Genetic Diversity in Mycobacterium tuberculosis Clinical Isolates and Resulting Outcomes of Tuberculosis Infection and Disease.

Tuberculosis claims more human lives than any other bacterial infectious disease and represents a clear and present danger to global health as new tools for vaccination, treatment, and interruption of transmission have been slow to emerge. Additionally, tuberculosis presents with notable clinical heterogeneity, which complicates diagnosis, treatment, and the establishment of nonrelapsing cure. How this heterogeneity is driven by the diversity ofclinical isolates of the causative agent, Mycobacterium tuberculosis, has recently garnered attention. Herein, we review advances in the understanding of how naturally occurring variation in clinical isolates affects transmissibility, pathogenesis, immune modulation, and drug resistance. We also summarize how specific changes in transcriptional responses can modulate infection or disease outcome, together with strain-specific effects on gene essentiality. Further understanding of how this diversity of M. tuberculosis isolates affects disease and treatment outcomes will enable the development of more effective therapeutic options and vaccines for this dreaded disease.

Inhibiting the Evolution of Antibiotic Resistance.

Efforts to battle antimicrobial resistance (AMR) are generally focused on developing novel antibiotics. However, history shows that resistance arises regardless of the nature or potency of new drugs. Here, we propose and provide evidence for an alternate strategy to resolve this problem: inhibiting evolution. We determined that the DNA translocase Mfd is an "evolvability factor" that promotes mutagenesis and is required for rapid resistance development to all antibiotics tested across highly divergent bacterial species. Importantly, hypermutator alleles that accelerate AMR development did not arise without Mfd, at least during evolution of trimethoprim resistance. We also show that Mfd's role in AMR development depends on its interactions with the RNA polymerase subunit RpoB and the nucleotide excision repair protein UvrA. Our findings suggest that AMR development can be inhibited through inactivation of evolvability factors (potentially with "anti-evolution" drugs)-in particular, Mfd-providing an unexplored route toward battling the AMR crisis.

The human proton pump inhibitors inhibit Mycobacterium tuberculosis rifampicin efflux and macrophage-induced rifampicin tolerance.

Tuberculosis treatment requires months-long combination chemotherapy with multiple drugs, with shorter treatments leading to relapses. A major impediment to shortening treatment is that Mycobacterium tuberculosis becomes tolerant to the administered drugs, starting early after infection and within days of infecting macrophages. Multiple lines of evidence suggest that macrophage-induced drug tolerance is mediated by mycobacterial drug efflux pumps. Here, using assays to directly measure drug efflux, we find that M. tuberculosis transports the first-line antitubercular drug rifampicin through a proton gradient-dependent mechanism. We show that verapamil, a known efflux pump inhibitor, which inhibits macrophage-induced rifampicin tolerance, also inhibits M.tuberculosis rifampicin efflux. As with macrophage-induced tolerance, the calcium channel-inhibiting property of verapamil is not required for its inhibition of rifampicin efflux. By testing verapamil analogs, we show that verapamil directly inhibits M. tuberculosis drug efflux pumps through its human P-glycoprotein (PGP)-like inhibitory activity. Screening commonly used drugs with incidental PGP inhibitory activity, we find many inhibit rifampicin efflux, including the proton pump inhibitors (PPIs) such as omeprazole. Like verapamil, the PPIs inhibit macrophage-induced rifampicin tolerance as well as intramacrophage growth, which has also been linked to mycobacterial efflux pump activity. Our assays provide a facile screening platform for M. tuberculosis efflux pump inhibitors that inhibit in vivo drug tolerance and growth.

Phase variation in Mycobacterium tuberculosis glpK produces transiently heritable drug tolerance.

