Isoprenoid biosynthesis inhibition disrupts Rab5 localization and food vacuolar integrity in Plasmodium falciparum - PubMed (original) (raw)

Isoprenoid biosynthesis inhibition disrupts Rab5 localization and food vacuolar integrity in Plasmodium falciparum

Ruth Howe et al. Eukaryot Cell. 2013 Feb.

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.

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Figures

Fig 1

Fig 1

Bypass of electron transport does not confer fosmidomycin resistance. Shown are data for growth inhibition by the isoprenoid biosynthesis inhibitor fosmidomycin (FSM) in P. falciparum parasites (control) compared to parasites that heterologously express yeast dihydroorotate dehydrogenase, which does not require ubiquinone (control + yDHODH). Results are the means and standard deviations from three independent biological replicates.

Fig 2

Fig 2

Fosmidomycin treatment inhibits protein prenylation. (A) Antifarnesyl immunoblot of extracts with or without treatment with 5 μM fosmidomycin (FSM) for 24 h. (B) Blot from panel A reprobed with antibodies to Pf-EIF1α to indicate equivalent protein loading. Results are representative of at least three independent biological replicates.

Fig 3

Fig 3

Mislocalization of Rab5 proteins by fosmidomycin treatment. (A and B) Confocal immunofluorescence with either anti-PfRab5a (A) or anti-PfRab5c (B) antibody in untreated parasites (control) compared to fosmidomycin-treated (+FSM) and fosmidomycin- and geranylgeraniol-treated (+FSM +GG-ol) parasites. FITC, fluorescein isothiocyanate. (C) Blinded scoring of >50 cells under each condition for severity of mislocalization of Rab5 (1, typical cellular punctae within parasite; 2, partial mislocalization to erythrocyte or erythrocyte membrane; 3, severe mislocalization to erythrocyte or erythrocyte membrane). ∗, P < 0.001 compared to untreated conditions.

Fig 4

Fig 4

Fosmidomycin treatment causes growth arrest of malaria parasites during schizogony. Shown are Giemsa-stained light micrographs of synchronized ring-stage parasites at the indicated time points of culture for comparison of untreated parasites (A) to those treated with 5 μM fosmidomycin (B) or 5 μM fosmidomycin plus 5 μM the downstream isoprenol geranylgeraniol (C). Images of representative cells are indicative of results from at least five independent biological replicates.

Fig 5

Fig 5

Fosmidomycin-treated parasites arrest during S phase. Shown are proportions of cells with unreplicated DNA (pre-S phase, morphologically early-ring-stage parasites, 12 h after invasion) compared to those in which DNA replication has begun (S phase), as determined by acridine orange staining and flow cytometric evaluation. Untreated parasites (A) are compared to fosmidomycin-treated parasites (B), fosmidomycin- and geranylgeraniol-treated parasites (C), and parasites with geranylgeraniol treatment alone (D). The cell cycle duration under these conditions is approximately 48 h; untreated parasites return to pre-S phase at 36 h.

Fig 6

Fig 6

Food vacuolar defect in fosmidomycin- and wortmannin-treated parasites. (A to C) Transmission electron microscopic evaluation of control parasites (A) compared to parasites treated for 24 h with either the isoprenoid inhibitor fosmidomycin (B) or the PI3-K inhibitor wortmannin (C). On the right is a magnified view of a hemozoin-containing FV. (D) Scoring of electron micrographs of control versus fosmidomycin (FSM)- and wortmannin-treated cells. Abnormal FVs were defined as FVs that either lacked an FV membrane or contained more than one discontiguous membrane-bound collection of hemozoin (n > 25 under each condition). ∗, P < 0.001 compared to untreated conditions (Fisher's 2-tailed test).

Fig 7

Fig 7

Loss of food vacuolar integrity upon fosmidomycin and wortmannin treatment. Shown is the confocal fluorescence microscopic localization of a plasmepsin II-GFP (P2-GFP) construct (left) or live fluorescence imaging of Lysotracker Red-stained malaria parasites (right). Control parasites (A) are compared to parasites treated for 24 h with either fosmidomycin (B), fosmidomycin plus geranylgeraniol (C), or wortmannin (D). Images are representative of at least three independent biological experiments. Visualization of PMII-GFP in FSM- and wortmannin-treated parasites required higher detector gain levels, resulting in increased observed erythrocyte autofluorescence.

Fig 8

Fig 8

Apicoplast and parasitophorous vacuolar targeting in fosmidomycin- and wortmannin-treated parasites. Shown is live-cell fluorescence of malaria parasites that express either the leader sequence (ACPL-GFP) (A) or signal sequence (ACPs-GFP) (B) from P. falciparum acyl carrier protein, fused to GFP, which traffic to the apicoplast or parasitophorous vacuole, respectively (27). Untreated parasites (control) are compared to fosmidomycin (+FSM)- and wortmannin (+wort)-treated parasites. Images are representative of at least three independent biological experiments.

Fig 9

Fig 9

Model of fosmidomycin effects on malaria parasites. Fosmidomycin blocks isoprenoid biosynthesis and causes a defect in growth and vesicular trafficking to the food vacuole (FV). These effects are rescued by geranylgeraniol (GG-ol), indicating that the essential isoprenoids in malaria are metabolically derived from geranylgeranyl pyrophosphate (GG-PP). GG-PP is the substrate for geranylgeranyltransferase (GGTase), which modifies the endocytosis regulator Rab5 (a small GTPase) in most eukaryotes. Blocking of isoprenoid biosynthesis (with fosmidomycin) decreases protein prenylation, causes Rab5 mislocalization, and alters FV morphology. IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate.

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