Western blot analyses of gametocyte lysates prepared from the transgenic
lines, using mouse anti-GFP antibody, demonstrated the expression of the
respective fusion proteins (Fig. S5A).
The proteins MDV1-TurboID-GFP and
G377-TurboID-GFP migrated at the expected molecular weights of 91 and
439 kDa, respectively. For PPLP2, a band of approximately 120 kDa was
detected in addition to the expected band of 187 kDa, indicating
processing of the protein. No protein bands were detected in lysates of
either the asexual blood stages of the transgenic lines nor of WT NF54
mixed asexual blood stages and gametocytes (Fig. S5A). Similarly,
immunolabeling with anti-GFP antibodies demonstrated the expression of
PPLP2-GFP-BirA, MDV1-TurboID-GFP, and G377-TurboID-GFP proteins in
gametocytes of the respective transgenic lines and confirmed the
presence of the tagged fusion proteins in vesicular structures (Fig.
2A). In WT NF54 parasites, no GFP-labeling was detected.
Subsequent Western blotting was employed to highlight biotinylated
proteins in the transgenic lines. For this, gametocyte cultures of each
line were treated with 50 µM biotin for 15 min (TurboID) or 20 h
(BioID). Immunoblotting of the respective gametocyte lysates using
streptavidin conjugated to alkaline phosphatase detected multiple
protein bands indicative of biotinylated proteins, including protein
bands of 91 kDa and 187 kDa, likely representing the biotinylated fusion
proteins MDV1-TurboID-GFP and PPLP2-GFP-BirA, respectively, while the
fusion protein G377-TurboID-GFP could not be identified uniquely due to
the high molecular weight (Fig. 2B). In gametocytes of the transgenic
lines that were not treated with biotin, minor protein bands were
detected, indicating endogenous biotin has been present in the
gametocytes, which triggered the activity of the biotin ligase. No
biotin-positive protein bands were detected in WT NF54 samples (Fig.
2B). IFA analyses of biotin-treated gametocytes of the transgenic lines,
using fluorophore-conjugated streptavidin, confirmed the presence of
biotinylated proteins, which localized in vesicular structures, while no
biotinylated proteins were detected in biotin-treated WT NF54
gametocytes (Fig. S5B).
BioID analyses were subsequently employed to analyze the proteomes of
the OBs and g-exonemes of P. falciparum . For this, gametocytes of
the respective transgenic lines were treated with biotin as described
above, and equal amounts of gametocytes per sample were harvested. Three
independent samples were collected from each of the three lines
(MDV1-TurboID-GFP, G377-TurboID-GFP and PPLP2-GFP-BirA); two additional
independent samples from the PPLP2-GFP-BirA line were included. Mass
spectrometric analysis was performed on streptavidin-purified protein
samples with three technical replicas for each sample. This resulted in
the identification of 636 (MDV1-TurboID-GFP), 189 (G377-TurboID-GFP),
and 298 (PPLP2-GFP-BirA) significantly enriched proteins, respectively
(Fig. 3A; Table S1). For each transgenic parasite line, the respective
bait protein, i.e. G377, MDV1, and PPLP2 was detected among these
proteins (marked in Table S1). In a first analysis step proteins without
a putative signal peptide and/or transmembrane domains (with no
C-terminal ER retention signal) were excluded, reducing the potential
interactors to 169 proteins (MDV1-TurboID-GFP), 50 proteins
(G377-TurboID-GFP), and 64 proteins (PPLP2-GFP-BirA) (Fig. 3A; Table
S2). Subsequently, proteins with defined known functions not related to
OBs or g-exonemes (e.g. nucleoporins, chaperons) were removed from the
list, eventually resulting in the following numbers of putative proteins
of egress vesicles: 132 proteins (MDV1-TurboID-GFP), 38 proteins
(G377-TurboID-GFP), and 44 proteins (PPLP2-GFP-BirA) (Fig. 3A; Table
S2).
The comparison of the filtered lists of proteins identified 13 proteins
as putative interactors of both OB proteins, G377 and MDV1. It also
revealed 16 proteins as putative interactors of MDV1 and PPLP2, while
only 2 proteins were shared between G377 and PPLP2 (Fig. 3B; Table S2).
In total, 20 proteins were shared between three bait proteins. This
group of proteins includes among others five members of the LCCL protein
family, as well as P230 and P230p, and P47, hence adhesion proteins
known to locate in the PV of gametocytes, where they form protein
complexes that are linked to the GPM (, ).
