A "suicide" BCG strain provides enhanced immunogenicity and robust protection against Mycobacterium tuberculosis in macaques.

Intravenous (IV) BCG delivery provides robust protection against Mycobacterium tuberculosis (Mtb) in macaques but poses safety challenges. Here, we constructed two BCG strains (BCG-TetON-DL and BCG-TetOFF-DL) in which tetracyclines regulate two phage lysin operons. Once the lysins are expressed, these strains are cleared in immunocompetent and immunocompromised mice, yet induced similar immune responses and provided similar protection against Mtb challenge as wild type BCG. Lysin induction resulted in release of intracellular BCG antigens and enhanced cytokine production by macrophages. In macaques, cessation of doxycycline administration resulted in rapid elimination of BCG-TetOFF-DL. However, IV BCG-TetOFF-DL induced increased pulmonary CD4 T cell responses compared to WT BCG and provided robust protection against Mtb challenge, with sterilizing immunity in 6 of 8 macaques, compared to 2 of 8 macaques immunized with WT BCG. Thus, a "suicide" BCG strain provides an additional measure of safety when delivered intravenously and robust protection against Mtb infection.

Synthetic lethality of Mycobacterium tuberculosis NADH dehydrogenases is due to impaired NADH oxidation.

IMPORTANCE: In 2022, it was estimated that 10.6 million people fell ill, and 1.6 million people died from tuberculosis (TB). Available treatment is lengthy and requires a multi-drug regimen, which calls for new strategies to cure Mycobacterium tuberculosis (Mtb) infections more efficiently. We have previously shown that simultaneous inactivation of type 1 (Ndh-1) and type 2 (Ndh-2) NADH dehydrogenases kills Mtb. NADH dehydrogenases play two main physiological roles: NADH oxidation and electron entry into the respiratory chain. Here, we show that this bactericidal effect is a consequence of impaired NADH oxidation. Importantly, we demonstrate that Ndh-1/Ndh-2 synthetic lethality can be achieved through simultaneous chemical inhibition, which could be exploited by TB drug development programs.

Development of an Engineered Mycobacterium tuberculosis Strain for a Safe and Effective Tuberculosis Human Challenge Model.

Human challenge experiments could greatly accelerate the development of a tuberculosis (TB) vaccine. Human challenge for tuberculosis requires a strain that can both replicate in the host and be reliably cleared. To accomplish this, we designed Mycobacterium tuberculosis (Mtb) strains featuring up to three orthogonal kill switches, tightly regulated by exogenous tetracyclines and trimethoprim. The resultant strains displayed immunogenicity and antibiotic susceptibility similar to wild-type Mtb under permissive conditions. In the absence of supplementary exogenous compounds, the strains were rapidly killed in axenic culture, mice and nonhuman primates. Notably, the strain that contained three kill switches had an escape rate of less than 10 -10 per genome per generation and displayed no relapse in a SCID mouse model. Collectively, these findings suggest that this engineered Mtb strain could be a safe and effective candidate for a human challenge model.

Differentiating the roles of Mycobacterium tuberculosis substrate binding proteins, FecB and FecB2, in iron uptake.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, mechanisms through which Mtb acquires host iron represent attractive therapeutic targets. Mtb scavenges host iron via Mtb siderophore-dependent and heme iron uptake pathways. While multiple studies describe the import of heme and ferric-siderophores and the export of apo-siderophores across the inner membrane, little is known about their transport across the periplasm and cell-wall environments. Mtb FecB and FecB2 are predicted periplasmic binding proteins implicated in host iron acquisition; however, their precise roles are not well understood. This study sought to differentiate the roles FecB and FecB2 play in Mtb iron acquisition. The crystallographic structures of Mtb FecB and FecB2 were determined to 2.0 Å and 2.2 Å resolution, respectively, and show distinct ligand binding pockets. In vitro ligand binding experiments for FecB and FecB2 were performed with heme and bacterial siderophores from Mtb and other species, revealing that both FecB and FecB2 bind heme, while only FecB binds the Mtb sideophore ferric-carboxymycobactin (Fe-cMB). Subsequent structure-guided mutagenesis of FecB identified a single glutamate residue-Glu339-that significantly contributes to Fe-cMB binding. A role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis and Mtb pull-down assays, which revealed interactions between FecB and members of the mycobacterial siderophore export and import machinery. Similarly, pull-down assays with FecB2 confirms its role in heme uptake revealing interactions with a potential inner membrane heme importer. Due to ligand preference and protein partners, our data suggest that Mtb FecB plays a role in siderophore-dependent iron and heme acquisition pathways; in addition, we confirm that Mtb FecB2 is involved in heme uptake.

