A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites

Area of Science:

• Biochemistry
• Parasitology
• Drug Discovery

Background:

• The methylerythritol phosphate (MEP) pathway is crucial for producing essential compounds and is a target for antimicrobial drug development.
• Understanding MEP pathway regulation in microorganisms, particularly in the malaria parasite Plasmodium falciparum, is critical but remains incomplete.
• The antimalarial drug fosmidomycin targets deoxyxylulose 5-phosphate reductoisomerase (DXR), an enzyme within the MEP pathway.

Purpose of the Study:

• To investigate the regulatory mechanisms of the MEP pathway in Plasmodium falciparum using a forward genetics approach.
• To identify genetic factors contributing to resistance against the MEP pathway inhibitor fosmidomycin.
• To elucidate the function and structural basis of the identified resistance-conferring protein.

Main Methods:

• Forward genetics screen to identify fosmidomycin-resistant Plasmodium falciparum mutants.
• Genetic analysis of mutations in the PF3D7_1033400 locus, encoding PfHAD1.
• Biochemical characterization of PfHAD1 enzyme activity, including substrate specificity and structural analysis.
• Metabolomic analysis of MEP pathway intermediates in wild-type and PfHAD1-deficient parasites.

Main Results:

• Fosmidomycin-resistant P. falciparum exhibited mutations in the PF3D7_1033400 locus, encoding the haloacid dehalogenase (HAD)-like protein PfHAD1.
• Loss-of-function PfHAD1 alleles were necessary for fosmidomycin resistance.
• PfHAD1 dephosphorylates various sugar phosphates, including glycolytic intermediates.
• Parasites lacking PfHAD1 showed elevated levels of MEP pathway metabolites, specifically the DXR substrate deoxyxylulose 5-phosphate.
• Structural analysis provided insights into the loss-of-function mutations.

Conclusions:

• PfHAD1 acts as a negative regulator of the MEP pathway by controlling substrate availability.
• The dephosphorylation activity of PfHAD1 influences the flux through the MEP pathway.
• PfHAD1 is a key factor in regulating fosmidomycin sensitivity in Plasmodium falciparum.
• The discovery of PfHAD1's role suggests that other HAD family proteins may also regulate the MEP pathway in diverse organisms.