Fig 1.
Cls activity and PfCls domain organization.
(A) Chemical structure of cardiolipin. (B) Reactions catalyzed by bacterial-type cardiolipin synthases. PG or PE can transfer a phosphatidyl group to PG and form CL. Cls can also catalyze PG formation from PE and glycerol. Phospholipase D activity of Cls leads to formation of PA by hydrolyzing CDP-DAG. Cls can also use CDP-DAG with EA for PE formation, as demonstrated for P. syringae and X. campestris Cls by radio-labelling in vivo and in extracts of E. coli expressing the enzymes. EA, cytidine monophosphate (CMP) and glycerol (Gly) are by products in these reactions. (C) Phylogeny of Cls homologs from Bacteria, Alveolata and Kinetoplastida generated by PhyML 3.0. Bootstrap values of 1000 replicates are indicated at each branch. Putative Cls of Plasmodium spp. are in blue. Sequences used in phylogeny are provided in S1 Table. (D) Domain organization of the PfCls. The characteristic HKD motifs are indicated in red. The hydrophobic region suggesting presence of a putative TMD is marked with green hatched box. The PfCls stretches expressed as recombinant proteins are indicated by blue arrows. (E) PfCls AlphaFold model with the HKD motif residues H187, K189, D194 (HKD motif 1), H478, K480, D485 (HKD motif 2) shown as red sticks. The extended HKD motif residues R185, R188, G200, N203, G492, N495, D497, S500, E506 are in cyan. (F) Anti-PfCls serum recognizes a band at the expected size of full-length PfCls (~71 kDa) and a major processed form of ~68 kDa in western blot of parasite lysate from blood-stage trophozoites. I, immune serum, PI, preimmune serum.
Fig 2.
Subcellular localization of PfCls and distribution of CL through asexual and sexual blood stages.
(A, B) Immunofluorescence confocal microscopy for localization of PfCls in asexual blood stages using anti-PfCls antisera. MitoTracker Red is the mitochondrial marker dye in (A). PfHU is the apicoplast marker in (B), DAPI is the nuclear dye. DIC, differential interference contrast; MT, mid-trophozoites; LT, late-trophozoites; S, schizonts; MS, Mid-schizonts; LS, late-schizonts. The co-localization rate (CR, %) and Pearson’s coefficient (PC) between PfCls and MitoTracker Red is given in (A). The scale bar is 4 µm. The images are representative of >20 images scanned from three sets each for (A) and (B). (C) Localization of PfCls at different gametocyte stages (II-V) with MitoTracker Red as the mitochondrial marker dye. The images are representative of >50 scans from two experiments. (D) Partial overlap of PfCls with α-tubulin signal below the parasite plasma membrane in gametocytes. The images are representative of >50 scans from two experiments. (E) Blood stage trophozoites stained with NAO and MitoTracker Red. The scale bar is 2 µm. The images are representative of >30 images. (F) Gametocytes (stage III-V) stained with NAO and MitoTracker Red. The scale bar is 4 µm. The images are representative of >90 images scanned for gametocyte stages.
Fig 3.
Biochemical characterization of recombinant PfCls.
(A) Coomassie-stained SDS-PAGE of purified recombinant PfCls (i), and the corresponding western blot probed with anti-PfCls serum (ii). (B) Protein-lipid overlay assay to check binding of recombinant PfCls with different concentrations of phospholipids: PG, PE, CDP-DAG and CL. Buffer alone served as negative control; the buffer column is shown in both panels of the blot to show similar exposure. The blot was probed with anti-6XHis Ab. The corresponding graph shows quantification of concentration-dependent binding from three repeat protein-lipid overlay experiments. Mean ± SD are plotted. * indicate P = 0.045 and 0.029 for comparison of binding to CDP-DAG versus PG and PE, respectively. (C) Co-sedimentation of PfCls with different phospholipid (PL) liposomes. The supernatant (S) and pellet (P) were probed with anti-6XHis Ab for detection of PfCls. The graph shows quantification of lipid-bound PfCls (%). Mean ± SD from three repeat experiments is plotted. (D) Conversion of CDP-DAG to PA indicates phospholipase D activity of PfCls. Lanes 1-5 of the TLC are lipid standards. P. syringae Cls was used as positive control. CMP, cytidine monophosphate. (E) PfCls catalyzes formation of PG from PE and glycerol. PsCls was the positive control enzyme. Representative images from three repeat experiments are shown for (D) and (E). (F) CL synthesis by PfCls in E. coli. Fluorescence confocal microscopy of late exponential phase bacteria transformed with pET23a(+)-PfCls (PfCls) or the empty vector pET23a(+) (EV) under identical scan settings. CL was detected by NAO staining. CL-rich regions in PfCls expressing cells are shown by solid arrows. Scale bar is 2 µm. (G) Mean fluorescence intensity calculated for CL-rich regions-- cell septum and cell poles of bacteria in (F). Mean ± SD are plotted for 40 cells each. P value (P < 0.0001) was calculated by the Mann-Whitney non-parametric two-tailed test. (H) 2D-TLC for detection of CL levels in E. coli expressing PfCls or transformed with the empty vector. CL, PE and PG were used as phospholipid standards. Dimensions of the mobile phase are indicated by solid arrows. CL levels are plotted as % of total phospholipids from three experiments; P < 0.01.