Metabolically distinct roles of NAD synthetase and NAD kinase define the essentiality of NAD and NADP in Mycobacterium tuberculosis.

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated derivative (NADP) are essential cofactors that participate in hundreds of biochemical reactions and have emerged as therapeutic targets in cancer, metabolic disorders, neurodegenerative diseases, and infections, including tuberculosis. The biological basis for the essentiality of NAD(P) in most settings, however, remains experimentally unexplained. Here, we report that inactivation of the terminal enzyme of NAD synthesis, NAD synthetase (NadE), elicits markedly different metabolic and microbiologic effects than those of the terminal enzyme of NADP biosynthesis, NAD kinase (PpnK), in Mycobacterium tuberculosis (Mtb). Inactivation of NadE led to parallel reductions of both NAD and NADP pools and Mtb viability, while inactivation of PpnK selectively depleted NADP pools but only arrested growth. Inactivation of each enzyme was accompanied by metabolic changes that were specific for the affected enzyme and associated microbiological phenotype. Bacteriostatic levels of NAD depletion caused a compensatory remodeling of NAD-dependent metabolic pathways in the absence of an impact on NADH/NAD ratios, while bactericidal levels of NAD depletion resulted in a disruption of NADH/NAD ratios and inhibition of oxygen respiration. These findings reveal a previously unrecognized physiologic specificity associated with the essentiality of two evolutionarily ubiquitous cofactors. IMPORTANCE The current course for cure of Mycobacterium tuberculosis (Mtb)-the etiologic agent of tuberculosis (TB)-infections is lengthy and requires multiple antibiotics. The development of shorter, simpler treatment regimens is, therefore, critical to the goal of eradicating TB. NadE, an enzyme required for the synthesis of the ubiquitous cofactor NAD, is essential for survival of Mtb and regarded as a promising drug target. However, the basis of this essentiality was not clear due to its role in the synthesis of both NAD and NADP. Here, we resolve this ambiguity through a combination of gene silencing and metabolomics. We specifically show that NADP deficiency is bacteriostatic, while NAD deficiency is bactericidal due to its role in Mtb's respiratory capacity. These results argue for a prioritization of NAD biosynthesis inhibitors in anti-TB drug development.

Synthetic lethality of Mycobacterium tuberculosis NADH dehydrogenases is due to impaired NADH oxidation.

Type 2 NADH dehydrogenase (Ndh-2) is an oxidative phosphorylation enzyme discussed as a promising drug target in different pathogens, including Plasmodium falciparum and Mycobacterium tuberculosis (Mtb). To kill Mtb, Ndh-2 needs to be inactivated together with the alternative enzyme type 1 NADH dehydrogenase (Ndh-1), but the mechanism of this synthetic lethality remained unknown. Here, we provide insights into the biology of NADH dehydrogenases and a mechanistic explanation for Ndh-1 and Ndh-2 synthetic lethality in Mtb. NADH dehydrogenases have two main functions: maintaining an appropriate NADH/NAD+ ratio by converting NADH into NAD+ and providing electrons to the respiratory chain. Heterologous expression of a water forming NADH oxidase (Nox), which catalyzes the oxidation of NADH, allows to distinguish between these two functions and show that Nox rescues Mtb from Ndh-1/Ndh-2 synthetic lethality, indicating that NADH oxidation is the essential function of NADH dehydrogenases for Mtb viability. Quantification of intracellular levels of NADH, NAD, ATP, and oxygen consumption revealed that preventing NADH oxidation by Ndh-2 depletes NAD(H) and inhibits respiration. Finally, we show that Ndh-1/ Ndh-2 synthetic lethality can be achieved through chemical inhibition.

Cyclic AMP is a critical mediator of intrinsic drug resistance and fatty acid metabolism in M. tuberculosis.

