Malaria is caused by members of the genus Plasmodium, which belong to the large monophyletic assemblage of parasitic organisms that make up the phylum Apicomplexa. This phylum includes a number of other pathogens such as Cryptosporidium, Toxoplasma, and Theileria and is distinguished in morphological terms by the presence of an apical organellar complex specialized for host cell invasion. Plasmodium spp. belong to the class Hemosporidia and are obligate intracellular parasites, switching between an arthropod vector and a vertebrate host, where they undergo cycles of asexual reproduction in blood cells.

Plasmodium falciparum causes the most severe form of malaria in humans, with several hundred million infections each year and 1–2 million deaths. Most of the clinical symptoms and the associated mortality and morbidity of P.falciparum malaria result from the parasite undergoing continuous cycles of asexual reproduction within human erythrocytes. Infected erythrocytes become rigid and develop the ability to cytoadhere to a number of cell types such as vascular endothelial cells, a mechanism that prevents the parasitized host cells from passing through to the spleen where they would be cleared from the bloodstream. These cellular modifications of infected erythrocytes are the result of a dramatic remodeling process induced by the parasite, a process that brings about cytoadherence by exposing various ligands on the surface for host cell receptors and facilitating nutrient import into the infecting parasite.

Host Cell RemodelingSurface protrusions of P. falciparum–infected erythrocytes, called knobs, display focal aggregates of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the adhesion ligand binding to endothelial cell receptors. The resulting sequestration of infected erythrocytes in tissues represents an important factor in fatalities of malaria patients. The main component of knobs is the knob-associated histidine-rich protein (KAHRP), which contributes to altered mechanical properties of parasite-infected erythrocytes. The role of KAHRP protein domains in these processes remains elusive. We generated stable transgenic P. falciparum–infected erythrocytes expressing mutant versions of KAHRP. Using atomic force and electron microscopy, we showed that the C-terminal repeat region is critical for the formation of functional knobs. The elasticity of the membrane differs dramatically in cells with different KAHRP mutations. We have proposed that the 5′ repeat region of KAHRP is important for the cross-linking to the host cell cytoskeleton required for knob protrusion and efficient adhesion under physiological flow conditions.

To establish infection in the host, malaria parasites of Plasmodia spp. must export remodeling and virulence proteins into the erythrocyte. These proteins traverse a series of membranes, including the parasite membrane, the parasitophorous vacuole membrane, and, for a subset, the erythrocyte membrane. We have shown that protein export into the host erythrocyte is mediated by PEXEL (Plasmodium export element), a unique pentameric sequence. This signal is functionally conserved in Plasmodia spp. and suggests a novel translocase for export across the parasitophorous vacuole membrane into the erythrocyte. We have characterized the exported proteome using the genome sequences of these parasites. In P. falciparum, approximately 400 putative erythrocyte-targeted proteins, encoded by 8 percent of the genome, have been identified, with 225 corresponding to virulence proteins; a further 160 are unique and likely to be involved in remodeling the host erythrocyte. We used green fluorescent protein chimeras and fluorescence photobleaching experiments to follow export of soluble and membrane-associated proteins through the infected erythrocyte. Our data show that the export signal is used by soluble and transmembrane proteins such as the virulence factor PfEMP1. The physical state of the exported proteins suggests trafficking as a complex rather than in vesicles. This study has identified the sequences required for expression of proteins on the outside of the P. falciparum–infected erythrocyte membrane.

A small number of proteins exported to the parasite-infected erythrocyte do not contain an apparent PEXEL, and it is not clear how they are translocated to this compartment. One of these molecules, the 48-kDa P. falciparum skeleton binding protein 1 (PfSBP1) is a type 2 integral membrane protein localized to Maurer's clefts and expressed early in the erythrocyte life cycle, with highest expression in the ring stages. The absence of a recognizable signal sequence and the inhibition of its export with brefeldin A suggests that PfSBP1 is transported via the endoplasmic reticulum–Golgi pathway and post-translationally inserted into the membrane of the Maurer's clefts. The function of PfSBP1 was not known, although it has been suggested that it either anchors Maurer's clefts to the erythrocyte cytoskeleton or prevents early rupture of the erythrocyte membrane by interacting with host cell proteins. We have shown that PfSBP1 is required for transport of PfEMP1 to the P. falciparum–infected erythrocyte surface. PfSBP1-deficient parasite cells express no detectable PfEMP1 on the erythrocyte surface and consequently do not adhere to the endothelial receptor chondroitin sulfate. Furthermore, we have used these parasites to show that the major reactivity of antibodies from multigravid malaria-infected women is directed toward PfEMP1, given that this reactivity is abolished in the absence of PfSBP1.

Invasion of Human Erythrocytes
A common mechanism appears to underlie motility and invasion in Apicomplexa. The prevailing molecular model for motility is based on observations that motile Apicomplexan parasites can translocate surface antigens and other synthetic substrates to their posterior end. Like gliding, this translocation is sensitive to cytochalasin, an actin filament–disrupting compound, and to butanedione monoxime, an inhibitor of myosin heavy-chain ATPase. Thus, gliding is driven by an actomyosin motor, housed in the pellicle of the zoite and linked to both to an inner membrane complex and the parasite plasma membrane. This allows parasite adhesion molecules on the surface and actin filaments in the pellicle to be moved progressively rearward by the force of the myosin motor as the parasite is driven forward. In support of the model, actin localizes broadly to the polar regions of zoites and to the subpellicular network, and myosin localizes between the inner membrane complex and the outer plasma membrane.

The liver stage sporozoites of Plasmodium spp. and tachyzoites of Toxoplasmagondii, the causative agents of malaria and toxoplasmosis, respectively, use a unique mode of locomotion termed gliding motility to invade host cells and cross cell substrates. This ameboid-like movement uses a parasite adhesin from the thrombospondin-related anonymous protein (TRAP) family and a set of proteins linking the extracellular adhesin, via an actinomyosin motor, to the inner membrane complex. However, the blood-stage merozoite of Plasmodium does not exhibit gliding motility. We have shown that homologues of the key proteins that make up the motor complex, including the recently identified glideosome-associated proteins (GAP) 45 and GAP50, are present in P. falciparum merozoites and appear to function in erythrocyte invasion. Furthermore, we have identified a merozoite TRAP homologue, termed MTRAP, a micronemal protein that shares key features with TRAP, including a thrombospondin repeat domain, a putative rhomboid-protease cleavage site, and a cytoplasmic tail that, in vitro, binds to the actin-binding protein aldolase. Analysis of other parasite genomes shows that the components of this motor complex are conserved across diverse Apicomplexan genera. Conservation of the motor complex suggests that a common molecular mechanism underlies all Apicomplexan motility, which, given its unique properties, highlights a number of novel targets for drug intervention to treat major diseases of humans and livestock.

Last updated September 2009