Mycobacterium tuberculosis and the closely related organism Mycobacterium bovis can survive and replicate inside macrophages. Intracellular survival is at least in part attributed to the failure of mycobacterial phagosomes to undergo fusion with lysosomes. The transformation of phagosomes into phagolysosomes involves gradual acquisition of markers from the endosomal compartment. Members of the rab family of small GTPases which confer fusion competence in the endocytic pathway are exchanged sequentially onto the phagosomal membranes in the course of their maturation. To identify the step at which the fusion capability of phagosomes containing mycobacteria is compromised, we purified green fluorescent protein-labeled M. bovis BCG phagosomal compartments (MPC) and compared GTPbinding protein profiles of these vesicles with latex bead phagosomal compartments (LBC). We report that the MPC do not acquire rab7, specific for late endosomes, even 7 days postinfection, whereas this GTP-binding protein is present on the LBC within hours after phagocytosis. By contrast, rab5 is retained and enriched with time on the MPC, suggesting fusion competence with an early endosomal compartment. Prior infection of macrophages with M. bovis BCG also affected the dynamics of rab5 and rab7 acquisition by subsequently formed LBC. Selective exclusion of rab7, coupled with the retention of rab5 on the mycobacterial phagosome, may allow organisms from the M. tuberculosis complex to avert the usual physiological destination of phagocytosed material.

A putative two-component system, mtrA-mtrB, was isolated from M. tuberculosis H37Rv by using phoB from Pseudomonas aeruginosa as a hybridization probe. The predicted gene product of mtrA displayed high similarity with typical response regulators, including AfsQ1, PhoB, PhoP, and OmpR. The predicted gene product of mtrB displayed similarities with the histidine protein kinases AfsQ2, PhoR, and EnvZ and other members of this class of proteins. Expression analysis in the T7 system showed that mtrA encoded a polypeptide with an apparent molecular mass of 30 kDa. MtrA was overproduced, purified, and demonstrated to participate in typical phosphotransfer reactions using a heterologous histidine protein kinase, CheA, as a phosphoryl group donor. Mycobacterium bovis BCG, harboring an mtrA-gfp (green fluorescent protein cDNA) transcriptional fusion, was used to monitor mtrA expression in infected J774 monolayers. Flow cytometric and fluorescence microscopic analyses indicated that the mtrA promoter was activated upon entry and incubation in J774 macrophages. In contrast, the hsp60-gfp fusion displayed no change in expression under the growth conditions tested. These results suggest a potential role for mtrA in adaptation of the M. tuberculosis complex organisms to environmental changes which may include intracellular conditions. Mycobacterium tuberculosis is notorious for both its slow growth and global importance as a human pathogen (4). Although there is a relatively safe vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG), against tuberculosis, its effectiveness appears to be variable (29, 32). Efforts to develop new vaccines and therapeutic approaches are hampered by the paucity of information regarding potential virulence factors of M. tuberculosis (31). Improved knowledge and identification of genes and functions critical for host-pathogen interactions that render this organism such a successful human pathogen are needed in order to refine the existing treatments or provide novel targets for intervention. In this context, and in the area of M. tuberculosis fundamental biology, the perception of this bacterium as an inert participant in pathogenic processes continues to be challenged by the latest advances in molecular genetic analyses. However, in some instances the views of M. tuberculosis as a static organism have received affirmation. For example, it has been recently reported that at least in one case, M. tuberculosis has a nonfunctional regulator of oxidative stress, oxyR (11, 35), whose homologs in other organisms are critical to their adjustments to endogenous and exogenous reactive oxygen intermediates (13). The oxyR gene is inactivated by multiple lesions in all strains of M. tuberculosis tested and in all members of the M. tuberculosis complex, representing a phenomenon that has been linked to the exquisite sensitivity of this organism to isonicotinic acid hydrazide (11). This finding appears to support the notion of preset expression levels for at least some subsets of genes in M. tuberculosis.In ...