A Mycobacterium tuberculosis Effector Targets Mitochondrion, Controls Energy Metabolism, and Limits Cytochrome c Exit.

Host metabolism reprogramming is a key feature of Mycobacterium tuberculosis (Mtb) infection that enables the survival of this pathogen within phagocytic cells and modulates the immune response facilitating the spread of the tuberculosis disease. Here, we demonstrate that a previously uncharacterized secreted protein from Mtb, Rv1813c, manipulates the host metabolism by targeting mitochondria. When expressed in eukaryotic cells, the protein is delivered to the mitochondrial intermembrane space and promotes the enhancement of host ATP production by boosting the oxidative phosphorylation metabolic pathway. Furthermore, the release of cytochrome c from mitochondria, an early apoptotic event in response to short-term oxidative stress, is delayed in Rv1813c-expressing cells. This study reveals a novel class of mitochondria targeting effectors from Mtb that might participate in host cell metabolic reprogramming and apoptosis control during Mtb infections. IMPORTANCE In this article, using a combination of techniques (bioinformatics, structural biology, and cell biology), we identified and characterized a new class of effectors present only in intracellular mycobacteria. These proteins specifically target host cell mitochondria when ectopically expressed in cells. We showed that one member of this family (Rv1813c) affects mitochondria metabolism in a way that might twist the immune response. This effector also inhibits the cytochrome c exit from mitochondria, suggesting that it might alter normal host cell apoptotic capacities, one of the first defenses of immune cells against Mtb infection.

A Persistent Tuberculosis Outbreak in the UK Is Characterized by Hydrophobic fadB4-Deficient Mycobacterium tuberculosis That Replicates Rapidly in Macrophages.

The genetic diversity of Mycobacterium tuberculosis can influence disease severity and transmissibility. To better understand how this diversity influences individuals and communities, we phenotyped M. tuberculosis that was causing a persistent outbreak in the East Midlands, United Kingdom. Compared to nonoutbreak isolates, bacilli had higher lipid contents and more hydrophobic cell surfaces. In macrophage infection models, the bacteria increased more rapidly, provoked the enhanced accumulation of macrophage lipid droplets and enhanced the secretion of IL-1ß. Natural deletions in fadB4, nrdB, and plcC distinguished the outbreak isolates from other lineage 3 isolates in the region. fadB4 is annotated with a putative role in cell envelope biosynthesis, so the loss of this gene has the potential to alter the interactions of bacteria with immune cells. Reintroduction of fadB4 to the outbreak strain led to a phenotype that more closely resembled those of nonoutbreak strains. The improved understanding of the microbiological characteristics and the corresponding genetic polymorphisms that associate with outbreaks have the potential to inform tuberculosis control. IMPORTANCE Tuberculosis (TB) killed 1.5 million people in 2020 and affects every country. The extent to which the natural genetic diversity of Mycobacterium tuberculosis influences disease manifestation at both the individual and epidemiological levels remains poorly understood. Insights into how pathogen polymorphisms affect patterns of TB have the potential to translate into clinical and public health practice. Two distinct lineage 3 strains isolated from local TB outbreaks, one of which (CH) was rapidly terminated and the other of which (Lro) persistently transmitted for over a decade, provided us with an opportunity to study these issues. We compared genome sequences, microbiological characteristics, and early immune responses that were evoked upon infection. Our results indicate that the natural lack of fadB4 in the Lro strain contributes to its unique features.

Dimethyl fumarate eliminates differentially culturable Mycobacterium tuberculosis in an intranasal murine model of tuberculosis.

Tuberculosis (TB) claims nearly 1.5 million lives annually. Current TB treatment requires a combination of several drugs administered for at least 6 months. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can persist in infected humans and animals for decades. Moreover, during infection, Mtb produces differentially culturable bacteria (DCB) that do not grow in standard media but can be resuscitated in liquid media supplemented with sterile Mtb culture filtrates or recombinant resuscitation-promoting factors (Rpfs). Here, we demonstrate that, in an intranasal murine model of TB, Mtb DCB are detectable in the lungs after 4 weeks of infection, and their loads remain largely unchanged during a further 8 weeks. Treatment of the infected mice with dimethyl fumarate (DMF), a known drug with immunomodulatory properties, for 8 weeks eliminates Mtb DCB from the lungs and spleens. Standard TB treatment consisting of rifampicin, isoniazid, and pyrazinamide for 8 weeks reduces Mtb loads by nearly four orders of magnitude but does not eradicate DCB. Nevertheless, no DCB can be detected in the lungs and spleens after 8 weeks of treatment with DMF, rifampicin, isoniazid, and pyrazinamide. Our data suggest that addition of approved anti-inflammatory drugs to standard treatment regimens may improve TB treatment and reduce treatment duration.

