Conoid

Some apicomplexan parasites (Toxoplasma, Eimeria, and Sarcocystis) contain an additional cytoskeletal structure, the conoid, not found in other apicomplexans (Plasmodium and Theileria). It has been suggested that the conoid plays a mechanical role in invasion by parasites that must penetrate the robust barrier of the intestinal epithelium of vertebrates (26, 108, 121, 136, 138). The conoid consists of a set of counterclockwise spiraling filaments that create a pointed or cone-shaped structure at the extreme apex of these parasites (26, 64, 136, 138, 153). Extrusion of the conoid can be stimulated by ionomycin-triggered calcium influx, whereas pretreatment with cytochalasin D inhibits conoid extrusion (108). The filamentous subunits of the conoid resemble microtubules but are curled into an extremely tight coil, suggesting that if they are microtubules they are unusually constructed or deformed. (Recent work by K. Hu, D. Roos, and J. Murray [submitted for publication] suggests that the Toxoplasma conoid is constructed from tubulin organized into a novel polymer form consisting of a comma-shaped sheet of nine protofilaments.) The conoid is approximately 250 nm in diameter and can be extended beyond or retracted into the apical polar ring. There are two ~400-nm-long, closely associated microtubules in the middle of the conoid. These microtubules are tightly bound to each other and are eccentric to the longitudinal axis of the conoid. This arrangement may be due to contact with the conoid and preconoidal rings via lateral projections (120). The course of the conoid subunits parallels the counterclockwise spiral of the subpellicular microtubules.

Role in apicomplexan replication

Apicomplexan parasites replicate by internal budding to create either two daughter cells or multiple progeny. Nuclear divisions within the Apicomplexa are cryptomitoses (the nuclear membrane remains intact throughout), and kariokinesis occurs without chromosomal condensation (26, 79, 130, 157). Replication in Toxoplasma and Neospora occurs by endodyogeny, which creates two daughter parasites (63, 71, 91, 154). Replication by Plasmodium, Theileria, Eimeria, and Babesia occurs by schizogeny, which can create 64 daughter parasites (3, 12, 72, 79, 150, 157). The processes of endodyogeny and schizogeny are quite similar, differing mainly in the preservation or loss of maternal cell specialization. In endodyogeny, two daughter parasites are formed within an intact, fully polarized mother parasite (Fig. ​(Fig.4A).4A). This preserves the ability of replicating tachyzoites to invade throughout the cell cycle. The internal daughter cells are delimited by an IMC and associated subpellicular microtubules, and each contains (in addition to the nucleus, mitochondrion, Golgi, and plastid) a complete set of apical organelles (100, 154, 183). When the daughter cells are fully mature, the maternal apical complex is disassembled and the daughter parasites bud from the mother, adopting her plasma membrane. In schizogeny, after host cell invasion, the parasite subpellicular microtubules and apical complex are disassembled (Fig. ​(Fig.4B).4B). After growth and multiple nuclear divisions, polarized parasites are regenerated when the nuclei move to the schizont periphery and associate with the assembling IMC, subpellicular microtubules, and apical organelles (3, 12, 72, 157). The newly repolarized parasites then bud out of the maternal cell as merozoites. Due to their large round shape and lack of apical specialization, replicating parasites are noninvasive during schizogeny.

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FIG. 4.

Replication by the Apicomplexa. (A) Endodyogeny proceeds without loss of maternal cell shape and apical polarity. Formation of a curved nucleus is coupled to construction of two buds composed of nascent daughter IMC and subpellicular microtubules. The daughter cells develop surrounded by their own subpellicular microtubules and IMC in the fully polarized mother cell. Once the daughter cells are mature, they bud out of the remnants of the mother cell. (B) Schizogeny proceeds with loss of apical polarity. Invasion of a polarized elongated parasite is followed by disassembly of the subpellicular microtubules and IMC and by extensive cell growth and nuclear divisions. To reassemble polarized daughter cells, the multiple nuclei align with individual sets of apical organelles, subpellicular microtubules, and IMC at the periphery of the replicating cell.

