Phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection

The phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection has been studied. Parasitized and nonparasitized erythrocytes from malaria-infected blood were separated and pure erythrocyte membranes from parasitized cells were isolated using Affi-Gel beads. In this way, the phospholipid content and composition of the membrane of nonparasitized cells, the erythrocyte membrane of parasitized cells and the parasite could be determined. The phospholipid content and composition of the erythrocyte membranes of nonparasitized and parasitized cells and erythrocytes from chloroquine-treated monkeys cured from malaria, were the same as in normal erythrocytes. The phospholipid content of the parasite increased during its development, but its composition remained unchanged. Three independent techniques, i.e., treatment of intact cells with phospholipase A2 and sphingomyelinase C, fluorescamine labeling of aminophospholipids and a phosphatidylcholine-transfer protein-mediated exchange procedure have been applied to assess the disposition of phospholipids in: erythrocytes from healthy monkeys, nonparasitized and parasitized erythrocytes from monkeys infected with Plasmodium knowlesi, and erythrocytes from monkeys that had been cured from malaria by chloroquine treatment. The results obtained by these experiments do not show any abnormality in phospholipid asymmetry in the erythrocyte from malaria-infected (splenectomized) monkeys, neither in the nonparasitized cells, nor in the parasitized cells at any stage of parasite development. Nevertheless, a considerable degree of lipid bilayer destabilization in the membrane of the parasitized cells is apparent from the enhanced exchangeability of the PC from those cells, as well as from their increased permeability towards fluorescamine.     

Characterization of permeation pathways in the plasma membrane of human erythrocytes infected with early stages of Plasmodium falciparum: association with parasite development

Human intraerythrocytic malarial parasites (Plasmodium falciparum) induce permeability changes in the membrane of their host cells. The differential permeability of infected erythrocytes at various stages of parasite growth, in combination with density gradient centrifugation, was used to fractionate parasitized cells according to their developmental stage. By this method it was possible to obtain cell fractions consisting essentially of erythrocytes infected with the youngest parasite stage (i.e., rings). These preparations were used for the measurement of transport of various solutes. It is shown that permeabilization of host erythrocyte membrane appears as early as 6 h after parasite invasion of the erythrocyte and increases gradually with parasite maturation. Since the selectivity for several different solutes and the enthalpy of activation of transport remain unaltered with maturation-related increase of permeability, it is concluded that the number of transport agencies in the host cell membrane increases with parasite maturation. Evidence is presented to indicate the need for parasite protein synthesis as an essential factor for the generation of the new permeability pathways.     

Possible mechanisms responsible for the increased ascorbic acid content of Plasmodium vinckei-infected mouse erythrocytes

The possible mechanisms underlying the acquisition of an increased ascorbic acid content by mouse erythrocytes containing the malarial parasite Plasmodium vinckei were investigated. Ascorbic acid was taken up readily by parasitized red blood cells but not by controls, whilst its partly oxidized form, dehydroascorbic acid, entered both. The uptake of both ascorbic acid and dehydroascorbic acid into erythrocytes was increased as a result of malarial infection. Lysates prepared from parasitized red blood cells reduced exogenous dehydroascorbic acid to ascorbic acid at a higher rate than control red blood cell lysates; this difference was abolished following dialysis of the lysates, a process which removes endogenous reduced glutathione (GSH). The rates of chemical and enzymatic reduction of dehydroascorbic acid to ascorbic acid by GSH were of similar magnitude, thus calling into question the existence of a specific dehydroascorbate reductase in erythrocytes and parasites. These observations suggest that the increased uptake of dehydroascorbic acid into parasitized red blood cells may be a result of enhanced dehydroascorbate-reducing capacity, whilst the presence of the parasite induces a selective increase in the permeability of the erythrocyte plasma membrane to ascorbic acid. The endogenous ascorbic acid content of livers obtained from infected mice was 55% below the normal concentration and its relative rate of destruction during incubation in vitro was enhanced in comparison with that of control livers. Furthermore, the capacity of liver homogenates to synthesize ascorbic acid from glucuronic acid was greatly reduced in infected mice. Therefore it is unlikely that the increase in ascorbic acid content of parasitized red blood cells is a consequence of increased biosynthesis and release of ascorbic acid by the host liver. We have not been able to exclude the possibility that the malarial parasite itself may be capable of de novo synthesis of ascorbic acid.     

