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The bacterium responsible for tuberculosis (TB), mycobacterium tuberculosis
(Mtb), is notoriously difficult to kill. The most commonly used antibiotics, rifampicin and isoniazid, need to be used for extended periods of time (typically 6-24 months) to effectively eliminate infection. In addition, emergence of antibiotic-resistant strains is an increasing problem.
Researchers at Albert Einstein College of Medicine of Yeshiva University have now identified a new biochemical pathway in Mtb and two novel ways to kill the bacterium. The pathway involves four enzymatic steps in the conversion of the disaccharide, trehalose, to α-glucan mediated by TreS, Pep2, GlgE (which has been identified as a maltosyltransferase that uses maltose 1-phosphate) and GlgB. Focusing on GlgE, the researchers found that blocking the enzyme induced toxic accumulation of maltose-1-phosphate, killing the bacteria in vitro and in a mouse model of infection. Inhibition of another enzyme in the pathway was non-lethal until combined with inactivation of Rv3032, a glucosyltransferase involved in a distinct α-glucan pathway. Inhibition of Rv3032 alone was also non-lethal to the bacteria.
The research validates inhibition of GlgE as therapy for TB but also highlights the potential for targeting two α-glucan pathways – a strategy that potentially leads to reduced incidence of resistance. Both approaches are also distinct from the mechanisms of currently used antibiotics.
The study is published in Nature Chemical Biology.
The ability of Mycobacterium tuberculosis (MTB) to establish latent infection poses significant problems in the treatment of TB, but a team of scientists led by researchers at Weill Cornell Medical College have now discovered compounds that are able to kill the bacterium in its dormant state. Unlike most other antibacterials, which inhibit the synthesis of bacterial proteins, the newly identified compounds inhibit protein degradation. Oxathiazol-2-ones, such as GLR5 and HT1171, were shown to selectively and irreversibly inhibit the proteasome of MTB, whilst having little effect on the human proteasome and showing no apparent toxicity to mammalian cells. The compounds were further shown to act as suicide-substrates which cyclocarbonylate the active site threonine of the bacterial proteasome. X-ray crystallographic studies revealed major conformational changes that protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Some of the many amino acid residues involved in the conformational changes are remote from the active site pocket and are different from those in the human proteasome, which may account for the selectivity of oxathiazol-2-ones for the bacterial system.
The study was published online on September 16th in the journal Nature.
The World Health Organisation estimates that one third of the world’s population is latently infected with Mycobacterium tuberculosis (MTB), and that ten per cent of infected individuals will develop active disease. Current treatments for tuberculosis are effective only during active infection, and the emergence of drug-resistant strains of MTB is compromising the efficacy of existing drugs. Nicotinamide adenine dinucleotide (NAD+) synthetase is an attractive target for control of MTB since the enzyme is essential for survival of both active and latent mycobacteria, but drug discovery efforts against this enzyme have so far been hampered by a lack of structural information.
University of Maryland scientists have now characterised the structure and mechanism of the MTB synthetase and hope that their work will facilitate the discovery of new drugs that will be able to combat both latent and active MTB infections. All living cells need the coenzyme, NAD+, which regulates many physiological processes including redox reactions. A number of biochemical pathways are able to synthesise NAD+ but, since MTB has only two pathways – both involving NAD+ synthetase – and humans have pathways that avoid NAD+ synthetase, an inhibitor of this enzyme should be effective against MTB but have minimal side effects.
The MTB NAD+ synthetase is a multifunctional enzyme which catalyzes the ATP-dependent formation of NAD+ at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. The study, which is published in full in Nature Structural & Molecular Biology, revealed a homooctameric subunit organization, suggesting a tight dependence of catalysis on the quaternary structure.
Tuberculosis is a major global cause of death and disease, with around one third of the world’s population believed to be infected with the M. Tuberculosis bacterium. Tuberculosis is known to have infected mankind since ancient times, and a new analysis of bone samples from the now submerged site of Atlit-Yam in the Eastern Mediterranean places the origins of human tuberculosis at least 9000 years ago. The emergence of infectious human diseases has been linked to changes in population density that occurred as a result of moving from a hunter-gatherer lifestyle to settled farming communities. Atlit-Yam is one of the earliest villages with evidence of agriculture and animal husbandry, and is believed to have been submerged shortly after abandonment, thus providing excellent conditions for preservation.
Bone samples from a woman and child, with skeletal changes consistent with a diagnosis of tuberculosis, were examined for evidence of M. Tuberculosis infection. Rigorous precautions were taken to prevent contamination, and independent centres were used to confirm the authenticity of findings. Analysis of DNA and bacterial cell wall lipid biomarkers confirmed that both woman and child had been infected with the modern strain of M. Tuberculosis, with evidence suggesting that the child had been infected shortly after birth. The fact that the 9000-year old samples closely resemble today’s prevalent strains suggests that tuberculosis has infected humans far longer than previously thought.
Since no evidence was found for M. Bovis, the form of the bacterium that infects cattle, the study also supports the current view that M. Tuberculosis did not evolve from M. Bovis.