The current tuberculosis (TB) vaccine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), provides insufficient protection against pulmonary TB. Previously, we generated a listeriolysin-expressing recombinant BCG strain, which to date has successfully completed phase I and phase IIa clinical trials. In an attempt to further improve efficacy, we deleted the antiapoptotic virulence gene nuoG, encoding NADH dehydrogenase 1 subunit G, from BCG ΔureC::hly. In vitro, deletion of nuoG unexpectedly led to strongly increased recruitment of the autophagosome marker LC3 to the engulfed vaccine, suggesting that nuoG also affects xenophagic pathways. In mice, BCG ΔureC::hly ΔnuoG vaccination was safer than BCG and improved protection over that of parental BCG ΔureC::hly, significantly reducing TB load in murine lungs, ameliorating pulmonary pathology, and enhancing immune responses. Transcriptome analysis of draining lymph nodes after vaccination with either BCG ΔureC::hly or BCG ΔureC::hly ΔnuoG demonstrated earlier and stronger induction of immune responses than that with BCG SSI and suggested upregulation of inflammasome activation and interferon-induced GTPases. In summary, BCG ΔureC::hly ΔnuoG is a promising next-generation TB vaccine candidate with excellent efficacy and safety.

Mycofactocin (MFT) belongs to the class of ribosomally synthesized and posttranslationally modified peptides conserved in many Actinobacteria. Mycobacterium tuberculosis assimilates cholesterol during chronic infection, and its in vitro growth in the presence of cholesterol requires most of the MFT biosynthesis genes (mftA, mftB, mftC, mftD, mftE, and mftF), although the reasons for this requirement remain unclear. To identify the function of MFT, we characterized MFT biosynthesis mutants constructed in Mycobacterium smegmatis, M. marinum, and M. tuberculosis. We found that the growth deficit of mft deletion mutants in medium containing cholesterol—a phenotypic basis for gene essentiality prediction—depends on ethanol, a solvent used to solubilize cholesterol. Furthermore, functionality of MFT was strictly required for growth of free-living mycobacteria in ethanol and other primary alcohols. Among other genes encoding predicted MFT-associated dehydrogenases, MSMEG_6242 was indispensable for M. smegmatis ethanol assimilation, suggesting that it is a candidate catalytic interactor with MFT. Despite being a poor growth substrate, ethanol treatment resulted in a reductive cellular state with NADH accumulation in M. tuberculosis. During ethanol treatment, mftC mutant expressed the transcriptional signatures that are characteristic of respirational dysfunction and a redox-imbalanced cellular state. Counterintuitively, there were no differences in cellular bioenergetics and redox parameters in mftC mutant cells treated with ethanol. Therefore, further understanding of the function of MFT in ethanol metabolism is required to identify the cause of growth retardation of MFT mutants in cholesterol. Nevertheless, our results establish the physiological role of MFT and also provide new insights into the specific functions of MFT homologs in other actinobacterial systems. IMPORTANCE Tuberculosis is caused by Mycobacterium tuberculosis, and the increasing emergence of multidrug-resistant strains renders current treatment options ineffective. Although new antimycobacterial drugs are urgently required, their successful development often relies on complete understanding of the metabolic pathways—e.g., cholesterol assimilation—that are critical for persistence and for pathogenesis of M. tuberculosis. In this regard, mycofactocin (MFT) function appears to be important because its biosynthesis genes are predicted to be essential for M. tuberculosis in vitro growth in cholesterol. In determining the metabolic basis of this genetic requirement, our results unexpectedly revealed the essential function of MFT in ethanol metabolism. The metabolic dysfunction thereof was found to affect the mycobacterial growth in cholesterol which is solubilized by ethanol. This knowledge is fundamental in recognizing the bona fide function of MFT, which likely resembles the pyrroloquinoline quinone-dependent ethanol oxidation in acetic acid bacteria exploited for industrial production of vinegar.

