Discussion
The exit of activated gametocytes from the RBC is mediated by molecules that are discharged by specialized secretory vesicles following the perception of the egress signal. Activation triggers the exocytosis of OBs needed to rupture the PVM as well as of recently identified PPLP2-positive g-exonemes that are important for the perforation of the RBCM.
Two previous proteomics analyses were performed, using the rodent malaria model P. berghei , to unveil on the egressome of gametocytes . In both approaches, the entity of proteins released during gametogenesis was collected and determined. In addition, a MDV1-specific interactome was established. The two studies described a variety of secreted proteases, like serine-rich antigens SERA1, SERA2, SERA3, the subtilisin-like proteases SUB1, SUB2, MiGS, and PMX as egressome components. Additional proteins included GEST, MDV1, PPLP2, G377, MSP1 and MSP9, and MTRAP as well as PSOP1, PSOP12 and PSOP17.
The aim of the present study was to evaluate the types and proteomes of gametocyte-specific egress vesicles in the human malaria speciesP. falciparum . We firstly demonstrated that OBs and g-exonemes are two different types of vesicles and described the consecutive exocytosis of the two vesicle types following gametocyte activation, with the latter exocytosis being dependent on intracellular calcium. These observations are in accord with our previous findings reporting that OBs discharge at 1 min post-activation, while PPLP2-resident vesicles discharge approximately 6 min later and that PPLP2 release, but not OB exocytosis, can be impaired by calcium chelators .
We then analyzed interactors of the OB-specific proteins G377 and MDV1 and of the g-exoneme component PPLP2 in order to characterize the gametocyte egressome. On the one hand, we found several previously described mediators of egress, including ones identified in the above mentioned proteomics analysis of egressing P. bergheigametocytes. These egress proteins comprise, in addition to the bait proteins, among others Pf11-1, GEST, GEP, MiGS, GEXP02, SUB2, EPF1, and PMX . Noteworthy, the latter two were so far assigned to egressing merozoites, but appear to play further roles in RBC exit by gametocytes. On the other hand, we identified novel constituents or proteins previously assigned to other functions and lifecycle stages, e.g. merozoite proteins like RhopH2, MaTrA, MSP8, and P38, all of which are known to be involved in RBC invasion or the PSOP members PSOP1, PSOP12, PSOP13, PSOP17 and GAMA (the ortholog of P. berghei PSOP9) originally described in the ookinete micronemes and assigned to mosquito midgut penetration . The fact that the here identified egressome includes proteins important for merozoite invasion, as well as ookinete proteins assigned to midgut extravasation, suggests that such proteins may have multiple functions related to host cell membrane modification in different lifecycle stages.
We also identified various proteases, e.g. SUB2, falstatin, DPAP1, DPAP2, M1AAP, M16, as well as PMI, PMIII, PMX, the majority of which were so far linked to hemoglobin processing (reviewed in e.g. Importantly, a high number of further interactors was previously associated to the Maurer´s clefts, like the gametocyte exported protein family members GEXP02, GEXP17, GEXP12, GEXP21, and GEXP22, further EPF1, PTP5, ETRAMP10.2, MSRP5, and PV1 as well as various PHIST proteins and unnamed exported proteins. We hypothesize that these proteins function in remodeling the erythrocyte cytoskeleton as a preceding step of RBCM rupture.
Noteworthy, the majority of the identified proteins were interactors of MDV1, which could be assigned, in equal parts, to male and female gametocytes or were considered to be sex-unspecific. MDV1 was originally described to function during male gametogenesis (); however, other reports neither confirmed its male-specificity in natural infections (; ) or assigned it to female gametocytes . This discrepancy in the sex assignment of MDV1 would explain, why no link between its interactors and any potential male-specific expression could be detected.
The identification of PPLP2-specific interactors proved difficult, even after repetition of the BioID analysis using another independent duplicate. The majority of PPLP2 interactors can be assigned to vesicle biogenesis, particularly transmembrane transporters. To be highlighted is the interaction of PPLP2 with the v-SNARE protein Vti1, which is one of three identified P. falciparum orthologs of eukaryotic Vti1 (). Indeed, in silico and transcript and protein expression analyses confirmed that many factors of the vesicle trafficking and fusion machinery, including SNAREs and associated proteins, are conserved in P. falciparum and expressed in gametocytes (reviewed in ). The role of these molecules in the dynamics of egress vesicles, however, is yet unknown and needs to be investigated in more detail.
Several of the identified candidates were interactors shared by PPLP2 with G377 and/or MDV1. The majority of these proteins belong to the LCCL domain protein-based multi-adhesion domain protein complex, including CCp1, CCp2, CCp3, CCp5 and FNPA as well as the associated proteins P48/45, and P230, plus the paralogs P47 and P230p. It has previously been shown that the components of this multi-adhesion domain protein complex are expressed throughout gametocyte maturation and localize in the PV, where the complex is anchored via P48/45 in the GPM (). IFAs confirmed the presence of CCp2 in vesicles and the PV and showed that CCp2 does neither co-label with G377 nor PPLP2. These results suggest that the components of the multi-adhesion domain protein complex have met the biotin ligase-tagged bait proteins in the secretory pathway, hence during endomembrane trafficking. This would also explain why PPLP2 was previously identified as an interactor of MDV1 .
In a last step of our study, we selected five known and yet uncharacterized components of the egress vesicle interactomes, including the novel v-SNARE Vti1, and verified their vesicular localization. In addition, we confirmed an OB-specific localization of PSOP1 and the unknown gene product PF3D7_0811600. The verification of PSOP1 as an OB-resident protein strengthens our hypothesis that PSOP proteins have additional functions distinct from ookinete biology. In this context, a recent study identified naturally acquired antibodies against PSOP1 in blood samples of various malaria cohorts, verifying the presence of the protein in gametocytes . Similarly, the presence of PSOP12, another egress molecule identified in this study that is a member of the 6-cys family, was demonstrated to be expressed in gametocytes in addition to ookinetes . The expression of such PSOP members from gametocyte maturation until ookinete development makes them promising candidates of transmission blocking vaccines .
In conclusion, we demonstrated the presence of two different types of egress vesicles with important functions during RBC exit by activated gametocytes. We further verified the existence of various OB components in P. falciparum , which were previously identified inP. berghei . In addition, we identified novel components of the two egress vesicle types, including Maurer´s cleft-associated proteins, proteases, transmembrane transporters, and vesicle trafficking proteins, but also various yet unknown proteins. Follow-up studies will need to verify the role of these candidates in gametogenesis in order to shed more light on the molecular machinery of the inside-out egress of gametocytes.