Virtual Screening Identifies Novel and Potent Inhibitors of Mycobacterium tuberculosis PknB with Antibacterial Activity.

Mycobacterium tuberculosis protein kinase B (PknB) is essential to mycobacterial growth and has received considerable attention as an attractive target for novel anti-tuberculosis drug development. Here, virtual screening, validated by biological assays, was applied to select candidate inhibitors of M. tuberculosis PknB from the Specs compound library (www.specs.net). Fifteen compounds were identified as hits and selected for in vitro biological assays, of which three indoles (2, AE-848/42799159; 4, AH-262/34335013; 10, AP-124/40904362) inhibited growth of M. tuberculosis H37Rv with minimal inhibitory concentrations of 6.2, 12.5, and 6.2 µg/mL, respectively. Two compounds, 2 and 10, inhibited M. tuberculosis PknB activity in vitro, with IC50 values of 14.4 and 12.1 µM, respectively, suggesting this to be the likely basis of their anti-tubercular activity. In contrast, compound 4 displayed anti-tuberculosis activity against M. tuberculosis H37Rv but showed no inhibition of PknB activity (IC50 > 128 µM). We hypothesize that hydrolysis of its ethyl ester to a carboxylate moiety generates an active species that inhibits other M. tuberculosis enzymes. Molecular dynamics simulations of modeled complexes of compounds 2, 4, and 10 bound to M. tuberculosis PknB indicated that compound 4 has a lower affinity for M. tuberculosis PknB than compounds 2 and 10, as evidenced by higher calculated binding free energies, consistent with experiment. Compounds 2 and 10 therefore represent candidate inhibitors of M. tuberculosis PknB that provide attractive starting templates for optimization as anti-tubercular agents.

Culture-Free Enumeration of Mycobacterium tuberculosis in Mouse Tissues Using the Molecular Bacterial Load Assay for Preclinical Drug Development.

BACKGROUND: The turnaround times for phenotypic tests used to monitor the bacterial load of Mycobacterium tuberculosis, in both clinical and preclinical studies, are delayed by the organism's slow growth in culture media. The existence of differentially culturable populations of M.tuberculosis may result in an underestimate of the true number. Moreover, culture methods are susceptible to contamination resulting in loss of critical data points. OBJECTIVES: We report the adaptation of our robust, culture-free assay utilising 16S ribosomal RNA, developed for sputum, to enumerate the number of bacteria present in animal tissues as a tool to improve the read-outs in preclinical drug efficacy studies. METHODS: Initial assay adaptation was performed using naïve mouse lungs spiked with known quantities of M. tuberculosis and an internal RNA control. Tissues were homogenised, total RNA extracted, and enumeration performed using RT-qPCR. We then evaluated the utility of the assay, in comparison to bacterial counts estimated using growth assays on solid and liquid media, to accurately inform bacterial load in tissues from M. tuberculosis-infected mice before and during treatment with a panel of drug combinations. RESULTS: When tested on lung tissues derived from infected mice, the MBL assay produced comparable results to the bacterial counts in solid culture (colony forming units: CFU). Notably, under specific drug treatments, the MBL assay was able to detect a significantly higher number of M. tuberculosis compared to CFU, likely indicating the presence of bacteria that were unable to produce colonies in solid-based culture. Additionally, growth recovery in liquid media using the most probable number (MPN) assay was able to account for the discrepancy between the MBL assay and CFU number, suggesting that the MBL assay detects differentially culturable sub-populations of M. tuberculosis. CONCLUSIONS: The MBL assay can enumerate the bacterial load in animal tissues in real time without the need to wait for extended periods for cultures to grow. The readout correlates well with CFUs. Importantly, we have shown that the MBL is able to measure specific populations of bacteria not cultured on solid agar. The adaptation of this assay for preclinical studies has the potential to decrease the readout time of data acquisition from animal experiments and could represent a valuable tool for tuberculosis drug discovery and development.

