Malaria Parasites And Disease

Malaria Parasites and Disease Perlmann P, Troye-Blomberg M (eds): Malaria Immunology. Chem Immunol. Basel, Karger, 2002, vol 80, pp 1–26

Structure and Life Cycle Hisashi Fujiokaa, Masamichi Aikawab a b

Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, USA, and Institute of Medical Sciences, Tokai University, Kanagawa, Japan

Introduction

Plasmodium species, as members of Apicomplexa, share many common morphological features. Each of the developmental stages in the life cycle of malaria parasites exhibits a remarkable conservation and distinct patterns of structural organization [1, 2]. These conservations are supposed to have originated in the special adaptation to the tissues and/or cells in the different hosts of malaria parasites. As the technology of electron microscopy has improved, more detailed electron microscopic observations of the various stages of malaria parasites have been carried out and greatly advanced our knowledge of the life cycle and the fine structure of malaria parasites. Although the significance of the morphological changes is not fully understood, the introduction of the techniques of immunoelectron microscopy to the field of malaria parasites [3] has helped us in the meaningful and dynamic analysis of parasite morphology and cell biology, and our knowledge of the subcellular localization of malaria antigens and their functions in specific parasite organelles has been accumulated. Structural, biochemical and molecular biological aspects are different among the complex cycle comprising the erythrocytic schizogony, mosquito stages, and preerythrocytic (exoerythrocytic) schizogony. In this chapter, we will describe the ultrastructure of each specific stage, and the morphological and functional changes of the host cells induced by malaria parasites.

Life Cycle

The life cycle of the malaria parasite is complex (fig. 1). The sporozoites are transmitted to the vertebrate host by the bite of infected female mosquitoes

Fig. 1. Schematic drawing of the life cycle of malaria parasites.

of the genus Anopheles. The sporozoites enter hepatocytes shortly after inoculation into the blood circulation. This process has demonstrated that sporozoite invasion of hepatocytes involves surface proteins of the sporozoite and host cell surface molecules. Sporozoites infected in the hepatocytes develop into preerythrocytic (exoerythrocytic, EE stage; fig. 9) schizonts during the next 5–15 days depending on the Plasmodium species. Plasmodium vivax, Plasmodium ovale and Plasmodium cynomolgi have a dormant stage, named hypnozoite [4, 5], that may remain in the liver for weeks to many years before the development of preerythrocytic schizogony. This results in relapses of malaria infection. Plasmodium falciparum and Plasmodium malariae have no persistent phase. A preerythrocytic schizont contains 10,000 to 30,000 merozoites, which are

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a

b

c

d

Fig. 2. Malaria merozoite invasion process. a Apical end of a P. knowlesi merozoite attaches to an erythrocyte (E). The erythrocyte membrane becomes thick at the attachment site (arrow). b Further advanced stage of erythrocyte entry by a P. knowlesi merozoite. The junction (arrow), formed between the thickened erythrocyte membrane and the merozoite, is always located at the orifice of the merozoite entry. No surface coat is visible on the portion of the merozoite surface, which has invaginated the erythrocyte membrane, while the surface coat is present behind the junction (arrow) site. c Erythrocyte entry by a P. knowlesi merozoite is almost complete. The junction (arrow) has now moved to the posterior end of the merozoite. An electron-opaque projection connects the merozoite’s apical end and erythrocyte membrane. d A trophozoite (ring form) stage of P. falciparum is surrounded by the parasitophorous vacuole membrane (PVM). R ⫽ Rhoptry; D ⫽ dense granules; Mn ⫽ micronemes; E ⫽ erythrocyte; N ⫽ nucleus. Bars ⫽ 0.5 ␮m. a and c are reprinted with permission from Fujioka and Aikawa (1999) [60]; courtesy of Harwood Academic Publisher.