P. falciparum replication has been studied by observing microtubule structures during replication in red cells (130). Premitotic intracellular parasites have a diffuse distribution of unpolymerized tubulin. The first visible structure is a spindle pole plaque that is localized within the nuclear membrane. This nucleates microtubules to give rise to a hemispindle, which partitions into two. Partitioning of the spindle pole plaque has also been observed by electron microscopy. A band of striated fibers termed the couche fibrillaire spans the separating plaques (42, 140, 161). This structure may represent centrin fibers, since similar structures are observed in algae and yeast (89, 164, 192). The spindle halves segregate in opposite directions by migration within the nuclear membrane to ultimately produce a full spindle. The multiple nuclear divisions of schizogeny are not completely synchronous; spindles in various stages of assembly are often present in a single late-stage schizont. Distinct postmitotic microtubule structures appear in late schizogeny. Each structure is associated with an individual nucleus and consists of extranuclear microtubules arranged in a regular radial array like the spokes on a cartwheel. These microtubules extend beyond the nucleus toward the center of the schizont. Remains of this microtubule organization persist as a rod-like structure in budding merozoites. This remnant may represent f-MAST, the reduced set of three or four subpellicular microtubules that occurs in P. falciparum merozoites (12, 59).

The microtubules of extracellular apicomplexans are not dynamic and are therefore impervious to microtubule-disrupting drugs (135). Assembly of microtubules occurs in the course of replication; therefore, intracellular parasites are susceptible to microtubule-depolymerizing drugs (Table ​(Table1)1) (168). In Toxoplasma and Plasmodium, the spindle and subpellicular microtubule populations are differentially stable to disruption by oryzalin or colchicine (14, 115). Lower concentrations (0.5 μM oryzalin or 1.0 mM colchicine) shorten microtubules. Under these conditions, Toxoplasma and Plasmodium continue to undergo nuclear division and budding but lack functional subpellicular microtubules and are incapable of invading new host cells. When removed from 0.5 μM oryzalin, Toxoplasma recovers normal morphology and is invasive (115). In contrast, higher concentrations of drug (2.5 μM oryzalin or 5.0 to 10.0 mM colchicine) disrupt both subpellicular and spindle microtubules (149, 168). Parasites under these conditions are incapable of nuclear division or budding, although cell growth, DNA synthesis, and centriole replication continue unchecked (115, 149, 168). When removed from 2.5 μM oryzalin, Toxoplasma tachyzoites attempt to bud as crescent-shaped parasites. Since the polyploid nuclear mass cannot be correctly segregated, daughter parasites are made that lack nuclei altogether (115).

TABLE 1.

