The ins, outs and roundabouts ofmalariaLawrence Bannister1 and Graham Mitchell2

1Department of Anatomy, Cell and Human Biology, Guy’s, King’s and St. Thomas’ School of Biomedical Science, Guy’s Hospital,

London SE1 1UL, UK2Department 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 falciparumis one of the world’s most pathogenic microbes. It killsmillions 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 controlmalaria are becoming decreasingly successful because ofantimalarial drug resistance in the parasite, insecticideresistance in mosquitoes, and socio-economic deficits andwarfare in human populations [1,2]. The defining problem,however, is the unusual biology of this organism. Plasmo-dium falciparum is an exceedingly small, haploid, butgenomically complicated eukaryote, able to constantlychange its gene expression to generate a sequence of formsthat exploit most efficiently quite different environments:liver and red blood cells in humans; gut, vascular systemand salivary glands in the mosquito (Fig. 1). In humans,the parasite lives mainly within cells, protected there frommost circulating antibodies, and outwitting the host’simmune attack on accessible parasite antigens by vary-ing the expression of their genes [3]. The destruction ofparasite-infected red blood cells by the spleen and liver isminimized, the parasites causing these cells to adhere toblood vessel walls, apparently out of harm’s way. There areother causes of the parasite’s success which are beyond theremit of this article, especially its ability to disseminateitself via a highly prolific insect vector which itself has ahigh breeding rate ensuring large populations and a highrate of evolution, for example, of insecticide resistance.

In humans, pathogenesis depends on the parasite’seffects on the red blood cell (RBC) population, an impactprogressively amplified by repeated 48-hour-cycles of inva-sion, intracellular growth, multiplication and re-invasion

(Fig. 2). In this article, the events of malaria infectionwithin the human bloodstream are outlined and illus-trated, related chiefly to P. falciparum though supple-mented with data from other species when data areotherwise lacking. The structural features of these stagesare of course constantly changing in life as the cycleproceeds, and the reader is encouraged to think beyond theimage to the myriad of underlying molecular processes atwork within the organism that the pictures reflect.

The stages of the cycle

The ring stage. Having invaded a RBC, the parasitespreads itself into a thin biconcave disc [4,5], thickeraround its perimeter where the elongated nucleus ispresent and thinner in the middle, giving it the appear-ance of a ring in Giemsa-stained blood smears. Theparasite fits snugly into a membrane-lined cavity, theparasitophorous vacuole (PV), within the RBC and feedson small aliquots of haemoglobin through its cytostome, aswell as taking up other nutrients transported in from theplasma. As the ring stage enlarges, it begins to synthesizemolecules specific to this stage [6], exporting some of theminto the RBC [7], and modifying the RBC membrane whichnow begins to adhere to the linings of visceral and otherblood vessels, including those of the placenta [8]. The ringeventually grows into the more rounded trophozoite stage.

The trophozoite. This is the period of most activefeeding, growth and RBC modification. New moleculesare exported into the RBC, some assembling into flatmembranous sacs of various forms, including those visiblein stained smears as Maurer’s clefts [9,10]. Others interactwith the RBC membrane and cytoskeleton to form smallknobs [10,11] on its surface, and some penetrate it, forexample, P. falciparum erythrocyte membrane protein(PfEMP)1 to stick the infected RBC to the endothelium ofblood vessels, thus reducing parasite removal from theblood stream by the defences of the body via the spleen. Ifthe 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 tonutrients. The parasite continues feeding on haemoglobin,and the haem products of haemoglobin digestion crystal-lize into particles of dark pigment, haemozoin, scatteredwithin the food (pigment) vacuole.Corresponding author: Lawrence Bannister (lawrence.bannister@kcl.ac.uk).

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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.

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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.

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The schizont. The parasite now undergoes a series ofnuclear divisions and intense synthesis and assemblyof molecules that are needed for RBC invasion. About16 nuclei are generated and these move into merozoitebuds formed around the schizont’s periphery. Merozoiteseventually pinch off from the residual body of cytoplasm,which is now full of compacted haemozoin crystals. Finally,the RBC membrane and parasitophorous vacuolar mem-brane (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 toinvade and establish itself in a new RBC. At the apex of theegg-shaped merozoite are three sets of secretory vesicles:(1) the twin pear-shaped rhoptries; (2) the more numerousbut smaller micronemes; and (3) small rounded vesiclescalled dense granules. The nucleus lies at the other end,and a plastid and a mitochondrion lie along one side ofthe merozoite, near a band of two or three microtubules.Apically, three dense cytoskeletal rings (polar rings) bracethe apical prominence. A flat sac of membrane underliesmost of the merozoite surface membrane, forming with itthe merozoite’s pellicle, which lines the whole cell exceptmost apically. The merozoite also contains numerous freeribosomes. Over the whole surface of the merozoite, thereis a thick, bristly adhesive coat (Fig. 2).

