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SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) play a central role in regulating and facilitating vesicular traffic in eukaryotic cells. While SNAREs have been well characterized in other eukaryotes, little is known about their role in the unique protein trafficking pathways in Plasmodium falciparum. We have identified seven Qa-SNAREs in the P. falciparum genome and confirmed the gene structure of all seven, which in one case differs from the predicted structure in the database. Based on comprehensive sequence alignments we made predictions for the intracellular locations of all seven P. falciparum Qa-SNAREs, and confirmed the predicted location for one Qa-SNARE, PfStx1, which is most closely related to other eukaryotic plasma membrane Qa-SNAREs such as syntaxin 1. This is the first identified trafficking component localized proximal to the P. falciparum plasma membrane.

Infection with the protozoan parasite Plasmodium falciparum results in the severest form of human malaria. The symptoms and pathology of malaria occur during the intra-erythrocytic stage of the parasite life-cycle, and virulence has been shown to be associated with parasite multiplication and the selectivity of erythrocyte invasion [1]. Understanding the parasite molecules that mediate erythrocyte selectivity may lead to new interventions aimed at limiting virulence. Invasion of erythrocytes by P. falciparum merozoites involves multiple ligand–receptor interactions, known as invasion pathways [2]. Several alternate erythrocyte surface receptors have been identified, including the major glycoproteins A and B, as well as the unrelated glycophorin C. Others have been described by their sensitivity to enzymes such as neuraminidase, chymotrypsin and trypsin, but the specific erythrocyte proteins involved are not known. Laboratory strains of P. falciparum vary in their dependence on these alternate receptors for invasion. Recognition of alternate receptors on the erythrocyte surface by P. falciparum merozoites is thought to be mediated by members of the reticulocyte-binding homolog (PfRh) and erythrocyte binding antigen (PfEBA) ligand families; each family contains five paralogous proteins [2]. Importantly, distinct laboratory lines express different members of the PfRh family

Host cell invasion by apicomplexan parasites is powered by an actin/myosin motor complex that has been most thoroughly described in Toxoplasma gondii tachyzoites. In T. gondii, two inner membrane complex (IMC) proteins, the peripheral protein TgGAP45 and the transmembrane protein TgGAP50, form a complex with the myosin, TgMyoA, and its light chain, TgMLC1. This complex, referred to as the glideosome, anchors the invasion motor to the IMC. We have identified and characterized orthologues of TgMLC1, TgGAP45 and TgGAP50 in blood-stages of the major human pathogen Plasmodium falciparum, supporting the idea that the same basic complex drives host cell invasion across the apicomplexan phylum. The P. falciparum glideosome proteins are transcribed, expressed and localized in a manner consistent with a role in erythrocyte invasion. Furthermore, PfMyoA interacts with PfMTIP through broadly conserved mechanisms described in other eukaryotes, and forms a complex with PfGAP45 and PfGAP50 in late schizonts and merozoites. P. falciparum is known to use multiple alternative invasion pathways to enter erythrocytes, hampering vaccine development efforts targeting erythrocyte invasion. Our data suggests that the same invasion motor underpins all alternative invasion pathways, making it an attractive target for the development of novel intervention strategies.

Plasmodium falciparum erythrocyte invasion is powered by an actin/myosin motor complex that is linked both to the tight junction and to the merozoite cytoskeleton through the Inner Membrane Complex (IMC). The IMC association of the myosin motor, PfMyoA, is maintained by its association with three proteins: PfMTIP, a myosin light chain, PfGAP45, an IMC peripheral membrane protein, and PfGAP50, an integral membrane protein of the IMC. This protein complex is referred to as the glideosome, and given its central role in erythrocyte invasion, this complex is likely the target of several specific regulatory effectors that ensure it is properly localized, assembled, and activated as the merozoite prepares to invade its target cell. However, little is known about how erythrocyte invasion as a whole is regulated, or about how or whether that regulation impacts the glideosome. Here we show that P. falciparum erythrocyte invasion is regulated by the release of intracellular calcium via the cyclic-ADP Ribose (cADPR) pathway, but that inhibition of cADPR-mediated calcium release does not affect PfGAP45 phosphorylation or glideosome association. By contrast, the serine/threonine kinase inhibitor, staurosporine, affects both PfGAP45 isoform distribution and the integrity of the glideosome complex. This data identifies specific regulatory elements involved in controlling P. falciparum erythrocyte invasion and reveals that the assembly status of the merozoite glideosome, which is central to erythrocyte invasion, is surprisingly dynamic.

Malarial merozoites use an array of ligands, including members of the Reticulocyte Binding Like (RBL) super-family of invasion proteins, to identify and invade erythrocytes. RBL family members are large Type I membrane anchored proteins expressed at the invasive end of merozoites that share homology with the Reticulocyte Binding Proteins 1 and 2 (PvRBP1 and 2) of Plasmodium vivax. Plasmodium species vary widely both in the number and sequence of their RBL genes, with the recently completed Plasmodium falciparum genome containing five RBL genes. Of these, three encode proteins shown to be involved in erythrocyte invasion, a fourth is a pseudogene, and the role of the fifth is as yet unclear. In order to identify sequence similarities and differences that may have functional implications for erythrocyte invasion as well as to gain insights into the recent evolutionary history of the P. falciparum RBL genes, we have sequenced all five corresponding RBL genes from the chimpanzee parasite Plasmodium reichenowi, which is the closest phylogenetic relative of P. falciparum, yet is unable to invade human erythrocytes. Two of the five P. falciparum RBL genes have highly conserved complete open reading frames in both species, while the other three genes show evidence of gene conversion and rapid evolution. The RBL super-family, therefore, appears to be surprisingly dynamic and divergent, implying that it is involved in species-specific aspects of erythrocyte recognition and invasion.