The length and complexity of tuberculosis (TB) therapy, as well as the propensity of Mycobacterium tuberculosis to develop drug resistance, are major barriers to global TB control efforts. M. tuberculosis is known to have the ability to enter into a drug-tolerant state, which may explain many of these impediments to TB treatment. We have identified a mechanism of genetically encoded but rapidly reversible drug tolerance in M. tuberculosis caused by transient frameshift mutations in a homopolymeric tract (HT) of 7 cytosines (7C) in the glpK gene. Inactivating frameshift mutations associated with the 7C HT in glpK produce small colonies that exhibit heritable multidrug increases in minimal inhibitory concentrations and decreases in drug-dependent killing; however, reversion back to a fully drug-susceptible large-colony phenotype occurs rapidly through the introduction of additional insertions or deletions in the same glpK HT region. These reversible frameshift mutations in the 7C HT of M. tuberculosis glpK occur in clinical isolates, accumulate in M. tuberculosis-infected mice with further accumulation during drug treatment, and exhibit a reversible transcriptional profile including induction of dosR and sigH and repression of kstR regulons, similar to that observed in other in vitro models of M. tuberculosis tolerance. These results suggest that GlpK phase variation may contribute to drug tolerance, treatment failure, and relapse in human TB. Drugs effective against phase-variant M. tuberculosis may hasten TB treatment and improve cure rates.

Bacterial Factors That Predict Relapse after Tuberculosis Therapy.

BACKGROUND: Approximately 5% of patients with drug-susceptible tuberculosis have a relapse after 6 months of first-line therapy, as do approximately 20% of patients after 4 months of short-course therapy. We postulated that by analyzing pretreatment isolates of Mycobacterium tuberculosis obtained from patients who subsequently had a relapse or were cured, we could determine any correlations between the minimum inhibitory concentration (MIC) of a drug below the standard resistance breakpoint and the relapse risk after treatment. METHODS: Using data from the Tuberculosis Trials Consortium Study 22 (development cohort), we assessed relapse and cure isolates to determine the MIC values of isoniazid and rifampin that were below the standard resistance breakpoint (0.1 µg per milliliter for isoniazid and 1.0 µg per milliliter for rifampin). We combined this analysis with clinical, radiologic, and laboratory data to generate predictive relapse models, which we validated by analyzing data from the DMID 01-009 study (validation cohort). RESULTS: In the development cohort, the mean (±SD) MIC of isoniazid below the breakpoint was 0.0334±0.0085 µg per milliliter in the relapse group and 0.0286±0.0092 µg per milliliter in the cure group, which represented a higher value in the relapse group by a factor of 1.17 (P=0.02). The corresponding MIC values of rifampin were 0.0695±0.0276 and 0.0453±0.0223 µg per milliliter, respectively, which represented a higher value in the relapse group by a factor of 1.53 (P<0.001). Higher MIC values remained associated with relapse in a multivariable analysis that included other significant between-group differences. In an analysis of receiver-operating-characteristic curves of relapse based on these MIC values, the area under the curve (AUC) was 0.779. In the development cohort, the AUC in a multivariable model that included MIC values was 0.875. In the validation cohort, the MIC values either alone or combined with other patient characteristics were also predictive of relapse, with AUC values of 0.964 and 0.929, respectively. The use of a model score for the MIC values of isoniazid and rifampin to achieve 75.0% sensitivity in cross-validation analysis predicted relapse with a specificity of 76.5% in the development cohort and a sensitivity of 70.0% and a specificity of 100% in the validation cohort. CONCLUSIONS: In pretreatment isolates of M. tuberculosis with decrements of MIC values of isoniazid or rifampin below standard resistance breakpoints, higher MIC values were associated with a greater risk of relapse than lower MIC values. (Funded by the National Institute of Allergy and Infectious Diseases.).

Nicotinamide Limits Replication of Mycobacterium tuberculosis and Bacille Calmette-Guérin Within Macrophages.

Novel antimicrobials for treatment of Mycobacterium tuberculosis are needed. We hypothesized that nicotinamide (NAM) and nicotinic acid (NA) modulate macrophage function to restrict M. tuberculosis replication in addition to their direct antimicrobial properties. Both compounds had modest activity in 7H9 broth, but only NAM inhibited replication in macrophages. Surprisingly, in macrophages NAM and the related compound pyrazinamide restricted growth of bacille Calmette-Guérin but not wild-type Mycobacterium bovis, which both lack a functional nicotinamidase/pyrazinamidase (PncA) rendering each strain resistant to these drugs in broth culture. Interestingly, NAM was not active in macrophages infected with a virulent M. tuberculosis mutant encoding a deletion in pncA. We conclude that the differential activity of NAM and nicotinic acid on infected macrophages suggests host-specific NAM targets rather than PncA-dependent direct antimicrobial properties. These activities are sufficient to restrict attenuated BCG, but not virulent wild-type M. bovis or M. tuberculosis.

