Abstract
Mycobacterium tuberculosis (Mtb) is known to produce wax esters (WE) when subjected to stress. However, nothing is known about the enzymes involved in biosynthesis of WE and their role in mycobacterial dormancy. We report that two putative Mtb fatty acyl-CoA reductase genes (fcr) expressed in E. coli display catalytic reduction of fatty acyl-CoA to fatty aldehyde and fatty alcohol. Both enzymes (FCR1/Rv3391) and FCR2/Rv1543) showed a requirement for NADPH as the reductant, a preference for oleoyl-CoA over saturated fatty acyl-CoA and were inhibited by thiol-directed reagents. We generated Mtb gene-knockout mutants for each reductase. Metabolic incorporation of 14C-oleate into fatty alcohols and WE was severely diminished in the mutants under dormancy-inducing stress conditions that are thought to be encountered by the pathogen in the host. The fatty acyl-CoA reductase activity in cell lysates of the mutants under nitric oxide stress was significantly reduced when compared with the wild type. Complementation restored the lost activity completely in the Δfcr1 mutant and partially in the Δfcr2 mutant. WE synthesis was inhibited in both Δfcr mutants. The Δfcr mutants exhibited faster growth rates, an increased uptake of 14C-glycerol suggesting increased permeability of the cell wall, increased metabolic activity levels and impaired phenotypic antibiotic tolerance under dormancy-inducing combined multiple stress conditions. Complementation of the mutants did not restore the development of antibiotic tolerance to wild-type levels. Transcript analysis of Δfcr mutants showed upregulation of genes involved in energy generation and transcription, indicating the inability of the mutants to become dormant. Our results indicate that the fcr1 and fcr2 gene products are involved in WE synthesis under in vitro dormancy-inducing conditions and that WE play a critical role in reaching a dormant state. Drugs targeted against the Mtb reductases may inhibit its ability to go into dormancy and therefore increase susceptibility of Mtb to currently used antibiotics thereby enhancing clearance of the pathogen from patients.
Highlights
Tuberculosis is a leading cause of preventable deaths [1,2]
We examined whether these previously uncharacterized Mycobacterium tuberculosis (Mtb) gene products, with amino acid sequence similarities to other acyl-CoA reductases, displayed such activity
Mtb is known to accumulate Wax esters (WE) under stress conditions that are thought to be encountered in the host by the pathogen [11]
Summary
Tuberculosis is a leading cause of preventable deaths [1,2]. The emergence of multi-drug resistant and nearly untreatable, extremely drug resistant Mycobacterium tuberculosis (Mtb) strains pose a great threat to public health [3]. The ability of the pathogen to survive in asymptomatic people in a drug-resistant, non-replicating state for decades and the inability of currently available drugs to eliminate the dormant pathogen make cure and eradication of tuberculosis extremely difficult. We showed that triacylglycerol (TAG) constitutes one storage lipid that probably serves as the source of the fatty acids [6,10,11,12]. Wax esters (WE) constitute another form of long term storage lipid used by living organisms including bacteria [13,14,15,16]. Virtually nothing is known about the biosynthesis of WE by Mtb [18], even though such lipids have long been known to be present in this pathogen [19]. The enzymes involved in wax synthesis and the genes that encode them in Mtb have not been identified
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