Opinion
TRENDS in Parasitology
Vol.19 No.5 May 2003
209
The ins, outs and roundabouts of
malaria
Lawrence Bannister1 and Graham Mitchell2
1
Department of Anatomy, Cell and Human Biology, Guy’s, King’s and St. Thomas’ School of Biomedical Science, Guy’s Hospital,
London SE1 1UL, UK
2
Department of Immunobiology, Guy’s, King’s and St. Thomas’ School of Medicine, Guy’s Hospital, London SE1 9RT, UK
The malaria parasite Plasmodium falciparum is a
complex eukaryote parasite with a dynamic pattern of
genomic expression, enabling it to exploit a series of
different habitats in human and mosquito hosts. In the
human bloodstream, the parasite grows and multiplies
within red blood cells and modifies them in various
ways to gain nutrients and combat the host’s defences,
before escaping and invading new red blood cells by a
multi-step process. These events are reflected in the
constantly changing structure of the organism during
the red blood cell cycle.
In its total impact on humanity, Plasmodium falciparum
is one of the world’s most pathogenic microbes. It kills
millions annually, and is especially lethal to young children.
It causes overt disease in many more millions of people,
and is steadily spreading to new lands. Efforts to control
malaria are becoming decreasingly successful because of
antimalarial drug resistance in the parasite, insecticide
resistance in mosquitoes, and socio-economic deficits and
warfare in human populations [1,2]. The defining problem,
however, is the unusual biology of this organism. Plasmodium falciparum is an exceedingly small, haploid, but
genomically complicated eukaryote, able to constantly
change its gene expression to generate a sequence of forms
that exploit most efficiently quite different environments:
liver and red blood cells in humans; gut, vascular system
and salivary glands in the mosquito (Fig. 1). In humans,
the parasite lives mainly within cells, protected there from
most circulating antibodies, and outwitting the host’s
immune attack on accessible parasite antigens by varying the expression of their genes [3]. The destruction of
parasite-infected red blood cells by the spleen and liver is
minimized, the parasites causing these cells to adhere to
blood vessel walls, apparently out of harm’s way. There are
other causes of the parasite’s success which are beyond the
remit of this article, especially its ability to disseminate
itself via a highly prolific insect vector which itself has a
high breeding rate ensuring large populations and a high
rate of evolution, for example, of insecticide resistance.
In humans, pathogenesis depends on the parasite’s
effects on the red blood cell (RBC) population, an impact
progressively amplified by repeated 48-hour-cycles of invasion, intracellular growth, multiplication and re-invasion
Corresponding author: Lawrence Bannister (lawrence.bannister@kcl.ac.uk).
(Fig. 2). In this article, the events of malaria infection
within the human bloodstream are outlined and illustrated, related chiefly to P. falciparum though supplemented with data from other species when data are
otherwise lacking. The structural features of these stages
are of course constantly changing in life as the cycle
proceeds, and the reader is encouraged to think beyond the
image to the myriad of underlying molecular processes at
work within the organism that the pictures reflect.
The stages of the cycle
The ring stage. Having invaded a RBC, the parasite
spreads itself into a thin biconcave disc [4,5], thicker
around its perimeter where the elongated nucleus is
present and thinner in the middle, giving it the appearance of a ring in Giemsa-stained blood smears. The
parasite fits snugly into a membrane-lined cavity, the
parasitophorous vacuole (PV), within the RBC and feeds
on small aliquots of haemoglobin through its cytostome, as
well as taking up other nutrients transported in from the
plasma. As the ring stage enlarges, it begins to synthesize
molecules specific to this stage [6], exporting some of them
into the RBC [7], and modifying the RBC membrane which
now begins to adhere to the linings of visceral and other
blood vessels, including those of the placenta [8]. The ring
eventually grows into the more rounded trophozoite stage.
The trophozoite. This is the period of most active
feeding, growth and RBC modification. New molecules
are exported into the RBC, some assembling into flat
membranous sacs of various forms, including those visible
in stained smears as Maurer’s clefts [9,10]. Others interact
with the RBC membrane and cytoskeleton to form small
knobs [10,11] on its surface, and some penetrate it, for
example, P. falciparum erythrocyte membrane protein
(PfEMP)1 to stick the infected RBC to the endothelium of
blood vessels, thus reducing parasite removal from the
blood stream by the defences of the body via the spleen. If
the infected RBC adheres to brain– blood vessel walls,
cerebral malaria can result [12], while in the placenta,
fetal growth can be affected by similar cytoadherence [13].