released into the blood circulation and invade the red blood cells. The merozoite develops within the erythrocyte through ring, trophozoite and schizont stages (erythrocytic schizogony; fig. 3a). The parasite modifies its host cell in several ways to enhance its survival. The erythrocyte containing the segmented schizonts eventually ruptures and releases the newly formed merozoites that invade new erythrocytes (fig. 2). Erythrocyte invasion by merozoites is dependent on the interactions of specific receptors on the erythrocyte membrane with ligands on the surface of the merozoite. The entire invasion process takes about 30 s. Concomitantly, a small portion of the parasites differentiate from newly invaded merozoites into sexual forms, which are macrogametocyte (female) and microgametocyte (male). What triggers this alternative developmental pathway leading to gametocyte formation is unknown. Mature macrogametocytes, taken into the midgut of the Anopheles mosquito, escape from the erythrocyte to form macrogametes. Microgametocytes exflagellate, each forming eight haploid motile microgametes after a few minutes in the mosquito midgut. The microgamete moves quickly to fertilize a macrogamete and forms a zygote. Within 18–24 h, the non-motile zygotes transform into motile ookinetes. The ookinetes have to cross two barriers: the peritrophic matrix (PM) and midgut epithelium (fig. 7a). After traversing the midgut epithelium, the ookinete reaches the extracellular space between the midgut epithelium and the overlaying basal lamina,

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and transforms into an oocyst (fig. 7b). Ten to 24 days after infection, depending on the Plasmodium species and ambient temperature, thousands of sporozoites (fig. 8b) are released into the hemocoel and the motile sporozoites invade the salivary gland epithelium. When an infected mosquito bites a susceptible vertebrate host, the Plasmodium life cycle begins again. Erythrocytic Schizogony Merozoites

The erythrocytic merozoite is an ovoid cell and measures approximately 1.5 ␮m in length and 1 ␮m in width (fig. 2a). The apical end of the merozoite is a truncated cone-shaped projection demarcated by the polar rings. Three types of membrane-bound organelles, namely, rhoptries (two prominent pear-shaped, 570 ⫻330 nm), micronemes (ovoid bodies, 100 ⫻ 40 nm), and dense granules (spheroid vesicles, 140 ⫻120 nm), are located at the anterior end of the merozoite [6]. The contents of these organelles play a role in the binding and entry of the merozoite into the host cells. Extracellular merozoites are intrinsically short-lived and must rapidly invade a new host erythrocyte. The merozoite is surrounded by a trilaminar pellicle that is composed of a plasma membrane and two closely aligned inner membranes [1]. The plasma membrane measures about 7.5 nm in thickness. Just beneath this inner membrane complex is a row of subpellicular microtubules which originate from the polar ring of the apical end and radiate posteriorly [2]. It has been suggested that the inner membrane complex and subpellicular microtubules function as a cytoskeleton giving rigidity to the merozoite and may be involved in invasion [7]. The presence of P. falciparum myosin A in the apex of the mature merozoites suggests its involvement in merozoite motility during invasion [8]. The outer membrane of the extracellular merozoite is covered with a surface coat of about 20 nm in thickness, and plays an important role in the early stages of merozoite invasion [9]. A mitochondrion is seen in the posterior portion of the merozoites [1]. Mammalian parasites appear to have a few cristate or acristate mitochondrion. An additional structure, referred to as a spherical body, has been identified. A recent study [10] described that the plastid of P. falciparum (or ‘apicoplast’) is the evolutionary homologue of the plant chloroplast. The apicoplast is surrounded by four membranes and is likely to contain many prokaryote-type pathways. Golgi complexes are inconspicuous in the merozoite. Host Cell Entry

Malaria merozoite invasion process is complex (fig. 2a–c) and involved in the multi-step sequence which can be divided into four phases: (1) initial

