doi:10.

1093/jmcb/mjp024 Published online October 4, 2009

Journal of Molecular Cell Biology (2010), 2, 11 13

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Research Highlight

Home Improvements: How the Malaria Parasite Makes the Red Blood Cell Home Sweet Home
G. Lucas Starnes and Andrew P. Waters*
Division of Infection and Immunity, Institute of Biomedical Life Sciences, Wellcome Centre for Molecular Parasitology, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK * Correspondence to: Andrew P. Waters, E-mail: a.waters@bio.gla.ac.uk

A recent study published in Nature by de Koning-Ward et al. provides further insights into the way in which the lethal malaria parasite, Plasmodium falciparum, remodels the host red blood cell during the pathogenic blood stage of development.
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Human malaria is an infectious disease caused by ve members of the protozoan parasite genus Plasmodium that annually aficts nearly half a billion individuals worldwide. Around one million of Plasmodium falciparum infections result in death, predominantly occurring in children in sub-Saharan Africa. In addition to the toll on human life, the resulting general morbidity contributes to a further depression in the gross domestic product of countries already struggling with destitute poverty. Therefore, among other goals, modern research into malaria parasites seeks to identify processes that are unique to the parasite as a result of its adaptations to a parasitic and, in this case, intracellular lifestyle. Once characterized, it is possible that such processes might then be targeted for drug design. During the pathogenic phase of their life cycle, the human Plasmodium parasites grow and replicate within a self-created, membrane-bound vacuole in a red blood cell (RBC). Occupation of the terminally differentiated RBCs offers a protected niche within the vertebrate host environment, but presents unique challenges to the parasite. In relation to other vertebrate cells, the RBC is a barren wasteland that lacks even an elementary infrastructure that might otherwise be subverted by the parasite to its own ends. The cell instead is streamlined as a result of its predominant role to carry oxygen to tissues and has jettisoned the protein-trafcking

machinery and target organelles present in other host cells. Malaria parasites therefore have a number of clear goals upon invading an RBC that require the cell to be extensively remodeled: they must establish a beachhead, i.e. the vacuole within which they develop; they must engage in dialog with the host cell cytoplasm importing nutrients and maintain the electrolyte balance; they must further modulate the infected RBC (iRBC) surface in order to import nutrients not readily provided by the RBCs (such as isoleucine) and engage in the linked processes of sequestration and antigenic variation. Therefore, Plasmodium has evolved to overcome these difculties and in fact generates its own secretory pathway(s) managing to export nearly 5% of proteins encoded by its genome (van Ooij et al., 2008). Bulk delivery of parasite encoded proteins to the cell surface occurs, of which some may combine to generate large adhesive knobs (Crabb et al., 1997). These knobs change the iRBCs plasticity and increase its ability to stick to other cells and the microvasculature, all of which are deleterious to the host and contribute to the clinical manifestations associated with malaria such as cerebral malaria, organ failure and pregnancy-associated malaria (Crabb et al., 1997). Although the secretory capabilities of this pathogen have been appreciated for quite some time, the logistics of transporting effector proteins across numerous

membranes has remained a black box in the understanding of Plasmodium biology. Faced with this question, researchers have spent signicant efforts toward understanding how the parasite accomplished such an amazing feat. Incrementally, the picture has come into focus as two studies independently identied a conserved secretory signal termed a PEXEL motif or a vacuolar transport signal (VTS) preceding proteins that are destined for delivery to the host cell surface (Hiller et al., 2004; Marti et al., 2004). Following a logical train of thought, de Koning-Ward et al. established a series of criteria in order to identify the machinery (a translocon) responsible for initiating the secretory process in Plasmodium (de Koning-Ward et al., 2009). These lofty criteria state: the components of the translocon should be limited to Plasmodium species; they should localize to the membrane of the parasitophorous vacuole, associate and/ or interact with secreted protein cargo and be essential for growth in the RBCs. Coupling these criteria with an established proteome from a detergentresistant membrane fraction of the ring stage parasites, de Koning-Ward et al. adeptly utilized biochemical and genetic approaches to identify the Plasmodium translocon of exported proteins (PTEX). The authors initially identied two protein components: a heat shock protein (HSP101) which in models may form a

