Characterization of the two component regulatory system PhoPR in Mycobacterium bovis.

Mycobacterium bovis is the causative agent of bovine tuberculosis and is a member of Mycobacterium tuberculosis complex, which causes tuberculosis in a number of mammals including humans. Previous studies have shown that the genes encoding the two-component system PhoPR, which regulates several genes involved in the virulence of M. tuberculosis, are polymorphic in M. bovis, when compared to M. tuberculosis, which results in a dysfunctional two-component system. In this study we investigated the role of PhoPR in two M. bovis strains with differing degrees of virulence. We found that the deletion of phoP in an M. bovis isolate reduced its capacity of inducing phagosomal arrest in bovine macrophages. By gene expression analysis, we demonstrated that, in both M. bovis strains, PhoP regulates the expression of a putative lipid desaturase Mb1404-Mb1405, a protein involved in redox stress AhpC, the sulfolipid transporter Mmpl8 and the secreted antigen ESAT-6. Furthermore, the lack of PhoP increased the sensitivity to acidic stress and alteration of the biofilm/pellicle formation of M. bovis. Both these phenotypes are connected to bacterial redox homeostasis. Therefore, the results of this study suggest a role of PhoPR in M. bovis to be linked to the mechanisms that mycobacteria display to maintain their redox balance.

Study of the in vivo role of Mce2R, the transcriptional regulator of mce2 operon in Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the agent of human tuberculosis, has developed strategies involving proteins and other compounds called virulence factors to subvert human host defences and damage and invade the human host. Among these virulence-related proteins are the Mce proteins, which are encoded in the mce1, mce2, mce3 and mce4 operons of M. tuberculosis. The expression of the mce2 operon is negatively regulated by the Mce2R transcriptional repressor. Here we evaluated the role of Mce2R during the infection of M. tuberculosis in mice and macrophages and defined the genes whose expression is in vitro regulated by this transcriptional repressor. RESULTS: We used a specialized transduction method for generating a mce2R mutant of M. tuberculosis H37Rv. Although we found equivalent replication of the MtΔmce2R mutant and the wild type strains in mouse lungs, overexpression of Mce2R in the complemented strain (MtΔmce2RComp) significantly impaired its replication. During in vitro infection of macrophages, we observed a significantly increased association of the late endosomal marker LAMP-2 to MtΔmce2RComp-containing phagosomes as compared to MtΔmce2R and the wild type strains. Whole transcriptional analysis showed that Mce2R regulates mainly the expression of the mce2 operon, in the in vitro conditions studied. CONCLUSIONS: The findings of the current study indicate that Mce2R weakly represses the in vivo expression of the mce2 operon in the studied conditions and argue for a role of the proteins encoded in Mce2R regulon in the arrest of phagosome maturation induced by M. tuberculosis.

Differential transcriptome profiles of attenuated and hypervirulent strains of Mycobacterium bovis.

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.