Isoprenoid Biosynthesis Inhibition Disrupts Rab5 Localization and Food Vacuolar Integrity in Plasmodium falciparum

Abstract

The antimalarial agent fosmidomycin is a validated inhibitor of the nonmevalonate isoprenoid biosynthesis (methylerythritol 4-phosphate [MEP]) pathway in the malaria parasite, Plasmodium falciparum. Since multiple classes of prenyltransferase inhibitors kill P. falciparum, we hypothesized that protein prenylation was one of the essential functions of this pathway. We found that MEP pathway inhibition with fosmidomycin reduces protein prenylation, confirming that de novo isoprenoid biosynthesis produces the isoprenyl substrates for protein prenylation. One important group of prenylated proteins is small GTPases, such as Rab family members, which mediate cellular vesicular trafficking. We have found that Rab5 proteins dramatically mislocalize upon fosmidomycin treatment, consistent with a loss of protein prenylation. Fosmidomycin treatment caused marked defects in food vacuolar morphology and integrity, consistent with a defect in Rab-mediated vesicular trafficking. These results provide insights to the biological functions of isoprenoids in malaria parasites and may assist the rational selection of secondary agents that will be useful in combination therapy with new isoprenoid biosynthesis inhibitors.

Isoprenoid Biosynthesis in Plasmodium falciparum

“…The P. falciparum geranylgeranyltransferase substrates PfRab5a and PfRab5c mislocalize from hemoglobincontaining vesicles to the host cell membrane upon treatment with fosmidomycin, and this mislocalization correlates with changes to food vacuole morphology and integrity. Proper localization is restored by geranylgeraniol supplementation (39). Geranylgeraniol supplementation also substantially rescues growth inhibition by fosmidomycin, suggesting that geranylgeranylation may be the only essential form of protein prenylation in P. falciparum (35,38).…”

“…Similar to metabolic labeling, probing for prenylation using a prenylation-specific antibody identifies proteins of 21 to 24 kDa and 50 kDa (39,68). Prenylation of these targets is reduced in fosmidomycin-treated parasites, confirming that prenyl groups are indeed products of the MEP pathway.…”

“…In contrast, fosmidomycin treatment inhibits intraerythrocytic growth of P. falciparum during the first cell cycle. Interestingly, fosmidomycin-treated parasites develop within the red blood cell, begin hemoglobin digestion, and initiate DNA replication prior to cell cycle arrest as multinucleate schizonts (39). The requirement of the new permeability pathways mentioned above for fosmidomycin import into P. falciparum cells may explain this delayed action, as these pathways are not fully developed until the trophozoite stage (31,32).…”

“…The 50% inhibitory concentration (IC 50 ) for fosmidomycin increases 10-fold when the medium is supplemented with farnesol or geranylgeraniol (35,38). Supplementation of the medium with geranylgeraniol also rescues protein mislocalization and the organelle disruption effects of fosmidomycin treatment (39). Treatment with high concentrations of fosmidomycin is not completely rescued by prenyl alcohol supplementation, perhaps due to the toxicity of these compounds at high concentrations (40).…”

Isoprenoid Precursor Biosynthesis Is the Essential Metabolic Role of the Apicoplast during Gametocytogenesis in Plasmodium falciparum

“…Once IPP and DMAPP are synthesized and transported out of the apicoplast, a myriad of isoprenoid products with different cellular localization and functions are synthesized in the malaria parasite. Many isoprenoid products have been identified as being present in the intraerythrocytic asexual stages of P. falciparum, such as ubiquinones and menaquinone (32,33,56), carotenoids and vitamin E (57-59), dolichols (50,60,61), and prenylated proteins (60,(62)(63)(64)(65). The presence and function of these isoprenoids in gametocytes remain to be confirmed and are under investigation.…”