# The Author (2009). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

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Starnes and Waters

hexameric structure and contains an AAA ATPase domain, both of which are commonly associated with translocon machinery in other systems, and a novel protein, PTEX150. In fulllment of the specied criteria, HSP101 and PTEX150 localize to the parasitophorous vacuole and are unique to Plasmodium species. Reciprocal protein pull-down experiments using both HSP101 and PTEX150 identied three additional components: exported protein 2 (EXP2), PTEX88 and thrioredoxin 2 (TRX2). The EXP2 protein has previously been identied to associate with the parasitophorous membrane vacuole and, in the model presented by the authors, co-operates with HSP101 to serve as the trans-membrane channel, thus anchoring the PTEX complex to the membrane. The specic role(s) during PTEX function of PTEX88/150 and TRX2 remain to be elucidated. However, they may participate in the recruitment and preparation of cargo prior to secretion. The identication of a secretory machinery was a signicant accomplishment, but interaction and/or association with PEXEL motif proteins, presumably secreted virulence factors, remained a criterion the authors sought to establish. To this end, the authors more closely examined their protein pull-down assays and were able to identify interactions with two native secreted PEXEL-motif containing proteins. In addition, the authors generated PEXEL-motif GFP fusion proteins, which were shown to specically interact with the three PTEX components: HSP101, EXP2 and PTEX150. Thus, the authors demonstrate that their novel protein export machinery, PTEX, does indeed interact with proteins harboring PEXEL-motifs and presumably participates in their export to the RBC surface. The authors convincingly establish the presence of a protein secretion system that participates in the vast remodeling of the host RBCs and the perpetuation of infection. Extensive homology searches using the entire HSP101 protein sequence suggest that this protein is in fact of ancient origin, in that beyond Plasmodium species the identied orthologs reside in plants. Perhaps this component (i.e. HSP101) of the secretion machinery has its origins in gene retention from an early endosymbiotic event in

which the engulfee was required to sample or communicate with its external environment. However, the PTEX proteins are clearly unique and restricted to Plasmodium and not shared even with other apicomplexan parasites (including Babesia spp. which also reside in the host erythrocyte). Eventually and as a result of continued adaptation, the machinery evolved to participate in the survival of the parasite in an unhelpful environment (i.e. the RBC). The nal criterion laid forth by the authors in their identication of a novel protein translocon for Plasmodium is that the gene or genes must be essential for growth in the RBC. The authors report an inability to recover parasites harboring disrupted gene products, which insinuates the HSP101 and PTEX150 genes are indeed essential to parasite growth. However, the specic criteria raise the chicken or the egg conundrum. Is it the cargo or the need for the cargo to be translocated (i.e. the translocon) that is essential? The answer is that the two cannot be separated; if the cargo is essential in its transported location, then the translocon responsible for its transport will be equally essential. Indeed, previous work has implied that 25% of PEXEL-motif containing proteins in fact are essential as attempts to disrupt their encoding genes have failed (Maier et al., 2008), which in the absence of inducible gene KO techniques is as good as Plasmodium researchers can do at present. The identication of the PTEX machinery elucidates one of the most fascinating dynamics in malaria biology and opens the door to an increased appreciation for how the parasite is able to remodel the RBC. Mutational analysis should allow a deeper understanding of the PTEX machinery. The dominant negative versions of the HSP101 ATPase could be expressed under the control of stage-specic promoters which may lead to a decrease in protein translocation efciency, thereby enabling the identication of other cargoes. Furthermore, efforts to generate a translocon logjam or backlog in an attempt to clog the secretion machinery with a large, bulky, fusion protein may permit purication of frozen complexes, so that their cargo might be more readily identied. This might help dene the cargo and

determine if there are different destinations for translocon-transported cargo outside of the parasitophorous vacuole (PV). This may in turn lead to a further classication of PEXEL/VTS-based translocons. How the cargo is recognized and loaded onto the translocon is at present another enigma, the zip code for export beyond the PV, the RxLxE/Q/D or PEXEL/ VTS motif, is known to be cleaved prior to engagement with the translocon in the parasites ER. Cleavage exposes xE/Q/D at the N-terminus of the cargo and how that interacts with the translocon (directly or via an unidentied chaperone) remains an open question. Lastly, many protein protein interactions have been predicted for a number of the translocon components through a yeast two-hybrid survey and these may warrant further investigation (LaCount et al., 2005). In reality, any detailed examination of the PTEX components in development will prove useful to understand parasite secretion across membranes. Encouragingly from the perspective of chemotherapy, the PTEX machinery is conserved in all Plasmodium species, although little of the cargo is (van Ooij et al., 2008). Therefore, with an increased appreciation of these basic biological processes, especially those unique to the pathogen itself, a greater opportunity for targeted therapeutics to all of malaria becomes more of a reality.

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References
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van Ooij, C., Tamez, P., Bhattacharjee, S., Hiller, N.L., Harrison, T., Liolios, K., Kooij, T., Ramesar, J., Balu, B., Adams, J., et al. (2008) The malaria secretome: from algorithms to essential function in blood stage infection. PLoS Pathog. 4, e1000084.

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