Generation of Distinct Differentially Culturable Forms of Burkholderia following Starvation at Low Temperature.

Bacteria have developed unique mechanisms to adapt to environmental stresses and challenges of the immune system. Here, we report that Burkholderia pseudomallei, the causative agent of melioidosis, and its laboratory surrogate, Burkholderia thailandensis, utilize distinct mechanisms for surviving starvation at different incubation temperatures. At 21°C, Burkholderia are present as short rods which can rapidly reactivate and form colonies on solid media. At 4°C, Burkholderia convert into coccoid forms that cannot be cultured on solid agar but can be resuscitated in liquid media supplemented with supernatant obtained from logarithmic phase cultures of B. thailandensis, or catalase and Tween 80, thus displaying characteristics of differentially culturable bacteria (DCB). These DCB have low intensity fluorescence when stained with SYTO 9, have an intact cell membrane (propidium iodide negative), and contain 16S rRNA at levels comparable with growing cells. We also present evidence that lytic transglycosylases, a family of peptidoglycan-remodeling enzymes, are involved in the generation of coccoid forms and their resuscitation to actively growing cells. A B. pseudomallei ΔltgGCFD mutant with four ltg genes deleted did not produce coccoid forms at 4°C and could not be resuscitated in the liquid media evaluated. Our findings provide insights into the adaptation of Burkholderia to nutrient limitation and the generation of differentially culturable bacteria. IMPORTANCE Bacterial pathogens exhibit physiologically distinct forms that enable their survival in an infected host, the environment and following exposure to antimicrobial agents. B. pseudomallei causes the disease melioidosis, which has a high mortality rate and is difficult to treat with antibiotics. The bacterium is endemic to several countries and detected in high abundance in the environment. Here, we report that during starvation at low temperature, B. pseudomallei produces coccoid forms that cannot grow in standard media and which, therefore, can be challenging to detect using common tools. We provide evidence that the formation of these cocci is mediated by cell wall-specialized enzymes and lytic transglycosylases, and that resuscitation of these forms occurs following the addition of catalase and Tween 80. Our findings have important implications for the disease control and detection of B. pseudomallei, an agent of both public health and defense interest.

Balance between Protection and Pathogenic Response to Aerosol Challenge with Mycobacterium tuberculosis (Mtb) in Mice Vaccinated with TriFu64, a Fusion Consisting of Three Mtb Antigens.

Tuberculosis vaccines capable of reducing disease worldwide have proven difficult to develop. BCG is effective in limiting childhood disease, but adult TB is still a major public health issue. Development of new vaccines requires identification of antigens that are both spatially and temporally available throughout infection, and immune responses to which reduce bacterial burden without increasing pathologic outcomes. Subunit vaccines containing antigen require adjuvants to drive appropriate long-lived responses. We generated a triple-antigen fusion containing the virulence-associated EsxN (Rv1793), the PPE42 (Rv2608), and the latency associated Rv2628 to investigate the balance between bacterial reduction and weight loss in an animal model of aerosol infection. We found that in both a low pattern recognition receptor (PRR) engaging adjuvant and a high PRR-engaging adjuvant (MPL/TDM/DDA) the triple-antigen fusion could reduce the bacterial burden, but also induced weight loss in the mice upon aerosol infection. The weight loss was associated with an imbalance between TNFα and IL-17 transcription in the lung upon challenge. These data indicate the need to assess both protective and pathogenic responses when investigating subunit vaccine activity.

A Virulence Associated Siderophore Importer Reduces Antimicrobial Susceptibility of Klebsiella pneumoniae.

