Fig. S1. Controls of transcript and IFA analyses.

A. RT-PCR control for possible contamination with genomic DNA. cDNA samples for trophozoites (TZ), schizonts (SZ), immature (GCII-IV) and mature (GCV) gametocytes were prepared lacking reverse transcriptase. Diagnostic PCR was performed with pfaldolase-specific primers and no PCR product was amplified. Arrow indicates expected size of aldolase-specific PCR product fragment of 378 base pairs (compare with Fig. 1B).

B. IFA control using sera from non-immunized mice for immunoblotting. Non-immunized mouse sera (NMS) were used to immunoblot fixed samples of schizonts (SZ) and mature gametocytes (mGC) (green). Schizonts were visualized by labelling with rabbit anti-MSP1 antisera and gametocytes were visualized by rabbit anti-Pfs230 antisera (red); nuclei were highlighted by Hoechst stain (blue). Bar, 5 μm.

C. Colocalization studies on PPLP2 and Pfg377. PPLP2-positive vesicles immunolabelled with mouse anti-PPLP2RP1 antisera (green) do not colocalize with Pfg377-positive osmiophilic bodies, shown using rat anti-Pfg377 antisera (red); nuclei were highlighted by Hoechst stain (blue). Bar, 2 μm. Results shown in A–C are representative for three independent experiments.

D. Fluorescence intensity measurements on labelled gametocytes. The fluorescence intensity along a longitudinal line of the gametocyte shown in C (right image) for PPLP2 (green) and Pfg377 (red) was analysed using Leica AF software.


Fig. S2. Relocalization of PPLP2-positive vesicles to the periphery of activated gametocytes is inhibited by BAPTA-AM. PPLP2-positive vesicles are present in BAPTA-AM-treated gametocytes, but do not relocalize to the periphery between 0–20 min p.a. In untreated gametocytes, PPLP2-positive vesicles relocalize upon activation and PPLP2 labelling disappears during gamete formation. Parasites were labelled with mouse anti-PPLP2RP1 antisera (green). Gametocytes were highlighted by labelling with rabbit anti-Pfs230 antisera (red), while nuclei were stained with Hoechst (blue). Results shown are representative for two independent experiments. Bar, 5 μm.


Fig. S3. The blood stage cycle of PPLP2(−) parasites.

A. Absence of PPLP2 in PPLP2(−) schizonts. Following immunolabelling with mouse anti-PPLP2RP2 antisera, no PPLP2 signal can be detected in PPLP2(−) schizonts via IFA (green). The schizonts were visualized by rabbit anti-MSP1 antisera (red), nuclei were highlighted with Hoechst stain (blue). Results shown are representative for two independent experiments. Bar, 5 μm.

B. The morphology of PPLP2(−) asexual blood stage parasites. Giemsa smears of rings, trophozoites and schizonts were microscopically analysed and compared with WT blood stage parasites. Bar, 5 μm.

C. Kinetics of long-term asexual WT and PPLP2(−) parasite replication in human erythrocytes in vitro. Synchronized cultures were maintained for 7 days at 2% hematocrit. After ∼ 18 h in culture parasitemia of WT and PPLP2(−) cultures was adjusted by addition of uninfected erythrocytes with the preservation of culture hematocrit. Parasitemia was assessed on days 1, 3, 5 and 7 by counting 600 to 3500 cells for each sample. A representative of four independent experiments is shown.


Fig. S4. The morphology of PPLP2(−) gametocytes and the superflagellum.

A. The morphology of PPLP2(−) gametocytes during development and following activation. Giemsa smears of gametocytes of stages I–V and of microgametocytes at 15 min p.a. were microscopically analysed and compared with WT gametocytes. Arrow indicates the superflagellum. Bar, 5 μm.

B. Immunolabelled activated PPLP2(−) microgametocyte (MiGC) forming a superflagellum. The activated microgametocyte was immunolabelled with rabbit anti-Pfs230 antisera (green), and counterstained with Evans Blue (red). The nucleus was highlighted with Hoechst stain (blue). Arrow indicates the superflagellum. Bar, 5 μm.

C. Ultrastructure of a PPLP2(−) macrogamete. Transmission electron microscopy demonstrates normal development of a PPLP2(−) macrogamete at 15 min p.a.

