Characterization of the Plasmodium falciparum and P. berghei glycerol 3-phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast.

Melanie J Shears,Vanessa Mollard,Geoffrey I Mcfadden,James I Macrae,Angelika Sturm,Manuel Llinás,Lindsey M Orchard,Cyrille Y Botté,Christopher D Goodman,Malcolm J Mcconville

doi:10.1111/cmi.12633

Abstract

SummaryMalaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti‐malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3‐phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N‐terminal targeting sequence to GFP and 3′ tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site‐directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3‐phosphate acyltransferase in malaria parasites.

Highlights

Malaria is caused by Plasmodium parasites, which have a complex life cycle involving multiple stages in the human and mosquito hosts (Greenwood et al, 2008; Aly et al, 2009)
A modest but significant decrease was observed between Pb apiG3PAT (À) and wild type at 66 h, with the mean proportion of merozoite surface protein 1 (MSP1) positive parasites found to be 64% and 78%, respectively (Fig. 9, Supplementary Fig. 8). As this equated to only an 18% reduction relative to wild type, the MSP1 phenotype observed for Pb apiG3PAT (À) parasites was still far more mild than reported for the P. berghei FASII null mutants (Yu et al, 2008; Annoura et al, 2012). This suggested that Pb apiG3PAT and FASII likely differed in their contribution to the synthesis of the MSP1 GPI anchor, and that the requirement for the enzyme for late liver stage development instead likely reflects its role in the synthesis of other essential lipid species
We find that disruption of the enzyme largely mirrors the phenotype of the FASII null mutants in each species, with no apparent defect observed for the Pf apiG3PAT mutant in blood stage parasites, but severe defects observed for the Pb apiG3PAT mutant in growth and merozoite formation in the late liver stage that result in markedly decreased parasite infectivity

Summary

Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti-malarial drug target, but surprisingly little is known about its role in lipid metabolism. We characterize the apicoplast glycerol 3-phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei

Introduction

Results and discussion

Conclusions

Experimental Procedures