Other exported molecules increase RBC permeability to
nutrients. The parasite continues feeding on haemoglobin,
and the haem products of haemoglobin digestion crystallize into particles of dark pigment, haemozoin, scattered
within the food (pigment) vacuole.
http://parasites.trends.com 1471-4922/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1471-4922(03)00086-2
210
Opinion
TRENDS in Parasitology
Vol.19 No.5 May 2003
Fig. 1. The lifecycle of Plasmodium falciparum. The main phases in the liver and in the red blood cells (asexual and sexual erythrocytic stages) of the human host, and in
the gut and in the salivary glands of the mosquito host are depicted.
http://parasites.trends.com
Opinion
TRENDS in Parasitology
Vol.19 No.5 May 2003
211
Fig. 2. The main stages of the asexual erythrocytic cycle of Plasmodium falciparum. For an animated version: see http://archive.bmn.com/supp/part/bannister.html.
Abbreviations: Hb, haemoglobin; MZ, merozoite; PV, parasitophorous vacuole; RBC, red blood cell. See Ref. [29] for further details and illustrations.
http://parasites.trends.com
212
Opinion
TRENDS in Parasitology
The schizont. The parasite now undergoes a series of
nuclear divisions and intense synthesis and assembly
of molecules that are needed for RBC invasion. About
16 nuclei are generated and these move into merozoite
buds formed around the schizont’s periphery. Merozoites
eventually pinch off from the residual body of cytoplasm,
which is now full of compacted haemozoin crystals. Finally,
the RBC membrane and parasitophorous vacuolar membrane (PVM) lyse by a protease-dependent process [14]
and the merozoites exit into a brief extracellular phase.
The merozoite. The free merozoite is very small,
, 1.2 mm long, but it contains all things necessary to
invade and establish itself in a new RBC. At the apex of the
egg-shaped merozoite are three sets of secretory vesicles:
(1) the twin pear-shaped rhoptries; (2) the more numerous
but smaller micronemes; and (3) small rounded vesicles
called dense granules. The nucleus lies at the other end,
and a plastid and a mitochondrion lie along one side of
the merozoite, near a band of two or three microtubules.
Apically, three dense cytoskeletal rings (polar rings) brace
the apical prominence. A flat sac of membrane underlies
most of the merozoite surface membrane, forming with it
the merozoite’s pellicle, which lines the whole cell except
most apically. The merozoite also contains numerous free
ribosomes. Over the whole surface of the merozoite, there
is a thick, bristly adhesive coat (Fig. 2).
Invasion
To succeed in getting into a fresh RBC, the merozoite has to
rapidly select and adhere to it, then enter and seal itself
inside. The sequence of events has been analyzed most
closely in Plasmodium knowlesi [15 – 18], but the evidence
we have of P. falciparum invasion [4] suggest that the
process is very similar in both species.
Adhesion and apical orientation. If any part of the
newly released merozoite contacts a new RBC, the merozoite adheres by means of its bristly coat. Then, ensues a
series of minor convulsions of the RBC surface as it is
pulled partially around the merozoite’s perimeter, then
released again. Now, the merozoite could lose its hold
and repeat the process elsewhere. However, if the apical
prominence touches the RBC, the merozoite re-orientates
itself vertically to the RBC surface and forms a close,
irreversible junction between the two cells. Just beneath
the RBC membrane, at this point, dense material appears,
thought to be a local concentration of the RBC cytoskeleton
and attached transmembrane molecules, bound externally
to ligands on the merozoite apex. Molecules responsible for
the initial attachment are likely to include merozoite
surface protein (MSP)1. Those engaging in apical junction
formation are uncertain, but are thought to include micronemal proteins such as apical membrane antigen (AMA)1
which is known to be secreted onto the merozoite’s apex
before invasion commences [19].
Parasitophorous vacuole formation. The formation of
the apical junction triggers the generation of a deep
membrane-lined pit in the RBC surface into which the
merozoite glides, becoming completely enclosed in a membranous bubble, the PV. Its lining membrane, the PVM,
persists through the erythrocytic cycle and grows as the
parasite enlarges [20]. The RBC changes result from the
http://parasites.trends.com
Vol.19 No.5 May 2003
secretion of material from rhoptries and probably micronemes onto its membrane at the centre of the zone of apical
contact. There is evidence that the secreted substances
are incorporated into the membrane of the invasion pit,
although how much parasite material is added is uncertain. Rhoptries contain several types of protein, and following the evidence from Toxoplasma rhoptries, they are
likely to contain lipid, so that the PVM could originate
substantially from the parasite itself. However, there is
also evidence that the PVM contains much RBC membrane lipid, so the origin of the PVM is at present still
unresolved [21].
