The experiments reported here indicate that the first 80 aminoacids of Pfg377 are sufficient to target the DsRed reporter to female gametocyte osmiophilic bodies. This is to our knowledge the first identification of a
P. falciparum aminoacid sequence able to mediate trafficking to such organelles, which are comparatively less characterized than similar electron dense organelles such as rhoptries, micronemes and dense granules in other parasite developmental stages. Molecular composition of osmiophilic bodies is also much less characterized than that of the above organelles, as only the gametocyte proteins Pfg377 and PfMdv-1/Peg3, the latter however abundantly present also in additional membrane compartments [
22,
23], have been so far localized in such organelles in
P. falciparum. Virtually nothing is known on biogenesis of and protein trafficking to osmiophilic bodies. In general, trafficking to organelles of the apical complex such as rhoptries, dense granules and micronemes have been mainly investigated in asexual stages of
Plasmodium and
T. gondii, and requirement of specific sequences in proper organelle localization is still poorly understood. From studies on vesicular trafficking in lysosome biogenesis in higher eukaryotes it results that C-terminal portions of lysosomal proteins are recognized in the Golgi by cytoplasmic adaptor proteins that mediate their trafficking to such organelles. In
T. gondii, the microneme proteins MIC2 and MIC6 and the rhoptry protein ROP2 reach their organelle destination with such a mechanism [
24‐
26]. At odds with the picture emerging from the above studies, functional analyses of fusion protein localization indicated instead that in
T. gondii the first 85 aminoacids of the rhoptry protein ROP1 are sufficient to ensure proper organelle targeting [
19], and in
P. falciparum the initial 24 residues of the rhoptry protein RhopH2 are able to drive localization of a fluorescent reporter to such organelles [
4]. The work presented here further supports the hypothesis that a signal peptide and a relatively limited N-terminal portion may be sufficient to specifically traffic a parasite protein to the gametocyte osmiophilic bodies.
As only three proteins have been so far positively localized in osmiophilic bodies (Pfg377, Pf Mdv-1/Peg3, PfGEST), a comparative sequence analysis to identify functionally conserved motifs is difficult. Nevertheless, both this approach and structural modeling analysis were undertaken to predict motifs functionally involved in trafficking to such organelles (see Additional file
4). However, the motifs predicted by computational analysis lack in specificity, thus suggesting that a higher number of osmiophilic body-associated proteins is needed to identify the determinants of osmiophilic body localization.
The positive identification presented here of a parasite sequence targeting proteins to female gametocyte osmiophilic bodies is a relevant improvement in the ongoing studies on protein trafficking in parasite sexual differentiation. In addition, the possibility to fluorescently tag such poorly characterized organelles opens novel avenues in cellular and imaging studies on their biogenesis and on their role in gamete egress.