Abstract
Several enzymes involved in central carbon metabolism and gluconeogenesisplay a critical role in survival and pathogenesis of Mycobacterium tuberculosis (Mtb). The only known functional fructose 1,6-bisphosphatase (FBPase) in Mtb is encoded by the glpX gene and belongs to the Class II sub-family of FBPase. We describe herein the generation of a ΔglpX strain using homologous recombination. Although the growth profile of ΔglpX is comparable to that of wild type Mtb when grown on the standard enrichment media, its growth is dysgonic with individual gluconeogenic substrates such as oleic acid, glycerol and acetate. In mice lung CFU titers of ΔglpX were 2–3 log10 lower than the wild-type Mtb strain. The results indicate that glpX gene encodes a functional FBPase and is essential for both in vitro and in vivo growth and survival of Mtb. Loss of glpX results in significant reduction of FBPase activity but not complete abolition. These findings verify that the glpX encoded FBPase II in Mtb can be a potential target for drug discovery.
Highlights
Mycobacterium tuberculosis (Mtb) grows on a variety of substrates in vitro but mounting evidence indicates that during infection most of its energy is derived from fatty acids [1, 2]
The growth profile of ΔglpX is similar to that of WT Mtb when grown in 7H9 medium with OADC enrichment, plus glycerol and Tween 80, a surfactant which can furnish Mtb with oleic acid through de-esterification [22]
While the ability of ΔglpX to grow on glycerol or acetate as a sole carbon source was severely compromised as compared to WT Mtb, ΔglpX grew twice as fast as WT Mtb on dextrose as a sole carbon source
Summary
Mtb grows on a variety of substrates in vitro but mounting evidence indicates that during infection most of its energy is derived from fatty acids [1, 2]. When bacterial metabolism is fueled by fatty acids, synthesis of sugars from intermediates of the TCA cycle ( the glyoxylate shunt) become important for growth and persistence [3,4,5,6]. Phosphoenolpyruvate carboxykinase (PEPCK), the enzyme linking the TCA cycle and gluconeogenesis, catalyses the reversible decarboxylation and phosphorylation of oxaloacetate (OAA) to form phosphoenolpyruvate (PEP). The PEPCK-encoding gene pckA is up-regulated by acetate or palmitate, but down-regulated by glucose. Deletion of the pckA gene of Mycobacterium bovis BCG led to a reduction in the capacity of the bacteria to infect and survive in macrophages [8]. PEPCK plays a pivotal role in the PLOS ONE | DOI:10.1371/journal.pone.0138436 September 23, 2015
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