During its erythrocytic asexual cycle the malaria parasite develops in a cell that is essentially a viscous solution of hemoglobin enclosed by a plasma membrane. However, despite the mature human erythrocyte being devoid of intracellular organelles and incapable of de novo protein synthesis, as well as lacking in the capacity for membrane turnover, upon infection it can be re-modeled through modifications of intrinsic membrane proteins and export of parasite-encoded proteins. The dramatic morphological changes of a Plasmodium falciparum-infected red cell, especially the appearance of knobs, are accomplished by trafficking parasite proteins across the host cell cytoplasm to the cell surface. Understanding the export pathways and molecular mechanisms involved could contribute to the development of novel therapeutic approaches to prevent sequestration and pathological sequelae. With the invasion of erythrocytes (as well as hepatocytes) there is invagination of the host cell plasma membrane and as a result the parasite becomes enclosed in a membrane-lined parasitophorous vacuole (PV). The parasitophorous vacuolar membrane (PVM), derived from the red cell membrane, is devoid of red cell membrane proteins, however, its lipid composition is similar to that of the host cell during invasion (Ward et al., 1993). Over time, the PVM enlarges to accommodate the growing parasite and is modified though the development of membranous structures, including a tubulovesicular network (TVN) and cisternae-shaped membrane vesicles termed Maurer’s clefts. The identification of the molecular characteristics of these various membrane structures progressed slowly largely due to difficulties associated with isolation and assurances of purity; however, in recent years significant advances have been made by the use of reporters such as green fluorescent protein (GFP),
Advances in Parasitology, Volume 67 ISSN 0065-308X, DOI: 10.1016/S0065-308X(08)00421-1
2008 Elsevier Ltd. All rights reserved.
Irwin W. Sherman
fluorescent lipids and transfection techniques combined with confocal fluorescence microscopy, immunoelectron microscopy and biochemical techniques. In 1902, using light microscopy and Giemsa-staining, Georg Maurer described stippling and dots in the red cell cytoplasm of P. falciparuminfected red blood cells. These structures, named Maurer’s clefts, are a feature of red cells infected with mature stages of P. falciparum (Haeggstrom et al., 2007) and, when such cells were examined using the transmission electron microscope, Maurer’s clefts were seen as membranous sacs with a translucent lumen and an electron-dense coat lying just below the red-cell surface (Atkinson and Aikawa, 1990; Kriek et al., 2003; Langreth et al., 1978; Trager et al., 1966). Initially they appear twisted and branched and near the PVM but later they relocate closer to the red cell membrane. The TVN consisting of interconnected tubules and vesicles grows during asexual development but does not undergo rapid movement. In addition, although Maurer’s clefts can be distinguished from the TVN by the presence of specific parasite proteins (Hawthorne et al., 2004; Taraschi et al., 2003; Vincensini et al., 2005) a physical connection to other membrane systems was the subject of controversy for several years. The TVN was first reported to be involved in nutrient uptake: it contains erythrocyte raft proteins whose import was restricted when tubule development was arrested by blocking sphingomyelin synthase. Indeed, using a threo-phospholipid analogue, 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, tubule formation was retarded, raft accumulation decreased and nutrient uptake blocked (Lauer et al., 1997). TEM (Langreth et al., 1978) and serial sections (Bannister et al., 2004), as well as fluorescence microscopy with lipid probes and serial sections of the P. falciparum-infected red cell, led to the postulate that Maurer’s clefts and the TVM