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Gene rv3722c of Mycobacterium tuberculosis is essential for in vitro growth, and encodes a putative pyridoxal phosphate-binding protein of unknown function. Here we use metabolomic, genetic and structural approaches to show that Rv3722c is the primary aspartate aminotransferase of M . tuberculosis , and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Rv3722c deficiency leads to virulence attenuation in macrophages and mice. Our results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in M . tuberculosis . Gene rv3722c is essential for in vitro growth of Mycobacterium tuberculosis , but its function is unclear. Here, Jansen et al. show that Rv3722c is the primary aspartate aminotransferase of this pathogen, mediates nitrogen distribution, and is important for virulence during infection of macrophages and mice.

Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5′-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.

Bacille Calmette-Guerin (BCG), an attenuated whole cell vaccine based on Mycobacterium bovis , is the only licensed vaccine against Mycobacterium tuberculosis ( Mtb ), but its efficacy is suboptimal and it fails to protect against pulmonary tuberculosis. We previously reported that Mtb lacking the virulence genes lprG and rv1410c ( ΔLprG) was highly attenuated in immune deficient mice. In this study, we show that attenuated ΔLprG Mtb protects C57BL/6J, Balb/cJ, and C3HeB/FeJ mice against Mtb challenge and is as attenuated as BCG in SCID mice. In C3HeB/FeJ mice, ΔLprG vaccination resulted in innate peripheral cytokine production and induced high polyclonal PPD-specific cytokine-secreting CD4 + T lymphocytes in peripheral blood. The ΔLprG vaccine afforded protective efficacy in the lungs of C3H/FeJ mice following both H37Rv and Erdman aerosolized Mtb challenges. Vaccine efficacy correlated with antigen-specific PD-1-negative CD4 + T lymphocytes as well as with serum IL-17 levels after vaccination. We hypothesize that induction of Th17 cells in lung is critical for vaccine protection, and we show a serum cytokine biomarker for IL-17 shortly after vaccination may predict protective efficacy.

Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide. Here, we evaluate a novel vaccine which induces a broad immune response to Mycobacterium tuberculosis including robust antibody responses and long-lived systemic and tissue-resident memory Th1, Th17, and cytotoxic CD4 + and CD8 + T cells. Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide. To date, the mainstay of vaccination involves the use of Mycobacterium bovis bacillus Calmette-Guérin (BCG), a live-attenuated vaccine that confers protection against extrapulmonary disease in infants and children but not against lung disease. Thus, there is an urgent need for novel vaccines. Here, we show that a multicomponent acellular vaccine (TB-MAPS) induces robust antibody responses and long-lived systemic and tissue-resident memory Th1, Th17, and cytotoxic CD4 + and CD8 + T cells, and promotes trained innate immunity mediated by γδT and NKT cells in mice. When tested in a mouse aerosol infection model, TB-MAPS significantly reduced bacterial loads in the lungs and spleens to the same extent as BCG. When used in conjunction with BCG, TB-MAPS further enhanced BCG-mediated protection, especially in the lungs, further supporting this construct as a promising TB vaccine candidate. IMPORTANCE Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide. Here, we evaluate a novel vaccine which induces a broad immune response to Mycobacterium tuberculosis including robust antibody responses and long-lived systemic and tissue-resident memory Th1, Th17, and cytotoxic CD4 + and CD8 + T cells. When tested in a mouse aerosol infection model, this vaccine significantly reduced bacterial loads in the lungs and spleens to the same extent as BCG. When used in conjunction with BCG, TB-MAPS further enhanced BCG-mediated protection, especially in the lungs, further supporting this construct as a promising TB vaccine candidate.

Understanding how genetic variation in Mycobacterium tuberculosis (Mtb) shapes its physical traits is essential to unraveling the evolution of this global pathogen. Here, we introduce a systematically optimized, high-throughput imaging platform for the comprehensive characterization of Mtb clinical strains. We demonstrate that Mtb’s phenotypic manifestation is shaped by both genetic background and culture density. By accounting for these factors, our analysis linked distinct cellular dynamics to specific lineages, sublineages, and even single nucleotide variations. Notably, we linked a recurring mutation to a unique cell-shortening phenotype, finding that it potentially acts by creating a cryptic antisense transcript. This platform provides a powerful framework for systematically dissecting the physiological dynamics underlying Mtb evolution and identifying new therapeutic vulnerabilities of this deadly pathogen.

New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100–150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical–genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical–genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery. A high-throughput chemical–genetic screening approach for the discovery of targets and chemicals to treat Mycobacterium tuberculosis yields tenfold more hit compounds than conventional whole-cell screening methods.