During active TB in humans a spectrum of pulmonary granulomas with central necrosis and hypoxia exists. BALB/c mice, predominantly used in TB drug development, do not reproduce this complex pathology thereby inaccurately predicting clinical outcome. We found that Nos2 −/− mice incapable of NO-production in immune cells as microbial defence uniformly develop hypoxic necrotizing lung lesions, widely observed in human TB. To study the impact of hypoxic necrosis on the efficacy of antimycobacterials and drug candidates, we subjected Nos2 −/− mice with TB to monotherapy before or after establishment of human-like pathology. Isoniazid induced a drug-tolerant persister population only when necrotic lesions were present. Rifapentine was more potent than rifampin prior to development of human-like pathology and equally potent thereafter, in agreement with recent clinical trials. Pretomanid, delamanid and the pre-clinical candidate BTZ043 were bactericidal independent of pulmonary pathology. Linezolid was bacteriostatic in TB-infected Nos2 −/− mice but significantly improved lung pathology. Hypoxic necrotizing lesions rendered moxifloxacin less active. In conclusion, Nos2 −/− mice are a predictive TB drug development tool owing to their consistent development of human-like pathology.Tuberculosis (TB) is a global health threat further worsened by increasing incidences of drug resistance and HIV co-infection 1 . New drugs to combat resistant TB and to shorten the current 6 to 9 month standard-of-care chemotherapy are urgently needed. In most cases, infection starts in the lungs where Mycobacterium tuberculosis induces well-structured granulomas composed of various hematopoietic cells in which the pathogen is contained 2, 3 . During progression to active disease, infected macrophages and neutrophils undergo necrosis at the core of these structure, resulting in the formation of a hypoxic caseum 2 . 18 F-fluorodeoxyglucose positron emission tomography/computed tomography has revealed that pulmonary TB is a highly dynamic disease: coexisting inflammatory hotspots in lungs of TB patients increase in size, shrink or even resolve over time while others are newly formed 4,5 . Recent studies have indicated that pro-and anti-inflammatory signals are spatially segregated within granulomas of humans suggesting that granuloma formation in TB is a direct consequence of the local activity of inflammatory pathways 6 . Among the available animal models, non-human primates mirror the facets of human TB best, but broad application is hampered by prohibitive cost and ethical concerns. To date, mice remain the most widely used

Human immune system mice are highly valuable for in vivo dissection of human immune responses. Although they were employed for analyzing tuberculosis (TB) disease, there is little data on the spatial organization and cellular composition of human immune cells in TB granuloma pathology in this model. We demonstrate that human immune system mice, generated by transplanted human fetal liver derived hematopoietic stem cells develop a continuum of pulmonary lesions upon Mycobacterium tuberculosis aerosol infection. In particular, caseous necrotic granulomas, which contribute to prolonged TB treatment time, developed, and had cellular phenotypic spatial-organization similar to TB patients. By comparing two recommended drug regimens, we confirmed observations made in clinical settings: Adding Moxifloxacin to a classical chemotherapy regimen had no beneficial effects on bacterial eradication. We consider this model instrumental for deeper understanding of human specific features of TB pathogenesis and of particular value for the pre-clinical drug development pipeline.

Copper (Cu) ions are critical in controlling bacterial infections, and successful pathogens like Mycobacterium tuberculosis (Mtb) possess multiple Cu resistance mechanisms. We report, as proof of concept, that a novel Cu hypersensitivity phenotype can be generated in mycobacteria, including Mtb, through a peptide, DAB-10, that is able to form reactive oxygen species (ROS) following Cu-binding. DAB-10 induces intramycobacterial oxidative stress in a Cu-dependent manner in vitro and during infection. DAB-10 penetrates murine macrophages and encounters intracellular mycobacteria. Significant intracellular Cu-dependent protection was observed when Mtb-infected macrophages were treated with DAB-10 alongside a cell-permeable Cu chelator. Treatment with the Cu chelator reversed the intramycobacterial oxidative shift induced by DAB-10. We conclude that DAB-10 utilizes the pool of phagosomal Cu ions in the host-Mtb interface to augment the mycobactericidal activity of macrophages while simultaneously exploiting the susceptibility of Mtb to ROS. DAB-10 serves as a model with which to develop next-generation, multifunctional antimicrobials.