Figure legends
Figure 1: Colocalization and dynamics of egress-related
vesicles during gametocyte activation. (A) Schematic of the
proteins MDV1, G377, and PPLP2. SP, signal peptide; MACPF, Membrane
Attack Complex/Perforin domain. (B) Vesicular localization of
MDV1, G377 and PPLP2 in gametocytes. WT NF54 gametocytes were
immunolabeled with rabbit anti-G377, rat anti-MDV1 and mouse anti-PPLP2
antisera (red and green) to investigate co-localization of the
respective proteins. Parasite nuclei were highlighted by Hoechst 33342
nuclear stain (blue). Bar; 2 µm. DIC, differential interference
contrast. Corresponding negative controls are provided in Fig. S2.(C) Quantification of protein colocalization. The Pearson´s
correlation coefficient (PCC) was calculated using Fiji ImageJ2.
Immunolabeling of G377 or MDV1 was defined as region of interest (n =
20). The error bars indicate mean ± SD. (D) Dynamics of G377-
and PPLP2-positive vesicles during gametocyte activation. WT NF54
gametocytes were collected at
0-20 min post-activation (p.a.) and immunolabeled, using rabbit
anti-G377 or mouse anti-PPLP2 antisera (green). Gametocytes were
counterstained with rabbit or mouse anti-P230 antisera (red). Parasite
nuclei were highlighted by Hoechst 33342 nuclear stain (blue). Bar; 2
µm. NRBS, neutral rabbit serum; NMS, neutral mouse serum. (E)Calcium-dependency of vesicle discharge following gametocyte activation.
WT NF54 gametocytes were treated with 25 µM BAPTA-AM prior to activation
and immunolabeled with as described in (D). Untreated gametocytes served
as control. A total of 50 activated (rounded) gametocytes per setting
were evaluated for the presence of the G377 or PPLP2 signal at 20 min
p.a. (n = 3). Corresponding IFA images are provided in Fig. S3. The
error bars indicate mean ± SD. ***p≤0.001 (One-Way ANOVA with Post-Hoc
Bonferroni Multiple Comparison test; C, E). Results (B, D) are
representative of three independent experiments.
Figure 2: Verification of the parasite lines to be used in
BioID. (A) Localization of the bait proteins in the BioID parasite
lines. Gametocytes of the PPLP2-GFP-BirA line and the G377-TurboID-GFP
and MDV1-TurboID-GFP lines were immunolabeled with mouse anti-GFP
antibody to highlight PPLP2, G377 and MDV1, fused to GFP and biotin
ligase (green). Gametocytes were counterstained with anti-P230 antisera
(red); parasite nuclei were highlighted by Hoechst 33342 nuclear stain
(blue). WT NF54 gametocytes served as a control. Bar; 5 µm. (B)Protein biotinylation in the BioID parasite lines. Gametocytes of the
PPLP2-GFP-BirA line and the G377-TurboID-GFP and MDV1-TurboID-GFP lines
were treated with biotin (+) for 20 h (PPLP2-GFP-BirA) and 15 min
(G377-, MDV-TurboID-GFP). Untreated parasite lines (-) and
biotin-treated and untreated NF54 WT gametocytes served as controls.
Gametocyte lysates were subjected to Western blot analysis and
biotinylated proteins were detected using streptavidin-conjugated AP.
Asteriks (*) highlight the bait proteins. Cloning strategy and
transfection verification are provided in Fig. S4. Results (A, B) are
representative of three independent experiments.