form a continuous meshwork (Wickert et al., 2003). Suggestive evidence for Maurer’s clefts being a domain of the TVN came from co-localization of a putative Maurer’s cleft protein, HRP2, to the tip of the TVN (Lauer et al., 1997). However others, using GFP chimeras of Maurer’s cleft cargo, provided evidence that the two were distinct entities (Knuepfer et al., 2005). HRP2 is a soluble protein found in the red cell cytosol and is released upon lysis of the red cells so it cannot act as a marker for a connection. Further, it was contended that a physical barrier prevented diffusion of protein between adjacent Maurer’s clefts and no evidence was found for a continuity of these structures (i.e. Maurer’s clefts, TVM with the PVM) (Knuepfer et al., 2005), however, more recent studies suggest that nascent Maurer’s clefts appear to form sub-domains of the PVM/TVN (Spycher et al., 2006). The rarity of finding connections between the TVN and the PVM (as well as Maurer’s clefts) may be due, as Kasturi Haldar claims (Haldar et al., 2005), to technical difficulties associated with electron microscopy as well as the undulating nature of
the TVN tubules, or more likely that such connections are only transitory. What recent experiments do make clear is that Maurer’s clefts bud from the PVM and diffuse within the cytoplasm of the red cell before ‘taking up residence at the cell periphery’ (Spycher et al., 2006). Kasturi Haldar (1957– ) grew up in India where she was first exposed to the devastating effects of parasitic diseases. She received her university education in the United States sponsored by a Government of India Merit Scholar and National Science Scholarship. From 1974 to 1978 she attended at Bryn Mawr College (Bryn Mawr, Pennsylvania) and received her bachelor of arts (BA) in 1978. Kasturi then moved to the Massachusetts Institute of Technology (Cambridge, Massachusetts) where in 1982 she completed a doctor of philosophy (PhD) in biochemistry working on Escherichia coli membrane vesicles (‘Proton translocation and quinone reduction catalyzed by D-amino acid dehydrogenase’) under the supervision of Christopher Walsh. In 1983–1984 she was a postdoctoral fellow with George Cross at the Rockefeller University (New York, New York) and after one year of leave to work in the Institute of Basic Medical Sciences at the Capitol Medical College (Beijing, China) she returned to Rockefeller as a research associate (1985–1987) working on identification of the transferrin receptor of P. falciparum, the transport of phospholipid (PL) analogues from the red cell to the parasite and the accumulation and metabolism of ceramides. In 1988, she moved to Stanford University (Palo Alto, California) where first as an assistant professor of microbiology and immunology, and later as an associate professor (1994–1998) she continued to carry out studies on intracellular transport: the movement of fluorescent tracers in P. falciparum, the inhibition of protein secretion by brefeldin A and discovery of the TVN. In 1998, she moved to the Northwestern University Feinberg School of Medicine (Chicago, Illinois) where she is presently professor of pathology and microbiology and immunology. Her current research is focused on how secretory pathways deliver proteins and lipids to organelles to support the growth of membranes especially the role of lipid rafts. Using the emerging genetic techniques in the development of functional assays she has mined databases for functional motifs of unique organelles to develop a high-throughput assay and to use microarrays to track global changes in secretory gene expression during intracellular development of Plasmodium and other pathogens such as Salmonella, Mycobacterium, Chlamydia and Toxoplasma. Her long-term research goals are to understand the common principles of vacuolar biogenesis in intracellular parasites in order to identify targets for immunological prophylaxis and/or chemotherapy.