The accessory genomes of many pathogenic bacteria include ABC transporters that scavenge metal by siderophore uptake and ABC transporters that contribute to antimicrobial resistance by multidrug efflux. There are mechanistic and recently recognized structural similarities between siderophore importer proteins and efflux pumps. Here we investigated the influence of siderophore importer YbtPQ on antimicrobial resistance of Klebsiella pneumoniae. YbtPQ is encoded in the yersiniabactin cluster in a prevalent mobile genetic element ICEKp, and is also common in pathogenicity islands of Escherichia coli and Yersinia species, where yersiniabactin enhances virulence. Deletion of ICEKp increased the susceptibility of K. pneumoniae to all antimicrobials tested. The mechanism was dependent on the yersiniabactin importer YbtPQ and may involve antimicrobial efflux, since it was affected by the inhibitor reserpine. The element ICEKp is naturally highly mobile, indeed the accessory genome of K. pneumoniae is recognized as a reservoir of genes for the emergence of hospital outbreak strains and for transfer to other Gram-negative pathogens. Introduction of ICEKp, or a plasmid encoding YbtPQ, to E. coli decreased its susceptibility to a broad range of antimicrobials. Thus a confirmed siderophore importer, on a rapidly evolving and highly mobile element capable of interspecies transfer, may have a secondary function exporting antimicrobials.

Coupling of Peptidoglycan Synthesis to Central Metabolism in Mycobacteria: Post-transcriptional Control of CwlM by Aconitase.

Mycobacterium tuberculosis causes human tuberculosis, and a better understanding of its biology is required to identify vulnerabilities that might be exploited in developing new therapeutics. The iron-sulfur cluster of the essential M. tuberculosis central metabolic enzyme, aconitase (AcnA), disassembles when exposed to oxidative/nitrosative stress or iron chelators. The catalytically inactive apo-AcnA interacts with a sequence resembling an iron-responsive element (IRE) located within the transcript of another essential protein, CwlM, a regulator of peptidoglycan synthesis. A Mycobacterium smegmatis cwlM conditional mutant complemented with M. tuberculosis cwlM with a disrupted IRE is unable to recover from combinations of oxidative, nitrosative, and iron starvation stresses. An equivalent M. tuberculosis cwlM conditional mutant complemented with the cwlM gene lacking a functional IRE exhibits a growth defect in THP-1 macrophages. It appears that AcnA acts to couple peptidoglycan synthesis and central metabolism, and disruption of this coupling potentially leaves mycobacteria vulnerable to attack by macrophages.

Difference in persistent tuberculosis bacteria between in vitro and sputum from patients: implications for translational predictions.

This study aimed to investigate the number of persistent bacteria in sputum from tuberculosis patients compared to in vitro and to suggest a model-based approach for accounting for the potential difference. Sputum smear positive patients (n = 25) provided sputum samples prior to onset of chemotherapy. The number of cells detected by conventional agar colony forming unit (CFU) and most probable number (MPN) with Rpf supplementation were quantified. Persistent bacteria was assumed to be the difference between MPNrpf and CFU. The difference in persistent bacteria between in vitro and human sputum prior to chemotherapy was quantified using different model-based approaches. The persistent bacteria in sputum was 17% of the in vitro levels, suggesting a difference in phenotypic resistance, whereas no difference was found for multiplying bacterial subpopulations. Clinical trial simulations showed that the predicted time to 2 log fall in MPNrpf in a Phase 2a setting using in vitro pre-clinical efficacy information, would be almost 3 days longer if drug response was predicted ignoring the difference in phenotypic resistance. The discovered phenotypic differences between in vitro and humans prior to chemotherapy could have implications on translational efforts but can be accounted for using a model-based approach for translating in vitro to human drug response.

Zebrafish Embryo Model for Assessment of Drug Efficacy on Mycobacterial Persisters.

Tuberculosis continues to kill millions of people each year. The main difficulty in eradication of the disease is the prolonged duration of treatment, which takes at least 6 months. Persister cells have long been associated with failed treatment and disease relapse because of their phenotypical, though transient, tolerance to drugs. By targeting these persisters, the duration of treatment could be shortened, leading to improved tuberculosis treatment and a reduction in transmission. The unique in vivo environment drives the generation of persisters; however, appropriate in vivo mycobacterial persister models enabling optimized drug screening are lacking. To set up a persister infection model that is suitable for this, we infected zebrafish embryos with in vitro-starved Mycobacterium marinumIn vitro starvation resulted in a persister-like phenotype with the accumulation of stored neutral lipids and concomitant increased tolerance to ethambutol. However, these starved wild-type M. marinum organisms rapidly lost their persister phenotype in vivo To prolong the persister phenotype in vivo, we subsequently generated and analyzed mutants lacking functional resuscitation-promoting factors (Rpfs). Interestingly, the ΔrpfAB mutant, lacking two Rpfs, established an infection in vivo, whereas a nutrient-starved ΔrpfAB mutant did maintain its persister phenotype in vivo This mutant was, after nutrient starvation, also tolerant to ethambutol treatment in vivo, as would be expected for persisters. We propose that this zebrafish embryo model with ΔrpfAB mutant bacteria is a valuable addition for drug screening purposes and specifically screens to target mycobacterial persisters.

