Fig 1.
RON11 is essential for intraerythrocytic growth.
(A) Schematic showing the integration of the repair plasmid that introduces the tetR-DOZI system in the genomic loci of RON11. (B) PCR confirming integration at the RON11 locus. Amplicons were amplified from genomic DNA isolated from the mutant and the parental line (NF54attB) [62]. Primers were designed to amplify 2 regions as shown. (C) Western blot of lysates from RON11apt schizont grown in the presence or absence of 0.5 μm aTc. Lysates were probed with antibodies against HA (RON11) and EF1α (loading control). The protein marker sizes are shown on the left (n = 4 biological replicates). (D) Growth of RON11apt parasites in the presence or absence of 0.5 μm aTc. Synchronous parasites were collected every 48 h, stained, and measured via flow cytometry. One representative data set of 3 biological replicates shown (n = 3 technical replicates; error bars = standard deviation (SD), the underlying data can be found in S1 Data). (E) IFAs showing the localization of RON11 in RON11apt schizont with respect to 2 rhoptry markers: RON4 (neck) and RAP1 (bulb). Images from left to right are phase-contrast, DAPI (nucleus, cyan), anti-HA (magenta), anti-RON4, or RAP1 (yellow), and fluorescence merge. Z stack images were deconvolved and projected as a combined single image. Representative images of 3 biological replicates. (F) Quantification of the colocalization of RON11apt with RON4 and RAP1 as determined by the PCC (n = 3 biological replicates, 4 schizonts per replicate, the underlying data can be found in S1 Data). Error bars = SEM; ****p < 0.0001 by unpaired two-tailed t test. HA, hemagglutinin; IFA, immunofluorescence assay; IgG, immunoglobulin G; MBP, maltose binding protein; PCC, Pearson’s correlation coefficient; SD, standard deviation; SEM, standard error of the mean.
Fig 2.
RON11 is required for merozoite invasion.
(A) Representative Hema-3-stained blood-smears showing the development of RON11apt parasites in the presence or absence of 0.5 μm aTc. Synchronous parasites were smeared, stained, and imaged by light microscopy. (B) Development of synchronous RON11apt parasites over 8 h post-egress in the presence or absence of 0.5 μm aTc. Schizonts were synchronized using 2 μm Compound 1 and collected every 2 h, stained, and measured via flow cytometry to distinguish between rings and schizonts. One representative data set of 3 biological replicates shown (n = 3 technical replicates; error bars = SD; the underlying data can be found in S1 Data). (C) In vitro neutralization assay was performed against 3D7 parasites using total IgG purified from rats immunized with rMBP-RON11 (N-term) or non-immune rat IgG (8 mg/ml) as the negative control (labeled Ctrl). Potential influence of anti-MBP antibodies was evaluated by pre-incubating IgG with rMBP (3 μm). Parasites were stained with SYBR green and parasitemia was measured by flow cytometry. Growth inhibitory activity is represented as the percentage (%) of inhibition relative to the “no-IgG” control (n = 2–4 biological replicates in duplicate; error bars = SEM; the underlying data can be found in S1 Data).
Fig 3.
RON11 is not required for merozoite attachment nor rhoptry secretion.
(A) Representative time-course images from S1 Movie (+RON11) and S2 Movie (-RON11) showing RON11apt merozoites (arrowhead) interacting with RBCs in the presence or absence of 0.5 μm aTc. One representative data set of 3 biological replicates shown. (B) Quantification of merozoites that induced deformation in RBCs that completed invasion or remained attached (n = 3 biological replicates, 35 merozoites for +RON11, and 53 for -RON11; the underlying data can be found in S1 Data). (C) Duration of merozoite-induced RBC deformation quantified using live video microscopy (n = 3 biological replicates, 14 merozoites for +RON11 and 12 for -RON11; error bars = SEM; **p < 0.01 by unpaired two-tailed t test; the underlying data can be found in S1 Data). (D) Duration between first contact of merozoites with RBCs and echinocytosis quantified using live video microscopy (n = 3 biological replicates, 35 merozoites for +RON11 and 53 for -RON11; error bars = SEM; ns = non-significant by unpaired two-tailed t test). (E) Duration of echinocytosis quantified using live video microscopy (n = 3 biological replicates, 23 merozoites for +RON11 and 29 for -RON11; error bars = SEM; ***p < 0.001 by unpaired two-tailed t test; the underlying data can be found in S1 Data). (F) Representative IFAs showing RON11apt attached-merozoites secreting RAP1 into RBCs in the presence or absence of aTc, after incubation with cytochalasin D. Images from left to right are phase-contrast, DAPI (nucleus, cyan), anti-HA (magenta), anti-RAP1 (yellow), and fluorescence merge. Z stack images were deconvolved and projected as a combined single image. Representative images of 3 biological replicates. (G) Quantification of RON11apt attached-merozoites secreting RAP1 into RBCs. Slides were blinded and secretion events were scored and represented as the percentage of events per 50 attached merozoites (n = 3 biological replicates, 50 events per replicate. Error bars = SEM; ns = non-significant by unpaired two-tailed t test; the underlying data can be found in S1 Data). RBC, red blood cell.