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recognition and reversible attachment of the merozoite to the erythrocyte membrane; (2) reorientation and junction formation between the apical end of the merozoite (irreversible attachment) and the release of rhoptry-microneme substances with parasitophorous vacuole formation; (3) movement of the junction and invagination of the erythrocyte membrane around the merozoite accompanied by removal of the merozoite’s surface coat, and finally (4) resealing of the parasitophorous vacuole membrane (PVM) and erythrocyte membrane after completion of merozoite invasion (fig. 2) [for review see 1, 11, 12]. The initial factor underlying recognition between merozoites and erythrocytes may occur between the merozoite surface coat filament and erythrocyte surface. Multiple different receptor-ligand interactions occur during the merozoite invasion process into an erythrocyte [13, 14]. Merozoite surface protein-1, with a glycosylphosphatidylinositol anchor, (MSP-1; also called MSA1, gp195 or PMMSA) could be involved in the initial recognition of the erythrocyte in a sialic acid-dependent way [15]. Herrera et al. [16] suggested that MSP-1 interacted with spectrin on the cytoplasmic face of the erythrocyte membrane. Three other P. falciparum-merozoite surface proteins, named MSP-2, MSP-3 and MSP-4, have been identified [17, 18]. A number of investigators concluded that sialic acid on glycophorins are involved in receptor recognition for merozoite invasion [15, 19–21] after initial attachment. The microneme derived 175-kD erythrocyte-binding antigen (EBA-175) [22] of P. falciparum also binds to sialic acids on glycophorin [23]. The gene structure of EBA-175 has striking similarities with the Duffy-binding proteins of P. vivax and P. knowlesi [23–26]. Phylogenetically distant malaria species, P. falciparum, P. vivax, P. knowlesi, and also rodent malaria parasites [27] maintain species-specific and biologically similar proteins. EBA-175 seems to be the most important ligand for binding of merozoites to glycophorin A on the erythrocytes; however, some P. falciparum merozoites can utilize alternative pathways for invasion. Dolan et al. [28] showed that glycophorin B can also act as an erythrocyte receptor. Furthermore malaria merozoites can utilize independent pathways for invasion without sialic acid [29]. Following reorientation of the apical end of the merozoite contacting the erythrocyte membrane, a junction is formed between the apical end of the merozoite and erythrocyte membrane, and moved from the apical end to the posterior end of the merozoite. The junction seems to selectively control internalization of host cell plasma membrane components into the PVM [30, 31]. The merozoite cap protein 1 (MCP-1) [32] with an oxidoreductase domain is localized to the junction. The positive charge cluster in the C-terminal domain of this protein resembles domains in some cytoskeleton-associated proteins, raising speculations that the C-terminal domain of MCP-1 interacts with the cytoskeleton in Plasmodium [33]. As the invasion progresses, the depression of the erythrocyte membrane deepens and conforms to the shape of

