Authors

T. D. Sirakova; C. Deb; J. Daniel; H. D. Singh; H. Maamar; V. S. Dubey;P. E. Kolattukudy

Comments

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Abbreviated Journal Title

PLoS One

Keywords

ACYL-COA REDUCTASE; FATTY ALCOHOL; DIACYLGLYCEROL ACYLTRANSFERASES; GENE; TRIACYLGLYCEROL; PURIFICATION; EXPRESSION; STARVATION; ACIDS; BIOSYNTHESIS; Multidisciplinary Sciences

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 C-14-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 Delta fcr1 mutant and partially in the Delta fcr2 mutant. WE synthesis was inhibited in both Delta fcr mutants. The Dfcr mutants exhibited faster growth rates, an increased uptake of C-14-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 Dfcr 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. Citation: Sirakova TD, Deb C, Daniel J, Singh HD, Maamar H, et al. (2012) Wax Ester Synthesis is Required for Mycobacterium tuberculosis to Enter In Vitro Dormancy. PLoS ONE 7(12): e51641. doi:10.1371/journal.pone.0051641

Journal Title

Plos One

Volume

7

Issue/Number

12

Publication Date

1-1-2012

Document Type

Article

Language

English

First Page

15

WOS Identifier

WOS:000312386800058

ISSN

1932-6203

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