Cyclic AMP (cAMP) is a ubiquitous second messenger that transduces signals from cellular receptors to downstream effectors. Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, devotes a considerable amount of coding capacity to produce, sense, and degrade cAMP. Despite this fact, our understanding of how cAMP regulates Mtb physiology remains limited. Here, we took a genetic approach to investigate the function of the sole essential adenylate cyclase in Mtb H37Rv, Rv3645. We found that a lack of rv3645 resulted in increased sensitivity to numerous antibiotics by a mechanism independent of substantial increases in envelope permeability. We made the unexpected observation that rv3645 is conditionally essential for Mtb growth only in the presence of long-chain fatty acids, a host-relevant carbon source. A suppressor screen further identified mutations in the atypical cAMP phosphodiesterase rv1339 that suppress both fatty acid and drug sensitivity phenotypes in strains lacking rv3645. Using mass spectrometry, we found that Rv3645 is the dominant source of cAMP under standard laboratory growth conditions, that cAMP production is the essential function of Rv3645 in the presence of long-chain fatty acids, and that reduced cAMP levels result in increased long-chain fatty acid uptake and metabolism and increased antibiotic susceptibility. Our work defines rv3645 and cAMP as central mediators of intrinsic multidrug resistance and fatty acid metabolism in Mtb and highlights the potential utility of small molecule modulators of cAMP signaling.

A periplasmic cinched protein is required for siderophore secretion and virulence of Mycobacterium tuberculosis.

Iron is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis. To acquire iron from the host, M. tuberculosis uses the siderophores called mycobactins and carboxymycobactins. Here, we show that the rv0455c gene is essential for M. tuberculosis to grow in low-iron medium and that secretion of both mycobactins and carboxymycobactins is drastically reduced in the rv0455c deletion mutant. Both water-soluble and membrane-anchored Rv0455c are functional in siderophore secretion, supporting an intracellular role. Lack of Rv0455c results in siderophore toxicity, a phenotype observed for other siderophore secretion mutants, and severely impairs replication of M. tuberculosis in mice, demonstrating the importance of Rv0455c and siderophore secretion during disease. The crystal structure of a Rv0455c homolog reveals a novel protein fold consisting of a helical bundle with a 'cinch' formed by an essential intramolecular disulfide bond. These findings advance our understanding of the distinct M. tuberculosis siderophore secretion system.

CinA mediates multidrug tolerance in Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis (Mtb) to resist and tolerate antibiotics complicates the development of improved tuberculosis (TB) chemotherapies. Here we define the Mtb protein CinA as a major determinant of drug tolerance and as a potential target to shorten TB chemotherapy. By reducing the fraction of drug-tolerant persisters, genetic inactivation of cinA accelerated killing of Mtb by four antibiotics in clinical use: isoniazid, ethionamide, delamanid and pretomanid. Mtb ΔcinA was killed rapidly in conditions known to impede the efficacy of isoniazid, such as during nutrient starvation, during persistence in a caseum mimetic, in activated macrophages and during chronic mouse infection. Deletion of CinA also increased in vivo killing of Mtb by BPaL, a combination of pretomanid, bedaquiline and linezolid that is used to treat highly drug-resistant TB. Genetic and drug metabolism studies suggest that CinA mediates drug tolerance via cleavage of NAD-drug adducts.

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy.

Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug­drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilized conditional Mtb knockdown mutants of essential genes as an experimentally tractable surrogate for drug treatment and probe the relationship between Mtb carbon metabolism and chemical­genetic interactions (CGIs). We examined the antitubercular drugs isoniazid, rifampicin, and moxifloxacin and found that CGIs are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivo­relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGIs could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of chemical­genetic­environmental interactions that can be used to optimize drug­drug interactions, as well as provide a framework for understanding in vitro correlates of in vivo efficacy.

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice.

The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits can be associated with genetic variants distributed across the mouse genome, making the CC a unique population for identifying specific host-pathogen genetic interactions that influence pathogenesis.

Multiple acyl-CoA dehydrogenase deficiency kills Mycobacterium tuberculosis in vitro and during infection.

