Tuberculosis (TB) remains a major global health threat, largely due to Mycobacterium tuberculosis (Mtb) resilience, which can be exacerbated by exposure to sublethal antibiotic concentrations arising from factors such as patient nonadherence and the granuloma structure which limits drug penetration. Within host granulomas, Mtb can exhibit both phenotypic tolerance and genotypic resistance, complicating curative treatments. This study aimed to determine whether sublethal rifampicin acts as a signaling molecule in Mycobacterium smegmatis (Msm) and the attenuated Mtb H37Ra strain, triggering phenotypic changes that promote tolerance to lethal drug levels. Msm exposed to half-MIC rifampicin showed an initial transient growth deceleration followed by a resumption of proliferation, indicating the acquisition of phenotypic tolerance. Deep data-independent acquisition (DIA) mass spectrometry-based proteomic profiling revealed that the early response (45 min) involved the upregulation of ribosomal proteins, DNA replication, and de novo purine biosynthesis. Proteins associated with phenotypic resistance (e.g., RpoZ, GidB, WhiB2) and efflux transporters were also upregulated. As Msm recovered (180 min), its proteome largely returned to baseline, but key resistance-associated pathways, including the Rifampin ADP-ribosyltransferase superfamily and certain efflux systems, remained dysregulated. Parallel studies on Mtb H37Ra also demonstrated a distinct proteomic shift, comprising conserved adaptive responses such as ribosomal perturbation and compensatory transcriptional activity, as well as species-specific dysregulation of drug influx/efflux pumps and cell envelope remodelling via the polyketide synthase family. These findings demonstrate that sublethal rifampicin exposure primes mycobacteria for enhanced tolerance to lethal drug concentrations, underscoring a significant challenge in current TB therapy.

Differentially culturable (DC) Mycobacterium tuberculosis (Mtb) phenotypes reduce the sensitivity of sputum culture and may be associated with adverse treatment outcomes in tuberculosis patients. Detecting and quantifying DC Mtb in clinical samples remains an important research objective; current approaches rely on culture filtrate (CF) supplemented growth assays that assume CF selectively resuscitates DC Mtb rather than broadly stimulating growth from low density inocula - an untested assumption for Mtb. We performed a half-logarithmic dilution series of Mtb from approximately 10,000-0 total bacilli, culturing each inoculum in either standard media (7H9) or CF and monitoring growth by optical density. Odds of growth increased sixfold for every tenfold increase in inoculum size. CF increased the odds of growth fivefold and reduced the time-to-positivity by 344 h (∼14 days) compared to 7H9 alone. However, CF's growth-promoting effects diminished with increasing inoculum size, becoming negligible at approximately 70,000 bacilli/mL. Notably, CF broadly altered Mtb cell physiology, producing shorter bacilli that were less likely to form aggregates or incorporate the mycomembrane probe, DMN-trehalose. These data indicate that Mtb is poorly culturable from low inoculum densities under conditions tested, with no growth occurring in wells with ≤30 total bacilli, a limitation only partially overcome by CF. Moreover, while CF promotes Mtb growth, its effects on bacillary physiology suggest an impact extending beyond simply resuscitation of DC Mtb. Improved methods are needed for detecting DC Mtb phenotypes, as current CF-based assays may not distinguish selective resuscitation from broader growth-promoting effects.

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. Pharmacokinetic and initial in vivo efficacy studies indicated that myxovalargin and analogues show potential for development as an antibacterial agent.

