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Early Events in the Invasion of Red Blood Cells by Plasmodium falciparum Merozoites
Summary: Brendan Crabb is studying the early events in the invasion of Plasmodium falciparum merozoites into human red blood cells, a poorly understood aspect of the malaria parasite's life cycle. In recent years, using newly developed genetic technologies, he has worked to identify the protein machinery involved in these events and characterize their function.
The merozoite is a short-lived extracellular form of the human malaria parasite Plasmodium falciparum, which, to survive, must rapidly attach to and invade a human red blood cell (RBC). Here, it resides and multiplies for several days in an environment that is largely protected from host immune responses. Merozoite surface proteins are involved in initial contact with target RBCs, a process that begins a cascade of events required for successful invasion of the cells. The surface proteins are considered prime targets for protective immune responses and, as a result, some of the proteins have become prominent vaccine targets. However, the identities of the primary attachment ligands and their RBC receptors remain unknown. Furthermore, early invasion events appear to promote a calcium-signaling event necessary for secondary invasion steps. Even though these processes are obligatory to the life cycle of the parasite, very little is known about the identity, architecture, and functioning of the molecular machinery involved. This is especially intriguing in the case of induction of a calcium signal, given that, in the P. falciparum genome, there are no obvious orthologues of common mammalian cell mediators of such events, such as tyrosine kinases and G protein–coupled receptors. Also perplexing is the paucity of surface proteins known to span the parasite membrane, at least one of which is presumed to be necessary to initiate the phosphorylation cascade that results in intracellular calcium release.
To identify surface components and underlying signaling machinery, we analyzed the protein content of detergent-resistant membranes (DRMs). In mammalian cells, signaling machinery is thought to congregate in such membranes, which are often referred to as lipid rafts. Our approach has proven successful in identifying many new glycosylphosphatidylinositol (GPI)-anchored surface proteins, some multimembrane-spanning surface and/or inner-membrane (a membrane found just underneath the parasite surface membrane) proteins and other adaptors and enzymes predicted to be involved in either initiating or receiving a calcium signal.
Our work has shown that there are at least nine GPI-anchored proteins on the merozoite surface of P. falciparum, and all are potential erythrocyte ligands. Such GPI-anchored proteins are not necessarily evenly spread over the surface, and, consistent with different roles in invasion, some tend to an apical localization. Besides a GPI-anchor, many surface proteins share similarities including cysteine-rich domains that are potentially significant in adherence. These domains include epidermal growth factor–like modules at the C terminus of four surface proteins and "6-Cys" modules in the surface proteins Pf12 and Pf38. Other 6-Cys family members are found on the surface of P. falciparum gametes and sporozoites. Modeling data by others have revealed that the structure of the 6-Cys proteins resembles that of the surface antigen (SAG)-related sequence (SRS) superfamily found on the surface of Toxoplasma gondii tachyzoites previously thought to be restricted to tissue-associated coccidian members of the Apicomplexan phylum. The dual Cys-rich domains in the prototype T. gondii SRS-family member SAG1 dimerize to form a receptor-binding site at the tip of the molecule. Members of the Plasmodium 6-Cys family are already recognized as playing adhesive roles on the surface of gametes and the liver-invasive sporozoite forms. The discovery of three different family members on the surface of blood stages suggests that they also function in this manner. The fact that these proteins possess an SRS-type fold adds weight to this argument; perhaps they represent the elusive adhesins that initiate contact with erythrocytes.
Peripheral proteins are also candidates for binding to erythrocyte receptors, and we have identified many in these studies. The proteins are secreted into the parasitophorous vacuole of schizont-stage parasites and bind to the surface of developing merozoites, at least to some degree, via an interaction with GPI-anchored proteins such as MSP-1. Most peripheral proteins belong to one of three families of proteins: the MSP-3/-6 group, the MSP-7 family, and the SERA protease family. Other peripheral proteins include the acidic-basic repeat antigen and Pf41, another 6-Cys family member. Despite the number of family members, it appears that only a few peripheral proteins, including MSP-7 family members and Pf41, are strongly associated with the surface and hence are likely to represent core structural components. These are the best candidates to mediate primary contact, but a role for more loosely associated proteins such as MSP-3 and MSP-6 and other unidentified proteins cannot be ruled out. It remains to be established which of the core surface proteins, whether membrane-associated or peripheral, have adhesive roles in the initial recognition and interaction of erythrocytes by merozoites. The difficulties in dissecting this issue relates to the likelihood that adhesion is of low affinity and probably requires a high concentration of correctly oligomerized surface ligands in a manner that is difficult to reproduce in vitro. We have also used blue native polyacrylamide gel electrophoresis on DRM material to isolate high-molecular-weight complexes for identification by mass spectrometry. This process has identified a number of previously unknown complexes on the merozoite surface, including a major new oligomer involving the dominant surface protein.
The next stage for this work is to characterize the role of the individual proteins described above in early invasion events. To do this, we are using both transfection technology, which allows the deletion or conditional disruption of target genes, and imaging. With respect to the latter, we are focusing on filming parasite invasion and characterizing changes in the kinetics of the defined steps in invasion in response to alterations of target genes. Here, we are also using in vivo fluorescence to monitor the location of affected proteins in real time. Our aim is to generate an understanding of the key mediators of the parasite's initiation of invasion at the level of both primary attachment and signal transduction. We anticipate that such insight will provide many potential avenues for the development of vaccines and therapeutic agents that interfere with this central aspect of malaria pathogenesis.
Last updated March 2007
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