Figure 3: In silico analysis of the egress vesicle
proteomes. (A) Schematic of candidate selection. Putative interactors
of MDV1, G377, and PPLP2 following BioID of lines MDV1-TurboID-GFP,
G377-TurboID-GFP and PPLP2-GFP-BirA were subjected to domain and
functional analysis, resulting in the identification of a total of 143
egress vesicle proteins. Signal peptides (SP) were predicted using
SignalP 4.1 & 5.0, transmembrane domains (TM) were predicted using
DeepTMHMM, and endoplasmic reticulum (ER) retention signals were
predicted using DeepLoc 2.0. Functional prediction was performed via
PlasmoDB. (B) Venn diagram depicting the egress vesicle
proteins grouped by bait protein. The final numbers of interactors for
each parasite line are depicted in bold, the predicted sex specificity
is indicated (; see PlasmoDB database). (C) Pie chart depicting
the egress vesicle proteins (percentage of total numbers) according to
predicted molecular function. (D) Pie chart depicting the
egress vesicle proteins (percentage of total numbers) grouped by stages
of peak expression (; see PlasmoDB database). RI, ring stage; TZ,
trophozoite; SZ, schizont; GC II, gametocyte stage II; GC V, gametocyte
stage V; OK, ookinete. Detailed information on the interactors before
and after the application of selection criteria is provided in Tables S1
and S2. The corresponding GO term and sex specificity analyses are
provided in Fig. S6.
Figure 4: Network
analysis of the egress vesicle proteins. The 143 egress vesicle
proteins were evaluated for potential interactions using the STRING
database. Based on the interaction among the query proteins, different
functional clusters were identified. (A) Adhesion protein/LCCL
domain protein cluster with 23 proteins; (B) the RBC invasion
and modification cluster with 59 proteins, including (B1) the
Maurer´s clefts subcluster and (B2) the rhoptry/microneme
subcluster; (C) the vesicle biogenesis cluster of 13 proteins.
Detailed information on individual proteins clustering in each
subnetwork is provided in Table S3.
Figure 5: Expression and localization of egress vesicle proteins
in blood stage parasites. Five pSLI-HA-glmS -based parasite lines
expressing select egress vesicle proteins tagged with HA were generated
for expression analysis. (A) Blood stage expression of egress
vesicle proteins. Lysates of asexual blood stages (ABS) and gametocytes
(GC) of the respective lines were immunoblotted with rat anti-HA
antibody to detect Sel1 (271 kDa), PSOP1 (53 kDa), the conserved protein
PF3D7_0811600 (144 kDa), Vti1 (49 kDa) and the conserved protein
PF3D7_1319900 (179 kDa). ABS and GC lysate of WT NF54 served as
negative controls, while immunoblotting with rabbit antibody against the
ER protein Pf39 (39 kDa) served as loading control. (B)Vesicular localization of egress vesicle proteins in gametocytes.
Gametocytes of the pSLI-HA-glmS -based parasite lines were
immunolabeled with rat anti-HA antibody (green) to detect the respective
HA-tagged egress vesicle protein. Gametocytes were counterstained with
anti-P230 antisera (red); parasite nuclei were highlighted by Hoechst
33342 nuclear stain (blue). Bar; 5 µm. Cloning strategy and transfection
verification are provided in Fig. S8. Results (A, B) are representative
of three independent experiments.
Figure 6: Localization and discharge of OB components.
(A) Localization of PSOP1 and
PF3D7_0811600 in OBs. Gametocytes of the PSOP1-HA-glmS line and
PF3D7_0811600-HA-glmS line were immunolabeled with rat anti-HA
antibody (green). OBs and g-exonemes were highlighted using rabbit
anti-G377 or mouse anti-PPLP2 antisera (red); parasite nuclei were
highlighted by Hoechst 33342 nuclear stain (blue). Samples were analyzed
via Airyscan super-resolution microscopy. Bar; 2 µm. DIC, differential
interference contrast. (B) Quantification of protein
colocalization. The Pearson´s correlation coefficient (PCC) was
calculated using Fiji ImageJ2. Immunolabeling of HA-tagged egress
proteins was defined as region of interest (n = 20). The error bars
indicate mean ± SD. (C) PSOP1 discharge following gametocyte
activation. Gametocytes of line PSOP1-HA-glmS were collected at
0-5 min post-activation (p.a.) and immunolabeled with rat anti-HA
antibody to highlight PSOP1 (green). Gametocytes were counterstained by
rabbit anti-P230 antisera (red); parasite nuclei were highlighted by
Hoechst 33342 nuclear stain (blue). Bar; 5 µm. DIC, differential
interference contrast. Results (A, C) are representative of three
independent experiments.