Drug disruption of cytoskeletal elements in the Apicomplexa

Drug and cytoskeletal element (drug mechanism)	Concn	Action	Comments
Actin microfilaments
Cytochalasin D (disrupter)a	0.1-0.2 μM	Inhibits actin-based gliding motility (extracellular) and invasion	Inhibits gliding motility in extracellular parasites; use resistant host cells to avoid host cell effects.
Latrunculin (disrupter)b	10 nM-5 μM	Inhibits motility and invasion	Will also show host cell actin effects.
Jasplakinolide (stabilizer)c	50 nM-2 μM	Filament polymerization inhibits gliding and invasion	Acts on extracellular parasites; will also act on host cell actin in intracellular settings.
Microtubules
Oryzalin (disrupter)d			Also other dinitroanilines such as trifluralin, ethafluralin
Subpellicular microtubules	0.5 μM	Parasites lack apical polarity	No effect on extracellular parasites; no effect on host cell microtubules, but intracellular parasites are affected.
Spindle microtubules	2.5 μM	Parasites are incapable of replication	As for subpellicular microtubules.
Colchicine (disrupter)e
Subpellicular microtubules	10 μM-1 mM	Parasites lack apical polarity	No drug effect on extracellular parasites; use resistant host cells to avoid host cell disruption (no microtubules in RBCs).
Spindle microtubules	5-10 mM	Parasites are incapable of replication	As for subpellicular microtubules.
Colcemid (disrupter)f	50 μM	Inhibits intracellular growth	No cell biological observations accompany study.
Vinblastine (disrupter)g	15-100 nM	Inhibits intracellular growth	No cell biological observations accompany study.
Vincristine (disrupter)h	7 nM	Inhibits intracellular growth	No cell biological observations accompany study.
Tubulozole (disrupter)i	10 μM	Parasites are killed	May inhibit parasite protein synthesis rather than microtubules
Taxol (stabilizer)j	0.1-0.5 μM	Parasites are incapable of budding	No drug effect on extracellular parasites; host cell microtubules will also be affected (no microtubules in RBCs).
Docetaxel/taxotere (stabilizer)k	10 nm-50 μM	Parasites are incapable of replication	No drug effect on extracellular parasites; host cell microtubules will also be affected (no microtubules in RBCs).
Epithalone A (stabilizer)l	0.1-0.5 μM	Parasites are incapable of budding	No drug effect on extracellular parasites; host cell microtubules will also be affected (no microtubules in RBCs).
Myosin
BDM (ATPase inhibitor)m	20-40 mM	Inhibits actin-based gliding motility (extracellular) and invasion	Will also act on host cell myosins (not present in red cells).
KT5926 (LC kinase inhibitor)n	1-5 μM	Inhibits actin-based gliding motility (extracellular) and invasion	Most probably inhibits secretion of TRAP family adhesins rather than myosin.

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^aData from references 8, 25, 28, 35, 37, 39, 54, 56, 57, 64, 69, 85, 103, 104, 129, 131, 140, 143-145, 148, 152, 156, 163, and 170.

^bData from reference 145.

^cData from references 123, 145, and 147.

^dData from references 11, 17, 22, 59, 78, 96, 110, 145, and 164.

^eData from references 15, 23, 59, 85, 131, 156, 173, 185, 188, and 189.

^fData from reference 16.

^gData from references 16, 35, 120, 131, 156, 174, 188, and 189.

^hData from references 35 and 174.

ⁱData from references 16, 35, and 36.

^jData from references 59, 69, 85, 144, 145, 159, and 168.

^kData from references 137 and 159.

^lData from reference 168.

^mData from references 37, 56, 57, 64, and 119.

ⁿData from reference 37.

Assessment of the activity of microtubule-destabilizing drugs on intracellular parasites is complicated by the activity of these drugs on host cells. In many cases, toxicity to host cells antedates any effects on parasite microtubules. In this situation, parasites fail to replicate, but this is due to adverse effects on the host cell rather than to direct inhibition of parasite functions. For these reasons, the most comprehensive examination of the effects of microtubule-disrupting drugs has been carried out with the P. falciparum erythrocytic stages in red cells (which lack microtubules). In vitro studies have established that replicating P. falciparum merozoites are susceptible to colchicine and colcemid, to vinblastine and vincristine, to the dinitroanilines trifluralin and pendimethalin, and to tubulozoles, including tubulozole-T, an isomer that is inactive in mammalian systems (15, 33, 34, 178). In contrast, merozoites show only low sensitivity to the benzimidazoles (albendazole, thiabendazole, mebendazole, and omeprazole) (33, 163). The results for benzimidazole are consistent with the deduced amino acid sequences of apicomplexan β-tubulins that lack Glu 198 and Phe 200, predictors of sensitivity to these compounds (44a, 81a). Plasmodium merozoites are also sensitive to microtubule-stabilizing drugs such as taxol, docetaxel, and epothilone A, which inhibit schizont nuclear division and budding (141, 162, 172). Colchicine, trifluralin, and taxol also perturb gametocyte and ookinete differentiation in Plasmodium (80, 88). Parallel experiments to examine the role of microtubules in other apicomplexans have been carried out with Eimeria, Toxoplasma, and Cryptosporidium by using dinitroaniline compounds such as oryzalin or trifluralin (10, 16, 168). These drugs specifically inhibit the microtubules of plants and protists and do not destabilize vertebrate microtubules. To assess the effects of other drugs such as colchicine and taxol, resistant host cells can be used. When colchicine- or taxol-resistant cells are used as host cells for Toxoplasma replication, the effects of colchicine and taxol are akin to what is observed in Plasmodium merozoites (115).