D. Ultrastructure of an activated PPLP2(−) microgametocyte with superflagellum. Transmission electron microscopy shows a PPLP2(−) microgametocyte at 15 min p.a., which has formed a superflagellum. The axonemes were cut longitudinally. E, erythrocyte; EC, erythrocyte cytoplasm; EM, erythrocyte membrane; IMC, inner membrane complex; N, nucleus; PDM, parasitophorous vacuole-derived membranes; PPM, parasite plasma membrane; SF, superflagellum. Bar, 1 μm.


Fig. S5. Ultrastructure of activated gametocytes chemically inhibited in egress.

A. Ultrastructure of activated microgametocytes treated with 1,10-phenanthroline. Transmission electron microscopy shows that at 20 min p.a. 1,10-phenanthroline-treated microgametocytes form superflagella containing bundles of axonemes. The superflagella were enveloped by the EM (left image modified from Sologub et al., 2011).

B. Ultrastructure of a gametocyte at 20 min p.a. following treatment with BAPTA-AM. The frame shown in the left image indicates the area enlarged in the right image.

C. Ultrastructure of a gametocyte at 20 min p.a. following treatment with cysteine/serine protease inhibitor TCLK.

D. Ultrastructure of a gametocyte at 20 min p.a. following treatment with cysteine inhibitor E64d. Results shown in A–D are representative for three independent experiments. Ax, axoneme; EM, erythrocyte membrane; IMC, inner membrane complex; N, nucleus; OB, osmiophilic body; PPM, parasite plasma membrane; PVM, parasitophorous vacuole membrane; SF, superflagellum. Bar, 1 μm.


Fig. S6. Effect of pore sealant on the blood stage cycle and exflagellation activity of activated PPLP2(−) gametocytes.

A. Purified recombinant full-length PPLP2. Full length PPLP2 (PPLP2RP3) was expressed in HEK-293 cells and His-tagged PPLP2RP3 was purified by Ni-NTA chromatography. SDS-PAGE followed by either silver staining or immunoblotting with anti-His antibody shows full-length PPLP2RP3 running with a molecular weight of 122 kDa as well as processing products at approximately 70 and 50 kDa.

B. Effect of Tetronic 90R4 on merozoite egress. WT and PPLP2(−) schizonts were resuspended in medium supplemented with 0.5% AlbuMax and Tetronic 90R4 in concentrations between 10–30 mg ml−1, and kept for 1 h at 37°C in the environmental chambers to accumulate sites of parasite egress. Egress was assessed by light microscopy and calculated as a fraction of schizonts that finished replication cycle and released during the incubation time. A total of 300–1000 schizonts per sites of egress were counted for each experimental condition. Results are presented as mean ± SEM of three independent experiments for WT parasites. One experiment was performed with PPLP2(−) parasites to confirm Tetronic 90R4 effect on egress observed with WT parasites.

C. Motility of PPLP2(−) microgametes during a 150-min time span. The numbers of microgametocytes forming actively moving microgametes were determined between 0–150 min p.a. for 10 optical fields at 400-fold magnification.

D. Ability of activated PPLP2(−) gametocytes to form exflagellation centres after prolonged cultivation. PPLP2(−) gametocytes of the same culture were harvested at days 12 and 23 of cultivation. The numbers of exflagellation centres were quantified at 15 min (15′) p.a. for 10 optical fields at 400-fold magnification and calculated as a percentage of all actively moving microgametes (the sum of trapped microgametes and exflagellation centres were set to 100%).

Results shown in C and D represent one experiment.


Fig. S7. Genomic DNA sequence of pplp2(−) gene locus with positively sequenced areas for 5′ and 3′ integration of the pCAM-BSD vector construct. The homologous pplp2 region used for generation of the gene disruption construct is highlighted in grey; the pCAM-BSD vector sequence is depicted as white letters on black background. Primers used for sequencing and the inserted stop codon are written in bold uppercase and underlined letters. Positively sequenced areas (sequences 1–4) are indicated with dotted lines underneath. Arrow heads (<; >) indicate 5′ to 3′ direction, double arrows (<<; >>) mark beginning and end of a sequence.


Movie S1. Red blood cell invasion by PPLP2(−) merozoites and transformation.


Movie S2. Egress of PPLP2(−) merozoites from infected red blood cell.


Movie S3. Influx of Alexa Fluor 488® phalloidin from the medium into an erythrocyte infected with a PPLP2(−) schizont during membrane permeabilization prior to egress.


Movie S4. Live cell recording of WT gametocyte egress from infected red blood cell in medium supplemented with fluorescent phalloidin.

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