Merozoite interiorization. As the invasion pit begins to
be formed, the merozoite begins to glide into it, maintaining at all times a small point of central attachment
between the two cells at the opening of the rhoptry ducts.
The larger zone of apical junction contact, however, now
changes as it becomes a ring moving backwards over the
merozoite surface, maintaining contact between the parasite and RBC around the rim of the enlarging invasion pit.
Merozoite movement is an active process, depending on
the interaction of actin and myosin, situated close to the
merozoite surface, beneath the moving ring of junctional
contact [22]. Similar gliding movements have been described in other apicomplexan species and they appear to be
typical of this group of organisms.
As the merozoite moves through the junctional ring, the
thick bristly merozoite coat disappears at the outer edge of
the moving junction to leave the front end of the merozoite
without a coat. It is known that most of the MSP1 molecule
is cleaved from the merozoite surface during invasion,
and the observed detachment of the coat bristles might
represent this molecular process [23].
Dense granule release and merozoite transition to the
ring stage. Eventually, the moving junction reaches the
posterior end of the merozoite, and the PVM closes over
and detaches from the RBC surface. At this point, another
secretory event occurs: the merozoite’s dense granules
move to the parasite’s surface and discharge their contents
into the PV at various points around its perimeter [17,24].
The effect of this is to cause further local enlargement of
the PVM, presumably as more parasite-derived material
is intercalated in its structure. Among molecules released
into this space are ring-infected erythrocyte antigen
(RESA) [25,26] and ring membrane antigen (RIMA) [27];
RESA crosses the PVM and moves to the RBC membrane
under the surface where it interacts with the RBC
cytoskeleton [28].
The merozoite now changes to a ring stage. This entails
the demolition of invasion-related structures: remnants
of rhoptries, micronemes, dense granules, microtubules,
polar rings and inner pellicular membranes; a change in
shape to a disc; and the beginning of haemoglobin feeding
via the cytostome (carried in as part of the invading
merozoite, but hitherto inactive). The enlargement of the
PVM caused by dense granule secretion presumably
makes way for the growth of the parasite as it begins to
feed actively. The parasite is now a ring stage, and the
cycle begins again.
Although this account touches on the main structural
changes of the cycle, there are of course many hidden
Opinion
TRENDS in Parasitology
and poorly understood events of profound importance, for
example, the switching expressions of various antigen
families such as PfEMP1 which create such difficulties for
the immune system (and for vaccine development), and the
diversion from the asexual cycle to the sexual stages. We
also have only a meagre understanding of even the most
basic processes of the parasite’s life: how it feeds, alters
and escapes from the RBC; the identities of the ligands and
receptors used during invasion; the signalling systems
related to invasion and multiplication; and so forth. The
availability of the P. falciparum genome database will
undoubtedly give a tremendous impetus to the study of
Plasmodium, but it still needs unpacking in terms of
the parasite’s total biology if rational approaches to
chemotherapy and vaccine development are to be achieved.
This is a major challenge for biomedical scientists in the
21st century, as we seek ways to curtail this most
fascinating, if ultimately most terrible, organism.
Vol.19 No.5 May 2003
10
11
12
13
14
15
16
17
18
19
Acknowledgements
L.B. and G.M. acknowledge support from the Wellcome Trust (Grant No.
059566).
20
References
1 World Health Organization (2003) Malaria – a global crisis. World
Health Organization
2 Trape, J.-F. et al. (2002) Combating malaria in Africa. Trends
Parasitol. 18, 224 – 230
3 Nogueira, P.A. et al. (2001) Variant antigens of Plasmodium
falciparum encoded by the var multigenic family are multifunctional
macromolecules. Res. Microbiol. 152, 141– 147
4 Langreth, S.G. (1978) Fine structure of human malaria in vitro.
J. Protozool. 25, 443 – 452
5 el-Shoura, S.M. (1994) Falciparum malaria in naturally infected
human patients: VIII. Fine structure of intraerythrocytic asexual
forms before and during chloroquine treatment. App. Parasitol. 35,
207 – 218
6 Spielmann, T. and Beck, H.P. (2000) Analysis of stage-specific
transcription in Plasmodium falciparum reveals a set of genes
exclusively transcribed in ring stage parasites. Mol. Biochem.