Motility and Invasion

Most members of the Apicomplexa are motile. In these species, locomotion is intimately associated with host cell invasion and probably employs the same underlying cellular machinery. In fact, gliding locomotion and invasion often appear continuous and occur without a discernible change in speed. Apicomplexans (including gregarines) move by gliding motility (38, 52, 77, 84, 133). This movement does not exploit a discrete organelle (such as a flagellum) or result from amoeboid deformations of the cell. It is, however, substrate dependent. Motility has been analyzed in T. gondii and consists of three behaviors: circular gliding, upright twirling, and helical gliding (61, 66). When a crescent-shaped parasite lies on its right side, it moves in counterclockwise circles. Twirling occurs when the parasite is attached to the substrate by its posterior end, producing a clockwise spinning. Lastly, helical gliding is similar to twirling but occurs in horizontal parasites (Fig. ​(Fig.5A).5A). This biphasic behavior consists of an 180° clockwise revolution (resulting in a corkscrewing forward movement) coupled to a parasite flip to return the parasite to its original face, so that it can initiate helical motion anew. Helical gliding is the only behavior that results in long-distance movement across a substrate. Actin-disrupting or -stabilizing drugs (cytochalasin D and jasplakinolide), as well as myosin inhibitors (butanedione monoxime [BDM]), disrupt Toxoplasma motility and invasion (35, 37, 126). This suggests that an actomyosin-based mechanism underlies these behaviors (Table ​(Table1).1). Cytochalasin inhibition of gliding motility and/or invasion has been demonstrated in C. parvum, T. gondii, E. tenella, E. acervulina, and Plasmodium species (37, 56, 77, 106, 133, 135). Motility and invasion are associated with translocation of secreted adhesive proteins from the apex to the parasite posterior and their shedding or deposition into a slime trail. Both adhesins and parasite surface antigens are deposited into a trail by gliding parasites (11, 22, 37, 48, 166, 167, 171). The presence of surface proteins in slime trails is likely to reflect the artificially sticky substrate used to capture adhesin trails.

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FIG. 5.

Motility and invasion by the Apicomplexa. (A) Helical gliding is the only behavior associated with motility that leads to long-distance movement across a substrate. During this biphasic behavior, the parasite moves forward a body length and then repositions to repeat this cycle. This sequence illustrates the path taken by a gliding Toxoplasma tachyzoite to move forward one complete cycle. The parasite travels forward by moving a contact zone from its apex to its posterior in a helical path. Because the parasite is crescent shaped, it moves in a “corkscrew” fashion (frames 1 to 5). The crescent shape of the tachyzoite prevents the continuation of this motion because the parasite cannot contact the substrate with its concave face. To rectify this, the parasite rotates 180° without forward motion (frames 6 and 7) before initiating another cycle of ‘corkscrewing’ translational movement (frames 8 and 9). (Modified from reference (66) with the permission of the publisher.) (B) Apicomplexan invasion consists of attachment and apical orientation, induction of a parasitophorous vacuole, and translocation of the parasite into the vacuole. Apically secreted adhesins are capped along the moving junction to the posterior of the parasite. The moving junction is associated with a constriction of parasite shape that moves from the apex to the posterior.