Fig 4.
RON11 knockdown generates merozoites with single rhoptries.
(A) Representative U-ExM images of RON11apt schizonts showing the structure of rhoptries within fully developed merozoites in the presence or absence of 0.5 μm aTc. ML10-arrested schizonts were stained with NHS-Ester (grayscale), anti-HA (magenta), and the DNA dye SYTOX (cyan). Selected Z stack images were projected as a combined single image. Number on image = Z-axis thickness of projection in μm. (B) Quantification of the percentage of merozoites per schizont with single, dual, or multiple rhoptries in the presence or absence of 0.5 μm aTc. Samples were blinded and the number of rhoptries per merozoite within each schizont were counted. Merozoites were scored based on the number of nuclei observed (n = 4 biological replicates, 28 schizonts for +RON11 and 36 for -RON11; error bars = SEM; ****p < 0.0001 by unpaired two-tailed t test; the underlying data can be found in S1 Data). HA, hemagglutinin; NHS, N-hydroxysuccinimide; U-ExM, ultrastructure expansion microscopy.
Fig 5.
RON11 knockdown does not affect the localization and processing of neck and bulb rhoptry proteins.
(A) Representative U-ExM images of RON11apt free merozoites and E64-arrested schizonts showing the localization of RON11, RON4, and RAP1 in the presence or absence of aTc. Parasites were stained with NHS-Ester (grayscale), anti-HA (magenta), and RON4 or RAP1 (yellow). Selected Z stack images were projected as a combined single image. Number on image = Z-axis thickness of projection in μm. (B) Western blot of parasite lysates isolated from E64-arrested RON11apt parasites in the presence or absence of aTc. Samples were probed with antibodies against RON4, RAP1, and EF1α (loading control). The protein marker sizes are shown on the left. Representative blot of 4 biological replicates shown. (C) Quantification of RON4 and RAP1 in E64-arrested RON11apt parasites in the presence or absence of aTc. Band intensities were normalized to the loading control, EF1α, and are presented as normalized arbitrary units (AU) (n = 4 biological replicates; error bars = SEM; **p < 0.01 by unpaired two-tailed t test; the underlying data can be found in S1 Data). (D) Quantification of RAP1 processing in E64-arrested RON11apt parasites in the presence or absence of aTc. Band intensities were normalized to the ratio of processed RAP1 (p67/p82) (n = 3 biological replicates; error bars = SEM; ns = non-significant by unpaired two-tailed t test; the underlying data can be found in S1 Data). HA, hemagglutinin; NHS, N-hydroxysuccinimide; U-ExM, ultrastructure expansion microscopy.
Fig 6.
RON11 knockdown phenotype is rescued by addition of aTc during the last 2 h of the asexual life cycle.
Growth of RON11apt parasites in the presence or absence of aTc. Tightly synchronous parasites were grown for 44 hpi and aTc was added back every 2 h. Samples were collected at 24, 44 and 56 hpi, and measured via flow cytometry to differentiate between rings and schizonts (n = 3 biological replicates; error bars = SEM; *ns = non-significant; ***p < 0.001; ****p < 0.0001 by one-way ANOVA compared to + RON11; the underlying data can be found in S1 Data). hpi, hours post-invasion.
Fig 7.
RON11 is required for the de novo biogenesis of the final rhoptry pair during merozoite segmentation.
(A) Schematic of the current model for de novo formation of rhoptries during schizogony. (B–D) Representative U-ExM images of different steps of rhoptry biogenesis in RON11apt late schizonts in the presence or absence of aTc. (B) Two rhoptries associated to each branch of a CP in the presence or absence of aTc. (C) Single rhoptries being segregated with each branch of a dividing CP during mitosis, and (D) final rhoptry pairs being segregated with a CP branch during the final mitotic event. Basal complexes are demarcated by a discontinuous line. Late-schizont stage parasites were stained with NHS-Ester (grayscale), anti-HA (magenta), and anti-RAP1 (yellow). Selected Z stack images were projected as a combined single image. Number on image = Z-axis thickness of projection in μm. CP, centriolar plaque; HA, hemagglutinin; NHS, N-hydroxysuccinimide; U-ExM, ultrastructure expansion microscopy.