Fig 1.
Untargeted metabolomic analysis of delayed death-parasites indicates isoprenoid defect and decreased haemoglobin turnover.
(A) Schematic of delayed death in P. falciparum. During the first IDC, parasites develop over approximately 48 hours from the ring-stage into a metabolically active trophozoite, before dividing into multiple daughter merozoites by schizogony. Parasites treated with an apicoplast inhibitor during this first IDC successfully complete schizogony, but the daughter merozoites inherit a defective apicoplast. The lethal effect of the drug manifests only after invasion of a new host cell, in which parasites fail to complete a second IDC. Delayed-death parasites in this study were sampled for metabolomic analysis by LC-MS at the indicated time intervals post drug administration. (B) Heat map summarising the metabolic effect of the delayed-death inhibitor indolmycin (50 μM) on P. falciparum-infected RBC cultures. Total metabolite pools (measured as ion counts) across two IDCs were determined relative to untreated controls. Samples were collected for analysis at 30, 58, 68, 78, and 88 hours post drug administration, equivalent to 30 hpi in the first IDC and then 14, 24, 34, and 44 hpi in the second IDC. Changes in total metabolite pools are expressed as log2 ratios of indolmycin-treated cultures compared to untreated controls. Data are presented as the means of three independent experiments. See S1 Data for raw metabolomics data underlying heat map. hpi, hours post invasion; IDC, intraerythrocytic developmental cycle; LC-MS, liquid chromatography mass spectrometry; RBC, red blood cell.
Fig 2.
Apicoplast inhibitors decrease global parasite prenylation in the second IDC after treatment.
(A) Immunoblot analysis of parasites treated with indolmycin (50 μM) with or without polyprenol rescue (5 μM GGOH), as indicated. Parasite lysates were collected after saponin isolation during their second IDC, and immunoblots were probed using anti-farnesyl (1:2,000) to analyse global parasite prenylation. Prenylated species are annotated as putative Rabs (23–27 kD) or HSP40 (40 kD) (assignment based on published P. falciparum prenylomes) [27,28]. Anti-Bip (1:1,000) were used as a loading control with an expected size of 65 kD. Immunoblot is representative of four independent experiments. (B) Quantification of the 27 kD Prenylated Rabs, normalised to average density of untreated. Data are presented as the means of four independent experiments ± SD. ***P < 0.001, two-tailed Student t test. See S2 Data for numerical data underlying figure. GGOH, geranylgeraniol; IDC, intraerythrocytic developmental cycle; ns, not significant.
Fig 3.
Polyprenol supplementation with GGOH protects parasites from the effect of apicoplast inhibitors in the second IDC after treatment.
(A) Dose-response curve from SYBR-Green susceptibility assay determined 48, 96, and 144 hours post indolmycin treatment, with polyprenol (5 μM GGOH) or isoprenoid (200 μM IPP) supplementation as indicated. Indolmycin causes a delayed-death effect (inhibition at 96 but not 48 hours) that is rescued by GGOH or IPP supplementation. Inhibition at 144 hours is rescued by IPP but not GGOH supplementation. Data are presented as the means of three independent experiments ± SEM. See S2 Data for numerical data underlying figure. (B) Summary table of EC50s for delayed-death antimalarials indolmycin, clindamycin, and chloramphenicol, determined at 48, 96, and 144 hours post treatment, with polyprenol (5 μM GGOH) or isoprenoid (200 μM IPP) rescue as indicated. Data are presented as the means of three independent experiments ± SD. *P < 0.05, two-tailed Student t test. (C) Thin-blood smears were collected for Giemsa-microscopy every 6 hours during the second IDC following indolmycin treatment. Scale bar = 5 μm. GGOH, geranylgeraniol; IDC, intraerythrocytic developmental cycle; IPP, isopentenyl pyrophosphate; ns, not significant.
Fig 4.
Indolmycin treated parasites aberrantly internalise F-dextran from preloaded RBCs in the second IDC after treatment.