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Plasmodial protein transport (i.e. trafficking) has been proposed to be a multi-step process: secretion, translocation across the PVM, movement and specific sorting in the red cell cytoplasm, followed by translocation/ insertion into the target site. Trafficking has recently been the subject of several reviews (Cooke et al., 2004a; Lanzer et al., 2006; Lingelbach and Przyborski, 2006; Marti et al., 2005; Przyborski et al., 2003; Przyborski and Lanzer, 2005; Tilley et al., 2007; Tonkin et al., 2006; van Ooij and Haldar, 2007). It has been shown that entry into the parasite’s default pathway (bulk flow) is via the endoplasmic reticulum mediated by a canonical (Burghaus and Lingelbach, 2001) or an unconventional N-terminal sequence (Wickham et al., 2001). The default pathway allows plasmodial proteins to be trafficked across the parasite plasma membrane (PPM) into the lumen of the PV. Additional sequences are required for proteins to be exported beyond the PVM (and into the red cell cytoplasm), and this is thought to occur via selective translocators in the PVM. Recent studies with knob-associated histidine-rich protein (KAHRP) have shown that although signal sequences with a recessed hydrophobic signal are necessary and sufficient for secretion into the PV, N-terminal signal sequences also mediate transport into the PV; however, a second signal is required for translocation across the PVM (Rug et al., 2006). Two laboratories have shown that a short plasmodial peptide motif is necessary for protein export across the PVM into the red cell cytoplasm. The motif, termed Plasmodium export element (PEXEL) by Marti et al. (2004) or host targeting (HT) by Hiller et al. (2004) has the consensus R/KxLxEQ with positions 1, 3 and 5 being critical. Identification of PEXEL has allowed a motif search of the P. falciparum genome at the website: http://www.plasmodb.org (last accessed 16 July 2008) that predicts an ‘exportome’ (called the ‘secretome’ by Hiller et al., 2004) of approximately 400 proteins (i.e. about 8% of the proteome) that are exported to the cell where they may be ‘involved in antigenic and structural modifications of the erythrocyte membrane and cytoplasm, mediate nutrient import from the red blood cell into the parasite and provide the machinery for protein export to the erythrocyte’ (Marti et al., 2004). Identification of signal motifs has allowed for the prediction of exported proteins, the ‘exportome’, for P. yoelii, P. berghei, P. chabaudi, P. vivax and P. knowlesi as well as P. falciparum; the ‘core’ exportome for the genus Plasmodium is postulated to be involved in the re-modelling of the host red cell, whereas the genes for P. falciparum ‘probably encode proteins directly or indirectly involved in the different properties of this parasite’ (Sargeant et al., 2006). Lingelbach and colleagues using biochemical methods, have shown that within the parasite P. falciparum erythrocyte surface protein-1 (PfEMP1) exists in a soluble form and becomes increasingly insoluble as it proceeds along the pathway to its final destination (Papakrivos
et al., 2005). These observations indicate that membrane insertion of PfEMP1 occurs after the protein is transported out of the endoplasmic reticulum. Moreover, the physical properties of PfEMP1 in the erythrocyte membrane resembled proteins that are part of larger complexes rather than integral membrane proteins. With a GFP-PfEMP1 chimera evidence was found for its presence in the red cell cytoplasm. Observation of diffusion of the fluorescent chimera was interpreted to mean that PfEMP1 was present as a soluble complex in red cell cytoplasm (Knuepfer et al., 2005) and membrane insertion took place only after export into the host cell. Together, these data suggest a trafficking pathway of PfEMP1 that differs distinctly from the transport pathways of surface proteins in eukaryotic cells. After PfEMP1 insertion into the Maurer’s clefts membrane, either within or attached to the cytoplasmic face of the cleft, the protein moves to the membrane cytoskeleton. Here PfEMP1 becomes anchored at the knob structure via the acidic terminal sequence (ATS) and its extracellular binding domains become exposed on the red-cell surface. The final translocation of PfEMP1 on to the surface of the red cell has been shown to involve a Maurer’s cleft protein, skeleton binding protein 1 (SBP1). SBP1 is a 48-kDa integral membrane protein that spans the cleft membrane with its N-terminus within the cleft and the C-terminus exposed to