Cobamides play a paradoxical but critical role in the biology of Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis. Although Mtb retains nearly all cobalamin (Cbl) biosynthetic genes and encodes multiple cobamide-requiring enzymes, experimental evidence indicates that Mtb is incapable of de novo Cbl synthesis under any tested conditions to date. Instead, an evolutionary shift appears to have occurred toward host dependency for biologically relevant cobamides or their precursors. This review highlights recent advances in our understanding of cobamide-related metabolism in Mtb , including: (i) the progressive erosion of de novo cobamide biosynthetic capacity across Mtb lineages; (ii) the role of host-derived cobamides in sustaining key mycobacterial metabolic pathways, including methionine synthesis and propionate catabolism; (iii) the impact of host immune pressures, including itaconate-mediated inhibition of methylmalonyl-CoA mutase; (iv) strategies employed by Mtb for cobamide and precursor acquisition; and (v) unique adaptations of Cbl-sensing riboswitches that regulate methionine synthesis, virulence-associated gene expression, and dormancy resuscitation. We also highlight unresolved questions, including possible niche-specific synthesis, utilization of alternate cobamide species, and the therapeutic potential of targeting cobamide-related metabolism. We review recent evidence of the centrality of cobamides in the metabolic flexibility of Mtb , virulence, and survival in the host environment, despite apparent loss of de novo biosynthetic capacity. Further mechanistic studies are required which may reveal vulnerabilities for the exploitation of cobamide acquisition, cobamide-related regulation, and the role of cobamides at the Mtb -host interface for innovative therapeutic interventions.

Differentially culturable (DC) Mycobacterium tuberculosis ( Mtb ) phenotypes reduce the sensitivity of sputum culture and may be associated with adverse treatment outcomes among tuberculosis patients. Accurately quantifying DC Mtb remains an important research objective, with current approaches tending to combine Most Probable Number (MPN) assays and culture filtrate (CF) supplementation. These assume that growth is equiprobable across all bacterial inoculum densities – an untested assumption for Mtb . We performed a half-logarithmic dilution series of Mtb from 70,000–0 CFU/mL, culturing each inoculum in either standard 7H9 or CF and monitoring growth by optical density. Inocula of ≥2,000 CFU/mL were 33 times more likely to grow than inocula of ≤700 CFU/mL. CF increased the odds of growth five-fold, and reduced the time-to-positivity by 294 hours (∼12 days) compared to 7H9 alone. However, CF’s growth-promoting effects diminished with increasing inoculum density, becoming negligible at 70,000 CFU/mL. Notably, CF broadly altered Mtb cell physiology, producing shorter bacilli that were less likely to incorporate the mycomembrane probe, DMN-trehalose. These data indicate that Mtb is poorly culturable from low inoculum densities – a limitation only partially overcome by CF. This non-uniform growth probability suggests that unsupplemented MPN assays may systematically underestimate Mtb CFU. Moreover, while CF promotes Mtb growth, its density-dependent activity and broader effects on cell physiology suggest that its influence extends beyond simply resuscitating DC Mtb . Improved methods are needed for detecting DC Mtb phenotypes, as these may support clinical care and shed light on factors that govern mycobacterial replication at different population densities. Highlights * CF : Culture filtrate CFU : Colony forming units CI : Confidence interval DC : Differentially culturable DCTB : Differentially culturable tubercle bacilli HIV : Human Immunodeficiency Virus MPN : Most probable number Mtb : Mycobacterium tuberculosis OR : Odds ratio Rpf : Resuscitation promoting factor TB : Tuberculosis TTP : Time-to-positivity

Tuberculosis (TB) imposes a major burden on global public health which is exacerbated by the escalating number of multidrug-resistant (MDR)-TB cases. There is consequently an urgent need for new anti-TB drugs and combination regimens. We have investigated the natural product antibiotic fusidic acid (FA) for repurposing against Mycobacterium tuberculosis , the causative agent of TB. Here, we report the results of synergy screens combining FA with a panel of approved anti-TB agents. Checkerboard and time-kill kinetics assays identified seven compounds from different chemical classes that synergized with FA in inhibiting the growth of M. tuberculosis in vitro : rifampicin (RIF), a rifamycin and frontline anti-TB drug; the macrolides, erythromycin (ERY), clarithromycin (CLR), and roxythromycin (ROX); the oxazolidinone, linezolid (LZD); the aminoglycoside, streptomycin (STR); and the aminocyclitol, spectinomycin (SPC). Among these, the strongest synergies were observed where FA was combined with SPC and ERY. Moreover, the FA-RIF combination was cidal, while all other FA combinations were bacteriostatic. These results provide in vitro evidence of the potential utility of FA-containing combinations against M. tuberculosis .