Conserved adhesins have been found in Plasmodium, Toxoplasma, Eimeria, Neospora, and Cryptosporidium spp. (22, 24, 92, 132, 155, 171, 185). The thrombospondin-related anonymous protein (TRAP) family adhesins have common structural motifs and presumably an underlying mechanism of action. They contain a conserved adhesive domain consisting of a thrombospondin type 1 repeat that occurs in different numbers and locations within the individual apicomplexan adhesins. TRAP family proteins are localized to micronemes and are apically secreted during motility and invasion (22, 171). TRAP proteins are located on the surface of motile parasites in a transmembrane form and are capped from anterior to posterior in gliding parasites. The short cytoplasmic tail of these proteins is conserved and is thought to interact with the actomyosin cytoskeleton in order to translocate the protein backward. Adhesins are released as soluble protein by proteolytic cleavage at the posterior end of locomoting parasites (23). When the sporozoite-specific TRAP gene is knocked out in P. berghei merozoites, parasites behave normally until they differentiate to sporozoites within the mosquito. TRAP knockout sporozoites are immotile and noninvasive (171). Moreover, expression of a TRAP construct missing the last 14 amino acids of the cytoplasmic tail permits surface localization of the protein in knockout sporozoites (81). However, these sporozoites also display atypical behavior, repeatedly gliding one-third of a circle and snapping back to their original position. This suggests that the inability of TRAP to be appropriately translocated and/or released at the posterior end of locomoting parasites prevents their continued forward movement (81, 101). Menard has recently written a comprehensive review of gliding motility and the adhesins in the Apicomplexa (101).

The existence of an actomyosin-based mechanism for invasion was first implied by observations of the effect of cytochalasin B on Plasmodium invasion. Cytochalasin B blocks Plasmodium knowlesii merozoite invasion of red cells (106). Merozoites will attach irreversibly to red cells and form a vestigial parasitophorous vacuole but are inhibited from moving into the cell. Since red cells are unequivocally nonphagocytic, the effects of cytochalasin on Plasmodium invasion are likely to represent drug disruption of parasite (not host cell) microfilaments. However, for other apicomplexans, cytochalasin inhibition of invasion was sometimes attributed to inhibition of induced phagocytosis by the host cell. Active invasion of Toxoplasma tachyzoites can be distinguished from the phagocytic uptake of parasites because invasion of host cells (including macrophages) does not induce host cell membrane ruffling, actin microfilament reorganization, or tyrosine phosphorylation, which are all indicative of phagocytosis (111). Invasion is three to four times faster than phagocytosis (occurring within 25 to 40 s) and is characterized by parasite penetration into a tight-fitting vacuole formed by invagination of the plasma membrane. In contrast, phagocytosis of Toxoplasma involves membrane ruffling and the parasite is captured in a loose-fitting phagosome that forms over 2 to 4 min (78, 111, 121). Phagocytosis involves both reorganization of the host cytoskeleton and tyrosine phosphorylation of host proteins (111).

Experiments with cytochalasin D-resistant T. gondii have definitively established that invasion of Toxoplasma is critically dependent on actin filaments in the parasite but not in the host cell (37). Invasion of cytochalasin D-resistant host cells by wild-type (cytochalasin-sensitive) Toxoplasma tachyzoites is blocked by cytochalasin D. As observed with Plasmodium, attachment and apical orientation of Toxoplasma is normal in the presence of cytochalasin D. Cytochalasin D-resistant Toxoplasma mutants were isolated by chemical mutagenesis and selection for growth in cytochalasin D in resistant host cells. These resistant parasites have a point mutation in the single-copy actin gene ACT1 (A136G) and can invade wild-type host cells in the presence of cytochalasin D. Transformation of the mutant act1 allele into wild-type Toxoplasma confers cytochalasin D-resistant motility and invasion either as an allelic replacement or as a nonhomologous integration, generating a pseudodiploid parasite (37).

Apicomplexan invasion consists of three phases: (i) attachment with apical orientation, (ii) induction of a parasitophorous vacuole, and (iii) translocation of the parasite into the vacuole (Fig. ​(Fig.5B).5B). Parasites attach to host cells and form an intimate connection through apical end contact (37, 53, 106). This results in sequential secretion from the parasite micronemes and rhoptries. Adhesins from the micronemes are translocated along the parasite length and are shed at the site of the moving junction; parasitophorous vacuole components from the rhoptries are secreted into this forming compartment (24). Both tight adhesion to the host cell and secretion into the host cell occur when parasites are immobilized early in invasion by cytochalasin treatment (37, 53, 65, 106).