Mycobacterium tuberculosis ( Mtb ) is the world’s most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene ( Rv3378c ) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid. Buter et al. elucidated the biological function of the terpene nucleoside 1-TbAd, which is made abundantly by virulent but not avirulent Mycobacterium tuberculosis strains, and demonstrate that 1-TbAd regulates the pH and function of host macrophage endolysosomes.

Whole-genome sequencing of (Mtb) populations from clinical samples has increasingly identified genes undergoing selection within and between hosts that drive differential infection and treatment outcomes. However, the intrahost Mtb mutational landscape-especially in the context of human immunodeficiency virus (HIV) coinfection and antiretroviral therapy (ART)-remains less clear, as do the potential impacts of such mutations on Mtb infection dynamics. Here, we performed whole-genome sequencing of Mtb populations isolated from 477 infected tissues across 20 non-human primates (NHP), including animals co-infected with simian immunodeficiency virus (SIV) with or without virological suppression by ART. We identified 116 mutations that emerged during infection, including those that are overrepresented within individual tissues and a subset that are shared across tissues during Mtb dissemination. We further find differential mutation trajectories across treatment groups, with higher mutation frequency and bacterial outgrowth in SIV-infected hosts and increased prevalence of oxidative damage-associated mutations in coinfected animals on ART. Finally, we demonstrate a common pattern of mutation in Mtb lipid metabolism and polyketide synthase genes and identify a subset of NHP-derived mutations that have also independently arisen in human clinical isolates. Together, our population-based sequencing uncovers Mtb diversification during early infection, captures discrete bacterial dissemination events, and infers differential immune pressures faced by Mtb in the setting of SIV-Mtb coinfection and ART suppression. Tuberculosis (TB) remains a leading cause of death worldwide, especially in people living with HIV (PLHIV). How HIV infection and antiretroviral therapy impact (Mtb) intrahost evolution remains unclear. Using whole-genome sequencing from hundreds of infected tissues from non-human primates, we find that simian immunodeficiency virus coinfected hosts and those receiving antiretroviral therapy exert different immune pressures on Mtb, leading to differences in mutation rates and types of DNA damage that are incurred. Mtb mutations were enriched in genes involved in lipid metabolism, and some of these are also seen in human TB strains. This work highlights the role of immune pressure in altering bacterial pathways that may enable Mtb adaptation to the host.

Human challenge experiments could accelerate tuberculosis vaccine development. This requires a safe Mycobacterium tuberculosis (Mtb) strain that can both replicate in the host and be reliably cleared. Here we genetically engineered Mtb strains encoding up to three kill switches: two mycobacteriophage lysin operons negatively regulated by tetracycline and a degron domain–NadE fusion, which induces ClpC1-dependent degradation of the essential enzyme NadE, negatively regulated by trimethoprim. The triple-kill-switch (TKS) strain showed similar growth kinetics and antibiotic susceptibilities to wild-type Mtb under permissive conditions but was rapidly killed in vitro without trimethoprim and doxycycline. It established infection in mice receiving antibiotics but was rapidly cleared upon cessation of treatment, and no relapse was observed in infected severe combined immunodeficiency mice or Rag −/− mice. The TKS strain had an escape mutation rate of less than 10 −10 per genome per generation. These findings suggest that the TKS strain could be a safe, effective candidate for a human challenge model. Engineered kill-switch-encoding Mycobacterium tuberculosis infects, elicits immune responses and is cleared from immunocompetent and immunocompromised mice, providing a model of controlled tuberculosis infection.

Whole genome sequencing of (Mtb) populations from clinical samples has increasingly identified genes undergoing selection within and between hosts that drive differential infection and treatment outcomes. However, the intrahost Mtb mutational landscape-especially in the context of human immunodeficiency virus coinfection and antiretroviral therapy (ART)-remains less clear, as do the potential impacts of such mutations on Mtb infection dynamics. Here, we performed whole genome sequencing of Mtb populations isolated from approximately 480 infected tissues across 20 non-human primates, including animals co-infected with simian immunodeficiency virus (SIV) with or without virological suppression by ART. We identified 116 mutations that emerged during infection, including those that are overrepresented within individual tissues and a subset that are shared across tissues during Mtb dissemination. We further find differential mutation trajectories across treatment groups, with higher mutation rate and bacterial outgrowth in SIV-infected hosts and increased prevalence of oxidative damage-associated mutations in coinfected animals on ART. Finally, we demonstrate a common pattern of mutation in Mtb lipid metabolism and polyketide synthase genes and identify a subset of NHP-derived mutations that have also independently arisen in human clinical isolates. Together, our population-based sequencing uncovers Mtb diversification during early infection, captures discrete bacterial dissemination events and infers differential immune pressures faced by Mtb in the setting of SIV-Mtb coinfection and ART suppression.