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

Screening of a library of small polar molecules against Mycobacterium tuberculosis (Mtb) led to the identification of a potent benzoheterocyclic oxime carbamate hit series. This series was subjected to medicinal chemistry progression underpinned by structure-activity relationship studies toward identifying a compound for proof-of-concept studies and defining a lead optimization strategy. Carbamate and free oxime frontrunner compounds with good stability in liver microsomes and no hERG channel inhibition liability were identified and evaluated in vivo for pharmacokinetic properties. Mtb-mediated permeation and metabolism studies revealed that the carbamates were acting as prodrugs. Toward mechanism of action elucidation, selected compounds were tested in biology triage assays to assess their activity against known promiscuous targets. Taken together, these data suggest a novel yet unknown mode of action for these antitubercular hits.

Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis. We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination partially restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S rRNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis. Moreover, this combination produced a modest potentiating effect against both FA-monoresistant and SPT-monoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant. These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds.

Phenotypic whole-cell screening against Mycobacterium tuberculosis (Mtb) in glycerol–alanine–salts supplemented with Tween 80 and iron (GASTE-Fe) media led to the identification of a 2-aminoquinazolinone hit compound, sulfone 1 which was optimized for solubility by replacing the sulfone moiety with a sulfoxide 2. The synthesis and structure–activity relationship (SAR) studies identified several compounds with potent antimycobacterial activity, which were metabolically stable and noncytotoxic. Compound 2 displayed favorable in vitro properties and was therefore selected for in vivo pharmacokinetic (PK) studies where it was found to be extensively metabolized to the sulfone 1. Both derivatives exhibited promising PK parameters; however, when 2 was evaluated for in vivo efficacy in an acute TB infection mouse model, it was found to be inactive. In order to understand the in vitro and in vivo discrepancy, compound 2 was subsequently retested in vitro using different Mtb strains cultured in different media. This revealed that activity was only observed in media containing glycerol and led to the hypothesis that glycerol was not used as a primary carbon source by Mtb in the mouse lungs, as has previously been observed. Support for this hypothesis was provided by spontaneous-resistant mutant generation and whole genome sequencing studies, which revealed mutations mapping to glycerol metabolizing genes indicating that the 2-aminoquinazolinones kill Mtb in vitro via a glycerol-dependent mechanism of action.

Background: Tuberculosis (TB) is predominantly an airborne disease. However, quantitative and qualitative analysis of bio-aerosols containing the aetiological agent, Mycobacterium tuberculosis (Mtb), has proven very challenging. Our objective is to sample bio-aerosols from newly diagnosed TB patients for detection and enumeration of Mtb bacilli. Methods: We monitored each of 35 newly diagnosed, GeneXpert sputum-positive, TB patients during 1 hour confinement in a custom-built Respiratory Aerosol Sampling Chamber (RASC). The RASC (a small clean-room of 1.4m 3) incorporates aerodynamic particle size detection, viable and non-viable sampling devices, real-time CO 2 monitoring, and cough sound-recording. Microbiological culture and droplet digital polymerase chain reaction (ddPCR) were used to detect Mtb in each of the bio-aerosol collection devices. Results: Mtb was detected in 27/35 (77.1%) of aerosol samples; 15/35 (42.8%) samples were positive by mycobacterial culture and 25/27 (92.96%) were positive by ddPCR. Culturability of collected bacilli was not predicted by radiographic evidence of pulmonary cavitation, sputum smear positivity. A correlation was found between cough rate and culturable bioaerosol. Mtb was detected on all viable cascade impactor stages with a peak at aerosol sizes 2.0-3.5μm. This suggests a median of 0.09 CFU/litre of exhaled air (IQR: 0.07 to 0.3 CFU/l) for the aerosol culture positives and an estimated median concentration of 4.5x10 7 CFU/ml (IQR: 2.9x10 7-5.6x10 7) of exhaled particulate bio-aerosol. Conclusions: Mtb was identified in bio-aerosols exhaled by the majority of untreated TB patients using the RASC. Molecular detection was more sensitive than mycobacterial culture on solid media, suggesting that further studies are required to determine whether this reflects a significant proportion of differentially detectable bacilli in these samples.