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112.

Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H-NS-like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H-NS, Lsr2 binds DNA in sequence-dependent and non-specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA-binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP-sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.

The lytic transglycosylase, LtgG, controls cell morphology and virulence in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of the tropical disease melioidosis. Its genome encodes an arsenal of virulence factors that allow it, when required, to switch from a soil dwelling bacterium to a deadly intracellular pathogen. With a high intrinsic resistance to antibiotics and the ability to overcome challenges from the host immune system, there is an increasing requirement for new antibiotics and a greater understanding into the molecular mechanisms of B. pseudomallei virulence and dormancy. The peptidoglycan remodeling enzymes, lytic transglycosylases (Ltgs) are potential targets for such new antibiotics. Ltgs cleave the glycosidic bonds within bacterial peptidoglycan allowing for the insertion of peptidoglycan precursors during cell growth and division, and cell membrane spanning structures such as flagella and secretion systems. Using bioinformatic analysis we have identified 8 putative Ltgs in B. pseudomallei K96243. We aimed to investigate one of these Ltgs, LtgG (BPSL3046) through the generation of deletion mutants and biochemical analysis. We have shown that LtgG is a key contributor to cellular morphology, division, motility and virulence in BALB/c mice. We have determined the crystal structure of LtgG and have identified various amino acids likely to be important in peptidoglycan binding and catalytic activity. Recombinant protein assays and complementation studies using LtgG containing a site directed mutation in aspartate 343, confirmed the essentiality of this amino acid in the function of LtgG.

Efficient Protein Digestion at Elevated Temperature in the Presence of Sodium Dodecyl Sulfate and Calcium Ions for Membrane Proteomics.

The growing significance of membrane proteins inspires continuous development and improvement of methods for robust membrane proteomics. Here, we developed a very simple and efficient method for membrane protein digestion using an ionic detergent, sodium dodecyl sulfate (SDS), at high temperature, conditions where trypsin is normally inactivated. Our results suggest that trypsin can be stabilized by a combination of calcium ions and sodium chloride, which enables protein digestion at elevated temperature in the presence of strong ionic detergents such as SDS. Finding the conditions for stabilization of trypsin offers novel opportunities for the application of detergents for the investigation of membrane proteins.

The hydrolase LpqI primes mycobacterial peptidoglycan recycling.

Growth and division by most bacteria requires remodelling and cleavage of their cell wall. A byproduct of this process is the generation of free peptidoglycan (PG) fragments known as muropeptides, which are recycled in many model organisms. Bacteria and hosts can harness the unique nature of muropeptides as a signal for cell wall damage and infection, respectively. Despite this critical role for muropeptides, it has long been thought that pathogenic mycobacteria such as Mycobacterium tuberculosis do not recycle their PG. Herein we show that M. tuberculosis and Mycobacterium bovis BCG are able to recycle components of their PG. We demonstrate that the core mycobacterial gene lpqI, encodes an authentic NagZ ß-N-acetylglucosaminidase and that it is essential for PG-derived amino sugar recycling via an unusual pathway. Together these data provide a critical first step in understanding how mycobacteria recycle their peptidoglycan.

Mycobacterial phosphatase PstP regulates global serine threonine phosphorylation and cell division.

Protein phosphatase PstP is conserved throughout the Actinobacteria in a genetic locus related to cell wall synthesis and cell division. In many Actinobacteria it is the sole annotated serine threonine protein phosphatase to counter the activity of multiple serine threonine protein kinases. We used transcriptional knockdown, electron microscopy and comparative phosphoproteomics to investigate the putative dual functions of PstP as a specific regulator of cell division and as a global regulator of protein phosphorylation. Comparative phosphoproteomics in the early stages of PstP depletion showed hyperphosphorylation of protein kinases and their substrates, confirming PstP as a negative regulator of kinase activity and global serine and threonine phosphorylation. Analysis of the 838 phosphorylation sites that changed significantly, suggested that PstP may regulate diverse phosphoproteins, preferentially at phosphothreonine near acidic residues, near the protein termini, and within membrane associated proteins. Increased phosphorylation of the activation loop of protein kinase B (PknB) and of the essential PknB substrate CwlM offer possible explanations for the requirement for pstP for growth and for cell wall defects when PstP was depleted.