Rapidly Correcting Frameshift Mutations in the Mycobacterium tuberculosis orn Gene Produce Reversible Ethambutol Resistance and Small-Colony-Variant Morphology.

We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosisorn gene. A frameshift mutation in orn produces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis However, the same orn mutation in a low-level EMB-resistant double embB-aftA mutant (MIC = 8 µg/ml) produces an SCV with an EMB MIC of 32 µg/ml. Reversible resistance is indistinguishable from a drug-persistent phenotype, because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size, and reversing the EMB MIC back to that (8 µg/ml) of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to that in the parental embB-aftA mutant, whereas large-colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to that of the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug resistance in human tuberculosis.

Rapid, direct detection of bacterial topoisomerase 1-DNA adducts by RADAR/ELISA.

Topoisomerases are proven drug targets, but antibiotics that poison bacterial Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and direct assay for quantification of Top1-DNA adducts that is suitable for high throughput assays. Adducts are recovered by "RADAR fractionation", a quick, convenient approach in which cells are lysed in chaotropic salts and detergent and nucleic acids and covalently bound adducts then precipitated with alcohol. Here we show that RADAR fractionation followed by ELISA immunodetection can quantify adducts formed by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens, Y. pestis and M. tuberculosis, expressed in E. coli or M. smegmatis, respectively. For both enzymes, quantification of adducts by RADAR/ELISA produces results comparable to the more cumbersome classical approach of CsCl density gradient fractionation. The experiments reported here establish that RADAR/ELISA assay offers a simple way to characterize Top1 mutants and analyze kinetics of adduct formation and repair. They also provide a foundation for discovery and optimization of drugs that poison bacterial Top1 using standard high-throughput approaches.

Cell envelope stress in mycobacteria is regulated by the novel signal transduction ATPase IniR in response to trehalose.

The cell envelope of mycobacteria is a highly unique and complex structure that is functionally equivalent to that of Gram-negative bacteria to protect the bacterial cell. Defects in the integrity or assembly of this cell envelope must be sensed to allow the induction of stress response systems. The promoter that is specifically and most strongly induced upon exposure to ethambutol and isoniazid, first line drugs that affect cell envelope biogenesis, is the iniBAC promoter. In this study, we set out to identify the regulator of the iniBAC operon in Mycobacterium marinum using an unbiased transposon mutagenesis screen in a constitutively iniBAC-expressing mutant background. We obtained multiple mutants in the mce1 locus as well as mutants in an uncharacterized putative transcriptional regulator (MMAR_0612). This latter gene was shown to function as the iniBAC regulator, as overexpression resulted in constitutive iniBAC induction, whereas a knockout mutant was unable to respond to the presence of ethambutol and isoniazid. Experiments with the M. tuberculosis homologue (Rv0339c) showed identical results. RNAseq experiments showed that this regulatory gene was exclusively involved in the regulation of the iniBAC operon. We therefore propose to name this dedicated regulator iniBAC Regulator (IniR). IniR belongs to the family of signal transduction ATPases with numerous domains, including a putative sugar-binding domain. Upon testing different sugars, we identified trehalose as an activator and metabolic cue for iniBAC activation, which could also explain the effect of the mce1 mutations. In conclusion, cell envelope stress in mycobacteria is regulated by IniR in a cascade that includes trehalose.

Incipient and Subclinical Tuberculosis: a Clinical Review of Early Stages and Progression of Infection.

Tuberculosis (TB) is the leading infectious cause of mortality worldwide, due in part to a limited understanding of its clinical pathogenic spectrum of infection and disease. Historically, scientific research, diagnostic testing, and drug treatment have focused on addressing one of two disease states: latent TB infection or active TB disease. Recent research has clearly demonstrated that human TB infection, from latent infection to active disease, exists within a continuous spectrum of metabolic bacterial activity and antagonistic immunological responses. This revised understanding leads us to propose two additional clinical states: incipient and subclinical TB. The recognition of incipient and subclinical TB, which helps divide latent and active TB along the clinical disease spectrum, provides opportunities for the development of diagnostic and therapeutic interventions to prevent progression to active TB disease and transmission of TB bacilli. In this report, we review the current understanding of the pathogenesis, immunology, clinical epidemiology, diagnosis, treatment, and prevention of both incipient and subclinical TB, two emerging clinical states of an ancient bacterium.

