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Apicomplexan parasites invade host cells by a conserved mechanism: parasite proteins are secreted from apical organelles, anchored in the host cell plasma membrane, and then interact with integral membrane proteins on the zoite surface to form the moving junction (MJ). The junction moves from the anterior to the posterior of the parasite resulting in parasite internalization into the host cell within a parasitophorous vacuole (PV). Conserved as well as coccidia-unique rhoptry neck proteins (RONs) have been described, some of which associate with the MJ. Here we report a novel RON, which we call RON12. RON12 is found only in Plasmodium and is highly conserved across the genus. RON12 lacks a membrane anchor and is a major soluble component of the nascent PV. The bulk of RON12 secretion happens late during invasion (after parasite internalization) allowing accumulation in the fully formed PV with a small proportion of RON12 also apparent occasionally in structures resembling the MJ. RON12, unlike most other RONs is not essential, but deletion of the gene does affect parasite proliferation. The data suggest that although the overall mechanism of invasion by Apicomplexan parasites is conserved, additional components depending on the parasite-host cell combination are required., (© 2013 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Unlabelled: Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca(2+) levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca(2+) increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca(2+) levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca(2+) in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca(2+)](c). Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca(2+)](c) in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells., Electronic Supplementary Material: The online version of this article (doi:10.1007/s11302-010-9202-y) contains supplementary material, which is available to authorized users.

A role for the Plasmodium falciparum cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of P. falciparum schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for > or =6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the P. falciparum PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria.

Members of the reticulocyte binding-like protein (RBL) family are merozoite-expressed proteins hypothesized to be essential for effective invasion of host erythrocytes. Proteins of the RBL family were first defined as merozoite invasion ligands in Plasmodium vivax, and subsequently in Plasmodium falciparum and other malaria parasite species. Comparative studies are providing insights regarding the complexity and evolution of this family and the existence of possible functionally alternative members. Here, we report the experimental and bioinformatic characterization of two new rbl genes in the simian malaria parasite species Plasmodium knowlesi. Experimental analyses confirm that a P. knowlesi gene fragment orthologous to P. vivax reticulocyte binding protein-1 (pvrbp1) represents a highly degenerated pseudogene in the H strain as well as two other P. knowlesi strains. Our data also confirm that a gene orthologous to pvrbp2 is not present in the P. knowlesi genome. However, two very diverse but related functional rbl genes are present and are reported here as P. knowlesi normocyte binding protein Xa and Xb (pknbpxa and pknbpxb). Analysis of these two rbl genes in Southern hybridizations and BLAST searches established their relationship to newly identified members of the RBL family in P. vivax and other species of simian malaria. Rabbit antisera specific for recombinant PkNBPXa and PkNBPXb confirmed expression of the prospective high molecular weight proteins and localized these proteins to the apical end of merozoites. Their precise location, as determined by immuno-electron microscopy (IEM), was found to be within the microneme organelles. Importantly, PkNBPXa and PkNBPXb are shown here to bind to host erythrocytes, and discussion is centered on the importance of these proteins in host cell invasion.

Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP1(19)), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP1(19) during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP1(19), fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP1(19) remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP1(19) and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP1(19) does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.

The most virulent form of malaria is caused by waves of replication of blood stages of the protozoan pathogen Plasmodium falciparum. The parasite divides within an intraerythrocytic parasitophorous vacuole until rupture of the vacuole and host-cell membranes releases merozoites that invade fresh erythrocytes to repeat the cycle. Despite the importance of merozoite egress for disease progression, none of the molecular factors involved are known. We report that, just prior to egress, an essential serine protease called PfSUB1 is discharged from previously unrecognized parasite organelles (termed exonemes) into the parasitophorous vacuole space. There, PfSUB1 mediates the proteolytic maturation of at least two essential members of another enzyme family called SERA. Pharmacological blockade of PfSUB1 inhibits egress and ablates the invasive capacity of released merozoites. Our findings reveal the presence in the malarial parasitophorous vacuole of a regulated, PfSUB1-mediated proteolytic processing event required for release of viable parasites from the host erythrocyte.

In Plasmodium falciparum, merozoite surface protein 7 (MSP7) was originally identified as a 22kDa protein on the merozoite surface and associated with the MSP1 complex shed during erythrocyte invasion. MSP7 is synthesised in schizonts as a 351-amino acid precursor that undergoes proteolytic processing. During biosynthesis the MSP1 and MSP7 precursors form a complex that is targeted to the surface of developing merozoites. In the sequential proteolytic processing of MSP7, N- and C-terminal 20 and 33kDa products of primary processing, MSP7(20) and MSP7(33) are formed and MSP7(33) remains bound to full length MSP1. Later in the mature schizont, MSP7(20) disappears from the merozoite surface and on merozoite release MSP7(33) undergoes a secondary cleavage yielding the 22kDa MSP7(22) associated with MSP1. In free merozoites, both MSP7(22) and a further cleaved product, MSP7(19) present only in some parasite lines, were detected; these two derivatives are shed as part of the protein complex with MSP1 fragments during erythrocyte invasion. Primary processing of MSP7 is brefeldin A-sensitive while secondary processing is resistant to both calcium chelators and serine protease inhibitors. Primary processing of MSP7 occurs prior to that of MSP1 in a post-Golgi compartment, whereas the secondary cleavage occurs on the surface of the developing merozoite, possibly at the time of MSP1 primary processing and well before the secondary processing of MSP1.