Uninfected RBCs were preloaded with F-dextran by gentle hypotonic lysis and resealing. Following this, enriched and synchronised schizont-stage parasites were added to the loaded RBCs and merozoites were allowed to reinvade. Newly invaded ring-stage parasites were treated with indolmycin (50 μM), with and without polyprenol (5 μM GGOH) rescue as indicated. (A) Representative live-cell images taken 72–78 hours after drug administration are shown (equivalent to 28–32 hpi in the second IDC after treatment). F-dextran, green signal; Hoechst: parasite nuclei, blue signal; merge: BF and blue signal. Scale bar = 5 μm. (B) The number of F-dextran compartments per iRBC were scored in three independent experiments. **P < 0.01, one-tailed Student t test. Scatter dot plot shows error bars with mean ± SD. See S2 Data for numerical data underlying figure. BF, bright field; F-dextran; fluorescein dextran; GGOH, geranylgeraniol; hpi, hours post invasion; IDC, intraerythrocytic developmental cycle; ns, not significant; RBC, red blood cell.
Fig 5.
Aberrant morphology of intracellular structures is prenylation dependent in delayed-death parasites.
Parasites were treated with indolmycin (50 μM), with and without polyprenol rescue (5 μM GGOH) as indicated. Enriched trophozoite-stage parasites were collected for reduced osmium fixation 72–78 hours post drug administration (equivalent to 28–32 hpi in the second IDC after treatment). (A) Representative images (top-down and cross-sectional), from SEM of each condition: untreated, indolmycin treated (+ indolmycin), and indolmycin treated with polyprenol rescue (+ indolmycin + GGOH). Structures indicated are N, DV, and CI. Scale bar = 3 μm. (B) 3D-rendered iRBCs using serial block-face scanning electron microscopy. Indicated compartments are RBC (white), parasite (blue), CIs (yellow), and DV (red). Scale bar = 3 μm. (C) Parasites were scored for number of DV compartments relative to number of nuclei in each treatment condition. Untreated: 1 nucleus n = 18, 2 nuclei n = 34, ≥ 3 nuclei n = 49; + indolmycin: 1 nucleus n = 16, 2 nuclei n = 29, ≥ 3 nuclei n = 14; + indolmycin + GGOH 1 nucleus n = 17, 2 nuclei n = 19, ≥ 3 nuclei n = 19. Scatter dot blots show error bars with mean ± SD. (D) The total volume (μm3) of the DVs and CIs were measured in each treatment condition. Bar graphs show error bars with mean ± SD. See S2 Data for numerical data underlying figure. CI, cytostomal invagination; DV, digestive vacuole; GGOH, geranylgeraniol; hpi, hours post invasion; IDC, intraerythrocytic developmental cycle; N, nucleus; RBC, red blood cell.
Fig 6.
Rab5a shows an aberrant localisation in the second IDC after clindamycin treatment.
(A) Representative live-cell images of untreated and clindamycin (5 μM) treated parasites in the second IDC following treatment are shown. Cell populations were divided into parasites with 6 or fewer nuclei (upper panel) and parasite with more than 6 nuclei (lower panel), as the disruption is clearly most pronounced in trophozoite stage parasites. GFP-Rab5a, green signal; DAPI: parasite nuclei, blue signal; merge of green and blue signal. Scale bar = 5 μm. (B) Graph showing the ratio of the brightest GFP focus fluorescence intensity to mean fluorescence intensity distributed in the parasite cytoplasm in untreated and clindamycin treated parasites. N = 38 for untreated, 1–6 nuclei; n = 52 for + clindamycin, 1–6 nuclei; n = 20 for control, > 6 nuclei and n = 16 for + clindamycin, > 6 nuclei. *P < 0.05; ***P < 0.001, Welch t test. Scatter dot plots show error bars with mean ± SD. See S2 Data for numerical data underlying figure. DAPI, 4′,6-diamidino-2-phenylindole; DIC, differential interference contrast; GFP, green fluorescent protein; IDC, intraerythrocytic developmental cycle; iRBC, infected red blood cell.
Fig 7.
IMC formation is disrupted in the second IDC after indolmycin treatment.
