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-enriched molecules are recruited to modulate this process in gametocytogenesis.

Fig. 1.

Fluorescent P. falciparum early gametocytes for proteomics analysis. A, stage I (top) and II (bottom) gametocytes expressing the Pfg27-GFP fusion protein (green) stained with a monoclonal antibody specific for the early gametocyte protein Pfs16 (red fluorescence). Nuclei were stained with Hoechst. Bar, 5 μm. B, sample of FACS-sorted parasites showing highly purified stage I and II gametocytes. Bar, 10 μm.

Fig. 2.

Proteomes of early and mature gametocytes and of asexual trophozoites. A, Venn diagram of the proteins identified in the proteomes of the three parasite stages. B, dendrogram from the SOTA clustering analysis on the relative emPAI profiles of 2211 proteins distributed across the three developmental stages. Vertical colored bars mark clusters of the proteins enriched in the three parasite stages (blue, trophozoites (troph); green, early gametocytes (gct I/II); red, mature gametocytes (gct V)). nr, number.

Fig. 3.

PfGEXP10 processing and export in gametocytogenesis. A, mass spectrometry identification of the N-acetylated PfGEXP10 polypeptide after cleavage at its PEXEL motif. The y-ion fragment series are color-coded in green, and the b-ions are in red. B, primary structure of the PfGEXP10 protein according to PlasmoDB and Eukaryotic Linear Motif (ELM) database annotations. The PfGEXP10 portion produced as the GST fusion for generating specific antibodies is shown. C, Western blot analysis with anti-PfGEXP10 antibody on gametocyte extracts. Soluble (S) and insoluble (P) fractions from gametocyte extracts were electrophoresed, blotted, and detected with Ponceau S staining (left side of each panel). Blots incubated with the control anti-GST and the anti-PfGEXP10 antibodies are shown on the right side of each panel. D, IFA with anti-PfGEXP10 antibody on early and mid-stage gametocytes. Fixed, permeabilized 3D7 gametocytes were simultaneously incubated with the serum against the PfGEXP10 protein or a control anti-GST antiserum (red fluorescence) and a serum against the gametocyte PVM marker PfMdv-1/peg3 (green fluorescence). Nucleus-specific Hoechst staining is shown on the right. 1 and 5, stage I; 2, stage II; 3 and 6, stage III; 4, late stage IV. Bar, 5 μm.