Trafficking of malarial proteins to the host cell cytoplasm and erythrocyte surface membrane involves multiple pathways

During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.     

Vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes

During the development of the asexual stage of the malaria parasite, Plasmodium falciparum, the composition, structure and function of the host cell membrane is dramatically altered, including the ability to adhere to vascular endothelium. Crucial to these changes is the transport of parasite proteins, which become associated with or inserted into the erythrocyte membrane. Protein and membrane targeting beyond the parasite plasma membrane must require unique pathways, given the parasites intracellular location within a parasitophorous vacuolar membrane and the lack of organelles and biosynthetic machinery in the host cell necessary to support a secretory system. It is not clear how these proteins cross the parasitophorous vacuolar membrane or how they traverse the erythrocyte cytosol to reach their final destinations. The identification of: (1) a P. falciparum homologue of the protein Sar1p, which is an essential component of the COPII-based secretory system in mammalian cells and yeast and (2) electron-dense, possibly coated, secretory vesicles bearing P. falciparum erythrocyte membrane protein 1 and P. falciparum erythrocyte membrane protein 3 in the host cell cytosol of P. falciparum infected erythrocytes recently provided the first direct evidence of a vesicle-mediated pathway for the trafficking of some parasite proteins to the erythrocyte membrane. The major advance in uncovering the parasite-induced secretory pathway was made by incubating infected erythrocytes with aluminium tetrafluoride, an activator of guanidine triphosphate-binding proteins, which resulted in the accumulation of the vesicles into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminium fluoride treatment, making their capture by electron microscopy possible. It appears that malaria parasites export proteins into the host cell cytosol to support a vesicle-mediated protein trafficking pathway.     

Plasmodium knowlesi induces alterations in phosphatidylcholine and phosphatidylethanolamine molecular species composition of parasitized monkey erythrocytes

Using high performance liquid chromatography and gas-liquid chromatography, we have characterized the phosphatidylcholine and phosphatidylethanolamine molecular species composition of trophozoite and schizont forms of Plasmodium knowlesi parasitized erythrocytes. Similarly, we determined these parameters in the erythrocyte membranes of trophozoite parasitized cells, unparasitized erythrocytes from infected monkeys before and after a chloroquine treatment and erythrocytes from monkeys that had never been infected. Plasma phosphatidylcholine molecular species composition was also studied. P. knowlesi parasitized erythrocytes presented higher amounts of 16:0/18:2-phosphatidylcholine than the various control cells, which appeared to be compensated for by a decrease in 18:0/20:4-, 16:0/20:3-, 16:0/18:1-, 18:0/18:2-, 18:0/20:3-, 16:0/16:0- and 16:0/18:0-phosphatidylcholines. In the case of phosphatidylethanolamine, the alterations were quantitatively of greater importance and consisted of an increase in, again, 16:0/18:2-phosphatidylethanolamine and a decrease in several species containing 20:4, namely 16:0/20:4-, 18:0/20:4- and 18:1/20:4-phosphatidylethanolamine; also the levels of alkoxy-phosphatidylethanolamines were markedly decreased. P. knowlesi development within monkey erythrocytes therefore appears to be associated with changes in phosphatidylcholine and phosphatidylethanolamine molecular species in the whole parasitized cell. These alterations are also exhibited by the host cell membrane, which provides the first experimental evidence that the parasite is able to manipulate the erythrocyte membrane lipid species composition. The consequences of these alterations on membrane physiology are discussed, as well as the implications that these data may have on the trafficking of phosphatidylcholine and phosphatidylethanolamine in the erythrocytes of P. knowlesi infected monkeys.     