the cytoplasm (Blisnick et al., 2005). In clonal transgenic parasite lines in which SBP1 was not expressed (i.e. SBP1 ‘knockouts’), PfEMP1 was not found on the red-cell surface (and the cells did not bind to CD36), yet the number of knobs appeared to be the same in the knockout as in the wild type; cleft number and structure were also unaffected. Moreover, other proteins associated with Maurer’s clefts such as P. falciparum erythrocyte surface protein-3 (PfEMP3), Pf332, KAHRP, REX (Hawthorne et al., 2004) and MAHRP (Spycher et al., 2006) were trafficked normally to the underside of the red cell membrane. It was hypothesized that the effect of SBP1 on PfEMP1 translocation is indirect and subtle, affecting cleft morphology and distance to the infected red-cell surface such that the final translocation step is inefficient (Cooke et al., 2006). Further, they reason, since other proteins were unaffected it appears these proteins have a larger margin of error for binding to the cytoskeleton. The final step in the delivery of PfEMP1 to the red cells may involve fusion with the cholesterol-rich microdomains in the red cell membrane (Frankland et al., 2006). Brian M. Cooke (1964– ) received an undergraduate degree from Bristol University (Bristol, United Kingdom) in 1989, and then moved to The University of Birmingham Medical School (Birmingham, United Kingdom) to begin a PhD in hematology with Gerard Nash and John Stuart. Under their guidance, he worked on malaria and sickle cell
Irwin W. Sherman
disease receiving his PhD in 1993. During the early 1990s, Brian spent time in The Gambia, West Africa, working with Brian Greenwood to extend his studies to isolates of malaria parasites in a clinical setting. There, he was the first to study the interactions of malaria-infected red blood cells with a variety of different vascular adhesion molecules and made an assessment of the role of different adhesion molecules in the development of clinical malaria. Cooke’s independent research career was launched properly when he moved to Australia in 1993 to join the malaria research group at WEHI in antigen discovery and vaccine research. During that time, under the supportive directorship of Sir Gustav Nossal, he was encouraged to set up a new laboratory with his colleague and mentor Ross Coppel at Monash University in Melbourne. Together they established a unique and internationally recognized molecular and cellular biorheology laboratory at Monash University. Over the past 15 years, Cooke’s work at Monash has focused on understanding the molecular basis by which malaria parasites cause disease in humans by modifying the properties of red blood cells in which the parasites reside, including the mechanisms involved in the trafficking of virulence proteins to the red blood cell surface. He and his group are part of a worldwide consortium studying the functional properties of novel genes identified in the genome of P. falciparum. The Cooke and Coppel groups at Monash University are supported by national and international research grants from Australia, the United Kingdom and the United States, including major funding from The Bill & Melinda Gates Foundation, to identify novel drug targets and to develop an effective malaria vaccine. Cooke is presently a National Health and Medical Research Council senior research fellow and associate professor in the Department of Microbiology at Monash University. In accord with the results of Cooke et al. (2006), Maier et al. (2007) found that a P. falciparum skeleton-binding protein 1 (PfSBP1) knockout resulted in marked reduction in surface-exposed PfEMP1 and because other cleft proteins such as KAHRP and MAHRP trafficked normally, PfSBP1 was required for trafficking of PfEMP1 from the parasite to Maurer’s clefts. But, in contrast to Cooke et al. (2006), they contend that PfSBP1 is responsible for the transfer of PfEMP1 from the PV to the Maurer’s clefts. Since PfPBSP1 lacks a PEXEL motif, it must be directly loaded into Maurer’s clefts, possibly crossing the PVM by being associated with a PEXELcontaining protein. PfEMP3, deposited on the cytoplasmic face of the erythrocyte cytoskeleton, is a component of the electron-dense plaque knob. Although truncation prevented its distribution at the red cell membrane (and knobs