Background: Tuberculosis (TB) is predominantly an airborne disease. However, quantitative and qualitative analysis of bio-aerosols containing the aetiological agent, Mycobacterium tuberculosis (Mtb), has proven very challenging. Our objective is to sample bio-aerosols from newly diagnosed TB patients for detection and enumeration of Mtb bacilli. Methods: We monitored each of 35 newly diagnosed, GeneXpert sputum-positive, TB patients during 1 hour confinement in a custom-built Respiratory Aerosol Sampling Chamber (RASC). The RASC (a small clean-room of 1.4m3) incorporates aerodynamic particle size detection, viable and non-viable sampling devices, real-time CO2 monitoring, and cough sound-recording. Microbiological culture and droplet digital polymerase chain reaction (ddPCR) were used to detect Mtb in each of the bio-aerosol collection devices. Results: Mtb was detected in 27/35 (77.1%) of aerosol samples; 15/35 (42.8%) samples were positive by mycobacterial culture and 25/27 (92.96%) were positive by ddPCR. Culturability of collected bacilli was not predicted by radiographic evidence of pulmonary cavitation, sputum smear positivity, or cough rate. Mtb was detected on all viable cascade impactor stages with a peak at aerosol sizes 2.0-3.5μm. This suggests a median of 0.09 CFU/litre of exhaled air (IQR: 0.07 to 0.3 CFU/l) for the aerosol culture positives and an estimated median concentration of 4.5x107 CFU/ml (IQR: 2.9x107-5.6x107) of exhaled particulate bio-aerosol. Conclusions: Mtb was identified in bio-aerosols exhaled by the majority of untreated TB patients using the RASC. Molecular detection was more sensitive than mycobacterial culture on solid media, suggesting that further studies are required to determine whether this reflects a significant proportion of differentially detectable bacilli in these samples.

ABSTRACT We deleted subunits I (cydA) and II (cydB) of the Mycobacterium tuberculosis cytochrome bd menaquinol oxidase. The resulting ΔcydA and ΔcydAB mutants were hypersusceptible to compounds targeting the mycobacterial bc1 menaquinol-cytochrome c oxidoreductase and exhibited bioenergetic profiles indistinguishable from strains deficient in the ABC-type transporter, CydDC, predicted to be essential for cytochrome bd assembly. These results confirm CydAB and CydDC as potential targets for drugs aimed at inhibiting a terminal respiratory oxidase implicated in pathogenesis.

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome bc1 complex, disruption of cell-wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylaminopyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure-activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties.

Knowledge of the airborne nature of respiratory disease transmission owes much to the pioneering experiments of Wells and Riley over half a century ago. However, the mechanical, physiological, and immunopathological processes which drive the production of infectious aerosols by a diseased host remain poorly understood. Similarly, very little is known about the specific physiological, metabolic and morphological adaptations which enable pathogens such as Mycobacterium tuberculosis (Mtb) to exit the infected host, survive exposure to the external environment during airborne carriage, and adopt a form that is able to enter the respiratory tract of a new host, avoiding innate immune and physical defenses to establish a nascent infection. As a first step towards addressing these fundamental knowledge gaps which are central to any efforts to interrupt disease transmission, we developed and characterized a small personal clean room comprising an array of sampling devices which enable isolation and representative sampling of airborne particles and organic matter from tuberculosis (TB) patients. The complete unit, termed the Respiratory Aerosol Sampling Chamber (RASC), is instrumented to provide real-time information about the particulate output of a single patient, and to capture samples via a suite of particulate impingers, impactors and filters. Applying the RASC in a clinical setting, we demonstrate that a combination of molecular and microbiological assays, as well as imaging by fluorescence and scanning electron microscopy, can be applied to investigate the identity, viability, and morphology of isolated aerosolized particles. Importantly, from a preliminary panel of active TB patients, we observed the real-time production of large numbers of airborne particles including Mtb, as confirmed by microbiological culture and polymerase chain reaction (PCR) genotyping. Moreover, direct imaging of captured samples revealed the presence of multiple rod-like Mtb organisms whose physical dimensions suggested the capacity for travel deep into the alveolar spaces of the human lung.

