Localization of ferrochelatase in Plasmodium falciparum

Abstract

Our previous studies have demonstrated de novo haem biosynthesis in the malarial parasite (Plasmodium falciparum and P. berghei). It has also been shown that the first enzyme of the pathway is the parasite genome-coded ALA (delta-aminolaevulinate) synthase localized in the parasite mitochondrion, whereas the second enzyme, ALAD (ALA dehydratase), is accounted for by two species: one species imported from the host red blood cell into the parasite cytosol and another parasite genome-coded species in the apicoplast. In the present study, specific antibodies have been raised to PfFC (parasite genome-coded ferrochelatase), the terminal enzyme of the haem-biosynthetic pathway, using recombinant truncated protein. With the use of these antibodies as well as those against the hFC (host red cell ferrochelatase) and other marker proteins, immunofluorescence studies were performed. The results reveal that P. falciparum in culture manifests a broad distribution of hFC and a localized distribution of PfFC in the parasite. However, PfFC is not localized to the parasite mitochondrion. Immunoelectron-microscopy studies reveal that PfFC is indeed localized to the apicoplast, whereas hFC is distributed in the parasite cytoplasm. These results on the localization of PfFC are unexpected and are at variance with theoretical predictions based on leader sequence analysis. Biochemical studies using the parasite cytosolic and organellar fractions reveal that the cytosol containing hFC accounts for 80% of FC enzymic activity, whereas the organellar fraction containing PfFC accounts for the remaining 20%. Interestingly, both the isolated cytosolic and organellar fractions are capable of independent haem synthesis in vitro from [4-14C]ALA, with the cytosol being three times more efficient compared with the organellar fraction. With [2-14C]glycine, most of the haem is synthesized in the organellar fraction. Thus haem is synthesized in two independent compartments: in the cytosol, using the imported host enzymes, and in the organellar fractions, using the parasite genome-coded enzymes.

Figure 1. Preparation of PfΔFC

RNA from the parasite was subjected to reverse transcriptase–PCR amplification with FC-specific primers, and the full-length cDNA obtained was sequenced. (A) Lanes: 1, PCR-amplified 750 bp fragment; 2, standards (1 kb ladder). (B) SDS/PAGE analysis of the C-terminal 32.5 kDa protein fragment expressed in E. coli and purified on an Ni²⁺-nitrilotriacetate column: Lanes: 1, Coomassie stain; 2, Western-blot analysis with anti-histidine tag antibodies. (C) Northern-blot analysis of parasite RNA with labelled PfΔFC cDNA showing an RNA band at 1.2 kb.

Figure 2. Specificity of anti-FC antibodies

Western-blot analysis was performed with antibodies against PfΔFC and hFC using purified FC preparations as well as parasite and red cell lysates. (A) Lanes: 1, 2, 5 and 6, Western-blot analysis with PfΔFC antibodies. (B) Lanes: 1, 2, 5 and 6, Western-blot analysis with hFC antibodies; 1, PfΔFC; 2, partially purified PfFC (top) and hFC (bottom); 3 and 5, parasite lysate; 4 and 6, red blood cell lysate; 3 and 4, preimmune sera from mice (top) and rabbit (bottom) controls.

Figure 3. Immunoprecipitation and SDS/PAGE analysis of [³⁵S]methionine-labelled P. falciparum in culture

(A) PfΔFC antibodies. (B) Preimmune sera; 1, organellar fraction; 2, cytosolic fraction.

Figure 4. Immunofluorescence analysis of FC localization in P. falciparum-infected red cells

(A) Smear analysis with PfΔFC antibodies followed by TRITC-conjugated (red) secondary antibodies; 1, bright field (the arrow indicates hemozoin pigment); 2, Hoescht stain indicating two parasites; 3, localized red fluorescence due to PfFC in the parasite. (B) Smear analysis with hFC antibodies followed by FITC-conjugated (green) secondary antibodies; 1, bright field (the arrow indicates hemozoin pigment); 2, Hoescht stain indicating two parasites; 3, broad green fluorescence covering the entire parasite cytoplasm due to hFC. (C) Analysis of the co-localization of PfFC with PfHsp60 using FITC-conjugated (green) secondary antibodies for the former and TRITC-conjugated (red) secondary antibodies for the latter; 1, bright field (dark spots represent the hemozoin pigment); 2, red fluorescence in the parasite due to PfHsp60; 3, green fluorescence in the parasite due to PfFC; 4, merge of 2 and 3 along with Hoescht stain (blue) showing lack of co-localization; 5, projection of two parasites showing lack of co-localization. (D) Analysis of co-localization of PfFC with MitoTracker; 1, bright field (dark spots represent the hemozoin pigment); 2, red fluorescence in the parasite due to MitoTracker; 3, green fluorescence in the parasite due to PfFC; 4, merge of 2 and 3 showing lack of co-localization; 5, projection of a single parasite showing lack of co-localization.

Figure 5. Immunoelectron-microscopic analysis of FC localization in parasite-infected red cells

The secondary antibodies were conjugated to 10 or 20 nm gold particles. (A) 1 and 3, uninfected and infected red cells probed with hFC antibodies respectively; 2, uninfected red cell probed with PfΔFC antibodies. (B) Three different fields showing discrete gold particles in typical apicoplast structures with multiple membranes. 1, the whole parasite; 2 (enlarged part from 1) and 3, elongating apicoplasts in a later-stage trophozoite; 4, a circular apicoplast in an early-stage tropozoite. The signals are localized close to the membrane. Scale bar, 100 μm. A_P, apicoplast; M, mitochondrion; R, red cell; MC, Maurer's cleft; P, parasite; Pv, parasite food vacuole; Pc, parasite cytosol.

Figure 6. Western-blot analysis of the parasite organellar and cytosolic fractions

Antibodies to (A) PfPP2C1, (B) PfNT1, (C) PfHsp60, (D) PfALAD and (E) PfFC were used. 1, Organellar fraction; 2, cytosolic fraction. The total parasite pellet isolated from 2 ml of culture (10% parasitaemia) was fractionated into organellar and cytosolic fractions and the entire fractions were analysed by SDS/PAGE for Western-blot analysis.

Figure 7. Incorporation of [2-¹⁴C]glycine and [4-¹⁴C]ALA into haem and the assay of FC enzymic activity in the organellar and cytosolic fractions

The cytosolic (shaded bars) or solubilized organellar (empty bars) fractions were incubated with [2-¹⁴C]glycine or [4-¹⁴C]ALA to measure incorporation into haem. FC enzymic activity was assayed in these fractions using ⁵⁹FeCl₃ and protoporphyrin as substrates. Results are expressed in terms of radioactivity in haem for the total cytosolic or solubilized organellar fraction obtained from 6 ml of parasite culture. (A) FC enzymic activity; 1, heat-denatured parasite fractions (control); 2, total enzymic activity. (B) [2-¹⁴C]Glycine incorporation into haem; 1, [1-¹⁴C]glycine incorporation (control); 2, [2-¹⁴C]glycine incorporation. (C) [4-¹⁴C]ALA incorporation into haem; 1, heat-denatured parasite fractions (control); 2, native parasite fractions; white bar, cytosol; shaded bar, solubilized organellar fraction.