3. P2 oligomerization in the parasite cytoplasm appears to be one of the prerequisite step for translocation to the host cells
Effector proteins in P. falciparum -infected iRBCs translocate to the host cells and render the host cell membrane rigid and promiscuous for solutes and other macromolecules (Goldberg et al., 2010; Hiller et al., 2004; Russo et al., 2010). A well-defined class of export proteins possesses a Plasmodium export element (PEXEL) or vacuolar targeting signal, a pentapeptide consensus sequence (RxLxE/Q/D) located 25-30 amino acids downstream of the host-targeting signal sequence (Marti et al., 2004; Spillman et al., 2015; Desai et al., 2012; Desai et al., 2014a). The cleavage of the PEXEL motif by the ER-resident aspartic proteases, plasmepsin V is required to destine the export proteins to the host cells (Russo et al., 2010; Boddey et al., 2010; Tarr et al., 2013; Osborne et al., 2010). However, the protease cleavage of plasmepsin V does not appear to be the sole deciding factor for translocation into the host cells as PI(3)P binding to the effector proteins in the ER upstream of the PEXEL motif seems to be a prerequisite step for translocation in addition to the plasmepsin V cleavage (Bhattacharjee et al., 2012a; Bhattacharjee et al., 2012b; Bhattacharjee et al., 2012c; Haldar et al., 2016; Hsiao et al., 2013). Another class of effector proteins, PEXEL negative exported proteins (PNEPs), do not contain PEXEL motif but still can translocate to the host cells (Pachlatko et al., 2010; Bhattacharjee et al., 2012a; Spycher et al., 2006; Saridaki et al., 2009; Haase et al., 2009; Heiber et al., 2013; Jani et al., 2008). The first 20 amino acids at the N terminus of PNEPs without a classical ‘N’ terminal signal sequence are sufficient to export non PEXEL effector proteins to the host cells (Saridaki et al., 2009; Haase et al., 2009) indicating a general export property of PNEPs. However, effector proteins of both the classes, PEXEL dependent and independent, do unfold in the vacuolar space before they traverse through the PTEX complex (Grüring et al., 2012; Desai et al., 2014b; Ho et al., 2018; Garten et al., 2018).
The process of oligomerization of P2 in the parasite cytoplasm precedes the localization on the iRBC surface. Out of several oligomeric species in the parasite cytoplasm at the trophozoite stage, only homo/hetero tetrameric species of P2 appear to translocate to the iRBC surface through iRBC cytoplasm (Das et al., 2012a). Neither ghost from iRBCs surface nor the iRBCs cytoplasm showed the presence of monomeric P2 indicating that the oligomerization in the parasite cytoplasm could be initial one of the prerequisite steps in the export mechanism of P2. In the 48h life cycle in iRBCs, the oligomerization window of P2 in the parasite cytoplasm approximately starts at around 22-24h post merozoite invasion (PMI) and continues till 34-36h PMI at late trophozoite to the early schizogonic stage (Das et al., 2012a). Before 22-24h and after 36-38h PMI, there were no oligomeric species of P2 observed in the western blot experiment (Das et al., 2012a; Das et al., 2012b) indicating tight regulation of oligomerization in that window of time and a possible link between oligomerization and export. After that window, oligomerization diminishes possibly due to the secretion of P2 into the culture supernatant or digestion. Immunofluorescence assay (IFA) using E2G12 after 34-36h PMI does not seem to suggest a possibility of P2 recycling back to the parasite from the host cells as iRBC cytosol did not show any P2 staining (Das et al., 2012a).
P2 does not contain any PEXEL motif and also it is devoid of classical export signal hence it comes under the PNEP class and possibly translocate through an unknown pathway. Transient transfection with P2-GFP followed by immunoblotting does not seem to suggest any protease processing of P2-GFP before export into the host cells as the molecular mass of P2-GFP did not deviate from the theoretical mass (Das et al., 2012a). In addition to oligomerization, the probable route of P2 translocation might have been PI(3)P binding and unfolding of the P2 tetramer/oligomer before PTEX traversing.