Food vacuole-associated lipid bodies and heterogeneous lipid environments in the malaria parasite, Plasmodium falciparum

The malaria parasite Plasmodium falciparum induces a sixfold increase in the phospholipid content of infected erythrocytes during its intraerythrocytic growth. We have characterized the lipid environments in parasitized erythrocyte using the hydrophobic probe, Nile Red. Spectral imaging with a confocal microscope revealed heterogeneous lipid environments in parasite-infected erythrocytes. An insight into the nature of these environments was gained by comparing these spectra with those of triacylglycerol/phospholipid emulsions and phospholipid membranes. Using this approach, we identified a population of intensely stained particles of a few hundred nanometers in size that are closely associated with the digestive vacuole of the parasite and appear to be composed of neutral lipids. Electron microscopy and isolation of food vacuoles confirmed the size of these particles and their intimate association respectively. Lipid analysis suggests that these neutral lipid bodies are composed of di- and triacylgycerols and may represent storage organelles for lipid intermediates that are generated during digestion of phospholipids in the food vacuole. Mono-, di- and triacylglycerol suspensions promote beta-haematin formation, suggesting that these neutral lipid bodies, or their precursors, may also be involved in haem detoxification. We also characterized other compartments of the infected erythrocyte that were stained less intensely with the Nile Red probe. Both the erythrocyte membrane and the parasite membrane network exhibit red shifts compared with the neutral lipid bodies that are consistent with cholesterol-rich and cholesterol-poor membranes respectively. Ratiometric imaging revealed more subtle variations in the lipid environments within the parasite membrane network.     

Lipid compartmentalization in erythrocytes parasitized by Plasmodium spp

Although reasonably well protected from the host immune system by the erythrocyte membrane, the intraerythrocytic malaria parasite has to make that membrane compatible with its own requirements for development and multiplication. The development of Plasmodium spp brings about major changes in the lipid composition of the host cell membrane, as well as in its physical properties. The parasite itself has a lipid composition that differs from that of the host cell and an intense lipid trafficking seems to occur between intracellular parasite and host cell membrane. Here, Ana Paula Sim?es, Ben Roelofsen and Jos Op den Kamp discuss how, despite serious methodological limitations and the existence of some conflicting results, an overall picture of lipid compartmentalization within the parasitized erythrocyte is perceived.     

Effect of hypervitaminosis A on hemolysis and lipid peroxidation in the rat

Erythrocytes from rats fed large doses of Vitamin A alone, or large doses of vitamin A and vitamin E or diphenyl-p-phenylene diamine (DPPD) were studied for H2O2-induced hemolysis. The vitamin A-dosed rats were more susceptible than normal rats to H2O2-induced hemolysis. Hemolysis was not accompanied by lipid peroxidation. Nevertheless, the antioxidants vitamin E and DPPD inhibited hemolysis in erythrocytes from vitamin A-dosed rats. These antioxidants had the same inhibitory effect when they were included in the diet or added to erythrocyte suspensions in vitro. Erythrocytes from vitamin A-dosed rats with or without added vitamin E or DPPD were less susceptible than the erythrocytes from normal rats to osmotic challenge, showing that vitamin A was present in levels sufficient to alter the structure of the erythrocyte membrane. These studies show that oxidative hemolysis occurs when the erythrocyte membrane is modified. Furthermore, this oxidative hemolysis is unrelated to lipid peroxidation.     

