Since the vast majority of species solely rely on innate immunity for host defense, it stands to reason that a critical evolutionary trait like immunological memory evolved in this primitive branch of our immune system. There is ample evidence that vaccines such as bacillus Calmette-Guérin (BCG) induce protective innate immune memory responses (trained immunity) against heterologous pathogens. Here we show that while BCG vaccination significantly reduces morbidity and mortality against influenza A virus (IAV), it fails to provide protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In contrast to IAV, SARS-CoV-2 infection leads to unique pulmonary vasculature damage facilitating viral dissemination to other organs, including the bone marrow (BM), a central site for BCG-mediated trained immunity. Finally, monocytes from BCG-vaccinated individuals mount an efficient cytokine response to IAV infection, while this response is minimal following SARS-CoV-2. Collectively, our data suggest that the protective capacity of BCG vaccination is contingent on viral pathogenesis and tissue tropism.

The emergence of multi-drug (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB) is a major threat to the global management of tuberculosis (TB) worldwide. New chemical entities are of need to treat drug-resistant TB. In this study, the mode of action of new, potent quinazoline derivatives was investigated against Mycobacterium tuberculosis (M. tb). Four derivatives 11626141, 11626142, 11626252 and 11726148 showed good activity (MIC ranging from 0.02–0.09 μg/mL) and low toxicity (TD₅₀ ≥ 5μg/mL) in vitro against M. tb strain H37Rv and HepG2 cells, respectively. 11626252 was the most selective compound from this series. Quinazoline derivatives were found to target cytochrome bc₁ by whole-genome sequencing of mutants selected with 11626142. Two resistant mutants harboured the transversion T943G (Trp312Gly) and the transition G523A (Gly175Ser) in the cytochrome bc₁ complex cytochrome b subunit (QcrB). Interestingly, a third mutant QuinR-M1 contained a mutation in the Rieske iron-sulphur protein (QcrA) leading to resistance to quinazoline and other QcrB inhibitors, the first report of cross-resistance involving QcrA. Modelling of both QcrA and QcrB revealed that all three resistance mutations are located in the stigmatellin pocket, as previously observed for other QcrB inhibitors such as Q203, AX-35, and lansoprazole sulfide (LPZs). Further analysis of the mode of action in vitro revealed that 11626252 exposure leads to ATP depletion, a decrease in the oxygen consumption rate and also overexpression of the cytochrome bd oxidase in M. tb. Our findings suggest that quinazoline-derived compounds are a new and attractive chemical entity for M. tb drug development targeting two separate subunits of the cytochrome bc₁ complex. Tuberculosis (TB) is the leading cause of death worldwide due to an infectious agent. Nowadays, the efficacy of mainstay anti-TB drugs is jeopardized due to the emergence of drug-resistant TB. New antitubercular drugs are needed for the treatment of TB. In this study, we decipher the mechanism of action of a new potent series of 2-Ethylthio-4-methylaminoquinazoline derivatives against Mycobacterium tuberculosis (M. tb). Out of 76 derivatives tested, 4 compounds (11626141, 11626142, 11626252 and 11726148) have good activity (MIC below 0.09 μg/mL) against M. tb and low toxicity in human hepatocytes. The lead compound 11626252 and two derivatives, 11626141 and 11626142, were specifically active against members of the M. tb complex and M. marinum. These derivatives target the cytochrome bc₁ oxidase, part of the mycobacterial electron-transport chain. Interestingly, we demonstrate that resistance to the quinazoline derivatives, as well as to other QcrB inhibitors, like Q203, AX-35 and lansoprazole sulfide, may emerge through a mutation in the Rieske iron-sulphur protein (QcrA). To our knowledge, this is the first report implicating the QcrA subunit in the pharmacological inhibition of cytochrome bc₁ activity.

