A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito

Abstract

The recent sequencing of several apicomplexan genomes has provided the opportunity to characterize novel antigens essential for the parasite life cycle that might lead to the development of new diagnostic and therapeutic markers. Here we have screened the Plasmodium falciparum genome sequence for genes encoding extracellular multidomain putative adhesive proteins. Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes. Orthologues were identified in the Cryptosporidium parvum genome sequence, indicating an evolutionary conserved function. Transcript and protein expression analysis shows sexual stage-specific expression of PfCCp1, PfCCp2, and PfCCp3, and cellular localization studies revealed plasma membrane-associated expression in mature gametocytes. During gametogenesis, PfCCps are released and localize surrounding complexes of newly emerged microgametes and macrogametes. PfCCp expression markedly decreased after formation of zygotes. To begin to address PfCCp function, the PfCCp2 and PfCCp3 gene loci were disrupted by homologous recombination, resulting in parasites capable of forming oocyst sporozoites but blocked in the salivary gland transition. Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine.

Figure 1.

Domain organization of the apicomplexan LCCL domain–containing gene family in P. falciparum (designated with prefix Pf) and orthologues present in C. parvum (designated with prefix Cp). All proteins have a signal peptide sequence represented by a black box at the beginning of the architectures. Ric, ricin domain; Disc, discoidin domain; A, ApicA domain; Anth, anthrax toxin NH₂-terminal region domain; FN2, fibronectin type II domain; LCCL, Limulus coagulation factor C domain; Lev, levanase domain; LH, lipoxygenase domain; NEC, neurexins and collagens domain; SR, scavenger receptor domain.

Figure 2.

Multiple sequence alignment of the NEC domain. Proteins are designated by their gene name followed by their species abbreviations and GenBank identifier numbers. The apicomplexan proteins are represented by their gene names only. The coloring reflects 85% consensus. The consensus abbreviations and coloring scheme are as follows: h, hydrophobic residues (LIYFMWACV) and l, aliphatic (LIAV) residues shaded yellow; c, charged (KERDH) residues and p, polar (STEDRKHNQ) residues colored blue; s, small (SAGDNPVT) residues colored green; C, conserved disulfide bonding cysteines are shaded red. In the secondary structure annotation, E represents a residue in a strand (extended) and H represents a residue in a helix. Species abbreviations are as follows: Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Ephmu, Ephydatia muelleri; Hd, Haliotis discus; Hs, Homo sapiens; Pf, Plasmodium falciparum.

Figure 3.

Phylogenetic trees showing affinity of apicomplexan versions to bacterial discoidin (A) and animal NEC (B) domains. Principal nodes supported by bootstrap values of >65% are shown with a filled green circle. Clades containing apicomplexan sequences are colored blue and apicomplexan sequences are boxed. Genes are designated by their gene names and the abbreviated source species names are as follows: Art, Arthrobacter spp.; At, Arabidopsis thaliana; Bh, Bacillus halodurans; Bsp, Bacillus spp. D-2; Ce, Caenorrhabditis elegans; Clpe, Clostridium perfringens; Cp, Cryptosporidium parvum; Dd, Dictyostelium discoidium; Dm, Drosophila melanogaster; Hs, Homo sapiens; Hyro, Hypomyces rosellus; Lal, Lactococcus lactis; Mivi, Micromonospora viridifaciens; Pf, Plasmodium falciparum; Scoe, Streptomyces coelicolor; Xf, Xylella fastidiosa; Yp, Yersinia pestis.

Figure 4.

Membrane-associated protein expression of PfCCps in mature gametocytes. (A) Western blot analysis of extracts from mixed asexual (lanes A) and mature gametocyte (lanes G) stages show gametocyte-specific PfCCp expression. As a control for protein loading, extracts were additionally screened with mouse sera recognizing Pf39, an abundant ER-resident protein (lanes marked Pf39). Lanes marked MBP indicate lack of reactivity with mouse control sera recognizing the recombinant fusion partner MBP. (B) Indirect IFA assay using mouse sera against PfCCp1, PfCCp2, and PfCCp3 revealed a punctate pattern associated to the parasite surface in mature gametocytes (green Alexa Fluor 488), whereas asexual stage parasites did not exhibit any labeling. Mouse sera directed against MBP shows no labeling of gametocytes. Erythrocytes were counterstained with Evans Blue (red). Arrows indicate asexual parasites visualized by TOTO-3 nuclear stain (blue). Bar, 5 μm.

Figure 5.

Ultrastructural PfCCp1 localization associates with the parasite membrane in the parasitophorous vacuole. Immunoelectron microscopic examination using anti-PfCCp1 primary antibody and either immunogold (A, enlarged in C) or alkaline phosphatase (B, enlarged in D) secondary labeling localize expression to the parasitophorous vacuole of mature gametocytes as well as in the gametocyte cytoplasm and attached to the outside of erythrocyte membranes. (E) No labeling was found in asexual parasites and erythrocytes (here shown for alkaline phosphatase labeling). Bars, 1 μm.

Figure 6.

PfCCps are present within mature gametocytes and localize extracellularly after gametogenesis, but expression ceases after fertilization. (A) PfCCp protein labeling in mature gametocytes is associated with the parasite surface, whereas (B) PfCCp protein is released during the emergence of gametes from within erythrocytes. (C) PfCCp expression markedly decreases in emerged gametes and protein instead localizes extracellularly (red Alexa Fluor 594), adhering to complexes of emerged macrogametes and adhering microgametes (green Alexa Fluor 488; detected with rabbit α-tubulin II antiserum). (D) Zygotes or unfertilized macrogametes detected with rabbit Pfs25 antiserum (red Alexa Fluor 594) exhibited a low remaining expression for PfCCps (green Alexa Fluor 488) 20 h after gamete emergence in vitro. Bloodmeal preparations isolated from mosquito midguts 24 h after feeding on gametocyte cultures revealed neither PfCCp expression (green Alexa Fluor 488) in (E) retort stages nor in (F) ookinetes (red Alexa Fluor 594, detected with Pfs25 antiserum), whereas gamete complexes in the bloodmeal preparations still showed associated PfCCp labeling. Labeling shown is for PfCCp1. Similar results were obtained for PfCCp2 and PfCCp3 (not depicted). Bars, 2 μm (A and B) and 10 μm (C–F).

Figure 7.

Schematic of the PfCCp2 locus, the disruption plasmid construct pPfCCp2-KO (using the pDT-Tg23 vector), and the organization of the PfCCp2 locus after integration by homologous recombination. The coding region of PfCCp2 (gray) is split into a region representing the portion homologous to the disruption plasmid (dark gray). The T. gondii DHFR gene and regulatory cassette are represented by a white box. Approximate location of restriction enzyme sites (B, BclI; Bg, BglII; S, SpeI; X, XbaI) and sizes of restriction digest fragments are indicated below the WT and disrupted PfCCp2 loci (refer to Southern blot analysis shown in Fig. S2). Amplification of a PCR product using the primers P1 and P2 was diagnostic of homologous integration. The PfCCp3 gene locus was similarly disrupted.

Figure 8.

Development of midgut oocysts and sporozoites after mosquito membrane feeding with gene-disrupted parasites. Ultrastructural examinations 12 d after feeding show oocysts in mosquito midguts after feeding PfCCp2-KO clone D-11H (A) and PfCCp3-KO clone I-9C (B) cultures. Mature oocysts exhibit fully developed sporozoites for PfCCp2-KO (C) and PfCCp3-KO (D). Bars, 1 μm.