P. Falciparum infected erythrocytes are capable of endocytosis

Summary P. falciparum, an intraerythrocytic parasite, obtains nourishment primarily through phagocytosis of the host cytosol but also through the incorporation of extracellular small molecules which enter through the parasitized red cell's membrane via pores. Normal mature erythrocytes are incapable of endocytosis. Several lines of evidence suggest that extracellular large molecules may be taken up when the mature red cell is parasitized byP. falciparum, but direct evidence has been lacking. We now report the use of ferritin, an electron dense protein, to demonstrate endocytosis inP. falciparum infected red cells. Parasitized red cells incubated with ferritin internalize that macromolecule as demonstrated by electron microscopy. While normal red cells incubated with ferritin took up none of the tracer molecule, parasitized red cells internalized substantial amounts. In addition both ferritin and apoferritin inhibited the growth ofP. falciparum in a dose dependent fashion, again indicating endocytosis of a macromolecule. These data indicate thatP. falciparum can somehow stimulate the mature erythrocyte to engage in endocytosis. We also note that both infected and non-infected red cells in a culture in whichP. falciparum is growing become abnormally sticky for ferritin. Moreover, parasitized red cells bind I¹²⁵-transferrin while non-parasitized erythrocytes do not. These observations suggest that a soluble parasite product alters the red cell membrane in a non-global manner, causing selective effects in relation to different proteins.     

脂质过氧化对人红细胞膜脂流动性的影响

研究枯稀过氧化氢/高铁血红素体系所产生的烷基过氧自由基对红细胞的损伤。测定了脂质过氧化的产物——丙二脂的生成,并证明阿魏酸钠对脂质过氧化的抑制。荧光偏振的结果指出,膜脂过氧化以后降低了膜脂的流动性。人红细胞用5DSA和16DSA标记并用ESR检测膜脂流动性,结果表明,序参数S几乎没有发生变化,旋转相关时间τ值的增加证明膜脂过氧化以后,疏水尾部的物理状态发生了改变。经脂质过氧化以后,红细胞膜中的不饱和脂防酸的减少,可能是降低膜脂流动性的原因之一。     

Low survival of parasite chicks may result from their imperfect adaptation to hosts rather than expression of defenses against parasitism

Host parents exhibit a variety of behaviors toward avian brood parasites, but not all of their actions have necessarily evolved in response to costs imposed by parasites. To investigate whether common waxbills (Estrilda astrild) have evolved defenses specifically against parasitic pin-tailed whydahs (Vidua macroura), I studied the specificity and flexibility of host behaviors toward nestlings at two sites that differed significantly in parasitism rates and intensities. I focused on documenting nestling survival because V. macroura young match the elaborate gape morphology of E. astrild nestlings, a pattern that suggests hosts may possess unique defenses against parasite chicks. Parasite young survived significantly worse than host young in mixed broods. However, this apparent discrimination was not associated with parasitism risk as would be expected if defenses had evolved specifically to counter parasitism. Parasite young may have survived poorly compared to host young because individual chicks were less able to stimulate sufficient care from foster parents or because they were more susceptible to nestling competition, disease, or reduced provisioning by hosts. Mortality may have also been exacerbated by poor timing of parasite egg laying. In nonparasitized and parasitized nests, rates of nestling survival were similar, further suggesting that parenting behaviors that result in chick mortality did not evolve solely in response to parasite young. In addition, orange-breasted waxbills (Amandava subflava) and zebra finches (Taeniopygia guttata), rarely parasitized and nonparasitized relatives of E. astrild, experience similar levels of nestling mortality presumably as a result of phylogenetically widespread parenting strategies. Despite the similarity of parasitic V. macroura nestlings and E. astrild nestlings, I found no evidence that E. astrild parents possess defenses that allow for specific discrimination against parasite chicks during the nestling period. Rather than being subject to host defenses evolved in an arms race, Vidua chicks may simply be imperfectly adapted to life in the nests of their hosts.     