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the merozoite. The junction is no longer observed at the initial attachment point but now appears at the orifice of the merozoite-induced invagination of the erythrocyte membrane. Cytochalasin B or D [34, 35] blocks the merozoite invasion step into erythrocyte. Staurosporine also blocks invasion at a step which is morphologically similar to the arrest seen with cytochalasin B or D [13, 14]. From these results, an actin-based motility system, probably within the parasite, might play an important role in the movement of junction during merozoite invasion into erythrocyte [35]. Several proteins have been identified and localized to the apical complex in Plasmodium species. The secretion-triggering mechanism seems to be similar to those of many other exocytotic cells. A calcium-dependent second-messenger system may be involved in the secretion of rhoptry-microneme contents [36]. Rhoptries contain high molecular weight proteins, 140-kD Rhop-1, 130-kD Rhop-2 and 110-kD Rhop-3 (Rhop-H) [for review see 37]. The Rhop-H proteins are localized in the electron-dense compartment of rhoptries of P. falciparum (fig. 8) [38]. Apical membrane antigen-1 (AMA-1 also called Pf83) is localized in the rhoptry organelles, and is processed to a 66-kD molecule with epitopes expressed on the surface of the merozoite. AMA-1 family proteins are homologous to the relatively well-conserved proteins in Plasmodium species [39– 41]. During host cell invasion, no surface coat is visible on the portion of the merozoite within the erythrocyte invagination (fig. 2b), whereas the surface coat on the portion of the merozoite still outside the erythrocyte appears similar to that seen on the free merozoites. Biochemical studies demonstrated that the 19-kD fragment is transported into the erythrocyte while other MSP-1 fragments were shed into supernatant during merozoite invasion [42, 43]. When the merozoite has completed entry, the junction fuses at the posterior end of the merozoite, closing the orifice in the fashion of an iris diaphragm. The merozoite still remains in close apposition to the thickened erythrocyte membrane at the point of final closure [1]. After completion of host cell entry, the merozoite is now surrounded by the PVM (fig. 2d). Fluorescent lipid probes have been used to demonstrate that PVM lipids are largely derived from the erythrocyte membrane [44]. This membrane serves as an interface between the parasite and host cell cytoplasm. Molecules such as nutrients must cross the PVM from the host cell to the parasite and other molecules such as metabolites and parasitesynthesized proteins must cross the PVM in the opposite direction. Dense granules of P. knowlesi merozoites were shown to move to the merozoite pellicle after merozoite entry into the erythrocyte [6]. These contents were released into the parasitophorous vacuole space and appeared to assist the formation of invaginations of the PVM. The ring-infected erythrocyte antigen (RESA; also called Pf155) [45, 46] is located in dense granules [47]. This antigen appears not to be transferred to the erythrocyte membrane during the initial formation of a

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junction between the apical end of a merozoite and the erythrocyte. The transportation process of the RESA/Pf155 protein from the dense granule to the infected erythrocyte membrane is unknown. This antigen is suggested to associate with the erythrocyte cytoskeleton mediated by spectrin [48, 49]. The organelle contents of the merozoite play a role in merozoite entry into the erythrocyte and also appear to have the additional roles of modification of the host cell membrane and PVM. These modifications seem to enable malaria parasites to survive and proliferate within the host erythrocytes. Recently subtilisin-like proteases, PfSUB1 [50] and PfSUB2 [51] from a subset of the dense granules were described. These enzymatically active proteases may function in the initial steps of erythrocyte invasion.

Trophozoites and Schizonts

When the extracellular merozoite invades the erythrocyte, it rounds up due to the rapid degradation of the inner membrane complex and subpellicular microtubules of the pellicular complex, and becomes a trophozoite. Dense granules within the merozoite move to the merozoite pellicle, and the contents of dense granules are released into the parasitophorous vacuole space [6]. The parasite in the erythrocyte is surrounded by the PVM (fig. 2d). This membrane serves as an interface between the parasite and host cell cytoplasm [for review see 52, 53]. Molecules such as nutrients must cross the PVM from the host cell to the parasite and other molecules, such as metabolites and parasite synthesized surface proteins (e.g. knob proteins), must cross the PVM in the opposite direction. The trophozoite survives intracellularly by ingesting host cell cytoplasm through a circular structure named the cytostome [54]. The cytostome (fig. 3a) possesses a double-membrane, consisting of an outer membrane (parasite plasmalemma) and an inner membrane (PVM). Malaria parasites use host hemoglobin as a source of amino acids; however, they cannot degrade the hemoglobin heme byproduct. Free heme is potentially toxic to the parasite. Therefore during hemoglobin degradation, most of the liberated heme is polymerized into hemozoin (malaria pigment), which is stored within the food vacuoles [55, 56]. The trophozoite of P. falciparum has several mitochondria with few cristae or acristate mitochondria. Cristate mitochondria, however, have also been observed in the erythrocytic trophozoites of P. malariae [57]. Immunoelectron microscopic analysis reveals the distinct mitochondrial localization of P. falciparum heat shock protein (PfHsp60) [58]. Ribosomes are abundant in the trophozoites, and most of them are of the free type. Various merozoite organelles, which had disappeared during trophozoite development, reappear at the segmented schizont (fig. 4a).

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