Shown are representative images of immunofluorescence assays using anti-GAP45 (1:1,000). Untreated, indolmycin (50 μM) treated and indolmycin treated with polyprenol rescue (5 μM GGOH) as indicated, collected in the second IDC after treatment. The IMC marker GAP45 localises atypically in indolmycin treated parasites. GGOH supplementation restores GAP45 localisation to the IMC equivalent to untreated. GAP45, green signal; DAPI, parasite nuclei, blue signal; merge of green and blue signal. Scale bar = 5 μm. BF, bright field; DAPI, 4′,6-diamidino-2-phenylindole; GGOH, geranylgeraniol; IDC, intraerythrocytic developmental cycle; IMC, inner membrane complex.
Fig 8.
Indolmycin-treated parasites have increased osmotic fragility in the second IDC after treatment.
(A) Parasites were treated with indolmycin (50 μM) and divided into two conditions, with and without polyprenol rescue (5 μM GGOH). Enriched trophozoite-stage parasites were collected 72–78 hours post drug administration (equivalent to 28–32 hpi in the second IDC after treatment) and incubated in solutions with tonicity varied by increasing the concentration of NaCl. Percentage iRBC lysis was calculated by measuring the absorbance at 415 nm (A415) of released haem normalised to a relative tonicity of one (isotonic). Representative of four independent experiments. Scatter plots show error bars with mean ± SEM. See S2 Data for numerical data underlying figure. (B) The concentration required for Lysis C50% for each of the three conditions: untreated, indolmycin treated (+ indolmycin), and polyprenol rescue (+ indolmycin + GGOH). The Lysis C50% for indolmycin treated parasites increased by 22.3% (n = 4, SD ± 6.07) whereas the Lysis C50% for the polyprenol rescued parasites did not significantly change. *P < 0.05; **P < 0.01; two-tailed Student t test. Box and whisker plots show the interquartile range. See S2 Data for numerical data underlying figure. GGOH, geranylgeraniol; hpi, hours post invasion; IDC, intraerythrocytic developmental cycle; Lysis C50%, half-maximal lysis; ns, not significant; RBC, red blood cell.
Fig 9.
Supplementing ubiquinone-independent yDHODH-expressing transgenic parasites with GGOH does not protect from delayed death in the third IDC after treatment.
Dose-response curve from SYBR-Green susceptibility assay determined 48, 96, and 144 hours post indolmycin treatment, with and without polyprenol (5 μM GGOH) rescue. Assays were performed in parallel with 3D7 and transgenic parasites expressing ubiquinone-independent yDHODH. yDHODH parasites are susceptible to delayed death equivalent to 3D7 (inhibition at 96 hours). GGOH supplementation does not further protect yDHODH parasites in the third IDC (inhibition at 144 hours). Representative of at least three independent experiments. Scatter plots show error bars with mean ± SEM. See S2 Data for numerical data underlying figure. GGOH, geranylgeraniol; IDC, intraerythrocytic developmental cycle; yDHODH, yeast dihydroorotate dehydrogenase.
Fig 10.
Multifaceted disruption of protein prenylation by isoprenoid fatigue during delayed death.
(1) IPP is synthesised by the apicoplast to produce the prenyl-group GGPP. Protein substrates are modified by prenylation through the covalent attachment of one or more prenyl groups to their C-terminal cysteine residues. Prenylated proteins in P. falciparum have canonical roles in endomembrane vesicle trafficking. (2) The early endosome marker Rab5a is a prenylated protein in P. falciparum putatively involved in haemoglobin-containing vesicle trafficking to the parasite DV. Depletion of IPP during delayed death disrupts protein prenylation and Rab5a mediated vesicle trafficking to the DV. (3) Prenylated Rab11a facilitates the assembly of the IMC by trafficking cargo, including the IMC protein GAP45 to the IMC during merozoites segmentation. Depletion of IPP during delayed death disrupts the formation of the IMC. DV, digestive vacuole; GAP45, glideosome-associated protein 45; GGPP, geranylgeranyl pyrophosphate; IMC, inner membrane complex; IPP, isopentyl pyrophosphate.