The influence of metmyoglobin and ferrylmyoglobin on the human erythrocyte membrane

Preliminary experiments revealed that ferrylmyoglobin decayed more slowly in the absence than in the presence of intact erythrocytes and erythrocyte membranes. This suggested the existence of interactions between FerrylMb and the erythrocyte membrane. Subsequent studies examined the influence of FerrylMb on the membrane of intact erythrocytes and on isolated erythrocyte membranes. The incubation of intact erythrocytes with FerrylMb did not influence their osmotic fragility or the fluidity of their membranes; the level of peroxidation of the membrane lipids increased only slightly (there was only a slight increase in the level of membrane lipid peroxidation). The activity of acetylcholinesterase significantly increased after 15 minutes of incubation, whereas longer incubation did not lead to any changes in the activity of this enzyme. The incubation of isolated erythrocyte membranes with FerrylMb resulted in an increase in their fluidity and a significant rise in the level of lipid peroxidation.     

Involvement of spectrin and ATP in infection of resealed erythrocyte ghosts by the human malarial parasite, Plasmodium falciparum

Resealed erythrocyte ghosts were prepared under different experimental conditions and were tested in vitro for susceptibility to infection with the human malarial parasite, Plasmodium falciparum. Resealed ghosts, prepared by dialyzing erythrocytes in narrow membrane tubing against low ionic strength buffer that was supplemented with magnesium ATP, were as susceptible to parasite infection as were normal erythrocytes. There was a direct correlation between intraerythrocytic ATP content and susceptibility to parasite infection. Neither MgCl2 nor sodium ATP could be substituted for magnesium ATP in maintaining high intraerythrocytic ATP concentration. When resealed ghosts were loaded with antispectrin IgG, malaria merozoite invasion was inhibited. At an average intracellular antispectrin IgG concentration of 3.5 micrograms/10(8) cells, there was a 35% inhibition of parasite invasion. This inhibition was due to spectrin crosslinking within the resealed ghosts, since the monovalent, Fab' fragments of antispectrin IgG had no inhibitory effect on invasion. These results indicate that the cytoskeleton plays a role in the complex process of merozoite entry into the host erythrocyte.     

Compartment analysis of ATP in malaria-infected erythrocytes

The ATP concentration of malaria-infected erythrocytes changes substantially with parasite development. These alterations have been attributed to a decline in host cell [ATP], but have not been tested critically hitherto. A method for the compartmental analysis of ATP in malaria (Plasmodium falciparum)-infected human red blood cells has been developed using Sendai virus to permeabilize the host erythrocyte membrane. Permeabilization and release of host cytosol was complete within 6 to 8 minutes and ATP was measured by the luciferin-luciferase bioluminescence assay in the lysate and in the pellet. Equal ATP concentrations were found in host and parasite compartments at the trophozoite and schizont stages. Both were lower than those detected in uninfected cells. Other methods for compartment analysis of ATP are presented and discussed.     

Human erythrocytes adhering to schistosomula of Schistosoma mansoni lyse and fail to transfer membrane components to the parasite

We studied the adherence of human erythrocytes to larvae of the intravascular parasite Schistosoma mansoni by transmission microscopy, freeze fracture, and fluorescence techniques. In addition, we used the adherent cells to investigate the problem of host antigen acquisition. Schistosomula were cultured for from 24 to 48 h after transformation in order to clear the remnants of the cercarial glycocalyx. In some cases, the worms were preincubated with wheat germ agglutinin to promote adherence of the erythrocytes. The results were similar with and without the lectin except that more cells attached to the lectin-coated parasites. Erythrocytes adhered within a few hours and, unlike neutrophils, did not fuse with the parasite. A layer of 10-20-nm electron dense material separated the outer leaflets of the tegumental and plasma membranes. In addition, many deformed and lysed cells were seen on the parasite surface. The ability of the worm to acquire erythrocyte membrane constituents was tested with carbocyanine dyes, fluorescein covalently conjugated to glycophorin, monoclonal antibodies against B and H blood group glycolipids, and rabbit alpha-human erythrocyte IgG. In summary, glycophorin, erythrocyte proteins, and glycolipids were not transferred to the parasite membrane within 48 h. Carbocyanine dyes were rapidly transferred to the parasite with or without lectin preincubation. Thus, the dye in the worm membrane came from both adherent and nonadherent cells. These studies suggest that, in the absence of membrane fusion, the parasite may acquire some lipid molecules similar in structure to host membrane glycolipids by simple transfer through the medium but that B and H glycolipids and erythrocyte membrane proteins are not transferred from adhering cells to the worm.     

