New drugs are urgently needed to combat the global TB epidemic. Targeting simultaneously multiple respiratory enzyme complexes of Mycobacterium tuberculosis is regarded as one of the most effective treatment options to shorten drug administration regimes, and reduce the opportunity for the emergence of drug resistance. During infection and proliferation, the cytochrome bd oxidase plays a crucial role for mycobacterial pathophysiology by maintaining aerobic respiration at limited oxygen concentrations. Here, we present the cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 Å. In conjunction with atomistic molecular dynamics (MD) simulation studies we discovered a previously unknown MK-9-binding site, as well as a unique disulfide bond within the Q-loop domain that defines an inactive conformation of the canonical quinol oxidation site in Actinobacteria. Our detailed insights into the long-sought atomic framework of the cytochrome bd oxidase from M. tuberculosis will form the basis for the design of highly specific drugs to act on this enzyme.

Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis. Here, we show that HM2-16F, a 6-substituted derivative of the FDA-approved drug amiloride, is an anti-tubercular inhibitor with bactericidal properties comparable to the FDA-approved drug bedaquiline (BDQ; Sirturo^®) and inhibits the growth of bedaquiline-resistant mutants. We show that HM2-16F weakly inhibits the F₁F_o-ATP synthase, depletes ATP, and affects the entry of acetyl-CoA into the Krebs cycle. HM2-16F synergizes with the cytochrome bcc-aa₃ oxidase inhibitor Q203 (Telacebec) and co-administration with Q203 sterilizes in vitro cultures in 14 days. Synergy with Q203 occurs via direct inhibition of the cytochrome bd oxidase by HM2-16F. This study shows that amiloride derivatives represent a promising discovery platform for targeting energy generation in drug-resistant tuberculosis.

A screening of more than 1,500 drug-resistant strains of Mycobacterium tuberculosis revealed evolutionary patterns characteristic of positive selection for three alanine racemase (Alr) mutations. We investigated these mutations using molecular modeling, in vitro MIC testing, as well as direct measurements of enzymatic activity, which demonstrated that these mutations likely confer resistance to d -cycloserine.

The New Zealand glowworm is the larva of a carnivorous fungus gnat that produces bioluminescence to attract prey. The bioluminescent system of the glowworm is evolutionarily distinct from other well-characterised systems, especially that of the fireflies, and the molecules involved have not yet been identified. We have used high throughput sequencing technology to produce a transcriptome for the glowworm and identify transcripts encoding proteins that are likely to be involved in glowworm bioluminescence. Here we report the sequencing and annotation of the first transcriptome of the glowworm, and a differential analysis of expression from the glowworm light organ compared with non-light organ tissue. The analysis identified six transcripts encoding proteins that are potentially involved in glowworm bioluminescence. Three of these proteins are members of the ANL superfamily of adenylating enzymes, with similar amino acid sequences to that of the luciferase enzyme found in fireflies (31 to 37 % identical), and are candidate luciferases for the glowworm bioluminescent system. The remaining three transcripts encode putative aminoacylase, phosphatidylethanolamine-binding and glutathione S-transferase proteins. This research provides a basis for further biochemical studies into how the glowworm produces light, and a source of genetic information to aid future ecological and evolutionary studies of the glowworm.

The mycobacterial cell wall frequently has been used as a target for drug development, andd-glutamate, synthesized by glutamate racemase (MurI), is an important component of peptidoglycan. While the essentiality of themurIgene has been shown in several bacterial species, includingEscherichia coli,Bacillus anthracis, andStreptococcus pneumoniae, studies in mycobacteria have not yet provided definitive results. This study aimed to determine whethermurIis indeed essential and can serve as a possible target for structure-aided drug design. We have achieved this goal by creating a ΔmurIstrain ofMycobacterium smegmatis, a close relative ofMycobacterium tuberculosis. The deletion of themurIgene inM. smegmatiscould be achieved only in minimal medium supplemented withd-glutamate, demonstrating that MurI is essential for growth and that glutamate racemase is the only source ofd-glutamate for peptidoglycan synthesis inM. smegmatis.