar-containing molecules, GlcN-Ins and mycothiol, which are derived from glutamate, were detected in the DeltapknG mutant. De novo glutamine synthesis was shown to be reduced by 50%. This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels. These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M. tuberculosis and translates them into metabolic adaptation.