The human pathogen Mycobacterium tuberculosis depends on host fatty acids as a carbon source. However, fatty acid ß-oxidation is mediated by redundant enzymes, which hampers the development of antitubercular drugs targeting this pathway. Here, we show that rv0338c, which we refer to as etfD, encodes a membrane oxidoreductase essential for ß-oxidation in M. tuberculosis. An etfD deletion mutant is incapable of growing on fatty acids or cholesterol, with long-chain fatty acids being bactericidal, and fails to grow and survive in mice. Analysis of the mutant's metabolome reveals a block in ß-oxidation at the step catalyzed by acyl-CoA dehydrogenases (ACADs), which in other organisms are functionally dependent on an electron transfer flavoprotein (ETF) and its cognate oxidoreductase. We use immunoprecipitation to show that M. tuberculosis EtfD interacts with FixA (EtfB), a protein that is homologous to the human ETF subunit ß and is encoded in an operon with fixB, encoding a homologue of human ETF subunit α. We thus refer to FixA and FixB as EtfB and EtfA, respectively. Our results indicate that EtfBA and EtfD (which is not homologous to human EtfD) function as the ETF and oxidoreductase for ß-oxidation in M. tuberculosis and support this pathway as a potential target for tuberculosis drug development.

Multiform antimicrobial resistance from a metabolic mutation.

A critical challenge for microbiology and medicine is how to cure infections by bacteria that survive antibiotic treatment by persistence or tolerance. Seeking mechanisms behind such high survival, we developed a forward-genetic method for efficient isolation of high-survival mutants in any culturable bacterial species. We found that perturbation of an essential biosynthetic pathway (arginine biosynthesis) in a mycobacterium generated three distinct forms of resistance to diverse antibiotics, each mediated by induction of WhiB7: high persistence and tolerance to kanamycin, high survival upon exposure to rifampicin, and minimum inhibitory concentration-shifted resistance to clarithromycin. As little as one base change in a gene that encodes, a metabolic pathway component conferred multiple forms of resistance to multiple antibiotics with different targets. This extraordinary resilience may help explain how substerilizing exposure to one antibiotic in a regimen can induce resistance to others and invites development of drugs targeting the mediator of multiform resistance, WhiB7.

Growth of Mycobacterium tuberculosis at acidic pH depends on lipid assimilation and is accompanied by reduced GAPDH activity.

Acidic pH arrests the growth of Mycobacterium tuberculosis in vitro (pH < 5.8) and is thought to significantly contribute to the ability of macrophages to control M. tuberculosis replication. However, this pathogen has been shown to survive and even slowly replicate within macrophage phagolysosomes (pH 4.5 to 5) [M. S. Gomes et al., Infect. Immun. 67, 3199-3206 (1999)] [S. Levitte et al., Cell Host Microbe 20, 250-258 (2016)]. Here, we demonstrate that M. tuberculosis can grow at acidic pH, as low as pH 4.5, in the presence of host-relevant lipids. We show that lack of phosphoenolpyruvate carboxykinase and isocitrate lyase, two enzymes necessary for lipid assimilation, is cidal to M. tuberculosis in the presence of oleic acid at acidic pH. Metabolomic analysis revealed that M. tuberculosis responds to acidic pH by altering its metabolism to preferentially assimilate lipids such as oleic acid over carbohydrates such as glycerol. We show that the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is impaired in acid-exposed M. tuberculosis likely contributing to a reduction in glycolytic flux. The generation of endogenous reactive oxygen species at acidic pH is consistent with the inhibition of GAPDH, an enzyme well-known to be sensitive to oxidation. This work shows that M. tuberculosis alters its carbon diet in response to pH and provides a greater understanding of the physiology of this pathogen during acid stress.

Genome-wide gene expression tuning reveals diverse vulnerabilities of M. tuberculosis.

Antibacterial agents target the products of essential genes but rarely achieve complete target inhibition. Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene "vulnerability" is a continuous, quantifiable trait that relates the magnitude of gene inhibition to the effect on bacterial fitness. We developed a CRISPR interference-based functional genomics method to systematically titrate gene expression in Mycobacterium tuberculosis (Mtb) and monitor fitness outcomes. We identified highly vulnerable genes in various processes, including novel targets unexplored for drug discovery. Equally important, we identified invulnerable essential genes, potentially explaining failed drug discovery efforts. Comparison of vulnerability between the reference and a hypervirulent Mtb isolate revealed incomplete conservation of vulnerability and that differential vulnerability can predict differential antibacterial susceptibility. Our results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.