Two Faces of CwlM, an Essential PknB Substrate, in Mycobacterium tuberculosis.

Tuberculosis claims >1 million lives annually, and its causative agent Mycobacterium tuberculosis is a highly successful pathogen. Protein kinase B (PknB) is reported to be critical for mycobacterial growth. Here, we demonstrate that PknB-depleted M. tuberculosis can replicate normally and can synthesize peptidoglycan in an osmoprotective medium. Comparative phosphoproteomics of PknB-producing and PknB-depleted mycobacteria identify CwlM, an essential regulator of peptidoglycan synthesis, as a major PknB substrate. Our complementation studies of a cwlM mutant of M. tuberculosis support CwlM phosphorylation as a likely molecular basis for PknB being essential for mycobacterial growth. We demonstrate that growing mycobacteria produce two forms of CwlM: a non-phosphorylated membrane-associated form and a PknB-phosphorylated cytoplasmic form. Furthermore, we show that the partner proteins for the phosphorylated and non-phosphorylated forms of CwlM are FhaA, a fork head-associated domain protein, and MurJ, a proposed lipid II flippase, respectively. From our results, we propose a model in which CwlM potentially regulates both the biosynthesis of peptidoglycan precursors and their transport across the cytoplasmic membrane.

A nested case-control study of predictors for tuberculosis recurrence in a large UK Centre.

BACKGROUND: Tuberculosis (TB) recurrence represents a challenge to control programs. In low incidence countries, the prevailing risk factors leading to recurrence are poorly characterised. METHODS: We conducted a nested case-control study using the Leicester TB service TBIT database. Cases were identified from database notifications between 1994 and 2014. Controls had one episode and were matched to cases on a ratio of two to one by the date of notification. Multiple imputation was used to account for missing data. Multivariate conditional logistic regression analysis was employed to identify clinical, sociodemographic and TB specific risk factors for recurrence. RESULTS: From a cohort of 4628 patients, 82 TB recurrences occurred (1.8%). Nineteen of 82 patients had paired isolates with MIRU-VNTR strain type profiles available, of which 84% were relapses and 16% reinfections. On multivariate analysis, smoking (OR 3.8; p = 0.04), grade 3/4 adverse drug reactions (OR 5.6; p = 0.02), ethnicity 'Indian subcontinent' (OR 8.5; p = <0.01), ethnicity 'other' (OR 31.2; p = 0.01) and receipt of immunosuppressants (OR 6.8; p = <0.01) were independent predictors of TB recurrence. CONCLUSIONS: Within this UK setting, the rate of TB recurrence was low, predominantly due to relapse. The identification of an elevated recurrence risk amongst the ethnic group contributing most cases to the national TB burden presents an opportunity to improve individual and population health.

Cmr is a redox-responsive regulator of DosR that contributes to M. tuberculosis virulence.

Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colonizes the human lung, often entering a non-replicating state before progressing to life-threatening active infections. Transcriptional reprogramming is essential for TB pathogenesis. In vitro, Cmr (a member of the CRP/FNR super-family of transcription regulators) bound at a single DNA site to act as a dual regulator of cmr transcription and an activator of the divergent rv1676 gene. Transcriptional profiling and DNA-binding assays suggested that Cmr directly represses dosR expression. The DosR regulon is thought to be involved in establishing latent tuberculosis infections in response to hypoxia and nitric oxide. Accordingly, DNA-binding by Cmr was severely impaired by nitrosation. A cmr mutant was better able to survive a nitrosative stress challenge but was attenuated in a mouse aerosol infection model. The complemented mutant exhibited a ∼2-fold increase in cmr expression, which led to increased sensitivity to nitrosative stress. This, and the inability to restore wild-type behaviour in the infection model, suggests that precise regulation of the cmr locus, which is associated with Region of Difference 150 in hypervirulent Beijing strains of Mtb, is important for TB pathogenesis.