Diverse Clinical Isolates of Mycobacterium tuberculosis Develop Macrophage-Induced Rifampin Tolerance.

The Mycobacterium tuberculosis lineage 4 strains CDC1551 and H37Rv develop tolerance to multiple antibiotics upon macrophage residence. To determine whether macrophage-induced tolerance is a general feature of clinical M. tuberculosis isolates, we assessed macrophage-induced drug tolerance in strains from lineages 1-3, representing the other predominant M. tuberculosis strains responsible for tuberculosis globally. All 3 lineages developed isoniazid tolerance. While lineage 1, 3, and 4 strains developed rifampin tolerance, lineage 2 Beijing strains did not. Their failure to develop tolerance may be explained by their harboring of a loss-of-function mutation in the Rv1258c efflux pump that is linked to macrophage-induced rifampicin tolerance.

The Mycobacterium tuberculosis regulatory network and hypoxia.

We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub.

Mycobacterium tuberculosis Ser/Thr protein kinase B mediates an oxygen-dependent replication switch.

The majority of Mycobacterium tuberculosis (Mtb) infections are clinically latent, characterized by drug tolerance and little or no bacterial replication. Low oxygen tension is a major host factor inducing bacteriostasis, but the molecular mechanisms driving oxygen-dependent replication are poorly understood. Here, we tested the role of serine/threonine phosphorylation in the Mtb response to altered oxygen status, using an in vitro model of latency (hypoxia) and reactivation (reaeration). Broad kinase inhibition compromised survival of Mtb in reaeration. Activity-based protein profiling and genetic mutation identified PknB as the kinase critical for surviving hypoxia. Mtb replication was highly sensitive to changes in PknB levels in aerated culture, and even more so in hypoxia. A mutant overexpressing PknB specifically in hypoxia showed a 10-fold loss in viability and gross morphological defects in low oxygen conditions. In contrast, chemically reducing PknB activity during hypoxia specifically compromised resumption of growth during reaeration. These data support a model in which PknB activity is reduced to achieve bacteriostasis, and elevated when replication resumes. Together, these data show that phosphosignaling controls replicative transitions associated with latency and reactivation, that PknB is a major regulator of these transitions, and that PknB could provide a highly vulnerable therapeutic target at every step of the Mtb life cycle-active disease, latency, and reactivation.

Mycobacterium tuberculosis supports protein tyrosine phosphorylation.

Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb. Here, using a combination of phospho-enrichment and highly sensitive mass spectrometry, we show extensive protein Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several STPKs function as dual-specificity kinases that phosphorylate Tyr in cis and in trans, suggesting that dual-specificity kinases have a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine site of the essential STPK PknB reduces its activity in vitro and in live Mtb, indicating that Tyr phosphorylation has a functional role in bacterial growth. These data identify a previously unrecognized phosphorylation system in a human pathogen that claims ∼ 1.4 million lives every year.

Integrated Modeling of Gene Regulatory and Metabolic Networks in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) is the causative bacterium of tuberculosis, a disease responsible for over a million deaths worldwide annually with a growing number of strains resistant to antibiotics. The development of better therapeutics would greatly benefit from improved understanding of the mechanisms associated with MTB responses to different genetic and environmental perturbations. Therefore, we expanded a genome-scale regulatory-metabolic model for MTB using the Probabilistic Regulation of Metabolism (PROM) framework. Our model, MTBPROM2.0, represents a substantial knowledge base update and extension of simulation capability. We incorporated a recent ChIP-seq based binding network of 2555 interactions linking to 104 transcription factors (TFs) (representing a 3.5-fold expansion of TF coverage). We integrated this expanded regulatory network with a refined genome-scale metabolic model that can correctly predict growth viability over 69 source metabolite conditions and predict metabolic gene essentiality more accurately than the original model. We used MTBPROM2.0 to simulate the metabolic consequences of knocking out and overexpressing each of the 104 TFs in the model. MTBPROM2.0 improves performance of knockout growth defect predictions compared to the original PROM MTB model, and it can successfully predict growth defects associated with TF overexpression. Moreover, condition-specific models of MTBPROM2.0 successfully predicted synergistic growth consequences of overexpressing the TF whiB4 in the presence of two standard anti-TB drugs. MTBPROM2.0 can screen in silico condition-specific transcription factor perturbations to generate putative targets of interest that can help prioritize future experiments for therapeutic development efforts.

