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
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, USA, and
b
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 origi-
nated 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 par-
asites 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 tech-
niques 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 anti-
gens 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 inocu-
lation 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 preeryth-
rocytic (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

Fujioka/Aikawa 2
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 mero-
zoite 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 erythro-
cyte 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 per-
mission 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 micro-
gamete 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 extra-
cellular space between the midgut epithelium and the overlaying basal lamina,

Structure and Life Cycle 3

and transforms into an oocyst (fig. 7b). Ten to 24 days after infection, depend-
ing on the Plasmodium species and ambient temperature, thousands of sporo-
zoites (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 mero-
zoite [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

Fujioka/Aikawa 4
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 junc-
tion and invagination of the erythrocyte membrane around the merozoite
accompanied by removal of the merozoite’s surface coat, and finally (4) reseal-
ing of the parasitophorous vacuole membrane (PVM) and erythrocyte mem-
brane 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 inva-
sion 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 inter-
acted 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 similar-
ities 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 biologi-
cally 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 mero-
zoite contacting the erythrocyte membrane, a junction is formed between the
apical end of the merozoite and erythrocyte membrane, and moved from the api-
cal 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

Structure and Life Cycle 5

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 inva-
sion 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 local-
ized 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 com-
partment 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 pro-
teins 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 frag-
ments 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 mero-
zoite 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 parasite-
synthesized proteins must cross the PVM in the opposite direction. Dense gran-
ules 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

Fujioka/Aikawa 6
junction between the apical end of a merozoite and the erythrocyte. The trans-
portation 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 par-
asites to survive and proliferate within the host erythrocytes. Recently subtil-
isin-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 gran-
ules 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 synthe-
sized surface proteins (e.g. knob proteins), must cross the PVM in the opposite
direction. The trophozoite survives intracellularly by ingesting host cell cyto-
plasm through a circular structure named the cytostome [54]. The cytostome
(fig. 3a) possesses a double-membrane, consisting of an outer membrane (para-
site plasmalemma) and an inner membrane (PVM). Malaria parasites use host
hemoglobin as a source of amino acids; however, they cannot degrade the hemo-
globin 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 micro-
scopic 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).

Structure and Life Cycle 7