The “moving junction” which forms during invasion is a circumferential zone of attachment at the orifice of the host cell invagination (7, 105). It is characterized by a markedly thickened host cell membrane with increased electron density and is frequently accompanied by a constriction in the parasite body. The parasite enters the nascent parasitophorous vacuole by capping the moving junction down its body. Ultimately, the parasite becomes enclosed within a cavity delimited by the invaginated host cell membrane. Formation of a moving junction that is capped to the posterior during invasion is likely to be a feature of invasion shared by many apicomplexans (Theileria is an exception [discussed below]). The moving junction is a highly specialized interface of the parasite with the host cell, presumably exploiting cytoskeletal proteins, signaling molecules, and receptors. Very little is known about this interface. P. falciparum MCP-1 (merozoite-capping protein 1) is a 60-kDa merozoite protein that moves from anterior to posterior with the moving junction during merozoite invasion of red cells (86). MCP-1 lacks both a signal sequence and transmembrane domain and is located in the parasite cytosol. The precise role of MCP-1 in invasion remains obscure. The protein has an amino-terminal domain that is conserved in bacterial and eukaryotic oxidoreductases (76). There are also a group of microneme proteins that may play a role in this junction that localize to the moving junction and the (exposed) posterior region during invasion (24, 57, 62).

Actin and Actin Binding Proteins

Genes encoding actin have been cloned in several members of the Apicomplexa (36, 83, 119, 189-191). In T. gondii and C. parvum, actin is encoded by a single-copy gene (36, 83, 119). In Cryptosporidium, the actin gene is intronless, and in Toxoplasma it has one intron. In P. falciparum, the situation is akin to that for α-tubulin. There are two genes encoding actin (189-191). One gene (actin I) is intronless and is expressed throughout the parasite life cycle. In contrast, the P. falciparum actin II gene has an intron and is transcribed only in the sexual stages (191). The amino acid sequence of actin II is divergent from that of previously characterized actins. Additionally, relative to the high degree of conservation shown by most actins, the 79% amino acid sequence similarity between Plasmodium actin I and actin II is quite low. Actin-related proteins (arps) have not been characterized in the Apicomplexa yet, but sequences in the Plasmodium genome are annotated as putative arps.

F-actin affinity chromatography has been used to isolate actin binding proteins from P. knowlesii and P. falciparum merozoites and from Toxoplasma tachyzoites (54, 125, 173). In P. knowlesii, five major proteins with molecular masses of 75, 70, 48, 40, and 32/34 kDa are eluted from F-actin columns (173). The 70-kDa protein has been identified as heat shock protein 70 (HSC70). The 32/34-kDa doublet coelutes with HSC70 from columns or in gel filtration chromatography; however, the identity of these proteins remains unknown. Highly enriched fractions of the Plasmodium HSC70-HSC32-HSC34 complex inhibit rabbit skeletal muscle actin polymerization in vitro. Biochemical experiments have established that this is due to a capping activity that is Ca²⁺ independent and is inhibited by PIP₂.

Homologs of two widely conserved actin-associated proteins, coronin and actin-depolymerizing factor (ADF), have been characterized in the Apicomplexa. Coronin is a WD repeat containing actin binding protein that was first characterized in Dictyostelium discoideum, where it is essential for phagocytosis and motility. WD repeats (a tryptophan-aspartic acid motif) are found in diverse proteins; this motif is thought to mediate protein-protein interactions. Homologs of coronin are found in a large variety of eukaryotes, ranging from humans to C. elegans to yeast. A coronin homolog has been described in P. falciparum and is encoded by a single-copy gene (174). Compared to Dictyostelium coronin, the Plasmodium protein has conserved residues throughout the entire protein. A monoclonal antibody to D. discoideum coronin detects a 42-kDa protein in Triton X-100-insoluble extracts of P. falciparum schizonts. ADF/actophorin/cofilin is a widely conserved low-molecular-weight actin monomer-sequestering protein with filament-severing activity. An ADF homolog has been characterized in T. gondii (9). The single-copy gene encodes a 13.4-kDa protein. Toxoplasma ADF has a high degree of sequence similarity to other ADF homologs, particularly Acanthamoeba actophorin and plant ADFs. Toxoplasma ADF localizes to cytoplasm, especially under the plasma membrane. Recombinant Toxoplasma ADF purified from E. coli binds actin monomers and depolymerizes microfilaments in a pH-independent, concentration-dependent fashion.

