Protein Export Marks the Early Phase of Gametocytogenesis of the Human Malaria Parasite Plasmodium falciparum

Abstract

Despite over a century of study of malaria parasites, parts of the Plasmodium falciparum life cycle remain virtually unknown. One of these is the early gametocyte stage, a round shaped cell morphologically similar to an asexual trophozoite in which major cellular transformations ensure subsequent development of the elongated gametocyte. We developed a protocol to obtain for the first time highly purified preparations of early gametocytes using a transgenic line expressing a green fluorescent protein from the onset of gametocytogenesis. We determined the cellular proteome (1427 proteins) of this parasite stage by high accuracy tandem mass spectrometry and newly determined the proteomes of asexual trophozoites and mature gametocytes, identifying altogether 1090 previously undetected parasite proteins. Quantitative label-free comparative proteomics analysis determined enriched protein clusters for the three parasite developmental stages. Gene set enrichment analysis on the 251 proteins enriched in the early gametocyte proteome revealed that proteins putatively exported and involved in erythrocyte remodeling are the most overrepresented protein set in these stages. One-tenth of the early gametocyte-enriched proteome is constituted of putatively exported proteins, here named PfGEXPs (P. falciparum gametocyte-exported proteins). N-terminal processing and N-acetylation at a conserved leucine residue within the Plasmodium export element pentamotif were detected by mass spectrometry for three such proteins in the early but not in the mature gametocyte sample, further supporting a specific role in protein export in early gametocytogenesis. Previous reports and results of our experiments confirm that the three proteins are indeed exported in the erythrocyte cytoplasm. This work indicates that protein export profoundly marks early sexual differentiation in P. falciparum, probably contributing to host cell remodeling in this phase of the life cycle, and that gametocyte-

SSP3 Is a Novel Plasmodium yoelii Sporozoite Surface Protein with a Role in Gliding Motility

“…SSP3 was not detected in lysates of mixed blood stages (see Fig. S4E in the supplemental material), which is in agreement with the lack of SSP3 in the previously published proteomes of asexual and sexual blood stages of P. falciparum and P. berghei (29)(30)(31)(32)(33). Because SSP3 contains 13 conserved cysteine residues, we tested whether these residues form disulfide bonds by analyzing the electrophoretic mobility of SSP3 under reducing and nonreducing (omitting the disulfide-reducing agent BME and with no boiling) conditions.…”

“…It is also possible that the lack of SSP3 on the sporozoite surface might alter the distribution or functionality of other proteins important for attachment and/or gliding in vitro. It has been shown that TRAP and S6, another TRAP family protein, are necessary for initial adhesion to glass slides (33). TRAP proteins are released from micronemes onto the sporozoite surface and are redistributed from the front to the back of the sporozoite.…”

Plasmodium falciparum STEVOR phosphorylation regulates host erythrocyte deformability enabling malaria parasite transmission

“…10,11,45,46 The rise in the knob density in mature asexual stages results in the increased number of vertical constraints between the spectrin network and the lipid bilayer, which further stiffens the membrane. 47 In GIEs, KAHRP and PfEMP1 are not expressed 48,49 and STEVORs may perform an analogous role by increasing vertical constraints between the cytoskeleton and the lipid bilayer. Protein-protein interactions essential for the erythrocyte membrane mechanical properties are modulated by posttranscriptional modifications of either cytoskeleton or parasite proteins.…”

Translational repression of the cpw-wpc gene family in the malaria parasite Plasmodium

“…0.5 ml was then separated from the ookinete culture and incubated with Hoechst 33342/PBS and imaged as above. Red blood cells in the remainder of the ookinete culture were lysed in a 140 mM NH 4 Cl solution on ice, ookinetes were collected by centrifugation and processed further for Western blotting with Roche monoclonal anti-GFP antibody (#11814460001).…”

“…Thus, many predicted sexual stage proteins remain uncharacterized and have unknown functions; specifically, of the 2430 proteins identified by mass-spectrometry in P. falciparum gametocytes (stages I-II, IV and V) [3,4], 830 (34%) are annotated with "unknown function" (www.plasmodb.org; June 5, 2014). Their molecular and cellular descriptions are vital to prioritize candidate surface proteins for transmission-blocking vaccine approaches that interfere with parasite development or inhibit infection of the mosquito vector by the ookinete [5].…”