appeared to be smaller), transfer of PfEMP1 to the surface was unaffected, as was cytoadherence (Waterkeyn et al., 2000). GFP-PfEMP3 chimeras were exported to the red cell cytoplasm as aggregates rather than in vesicles and were trafficked to Maurer’s clefts. Since PfEMP3 was not required for PfEMP1 trafficking its precise role remains unclear, but what is known is that it contributes to the reduced deformability of parasitized cells (Glenister et al., 2002). A variation on the trafficking of PfEMP1, KAHRP and PfEMP3 is seen with STEVOR. STEVOR proteins are expressed in gametocytes and sporozoites as well as on the surface of red cells bearing asexual stages (McRobert et al., 2004). The precise function of STEVOR variants is unknown. STEVOR proteins contain two transmembrane domains and a predicted signal sequence. The transmembrane domains are crucial for the targeting of STEVOR to Maurer’s clefts and when this is deleted it accumulates in the red cell cytoplasm in a soluble form. Although STEVOR and PfEMP1 are both transported to Maurer’s clefts, STEVOR traverses the secretory pathway as an integral membrane protein (Przyborski et al., 2005) and there is no co-regulation of the var and stevor gene families (Duffy and Tham, 2007; Sharp et al., 2006). Trafficking of PfCG2, a non-integral high-molecular-weight protein (320–330 kDa) located on the cytosolic face of the PVM to the digestive food vacuole (FV) has been described (Cooper et al., 2005). The levels of PfCG2, found in electron-dense patches along the PVM, the cytoplasm and the digestive FV, increase through the trophozoite stage and decline rapidly in the schizont. Since PfCG2 does not have a PEXEL-targeting sequence (necessary for secretion from the PV into the red cell cytoplasm), the mechanism for its trafficking to the digestive FV remains an open question. Once inside the FV, PfCG2 is subjected to proteolysis by plasmepsins, and becomes associated with the membranous material between hemozoin crystals, and could serve as a nucleation site for malaria pigment formation. Although a precise function for PfCG2 is unknown, its importance is suggested by the fact that attempts to knockout the gene have been unsuccessful. Leann Tilley (1958– ) grew up in the small town of Stawell in Victoria, Australia. She moved to Melbourne to start her university studies and obtained a bachelor of science (BSc) (Hons) from the University of Melbourne (Melbourne, Vic, Australia) (1978) and a PhD in biochemistry from the University of Sydney (Sydney, NSW, Australia) (1984). After post-doctoral fellowships at the University of Utrecht (Utrecht, The Netherlands) (1985) and the Colle`ge de France (Paris, France) (1986), she returned to Australia and the University of Melbourne (1987–1988) where she continued her studies of the proteins and lipids
Irwin W. Sherman
of the human erythrocyte membrane using fluorescence-based biophysical methods. This led quite naturally to collaborative studies with Robin Anders and Mick Foley at Walter and Eliza Hall Institute (WEHI) and the University of Melbourne on P. falciparum-red cell protein interactions. In 1989, she joined the Biochemistry Department at La Trobe University (Vic, Australia) and was appointed professor in 2004. At La Trobe, Tilley established a malaria research program using a series of fluorescence-based imaging techniques to ask questions about the cell biology of Plasmodium. A Wellcome grant helped establish a confocal fluorescence photobleaching facility and a Linkage Infrastructure Equipment and Facilities (LIEF, Australia) grant for a confocal fluorescence correlation facility. Currently, Tilley is deputy director of a new Centre of Excellence for Coherent X-ray Science (La Trobe University, Vic, Australia). The Centre brings physicists, chemists and biologists together to develop fundamentally new approaches to probing biological structures and processes. It combines world-class expertise in imaging, structural biology, laser science and molecular theory. The project will develop novel high-resolution imaging using the Australian Synchrotron, and ultimately x-ray lasers, to determine the structures of important drug targets whose molecular architecture cannot be determined with current techniques. Her laboratory is using a range of techniques to image the ultrastructure of malaria parasite-infected erythrocytes with particular emphasis on protein trafficking and the molecular mechanisms of anti-malarials. Although defining the molecular mechanisms involved in the regulation of trafficking of plasmodial proteins has been a study of errors slowly corrected it is now clear that this complex process will continue to provide challenges for biochemists and molecular biologists and a fuller understanding will require new methodologies and approaches. As a teenager, after