New drugs with new mechanisms of action are urgently required to tackle the global tuberculosis epidemic. Following the FDA-approval of the ATP synthase inhibitor bedaquiline (Sirturo^®), energy metabolism has become the subject of intense focus as a novel pathway to exploit for tuberculosis drug development. This enthusiasm stems from the fact that oxidative phosphorylation (OxPhos) and the maintenance of the transmembrane electrochemical gradient are essential for the viability of replicating and non-replicating Mycobacterium tuberculosis (M. tb), the etiological agent of human tuberculosis (TB). Therefore, new drugs targeting this pathway have the potential to shorten TB treatment, which is one of the major goals of TB drug discovery. This review summarises the latest and key findings regarding the OxPhos pathway in M. tb and provides an overview of the inhibitors targeting various components. We also discuss the potential of new regimens containing these inhibitors, the flexibility of this pathway and, consequently, the complexity in targeting it. Lastly, we discuss opportunities and future directions of this drug target space.

Mycobacterium abscessusis the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis.M.abscessusdisplays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such,M.abscessusis clinically resistant to the entire regimen of front-lineM.tuberculosisdrugs, and treatment with antibiotics that do inhibitM.abscessusin the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) inM.abscessus. EPT protected zebrafish from lethalM.abscessusinfection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater againstM.abscessusthanM.tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRS_Mabsand LeuRS_Mtbwith similar residues and dissociation constants. Since EPT-resistantM.abscessusmutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that whenM.abscessusLeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports ourin vitrodata that supplementation of media with norvaline reduced the emergence of EPT mutants in bothM.abscessusandM.tuberculosis. Furthermore, the combination of EPT and norvaline had improvedin vivoefficacy compared to EPT in a murine model ofM.abscessusinfection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogenM.abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial forM.abscessusand other mycobacterial infections like tuberculosis.

The production of itaconate by macrophages was only discovered in 2011. An increasing number of studies have since revealed essential biological functions for this small molecule, ranging from antimicrobial to immunomodulator. The antibacterial role of itaconate has however been questioned because the estimated concentration of itaconate in macrophages (low-millimolar) is lower than the minimum inhibitory concentration (MIC) of itaconate reported for several bacterial strains (low-to-mid-millimolar). We note that some of these investigations have tended to ignore the high acidity of this small diacid (pKas 3.85 and 5.45), thereby potentially biassing activity measurements. We measured the MIC of itaconate inEscherichia coli(not known to metabolize itaconate) and inSalmonella entericaserovar Typhimurium (known to metabolize itaconate) at varying pH values to probe the effect that pH has on itaconate toxicity. Herein, we demonstrate that the antimicrobial effect of itaconate is dependent upon the pH of the media and that itaconate does have antimicrobial activity at biologically relevant pH and concentrations. Under nutrient-poor conditions, the antimicrobial activity of itaconate in bothE. coliandS. Typhimurium increased approximately 200-fold when the pH was dropped by one unit, whereas itaconate was not found to be toxic under nutrient rich conditions. Our results also reveal that the activity of itaconate is synergistic with acidity, yet is not a function of increased permeability with protonation. Similar experiments performed with succinate (a pKa-matched diacid) yielded drastically different results, consistent with a target-based mechanism of action for itaconate. Overall, our work shows the importance of controlling the pH when performing experiments with itaconic acid.

The metabolic adaptability of intracellular pathogens, such as the Gram-negative Salmonella enterica serovar Typhimurium (STm), enables their survival in nutrient-restricted host environments, while also presenting an opportunity for selective antimicrobial targeting. Herein, we report the synthesis and screening of a small pantetheinamide library aimed at inhibiting the proliferation of STm within macrophages. Two lead compounds exhibited selective activity against intracellular STm and reduced bacterial burden in a murine colitis model. Mechanistic studies suggest their primary mode of action to be the inhibition of coenzyme A (CoA) biosynthesis via the PanD–PanZ regulatory complex, which is absent in host cells. Importantly, resistance pressure to our compound was shown to be significantly stronger in nutrient-limited media compared to nutrient-rich media, supporting the targeting of nutrient stress as a strategy to delay resistance. This work highlights the value of inhibiting pathogen-specific metabolic vulnerabilities in combination with host defence mechanisms to develop novel antimicrobials.