Genetic models of latent tuberculosis in mice reveal differential influence of adaptive immunity.

Studying latent Mycobacterium tuberculosis (Mtb) infection has been limited by the lack of a suitable mouse model. We discovered that transient depletion of biotin protein ligase (BPL) and thioredoxin reductase (TrxB2) results in latent infections during which Mtb cannot be detected but that relapse in a subset of mice. The immune requirements for Mtb control during latency, and the frequency of relapse, were strikingly different depending on how latency was established. TrxB2 depletion resulted in a latent infection that required adaptive immunity for control and reactivated with high frequency, whereas latent infection after BPL depletion was independent of adaptive immunity and rarely reactivated. We identified immune signatures of T cells indicative of relapse and demonstrated that BCG vaccination failed to protect mice from TB relapse. These reproducible genetic latency models allow investigation of the host immunological determinants that control the latent state and offer opportunities to evaluate therapeutic strategies in settings that mimic aspects of latency and TB relapse in humans.

Metabolic bifunctionality of Rv0812 couples folate and peptidoglycan biosynthesis in Mycobacterium tuberculosis.

Comparative sequence analysis has enabled the annotation of millions of genes from organisms across the evolutionary tree. However, this approach has inherently biased the annotation of phylogenetically ubiquitous, rather than species-specific, functions. The ecologically unusual pathogen Mycobacterium tuberculosis (Mtb) has evolved in humans as its sole reservoir and emerged as the leading bacterial cause of death worldwide. However, the physiological factors that define Mtb's pathogenicity are poorly understood. Here, we report the structure and function of a protein that is required for optimal in vitro fitness and bears homology to two distinct enzymes, Rv0812. Despite diversification of related orthologues into biochemically distinct enzyme families, rv0812 encodes a single active site with aminodeoxychorismate lyase and D-amino acid transaminase activities. The mutual exclusivity of substrate occupancy in this active site mediates coupling between nucleic acid and cell wall biosynthesis, prioritizing PABA over D-Ala/D-Glu biosynthesis. This bifunctionality reveals a novel, enzymatically encoded fail-safe mechanism that may help Mtb and other bacteria couple replication and division.

Rv0954 Is a Member of the Mycobacterial Cell Division Complex.

Proper control of cell division in the intracellular pathogen Mycobacterium tuberculosis is central to its growth, survival, pathogenesis, and resistance to antibiotics. Nevertheless, the divisome components and mechanisms by which mycobacteria regulate their cell cycle are not entirely understood. Here we demonstrate that the previously uncharacterized Rv0954 protein localizes to the mid-cell during cell division and interacts with the division-related proteins LamA, PbpA, and PknH. Deletion of rv0954 did not result in alterations in cell morphology or sensitivity to cell wall-targeting antibiotics but transposon mutagenesis demonstrated genetic interactions with genes related to cell division. This work suggests that Rv0954 participates in cell division and reveals potential components of the mycobacterial divisome for future investigation.

Nonredundant functions of Mycobacterium tuberculosis chaperones promote survival under stress.

Bacterial chaperones ClpB and DnaK, homologs of the respective eukaryotic heat shock proteins Hsp104 and Hsp70, are essential in the reactivation of toxic protein aggregates that occur during translation or periods of stress. In the pathogen Mycobacterium tuberculosis (Mtb), the protective effect of chaperones extends to survival in the presence of host stresses, such as protein-damaging oxidants. However, we lack a full understanding of the interplay of Hsps and other stress response genes in mycobacteria. Here, we employ genome-wide transposon mutagenesis to identify the genes that support clpB function in Mtb. In addition to validating the role of ClpB in Mtb's response to oxidants, we show that HtpG, a homolog of Hsp90, plays a distinct role from ClpB in the proteotoxic stress response. While loss of neither clpB nor htpG is lethal to the cell, loss of both through genetic depletion or small molecule inhibition impairs recovery after exposure to host-like stresses, especially reactive nitrogen species. Moreover, defects in cells lacking clpB can be complemented by overexpression of other chaperones, demonstrating that Mtb's stress response network depends upon finely tuned chaperone expression levels. These results suggest that inhibition of multiple chaperones could work in concert with host immunity to disable Mtb.