Fig 4.
PbCls is required for asexual blood-stage growth and mitochondrial function.
(A) Asexual growth of PbCls KO parasites is significantly reduced compared to WT-GFP and PbCls-complemented lines. Parasitemia was monitored daily following infection; KO parasites exhibited a marked proliferation defect (day 1, P = 0.0308; day 2, P = 0.4687; day 3, P = 0.0978; day 4, P = 0.0009; day 5, P = 0.0001; day 6, P = 0.0009; day 7, P = 0.0002). (B) Gametocytemia in PbCls KO parasites is significantly decreased on day 3 post-infection compared to WT-GFP (P < 0.0001), but recovers by day 4 (P = 0.1471). (C) Relative total CL levels in PbCls KO compared to WT-GFP parasites. P-value was determined by unpaired Student’s t-test (P = 0.0054). (D) Relative abundance of CL molecular species in WT-GFP and PbCls KO parasites. Mean ± SD of six independent biological replicates are plotted. Multiple t-test with 5% Benjamini-Krieger-Yekutieli FDR correction was applied; adjusted P-values are depicted. (E and F) Mitochondrial membrane potential was assessed using MitoTracker Red FM staining. PbCls KO parasites showed a significant reduction in relative fluorescence intensity compared to WT-GFP (P < 0.0001). Error bars represent mean ± SEM; data are representative of three independent experiments. Statistical comparisons were performed using one-way ANOVA. (G) Basal OCR of WT-GFP and PbCls KO parasites from S6F Fig showing mean ± SD of data points for basal respiration (between 1-20 min). P-value was determined by unpaired Student’s t-test (P < 0.0001).
Fig 5.
PbCls is critical for late liver-stage maturation but dispensable during early exo-erythrocytic development and mosquito stages.
(A) Significant reduction (P = 0.0002) in ookinete numbers in PbCls KO when mosquitoes fed on mice with low gametocytemia at day 3 post infection. (B) No significant difference in ookinete numbers (P = 0.4762) between WT-GFP, PbCls KO, and PbCls-comp parasites when mosquitoes fed on mice at day 4 post infection. (C) Oocyst quantification showed no significant differences among WT-GFP, PbCls KO, and PbCls-comp parasites (P = 0.5272). (D) Midgut sporozoite numbers were comparable between parasite lines (P = 0.0591). (E) Salivary gland sporozoite quantification revealed no significant differences between WT-GFP, PbCls KO, and PbCls-comp parasites (P = 0.1035). Data represent mean ± SEM from three independent experiments. (F and G) HepG2 cells were infected with WT-GFP or PbCls KO sporozoites and fixed at 24, 40, and 65 hpi and immunostained with anti-UIS4 or anti-MSP1 antibodies; nuclei were stained with Hoechst 33342. (H and I) Quantification of EEF number and size revealed no significant difference between WT-GFP and PbCls KO parasites (EEF number: 24 hpi, P = 0.1901; 40 hpi, P = 0.1557; 65 hpi, P = 0.0959; EEF area: 24 hpi, P = 0.0276; 40 hpi, P = 0.0163; 60 hpi, P = 0.0133). (J) Schizont maturation was severely impaired in PbCls KO parasites at 65 hpi, as evidenced by significantly reduced nuclear counts compared to WT-GFP (P < 0.0001). (K) Counting of EEF stages demonstrated an arrest at the cytomere stage in KO parasites, with a significant reduction in mature merozoites (P = 0.0003). Data represent mean ± SEM from three independent biological replicates. Statistical significance was determined using one-way ANOVA. (L) Detached cell formation is significantly reduced in PbCls KO parasites compared to WT-GFP (P = 0.0018).
Table 1.
Infectivity of PbCls KO sporozoites in C57BL/6 mice. PbCls KO parasites exhibited a significant delay in the pre-patent period compared to controls (Kruskal-Wallis test, P = 0.0313).
Table 2.
Infectivity of detached cells in Swiss mice.
Fig 6.
Mitochondrial branching and PVM rupture defects in PbCls KO parasites.
(A) Immunofluorescence analysis using an anti-IscU antibody revealed impaired mitochondrial branching in PbCls KO parasites compared to WT-GFP at 40 and 65 hpi. (B) Quantification of mitochondrial branching in EEFs confirmed a significant reduction in PbCls KO parasites compared to WT-GFP. Mitochondria exhibiting ≥5 at 40 hpi or ≥10 branched tubular networks at 65 hpi were classified as normal, while those below these numbers were considered unbranched. Statistical analysis demonstrated a significant decrease in mitochondrial branching in PbCls KO parasites (P = 0.0052 at 40 hpi; P = 0.0003 at 65 hpi). A total of 100 EEFs at 40 hpi and 50 EEFs at 65 hpi were analyzed per group, from two independent biological replicates. (C) HepG2 cells infected with WT-GFP or PbCls KO parasites were fixed at 65 hpi and immunostained with anti-UIS4 antibody to label the PVM. Nuclei were stained with Hoechst. PVM rupture in WT-GFP is indicated by a white line. (D) Quantification of liver-stage parasites shows a significantly higher proportion of EEFs with intact PVMs in PbCls KO parasites relative to WT-GFP (P < 0.005). A total of 120 EEFs were analysed for each group from two independent biological replicates.