Characterization of Mycobacterium tuberculosis Cor, a Protein Essential for Carbon Monoxide Resistance and Pathogenesis
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Tuberculosis, caused by Mycobacterium tuberculosis, is global health burden as it remains one of the most devastating human infectious diseases causing two million deaths annually and latently infecting a third of the world's population. We previously demonstrated that M. tuberculosis induces an enzyme, heme oxygenase (HO1), that produces carbon monoxide (CO) gas and that M. tuberculosis adapts its transcriptome during CO exposure. We now demonstrate that M. tuberculosis carries a novel resistance gene to combat CO toxicity. We screened an M. tuberculosis transposon library for CO-susceptible mutants and found that disruption of Rv1829 (carbon monoxide resistance, cor) leads to marked CO sensitivity. Heterologous expression of Cor and Cor homologue from Thermotoga maritima (TM0160) in Escherichia coli rescued it from CO toxicity, suggesting a conserved function across diverse microbial species. Importantly, the virulence of the cor mutant is attenuated in a mouse model of tuberculosis. Thus, Cor is necessary and sufficient to protect bacteria from host-derived CO. Evolutionary modeling suggested that Cor forms an active site, thus we predicted that Cor has enzymatic activity. To determine potential Cor enzymatic activity, we profiled the mycobacterial metabolome using liquid-chromatography mass spectrometry and, in vitro, monitored metabolite fluctuations in the presence and absence of recombinant Cor. Our activity-based metabolomic profiling data showed the accumulation of phosphatidic acid and multiple phospholipids in the presence of Cor, indicating its potential role in catalyzing a reaction involved in phospholipid biosynthesis. Our in vivo metabolomic analysis of a Cor mutant strain showed that in the presence of CO, levels of dihydroxyacetone phosphate is increased, whereas levels of glycerol-3-phosphate is reduced, which is required for phospholipid biosynthesis. Furthermore, we identified key metabolic enzymes in Mtb that physically interact with Cor using bioinformatics and immunoprecipitation techniques. Specifically, Cor interacted with glycerol-3-phosphate dehydrogenase 2, an enzyme that catalyzes the interconversion of glycerol-3-phosphate to dihydroxyacetone phosphate. Taken together, this represents the first report of a role for HO1-derived CO in controlling infection of an intracellular pathogen and the first identification of a CO resistance gene in a pathogenic organism, which may have a critical role in phospholipid biosynthesis.
Pages 1-101 of the dissertation are incorrectly numbered as pages 2-102.
Drug Resistance, Bacterial