The metabolic repair enzyme phosphoglycolate phosphatase regulates central carbon metabolism and fosmidomycin sensitivity inPlasmodium falciparum

Abstract

1213 Abstract 14 The asexual blood stages of the malaria parasite, Plasmodium falciparum are highly dependent 15 on glycolysis for ATP synthesis, redox balance and provision of essential anabolic precursors. 16 Recent studies have suggested that members of the haloacid dehalogenase (HAD) family of 17 metabolite phosphatases may play an important role in regulating multiple pathways in P.18 falciparum central carbon metabolism. Here, we show that the P. falciparum HAD protein, 19 phosphoglycolate phosphatase (PfPGP), which is homologous to yeast Pho13 and mammalian 20 PGP, regulates glycolysis in asexual blood stages by controlling intracellular levels of several 21 intermediates and novel end-products of this pathway. Deletion of the P. falciparum pgp gene 22 significantly attenuated asexual parasite growth in red blood cells, while comprehensive 23 metabolomic analysis revealed the accumulation of two previously uncharacterized metabolites, 24 as well as changes in a number of intermediates in glycolysis and the pentose phosphate 25 pathway. The two unknown metabolites were assigned as 2-phospho-lactate and 4-26 phosphoerythronate by comparison of their mass spectra with synthetic standards. 2-Phospho-27 lactate was significantly elevated in wildtype and ∆PfPGP parasites cultivated in the presence of 28 methylglyoxal and D-lactate, but not L-lactate, indicating that it is a novel end-product of the 29 methylglyoxal pathway. 4-Phosphoerythronate is a putative side product of the glycolytic 30 enzyme, glyceraldehyde dehydrogenase and the accumulation of both 4-phosphoerythronate and 31 2-phospho-D-lactate were associated with changes in glycolytic and the pentose phosphate 2 32 pathway fluxes as shown by 13 C-glucose labelling studies and increased sensitivity of the 33 ∆PfPGP parasites to the drug fosmidomycin. Our results suggest that PfPGP contributes to a 34 novel futile metabolic cycle involving the phosphorylation/dephosphorylation of D-lactate as 35 well as detoxification of metabolites, such as 4-phosphoerythronate, and both may have 36 important roles in regulating P. falciparum central carbon metabolism. 3738 Author summary 39 The major pathogenic stages of the malaria parasite, Plasmodium falciparum, develop in red 40 blood cells where they have access to an abundant supply of glucose. Unsurprisingly these 41 parasite stages are addicted to using glucose, which is catabolized in the glycolytic and the 42 pentose phosphate pathways. While these pathways also exist in host cells, there is increasing 43 evidence that P. falciparum has evolved novel ways for regulating glucose metabolism that 44 could be targeted by next-generation of anti-malarial drugs. In this study, we show the red blood 45 cell stages of P. falciparum express an enzyme that is specifically involved in regulating the 46 intracellular levels of two metabolites that are novel end-products or side products of glycolysis.47 Parasite mutants lacking this enzyme are viable but exhibit diminished growth rates in red blood 48 cells. These muta...

Metabolic proof-reading in Plasmodium berghei: essentiality of phosphoglycolate phosphatase

“…Our results are similar and indicate essentiality of PbPGP in asexual stages. This conclusion on the gene essentiality of PbPGP is in agreement with the findings of Dumont et al, on PfPGP where, Δpfpgp parasites show growth defect [16].…”

“…Our results are similar and indicate es-324 sentiality of PbPGP in asexual stages. This conclu-325 sion on the gene essentiality of PbPGP is in agree-326 ment with the findings of Dumont et al, on PfPGP 327where, ∆pfpgp parasites show growth defect(16).328 Further biochemical and structural studies on PGP 329 could pave way for rational design of inhibitors with 330 potent anti-malarial activity. 331 MATERIALS 332 All chemicals, molecular biology reagents, 333 and media components were from Sigma Aldrich, 334 New England Biolabs, Gibco, Invitrogen and US 335 biochemicals, USA; SRL, Spectrochem and Hi-336 media, India.…”

“…The authors speculate that although L-lactate is the major isomer that is produced in the parasite, it is D-lactate that is phosphorylated [16] and accumulates in ΔPfPGP cells. Our results on the purified enzyme clearly show that PbPGP acts only on 2-phospho L-lactate and not on 2-phospho D-lactate ( Fig.…”

“…Recent studies have identified and characterised HADSF members from the apicomplexan parasite, Plasmodium [ 4,10,13,14,15,16]. HADSF members from Plasmodium have been found to be involved in processes that lead to the development of resistance to the drug fosmidomycin, which inhibits isoprenoid biosynthesis [4].…”

“…A large scale study reported by Huang et al, has identified a HADSF member from Salmonella enterica that catalyzes dephosphorylation of more than 100 phosphorylated substrates [5]. This extended substrate specificity is a common observation in HADSF members and often leads to a confounding situation where determining the physiological substrate of such promiscuous enzymes becomes a challenging task.Recent studies have identified and characterised HADSF members from the apicomplexan parasite, Plasmodium [ 4,10,13,14,15,16]. HADSF members from Plasmodium have been found to be involved in processes that lead to the development of resistance to the drug fosmidomycin, which inhibits isoprenoid biosynthesis [4].…”