Parasitol. 111, 453 – 458
7 Das, A. (1994) Biosynthesis, export and processing of a 45 kDa protein
detected in membrane clefts of erythrocytes infected with Plasmodium
falciparum. Biochem. J. 302, 487 – 496
8 Pouvelle, B. et al. (2000) Cytoadhesion of Plasmodium falciparum
ring-stage-infected erythrocytes. Nat. Med. 6, 1264 – 1268
9 Aikawa, M. et al. (1986) Membrane-associated electron-dense
material of the asexual stages of Plasmodium falciparum: Evidence
21
22
23
24
25
26
27
28
29
213
for movement from the intracellular parasite to the erythrocyte
membrane. Am. J. Trop. Med. Hyg. 35, 30 – 36
Atkinson, C.T. and Aikawa, M. (1990) Ultrastructure of malariainfected erythrocytes. Blood Cells 16, 351 – 368
Cooke, B.M. (2000) Falciparum malaria: sticking up, standing out and
outstanding. Parasitol. Today 16, 416– 420
Adams, S. (2002) Breaking down the blood-brain barrier: Signaling a
path to cerebral malaria? Trends Parasitol. 18, 360– 366
Scherf, A. et al. (2001) Molecular mechanisms of Plasmodium
falciparum placental adhesion. Cell. Microbiol. 3, 125– 131
Salmon, B.L. et al. (2001) Malaria parasite exit from the host
erythrocyte: A two-step process requiring extraerythrocytic proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 271 – 276
Dvorak, J.A. et al. (1975) Invasion of erythrocytes by malaria
merozoites. Science 187, 748 – 750
Aikawa, M. (1978) Erythrocyte entry by malarial parasites. A moving
junction between erythrocyte and parasite. J. Cell Biol. 77, 72 – 82
Bannister, L.H. (1975) Structure and invasive behaviour of Plasmodium knowlesi merozoites in vitro. Parasitology 71, 483– 491
Bannister, L.H. and Dluzewski, A.R. (1990) The ultrastructure of red
cell invasion in malaria infections: a review. Blood Cells 16, 257 – 292
Narum, D.L. and Thomas, A.W. (1994) Differential localization of
full-length and processed forms of PF83/AMA-1 an apical membrane
antigen of Plasmodium falciparum merozoites. Mol. Biochem. Parasitol. 67, 59 – 68
Lingelbach, K. and Joiner, K.A. (1998) The parasitophorous vacuole
membrane surrounding Plasmodium and Toxoplasma: an unusual
compartment in infected cells. J. Cell Sci. 111, 1467 – 1475
Preiser, P. (2000) The apical organelles of malaria merozoites: host cell
selection, invasion, host immunity and immune evasion. Microbes
Infect. 2, 1 – 17
Pinder, J.C. (2000) Motile systems in malaria merozoites: how is the
red cell invaded? Parasitol. Today 16, 240 – 245
Holder, A.A. et al. (1994) Malaria parasites and erythrocyte invasion.
[Review]. Biochem. Soc. Trans. 22, 291 – 295
Torii, M. (1989) Release of merozoite dense granules during erythrocyte invasion by Plasmodium knowlesi. Infect. Immun. 57, 3230– 3233
Aikawa, M. (1990) Pf155/RESA antigen is localized in dense granules
of Plasmodium falciparum merozoites. Exp. Parasitol. 71, 326 – 329
Culvenor, J.G. (1991) Plasmodium falciparum ring-infected erythrocyte surface antigen is released from merozoite dense granules after
erythrocyte invasion. Infect. Immun. 59, 1183– 1187
Trager, W. (1992) Transfer of a dense granule protein of Plasmodium
falciparum to the membrane of ring stages and isolation of dense
granules. Infect. Immun. 60, 4656 – 4661
Da Silva, E. et al. (1994) The Plasmodium falciparum protein RESA
interacts with the erythrocyte cytoskeleton and modifies erythrocyte
thermal stability. Mol. Biochem. Parasitol. 66, 59 – 69
Bannister, L.H. et al. (2001) A brief illustrated guide to the
ultrastructure of Plasmodium falciparum asexual blood stages.
Parasitol. Today 16, 427 – 433
Do you want to reproduce material from a Trends journal?
This publication and the individual contributions within it are protected by the copyright of Elsevier Science. Except as outlined in
the terms and conditions (see p. ii), no part of any Trends journal can be reproduced, either in print or electronic form, without
written permission from Elsevier Science. Please address any permission requests to:
Rights and Permissions,
Elsevier Science Ltd,
PO Box 800, Oxford, UK OX5 1DX.
http://parasites.trends.com