Plasmodium berghei: oxidant defense system

Glutathione oxidant defense system protects the erythrocyte from oxidative damage. This defense system was studied in mouse erythrocytes infected with Plasmodium berghei and in isolated parasites. The efficiency of this system was found to be increased in parasitized erythrocytes compared to the normal erythrocytes. The increase in the components of the oxidant defense system in the parasitized cells could result from parasitic addition to these components. This defense system present in the parasite may protect the parasite from oxidative damage and help the parasite in its growth and development.     

A nonpermeant biotin derivative gains access to the parasitophorous vacuole in Plasmodium falciparum-infected erythrocytes permeabilized with streptolysin O

In its host erythrocyte, the malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole, the membrane of which forms a barrier between the host cell cytosol and the parasite surface. The vacuole is a unique compartment because it contains specific proteins that are believed to be involved in cell biological functions essential for parasite survival. As a prerequisite for the characterization of the vacuolar proteome, we have developed an experimental approach that allows the selective biotinylation of soluble vacuolar proteins. This approach utilizes nonpermeant biotin derivatives that can be introduced into infected erythrocytes after selective permeabilization of the erythrocyte membrane with the pore-forming protein streptolysin O. The derivatives gain access to the vacuolar lumen but not to the parasite cytosol, thus providing supportive evidence for the existence of nonselective pores within the vacuolar membrane that have been postulated based on electrophysiological studies. Soluble vacuolar proteins that are biotin-labeled can be isolated by affinity chromatography using streptavidin-agarose.     

Possible basis for membrane changes in nonparasitized erythrocytes of malaria-infected animals

Previous studies (Gupta et al. (1982) Nature 299, 259-261) have shown that nonparasitized erythrocytes of Plasmodium knowlesi-infected monkeys contain the procoagulant phospholipid phosphatidylserine (PS) in the outer-half of their membrane bilayer. A reinvestigation of this problem has now revealed that in acute P. knowlesi infection, at least 30% of the infected animals do not have this abnormality. However, PS externalization was a consistent feature in the uninfected red cells of chronically infected animals. Also, a similar membrane change was observed in the red cells of uninfected splenectomized monkeys. These results strongly suggest that spleen plays an important role in maintaining the exclusive inner distribution of PS in the normal erythrocyte membrane, and that partial migration of this lipid to the outer monolayer in nonparasitized erythrocytes could be attributed to an abnormal physiology of this organ in malarial infection.     

Host chaperones are recruited in membrane-bound complexes by Plasmodium falciparum.

The ability of malarial parasite to deploy proteins at the surface of infected erythrocytes is well known. After their synthesis within the parasite, the cargo proteins are exported from the parasite and carried across the erythrocyte cytoplasm to be delivered at the erythrocyte surface. Our knowledge about the mechanisms involved in this complex trafficking path is limited. We have addressed the involvement of chaperones in traffic across erythrocyte cytoplasm. Our analyses of the chaperones available to the parasite indicated that none of the reported chaperones of the parasite origin are present in the erythrocyte cytoplasm. The chaperones of the host (Hsp70, Hsp90, Hop60), on the other hand, were readily detected in the erythrocyte cytosol. Hypotonic lysis and detergent solubilization experiments indicated that unlike their soluble nature in normal erythrocytes, host chaperones are recruited in membrane-bound, detergent-resistant complexes in infected cells. The association of host-Hsp70 with detergent-resistant complexes was ATP-dependent. Importantly, host chaperones could be detected in knob-enriched fractions and could be cross-linked to the knob subunit, PfHRP1, in a large complex at the surface of the infected erythrocytes. Our results implicate host chaperones in the assembly of parasite proteins such as knob subunits at the erythrocyte surface.