One surprising finding is that a homolog of the actin monomer binding protein profilin has not been found in the Apicomplexa. In its place, Toxoplasma has apparently substituted a novel protein, toxofilin (125). Toxofilin is a 27-kDa actin monomer binding protein that was originally isolated from Toxoplasma extracts on G-actin affinity columns. In pyrene actin assays, toxofilin inhibits actin polymerization, acting as an actin-sequestering protein. It also slows microfilament disassembly through a filament end-capping activity. A single-copy gene encodes toxofilin. The protein has a pI of 9.63 and two coiled-coil domains and lacks consensus motifs or any similarity to known proteins. Overexpression of green fluorescent protein (GFP)-tagged toxofilin in vertebrate cells disrupts stress fibers and reduces microfilament levels by half. Toxofilin localizes to the apical cytoplasm in intracellular Toxoplasma but is found at the posterior of invading parasites. In motile parasites, toxofilin is localized throughout the entire cytoplasm.

Plasmodium Modification and Mimicry of Erythrocyte Cytoskeletal Proteins

Infection with Plasmodium merozoites results in dramatic changes to the shape and biochemical properties of the parasitized red cell. Red cells infected with Plasmodium have increased phosphorylation of band 4.1, and a cysteine protease from the parasite cleaves red cell ankyrin (96, 128). P. falciparum induces knob formation of the surface of red cells, and the normal discoid shape of these cells becomes spherical (176). These structural alterations contribute to sequestration of infected red cells in organ capillaries, preventing their circulation and exposure to the spleen (107). The Plasmodium proteins RESA, MESA, and HRP-1 are anchored to the red cell membrane by association with spectrin, band 4.1, and the band 3 binding domain of ankyrin, respectively (17, 94, 97, 122). P. falciparum growth is decreased in human erythrocytes containing abnormal spectrin or band 4.1 (142). Parasites invade these cells normally, suggesting that an intact red cell membrane skeleton is required for parasite growth.

Plasmodium erythrocytic stages also synthesize proteins which are similar to ankyrin and spectrin and which are hypothesized to play a role in reorganization of the cytoskeleton of red cells. Plasmodium chabaudi ROPE (repetitive organellar protein) has a structure similar to that of spectrin (188). This 229-kDa protein is localized to the apical end of merozoites, possibly in the rhoptries. ROPE has characteristics of a cytoskeletal protein. A 364-amino-acid repetitive region based on 32 11-mer repeats suggests that the protein forms an helical coiled-coil triple helix containing a leucine-histidine zipper. Strikingly, this three-dimensional arrangement resembles the structure of spectrin. It has been postulated that ROPE may be involved in invasion, by interacting with the erythrocyte cytoskeleton via molecular mimicry of spectrin. P. falciparum expresses an 88-kDa phosphoprotein that is nearly identical to the amino-terminal region of ankyrin, a region of the protein that binds band 3 (170). This protein may also help the parasite reorganize the membrane skeleton via molecular mimicry.

We thank Antonio Barragan, Audra Charron, John Cooper, Susan Dutcher, Olivia Giddings, Dan Goldberg, Wallace Marshall, Alissa Weaver, and Dawn Wetzel for commenting on the manuscript.

N.S.M. is supported by Individual NRSA fellowship F32 GM20484-01A1 and was previously supported by an NIH training grant in Infectious Disease held by the Washington University School of Medicine. (T32; AI-0717221).