The DosR Regulon Modulates Adaptive Immunity and Is Essential for Mycobacterium tuberculosis Persistence.

RATIONALE: Hypoxia promotes dormancy by causing physiologic changes to actively replicating Mycobacterium tuberculosis. DosR controls the response of M. tuberculosis to hypoxia. OBJECTIVES: To understand DosR's contribution in the persistence of M. tuberculosis, we compared the phenotype of various DosR regulon mutants and a complemented strain to M. tuberculosis in macaques, which faithfully model M. tuberculosis infection. METHODS: We measured clinical and microbiologic correlates of infection with M. tuberculosis relative to mutant/complemented strains in the DosR regulon, studied lung pathology and hypoxia, and compared immune responses in lung using transcriptomics and flow cytometry. MEASUREMENTS AND MAIN RESULTS: Despite being able to replicate initially, mutants in DosR regulon failed to persist or cause disease. On the contrary, M. tuberculosis and a complemented strain were able to establish infection and tuberculosis. The attenuation of pathogenesis in animals infected with the mutants coincided with the appearance of a Th1 response and organization of hypoxic lesions wherein M. tuberculosis expressed dosR. The lungs of animals infected with the mutants (but not the complemented strain) exhibited early transcriptional signatures of T-cell recruitment, activation, and proliferation associated with an increase of T cells expressing homing and proliferation markers. CONCLUSIONS: Delayed adaptive responses, a hallmark of M. tuberculosis infection, not only lead to persistence but also interfere with the development of effective antituberculosis vaccines. The DosR regulon therefore modulates both the magnitude and the timing of adaptive immune responses in response to hypoxia in vivo, resulting in persistent infection. Hence, DosR regulates key aspects of the M. tuberculosis life cycle and limits lung pathology.

A high-resolution network model for global gene regulation in Mycobacterium tuberculosis.

The resilience of Mycobacterium tuberculosis (MTB) is largely due to its ability to effectively counteract and even take advantage of the hostile environments of a host. In order to accelerate the discovery and characterization of these adaptive mechanisms, we have mined a compendium of 2325 publicly available transcriptome profiles of MTB to decipher a predictive, systems-scale gene regulatory network model. The resulting modular organization of 98% of all MTB genes within this regulatory network was rigorously tested using two independently generated datasets: a genome-wide map of 7248 DNA-binding locations for 143 transcription factors (TFs) and global transcriptional consequences of overexpressing 206 TFs. This analysis has discovered specific TFs that mediate conditional co-regulation of genes within 240 modules across 14 distinct environmental contexts. In addition to recapitulating previously characterized regulons, we discovered 454 novel mechanisms for gene regulation during stress, cholesterol utilization and dormancy. Significantly, 183 of these mechanisms act uniquely under conditions experienced during the infection cycle to regulate diverse functions including 23 genes that are essential to host-pathogen interactions. These and other insights underscore the power of a rational, model-driven approach to unearth novel MTB biology that operates under some but not all phases of infection.

Osmosensory signaling in Mycobacterium tuberculosis mediated by a eukaryotic-like Ser/Thr protein kinase.

Bacteria are able to adapt to dramatically different microenvironments, but in many organisms, the signaling pathways, transcriptional programs, and downstream physiological changes involved in adaptation are not well-understood. Here, we discovered that osmotic stress stimulates a signaling network in Mycobacterium tuberculosis regulated by the eukaryotic-like receptor Ser/Thr protein kinase PknD. Expression of the PknD substrate Rv0516c was highly induced by osmotic stress. Furthermore, Rv0516c disruption modified peptidoglycan thickness, enhanced antibiotic resistance, and activated genes in the regulon of the alternative σ-factor SigF. Phosphorylation of Rv0516c regulated the abundance of EspA, a virulence-associated substrate of the type VII ESX-1 secretion system. These findings identify an osmosensory pathway orchestrated by PknD, Rv0516c, and SigF that enables adaptation to osmotic stress through cell wall remodeling and virulence factor production. Given the widespread occurrence of eukaryotic-like Ser/Thr protein kinases in bacteria, these proteins may play a broad role in bacterial osmosensing.