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.