The putative interactors were then grouped by predicted function (Fig.
3C). The majority of proteins belonged to the categories of protein
trafficking, processing and adhesion as well as transmembrane transport
(~10 % in each category). A total of 8% of proteins
was previously assigned to host cell exit, e.g. EPF1 (exported protein
family 1), GEP (gamete egress protein), GEXP02 (gametocyte exported
protein 2), PMX (plasmepsin X), SUB2 (subtilisin-like protease 2), MiGS
(microgamete surface protein), and GEST (gamete egress and sporozoite
traversal protein) ( Further, 35% of proteins are of unknown function.
An additional gene ontology (GO) term analysis revealed main molecular
functions in peptidase activities and pyrophosphate hydrolysis and well
as transmembrane transport (Fig. S6A) and cellular localizations in host
cellular components and vesicles (Fig. S6B).
A comparative transcriptional analyses of these putative interactors
(according to table “Transcriptomes of 7 sexual and asexual life
stages”; ; see PlasmoDB database; ) showed that the majority of
proteins exhibited peaks in stage V gametocytes and in ookinetes or in
ring stages and trophozoites (Fig. 3D), suggesting that they represent
two groups of proteins, either present during gametocyte development,
e.g. P230, P48/45, or with roles in the mosquito midgut phase, e.g.
members of the PSOP family. When the sex specificity of the interactors
was evaluated (according to table “Gametocyte Transcriptomes”; ; see
PlasmoDB database; ), roughly one-third of proteins could be assigned to
either male or female gametocytes, while one third of proteins did not
exhibit sex-specific transcript expression (Fig. S6C).
The interactors were further subjected to STRING-based analyses to
investigate the protein-protein interaction networks (see string-db.org;
text mining included). The STRING analysis revealed three main clusters
(Fig. 4; Table S3). The first cluster involved proteins previously shown
to form multi-adhesion domain protein complexes like the LCCL domain
proteins, P48/45, and P230, plus the paralogs P47 and P230p (; reviewed
in ). Furthermore, the cluster includes three members of the PSOP
family, PSOP1, PSOP12 and PSOP13 and proteins linked to the PVM, i.e.
P16, Pfg17-744 and Pfg14-748 . In addition, the three vesicle markers
G377, MDV1 and PPLP2, are found in this cluster. Noteworthy, the
majority of these proteins are interactors of all three bait proteins.
A second cluster comprises proteins linked to vesicle biogenesis,
particularly transmembrane transporters including the previously
described ABCG2 transporter of female gametocytes . Further, the vesicle
trafficking-related protein clathrin (heavy chain) and sortilin are
found in this cluster (Fig. 4; Table S3). Four of the proteins of this
cluster belong to the group of PPLP2 interactors.
The third cluster resembles a megacluster that comprises various
proteins particularly linked to RBC invasion and modification. Within
the megacluster, two subclusters can be distinguished, one of which
includes proteins linked to the Maurer´s clefts, while the other one
comprises proteins linked to rhoptries and micronemes. Additional
proteins are associated with these subclusters, several of which are
proteases, e.g. SUB2, falstatin, the dipeptidyl aminopeptidases DPAP1
and DPAP2, the metalloprotease M16, and the plasmepsins PMI, PMIII, PMX.
Furthermore, known components of the merozoite surface like GAMA, MaTrA,
MSP8 or P38 as well as various exported proteins like GEXP08, GEXP21,
EXP1 and EXP3 are found within the megacluster (Fig. 4, Table S3). Since
the majority of the megacluster proteins were previously linked to rings
and trophozoites, they may have functions in both the asexual and sexual
blood stages.
The fact that several known proteins where found in both the OB and
g-exoneme interactomes suggests that these may have met during
endomembrane trafficking. In this context, we validated the vesicular
localization of CCp2, a component of the LCCL domain adhesion protein
complex known to be synthesized continuously and present at the GPM
(reviewed in ). CCp2 as well as other components of the adhesion protein
complex have been identified as interactors of all three bait proteins
(see above). IFA analyses confirmed the presence of CCp2 in vesicles and
in association with the GPM as well as the plasma membrane of gametes,
where it neither co-localizes with G377 nor PPLP2 (Fig. S7).
In conclusion, we identified various proteins as potential constituents
of egress vesicles. Among the candidates, previously described
components of OBs and g-exonemes as well as novel proteins were found.
Novel candidates particularly included peptidases, transmembrane
transporters and proteins involved in vesicle trafficking.