Abstract
The sexual phase of the malaria parasite Plasmodium falciparum is accompanied by the coordinated expression of stage-specific adhesive proteins. Among these are six secreted proteins with multiple adhesion domains, termed P. falciparum LCCL domain-containing protein (PfCCp) proteins, which are expressed in the parasitophorous vacuole of the differentiating gametocytes and which are later associated with macrogametes. Although the majority of the PfCCp proteins are implicated in parasite development in the mosquito vector, their functions remain unknown. In the present study we investigated the molecular interactions between the PfCCp proteins during gametocyte development and emergence. Using five different gene-disruptant parasite lines, we show that the lack of one PfCCp protein leads to the loss of other PfCCp family members. Co-immunoprecipitation assays on gametocyte lysates revealed formation of complexes involving all PfCCp proteins, and affinity chromatography co-elution binding assays with recombinant PfCCp domains further indicated direct binding between distinct adhesion domains. PfCCp-coated latex beads bind to newly formed macrogametes but not to gametocytes or older macrogametes 6 or 24 h post-activation. In view of these data, we propose that the PfCCp proteins form multi-protein complexes that are exposed during gametogenesis, thereby mediating cell contacts of macrogametes.
Highlights
The sexual phase of the malaria pathogen Plasmodium falciparum begins with the differentiation of predetermined blood stage parasites to gametocytes in the human host and continues with the formation of male and female gametes in the midgut of the blood-fed female mosquito vector
PfCCp1 transcripts were not detected by RT-PCR in the gametocyte stages of disruptant parasites, and the lack of PfCCp1 expression was confirmed by Western blot analysis of gametocyte protein extracts isolated from PfCCp1-KO parasites compared with a wild type control (Fig. 1A) and by immunofluorescence assay
During the last two decades a substantial number of proteins has been identified that are expressed in the sexual and mosquito stages of malaria parasites. The majority of these sexual stage proteins are initially expressed in the parasitophorous vacuole during gametocyte differentiation, and some are later exposed on the surface of emerged gametes and fertilized zygotes
Summary
Gene Identifications—The following gene identifiers are assigned to the proteins investigated in this study: Pf39, PF11_0098; PfCCp1, PF14_0723; PfCCp2, PF14_0532; PfCCp3, PF14_0067; PfCCp4, PFI0185w; PfCCp5, PFA0445w; PfFNPA, PF14_0491; and Pfs, PF10_0303. PCR was performed with 100 ng of genomic DNA from wild type or disruptant parasites for 35 cycles of amplification and with an annealing temperature of 50 °C, using the above mentioned primers a, b, bЈ, c, cЈ, and d (shown in supplemental Fig. S1). PCR with genomic DNA from wild type or disruptant parasites was performed for 33 cycles of amplification and with annealing temperatures ranging from 42 to 50 °C using the primers s, t, u, and v (shown in supplemental Fig. S2). The membranes were blocked for nonspecific binding by incubation in Tris-buffered saline containing 5% skim milk and 1% bovine serum albumin fraction V, followed by immune recognition for 2 h at room temperature with mouse immune sera specific to PfCCp proteins, Pf39, or to GST- and His6/SUMO fusion partner protein control. Binding of latex beads to macrogametes was subsequently verified by immunofluorescence assay using antibodies against Pfs in combination with Alexa Fluor 596-conjugated secondary antibodies
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