Ketogenesis: An Achilles' heel of starvation-selected persistent mycobacteria

Mycobacterial pathogens adapt to environmental stresses such as nutrient deprivation by entering a non-replicative antibiotic-tolerant state of persistence. Using a biochemically-validated data-driven approach, we identified an adaptive metabolic network underlying the mycobacterial response to starvation in M. tuberculosis, M. bovis BCG and M. smegmatis. All three species show a strong Mg+2-dependence for surviving complete nutrient deprivation, accompanied by a broad phenotypic antibiotic resistance. Multivariate analysis of RNA-seq, metabolic phenotyping and biochemical data revealed substantial metabolic remodelling involving a shift to triacylglycerol utilization with adaptation to the consequent ketoacidosis by upregulation of cytochrome P450s. Paradoxically, the ketosis-driven P450 upregulation generated substantial levels of reactive oxygen species (ROS) yet conferred hypersensitivity to killing by hydrogen peroxide-induced inactivation of the P450s that reduced ROS levels. This emergent property of starvation-induced mycobacterial persistence represents a potentially exploitable vulnerability. Profiles of Mycobacterium bovis BCG coding RNA, enriched through the depletion of rRNAs and tRNAs, were generated by deep sequencing, in biological triplicate, using Illumina MiSeq as bacterial cultures were starved and subsequently replenished with nutrients.