Multidrug-resistant tuberculosis (MDR-TB), caused by drug resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. In this study, we examined a dataset of 5,310 M. tuberculosis whole genome sequences from five continents. Despite great diversity with respect to geographic point of isolation, genetic background and drug resistance, patterns of drug resistance emergence were conserved globally. We have identified harbinger mutations that often precede MDR. In particular, the katG S315T mutation, conferring resistance to isoniazid, overwhelmingly arose before rifampicin resistance across all lineages, geographic regions, and time periods. Molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of pre-MDR polymorphisms, particularly katG S315, into molecular diagnostics will enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.
Biomarkers for tuberculosis treatment outcome will assist in guiding individualized treatment and evaluation of new therapies. To identify candidate biomarkers, RNA sequencing of whole blood from a well-characterized TB treatment cohort was performed. Application of a validated transcriptional correlate of risk for TB revealed symmetry in host gene expression during progression from latent TB infection to active TB disease and resolution of disease during treatment, including return to control levels after drug therapy. The symmetry was also seen in a TB disease signature, constructed from the TB treatment cohort, that also functioned as a strong correlate of risk. Both signatures identified patients at risk of treatment failure 1–4 weeks after start of therapy. Further mining of the transcriptomes revealed an association between treatment failure and suppressed expression of mitochondrial genes before treatment initiation, leading to development of a novel baseline (pre-treatment) signature of treatment failure. These novel host responses to TB treatment were integrated into a five-gene real-time PCR-based signature that captures the clinically relevant responses to TB treatment and provides a convenient platform for stratifying patients according to their risk of treatment failure. Furthermore, this 5-gene signature is shown to correlate with the pulmonary inflammatory state (as measured by PET-CT) and can complement sputum-based Gene Xpert for patient stratification, providing a rapid and accurate alternative to current methods.
The viability of Mycobacterium tuberculosis (Mtb) depends on energy generated by its respiratory chain. Cytochrome bc1-aa3 oxidase and type-2 NADH dehydrogenase (NDH-2) are respiratory chain components predicted to be essential, and are currently targeted for drug development. Here we demonstrate that an Mtb cytochrome bc1-aa3 oxidase deletion mutant is viable and only partially attenuated in mice. Moreover, treatment of Mtb-infected marmosets with a cytochrome bc1-aa3 oxidase inhibitor controls disease progression and reduces lesion-associated inflammation, but most lesions become cavitary. Deletion of both NDH-2 encoding genes (Δndh-2 mutant) reveals that the essentiality of NDH-2 as shown in standard growth media is due to the presence of fatty acids. The Δndh-2 mutant is only mildly attenuated in mice and not differently susceptible to clofazimine, a drug in clinical use proposed to engage NDH-2. These results demonstrate the intrinsic plasticity of Mtb’s respiratory chain, and highlight the challenges associated with targeting the pathogen’s respiratory enzymes for tuberculosis drug development.
The systems participating in detoxification of reactive oxygen intermediates in Mycobacterium tuberculosis are believed to play a dual role in the biology of this highly adapted human pathogen: (i) they may contribute to the survival of this bacterium in the host; and (ii) alterations in the gene encoding catalase/peroxidase have been linked to this organism's resistance to the front-line antituberculosis drug isoniazid. These relationships prompted us to extend investigations of the oxidative-stress-response systems in M. tuberculosis by analysing the alkyl hydroperoxide reductase gene ahpC and its putative regulator oxyR. Surprisingly, the oxyR gene was found to be inactivated by multiple lesions in M. tuberculosis H37Rv. These alterations were observed in all M. tuberculosis strains tested, and in members of the M. tuberculosis complex: Mycobacterium bovis BCG, Mycobacterium africanum, and Mycobacterium microti. The corresponding region carrying these genes in Mycobacterium leprae, an organism not sensitive to isoniazid, has a complete oxyR gene divergently transcribed from ahpC. An increase in minimal inhibitory concentration for isoniazid was observed upon transformation of M. tuberculosis H37Rv with cosmids carrying the oxyR-ahpC region of M. leprae. In keeping with the observed inactivation of oxyR, transcriptional activity of the corresponding region in M. tuberculosis was an order of magnitude lower than that of the oxyR gene from M. leprae. While the loss of this putative regulator of oxidative-stress response in M. tuberculosis is paradoxical considering the fact that survival in host macrophages is regarded as a critical feature of this pathogen, it offers a partial explanation for the exquisite sensitivity of M. tuberculosis to isoniazid.
The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co-factors are necessary, and fluorescence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shuttle-plasmid vector carrying gfp cDNA was constructed and used to generate transcriptional fusions with promoters of interest and to examine their expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters studied were: (i) ahpC from Mycoosis and Mycobacterium leprae, a gene encoding alkyl hydroperoxide reductase which, along with the divergently transcribed regulator oxyR, are homologues of corresponding stress-response systems in enteric bacteria and play a role in isoniazid sensitivity; (ii) mtrA, an M. tuberculosis response regulator belonging to the superfamily of bacterial two-component signal-transduction systems; (iii) hsp60, a previously characterized heat-shock gene from M. bovis; and (iv) tbprc3, a newly isolated promoter from M. tuberculosis. Expression of these promoters in mycobacteria was analysed using epifluorescence microscopy, laser scanning confocal microscopy, fluorescence spectroscopy, and flow cytometry. These approaches permitted assessment of fluorescence prior to and after macrophage infection, and analyses of promoter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence-activated cell sorting from cells harbouring the vector used to construct the fusion. In addition, the stable expression of mtrA-gfp fusion in M. bovis BCG facilitated localization and isolation of phagocytic vesicles containing mycobacteria. The experiments presented here suggest that GFP will be a useful tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophages.
Treatment of MDR tuberculosis involves substantial risk of acquired resistance to SLDs, increasing as baseline drug resistance increases. The risk was significantly lower in programs documented by the GLC to meet specific standards.
Protection against Mycobacterium tuberculosis (Mtb) infection requires CD4 T cells to migrate into the lung and interact with infected macrophages. In mice, less-differentiated CXCR3+ CD4 T cells migrate into the lung and suppress growth of Mtb, while CX3CR1+ terminally-differentiated Th1 cells accumulate in the blood vasculature and do not control pulmonary infection. Here we examine CD4 T cell differentiation and lung homing during primary Mtb infection of rhesus macaques. Mtb-specific CD4 T cells simultaneously appeared in the airways and blood ~21–28 days post-exposure, indicating that recently primed effectors are quickly recruited into the lungs after entering circulation. Mtb-specific CD4 T cells in granulomas display a tissue-parenchymal CXCR3+CX3CR1−PD-1hiCTLA-4+ phenotype. However, most granuloma CD4 T cells are found within the outer lymphocyte cuff, and few localize to the myeloid cell core containing the bacilli. Using the intravascular stain approach, we find essentially all Mtb-specific CD4 T cells in granulomas have extravasated across the vascular endothelium into the parenchyma. Therefore, it is unlikely that lung-homing defects introduced by terminal differentiation limit the migration of CD4 T cells into granulomas following primary Mtb infection of macaques. However, intralesional positioning defects within the granuloma may pose a major barrier to T cell-mediated immunity during tuberculosis.
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 ...
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