SummaryGrowth of bacteria and fungi on fatty acid substrates requires the catabolic b -oxidation cycle and the anaplerotic glyoxylate cycle. Propionyl-CoA generated by b -oxidation of odd-chain fatty acids is metabolized via the methylcitrate cycle. Mycobacterium tuberculosis possesses homologues of methylcitrate synthase (MCS) and methylcitrate dehydratase (MCD) but not 2-methylisocitrate lyase (MCL). Although MCLs share limited homology with isocitrate lyases (ICLs) of the glyoxylate cycle, these enzymes are thought to be functionally non-overlapping. Previously we reported that the M. tuberculosis ICL isoforms 1 and 2 are jointly required for growth on fatty acids, in macrophages, and in mice. ICL-deficient bacteria could not grow on propionate, suggesting that in M. tuberculosis ICL1 and ICL2 might function as ICLs in the glyoxylate cycle and as MCLs in the methylcitrate cycle. Here we provide biochemical and genetic evidence supporting this interpretation. The role of the methylcitrate cycle in M. tuberculosis metabolism was further evaluated by constructing a mutant strain in which prpC (encoding MCS) and prpD (encoding MCD) were deleted. The D prpDC strain could not grow on propionate media in vitro or in murine bone marrow-derived macrophages infected ex vivo ; growth under these conditions was restored by complementation with a plasmid containing prpDC . Paradoxically, bacterial growth and persistence, and tissue pathology, were indistinguishable in mice infected with wildtype or D prpDC bacteria.

Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.

SummaryTuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F420 deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F420 and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.

Cell-permeable small molecules that inhibit their targets on fast timescales are powerful probes of cell-division mechanisms. Such inhibitors have been identified using phenotype-based screens with chemical libraries. However, the characteristics of compound libraries needed to effectively span cell-division phenotype space, to find probes that target different mechanisms, are not known. Here we show that a small collection of 100 diaminopyrimidines (DAPs) yields a range of cell-division phenotypes, including changes in spindle geometry, chromosome positioning and mitotic index. Monopolar mitotic spindles are induced by four inhibitors, including one that targets Polo-like kinases (Plks), evolutionarily conserved serine/threonine kinases. Using chemical inhibitors and high-resolution live-cell microscopy, we found that Plk activity is needed for the assembly and maintenance of bipolar mitotic spindles. Plk inhibition destabilizes kinetochore microtubules while stabilizing other spindle microtubules, leading to monopolar spindles. Further testing of compounds based on 'privileged scaffolds', such as the DAP scaffold, could lead to new cell-division probes and antimitotic agents.