reading ‘Of Microbes and Life’ by Jacques Monod, which describes his discovery of the lactose operon in E. coli, Michael Lanzer (1959– ) decided he wanted to become a molecular biologist— and he did. From 1979 to 1984, Lanzer studied biology at the University of Heidelberg (Heidelberg, Germany). Hermann Bujard who, more than 20 years after the initial discovery by Jacob and Monod, had some unresolved questions regarding gene regulation of the lac operon, accepted Lanzer as a PhD student and 3 years later he published his first scientific paper on the thermodynamic parameters that determine the intricate interplay between the ribonucleic acid (RNA) polymerase, repressor, promoter and operator responsible for the tight
regulation of the operon. This study, which appeared in the Proceedings of the National Academy of Science USA, remains one of his most cited publications. During the period when he was pursuing his PhD (1984– 1987) he spent some time at the pharmaceutical company Hoffmann LaRoche in Basel, Switzerland. At that time, Hoffmann LaRoche had a small but effective malaria research team, and many prominent malariologists of the time, including John Scaife and Ruth and Victor Nussenzweig, came to visit, and their descriptions as well as a selfrealization led to Lanzer’s recognition of the beauty of P. falciparum with its many developmental stages and its complex life cycle. The unresolved questions of its biology, the humanitarian problem and the possibility of finding a niche in a research field that was small and less competitive convinced him to try his luck at conducting malaria research. At the Memorial Sloan Kettering Cancer Center in New York in the laboratory of Jeffrey Ravetch, he found the right environment. Ravetch had just published a number of seminal papers on P. falciparum biology and Lanzer joined his team to study gene regulation and chromosome structure and function in this parasite. It was a very productive and inspirational time (1988–1993). For example, together they were the first to clone an entire P. falciparum chromosome using yeast artificial chromosomes. In 1993, Lanzer was appointed assistant member at the University of Wu¨rzburg (Wu¨rzburg, Germany) and in 1999 full professor of parasitology at the University of Heidelberg. In succeeding years he has remained true to malaria with current research focusing on mechanisms of drug resistance and on protein targeting and trafficking pathways in the parasite.
Klaus Lingelbach (1955– ) studied biology at the universities of Tuebingen (Tuebingen, Germany) and Heidelberg with major subjects zoology/parasitology. Having acquired a solid basis in general parasitology, he became interested in the molecular basis of host–parasite interaction. Upon completion of his diploma thesis on tapeworms (1982, ‘The immune response to Echincoccus multilocularis’) he then joined the immunoparasitology unit of WEHI where he became involved in malaria research and in the efforts towards the development of a vaccine. At that time Lingelbach realized that very little was known about the cell biology of the parasite and the infected host cell. To gain more experience in basic cell biochemistry, he joined the Biochemistry Department at La Trobe University, where he completed a PhD (1986) on mitochondrial protein import under the supervision
Irwin W. Sherman
of Nick Hoogenraad. In retrospect, the scientific community in Melbourne provided an ideal environment for a young scientist to combine modern biochemistry with excellent parasitology. During that time, Lingelbach started to develop a scientific and experimental framework to study protein trafficking in the infected erythrocyte. In 1987, he joined Bernhard Dobberstein’s group at the European Molecular Biology Laboratory in Heidelberg (Heidelberg, Germany) where he became acquainted with the molecular principles of protein secretion. In 1989, Lingelbach was offered a position as a junior group leader at the University of Hamburg (Hamburg, Germany), where he began his work on protein secretion in P. falciparum. In 1992, he joined the Bernhard Nocht Institute for Tropical Medicine (Hamburg, Germany) first as an independent group leader and, later, as the co-ordinator of the Molecular Parasitology Program. In 1996, he was appointed professor for parasitology at the Philipps-Universita¨t Marburg (Marburg, Germany) Lingelbach’s research interests continue to be describing the mechanisms that underlie host cell modification by secreted proteins in the P. falciparum-infected erythrocyte.