Invasion

To succeed in getting into a fresh RBC, the merozoite has torapidly select and adhere to it, then enter and seal itselfinside. The sequence of events has been analyzed mostclosely in Plasmodium knowlesi [15–18], but the evidencewe have of P. falciparum invasion [4] suggest that theprocess is very similar in both species.

Adhesion and apical orientation. If any part of thenewly released merozoite contacts a new RBC, the mero-zoite adheres by means of its bristly coat. Then, ensues aseries of minor convulsions of the RBC surface as it ispulled partially around the merozoite’s perimeter, thenreleased again. Now, the merozoite could lose its holdand repeat the process elsewhere. However, if the apicalprominence touches the RBC, the merozoite re-orientatesitself vertically to the RBC surface and forms a close,irreversible junction between the two cells. Just beneaththe RBC membrane, at this point, dense material appears,thought to be a local concentration of the RBC cytoskeletonand attached transmembrane molecules, bound externallyto ligands on the merozoite apex. Molecules responsible forthe initial attachment are likely to include merozoitesurface protein (MSP)1. Those engaging in apical junctionformation are uncertain, but are thought to include micro-nemal proteins such as apical membrane antigen (AMA)1which is known to be secreted onto the merozoite’s apexbefore invasion commences [19].

Parasitophorous vacuole formation. The formation ofthe apical junction triggers the generation of a deepmembrane-lined pit in the RBC surface into which themerozoite glides, becoming completely enclosed in a mem-branous bubble, the PV. Its lining membrane, the PVM,persists through the erythrocytic cycle and grows as theparasite enlarges [20]. The RBC changes result from the

secretion of material from rhoptries and probably micro-nemes onto its membrane at the centre of the zone of apicalcontact. There is evidence that the secreted substancesare incorporated into the membrane of the invasion pit,although how much parasite material is added is uncer-tain. Rhoptries contain several types of protein, and fol-lowing the evidence from Toxoplasma rhoptries, they arelikely to contain lipid, so that the PVM could originatesubstantially from the parasite itself. However, there isalso evidence that the PVM contains much RBC mem-brane lipid, so the origin of the PVM is at present stillunresolved [21].

Merozoite interiorization. As the invasion pit begins tobe formed, the merozoite begins to glide into it, maintain-ing at all times a small point of central attachmentbetween the two cells at the opening of the rhoptry ducts.The larger zone of apical junction contact, however, nowchanges as it becomes a ring moving backwards over themerozoite surface, maintaining contact between the para-site and RBC around the rim of the enlarging invasion pit.Merozoite movement is an active process, depending onthe interaction of actin and myosin, situated close to themerozoite surface, beneath the moving ring of junctionalcontact [22]. Similar gliding movements have been des-cribed in other apicomplexan species and they appear to betypical of this group of organisms.

As the merozoite moves through the junctional ring, thethick bristly merozoite coat disappears at the outer edge ofthe moving junction to leave the front end of the merozoitewithout a coat. It is known that most of the MSP1 moleculeis cleaved from the merozoite surface during invasion,and the observed detachment of the coat bristles mightrepresent this molecular process [23].

Dense granule release and merozoite transition to thering stage. Eventually, the moving junction reaches theposterior end of the merozoite, and the PVM closes overand detaches from the RBC surface. At this point, anothersecretory event occurs: the merozoite’s dense granulesmove to the parasite’s surface and discharge their contentsinto the PV at various points around its perimeter [17,24].The effect of this is to cause further local enlargement ofthe PVM, presumably as more parasite-derived materialis intercalated in its structure. Among molecules releasedinto 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 membraneunder the surface where it interacts with the RBCcytoskeleton [28].

The merozoite now changes to a ring stage. This entailsthe demolition of invasion-related structures: remnantsof rhoptries, micronemes, dense granules, microtubules,polar rings and inner pellicular membranes; a change inshape to a disc; and the beginning of haemoglobin feedingvia the cytostome (carried in as part of the invadingmerozoite, but hitherto inactive). The enlargement of thePVM caused by dense granule secretion presumablymakes way for the growth of the parasite as it begins tofeed actively. The parasite is now a ring stage, and thecycle begins again.

Although this account touches on the main structuralchanges of the cycle, there are of course many hidden

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and poorly understood events of profound importance, forexample, the switching expressions of various antigenfamilies such as PfEMP1 which create such difficulties forthe immune system (and for vaccine development), and thediversion from the asexual cycle to the sexual stages. Wealso have only a meagre understanding of even the mostbasic processes of the parasite’s life: how it feeds, altersand escapes from the RBC; the identities of the ligands andreceptors used during invasion; the signalling systemsrelated to invasion and multiplication; and so forth. Theavailability of the P. falciparum genome database willundoubtedly give a tremendous impetus to the study ofPlasmodium, but it still needs unpacking in terms ofthe parasite’s total biology if rational approaches tochemotherapy and vaccine development are to be achieved.This is a major challenge for biomedical scientists in the21st century, as we seek ways to curtail this mostfascinating, if ultimately most terrible, organism.

AcknowledgementsL.B. and G.M. acknowledge support from the Wellcome Trust (Grant No.059566).

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