ABSTRACT The tuberculosis (TB) drug discovery pipeline is fueled by compounds identified in whole-cell screens against the causative agent, Mycobacterium tuberculosis. Phenotypic screening enables the selection of molecules that inhibit essential cellular functions in live, intact bacilli grown under a chosen in vitro condition. However, deducing the mechanism of action (MOA), which is important to avoid promiscuous targets, often requires significant biological resources in a lengthy process that risks decoupling medicinal chemistry and biology efforts. Therefore, there is a need to develop methods enabling rapid MOA assessment of putative “actives” for triage decisions. Here, we describe a modified version of a bioluminescence reporter assay that allows nondestructive detection of compounds targeting either of two macromolecular processes in M. tuberculosis: cell wall biosynthesis or maintenance of DNA integrity. Coupling the luxCDABE operon from Photorhabdus luminescens to mycobacterial promoters driving expression of the iniBAC operon (PiniB-LUX) or the DNA damage-inducible genes, recA (PrecA-LUX) or radA (PradA-LUX), provided quantitative detection in real time of compounds triggering expression of any of these promoters over an extended 10- to 12-day incubation. Testing against known anti-TB agents confirmed the specificity of each reporter in registering the MOA of the applied antibiotic in M. tuberculosis, independent of bactericidal or bacteriostatic activity. Moreover, profiles obtained for experimental compounds indicated the potential to infer complex MOAs in which multiple cellular processes are disrupted. These results demonstrate the utility of the reporters for early triage of compounds based on the provisional MOA and suggest their application to investigate polypharmacology in known and experimental anti-TB agents.

High-throughput screening of a library of small polar molecules against Mycobacterium tuberculosis led to the identification of a phthalimide-containing ester hit compound (1), which was optimized for metabolic stability by replacing the ester moiety with a methyl oxadiazole bioisostere. A route utilizing polymer-supported reagents was designed and executed to explore structure-activity relationships with respect to the N-benzyl substituent, leading to compounds with nanomolar activity. The frontrunner compound (5h) from these studies was well tolerated in mice. A M. tuberculosis cytochrome bd oxidase deletion mutant (ΔcydKO) was hyper-susceptible to compounds from this series, and a strain carrying a single point mutation in qcrB, the gene encoding a subunit of the menaquinol cytochrome c oxidoreductase, was resistant to compounds in this series. In combination, these observations indicate that this novel class of antimycobacterial compounds inhibits the cytochrome bc1 complex, a validated drug target in M. tuberculosis.

Whole-cell high-throughput screening of a diverse SoftFocus library against Mycobacterium tuberculosis (Mtb) generated a novel aminopyrazolo[1,5-a]pyrimidine hit series. The synthesis and structure activity relationship studies identified compounds with potent antimycobacterial activity. The SAR of over 140 compounds shows that the 2-pyridylmethylamine moiety at the C-7 position of the pyrazolopyrimidine scaffold was important for Mtb activity, whereas the C-3 position offered a higher degree of flexibility. The series was also profiled for in vitro cytotoxicity and microsomal metabolic stability as well as physicochemical properties. Consequently liabilities to be addressed in a future lead optimization campaign have been identified.

Mycobacterium tuberculosis is included among a select group of bacteria possessing the capacity for de novo biosynthesis of vitamin B12, the largest and most complex natural organometallic cofactor. The bacillus is also able to scavenge B12 and related corrinoids utilizing an ATP-binding cassette-type protein that is distinct from the only known bacterial B12-specific transporter, BtuFCD. Consistent with the inferred requirement for vitamin B12 for metabolic function, the M. tuberculosis genome encodes two B12 riboswitches and three B12-dependent enzymes. Two of these enzymes have been shown to operate in methionine biosynthesis (MetH) and propionate utilization (MutAB), while the function of the putative nrdZ-encoded ribonucleotide reductase remains unknown. Taken together, these observations suggest that M. tuberculosis has the capacity to regulate core metabolic functions according to B12 availability - whether acquired via endogenous synthesis or through uptake from the host environment - and, therefore, imply that there is a role for vitamin B12 in pathogenesis, which remains poorly understood.