Proteolytic shedding of surface proteins during invasion by apicomplexan parasites is a widespread phenomenon, thought to represent a mechanism by which the parasites disengage adhesin-receptor complexes in order to gain entry into their host cell. Erythrocyte invasion by merozoites of the malaria parasite Plasmodium falciparum requires the shedding of ectodomain components of two essential surface proteins, called MSP1 and AMA1. Both are released by the same merozoite surface "sheddase," but the molecular identity and mode of action of this protease is unknown. Here we identify it as PfSUB2, an integral membrane subtilisin-like protease (subtilase). We show that PfSUB2 is stored in apical secretory organelles called micronemes. Upon merozoite release it is secreted onto the parasite surface and translocates to its posterior pole in an actin-dependent manner, a trafficking pattern predicted of the sheddase. Subtilase propeptides are usually selective inhibitors of their cognate protease, and the PfSUB2 propeptide is no exception; we show that recombinant PfSUB2 propeptide binds specifically to mature parasite-derived PfSUB2 and is a potent, selective inhibitor of MSP1 and AMA1 shedding, directly establishing PfSUB2 as the sheddase. PfSUB2 is a new potential target for drugs designed to prevent erythrocyte invasion by the malaria parasite.

Six myosins genes are now annotated in the Plasmodium falciparum Genome Project. Malaria myosins have been named alphabetically; accordingly, we refer to the two latest additions as Pfmyo-E and Pfmyo-F. Both new myosins contain regions characteristic of the functional motor domain of "true" myosins and, unusually for P. falciparum myosins, Pfmyo-F encodes two consensus IQ light chain-binding motifs. Phylogenetic analysis of the 17 currently known apicomplexan myosins together with one representative of each myosin class clusters all but one of the apicomplexan sequences together in Class XIV. This refines the earlier definition of the Class XIV Subclasses XIVa and XIVb. RT-PCR on blood stage parasite mRNA amplifies a specific product for all six myosins and each shows developmentally regulated transcription. Thus: Pfmyo-A and Pfmyo-B genes are transcribed throughout development; Pfmyo-C is predominant in trophozoites; Pfmyo-D occurs in trophozoites and schizonts; Pfmyo-E though barely present in earlier stages is abundant in schizonts; Pfmyo-F increases steadily throughout development and maturation. It is known that Pfmyo-A and Pfmyo-B are synthesised during late schizogony and we now show that Pfmyo-D expression is also temporally regulated to late trophozoites and schizonts where it distributes close to segregating nuclei. Thus, in asexual stages myosin synthesis does not always parallel transcript accumulation, showing that translation is also regulated. The implication is that the mRNAs are either subjected to turnover, synthesised and degraded, or that they are sequestered in an inactivate form until required for protein synthesis., (Copyright 2005 Wiley-Liss, Inc.)

The three-dimensional structure of the Plasmodium falciparum ring stage has been explored by reconstruction from serial sections and stereoscopic examination of tilted sections. The ring-like light microscopic appearance is related to the shape and contents of the biconcave discoidal parasite at this stage, its thick perimeter containing most of the ribosomes and its thin center containing smooth membrane organelles. The shapes of rings vary between flat and curved cuplike forms. The rough endoplasmic reticulum is a branched network continuous with the nuclear envelope. Evidence for a simple Golgi complex is seen in the presence on the outer nuclear envelope of a locus of coated vesicle budding associated with a single membranous cisterna or cluster of smooth vesicles. In middle and late stage rings this complex migrates along an extension of the nuclear envelope continuous with the rough endoplasmic reticulum. Evidence is also presented for a mechanism of exporting membrane from the parasite into the parasitophorous vacuole membrane and beyond into the red blood cell, by means of double-membraned vesicle-based exocytosis.

During asexual development Plasmodium schizonts undergo a series of complex biochemical and structural changes. Using tightly synchronized cultures of 2 P. falciparum lines (clone C10 and strain ITO4) for light microscopy and fluorescence imaging we monitored the timing and sequence of expression of proteins associated with invasion-related organelles. Antibodies to rhoptry, micronemal and dense granule proteins (Rhoptry Associated Protein 1, Apical Membrane Antigen 1, Erythrocyte Binding Antigen 175, Ring-infected Erythrocyte Surface Antigen) and to pellicle-associated proteins (Merozoite Surface Protein 1, PfMyosin-A) were used. Clone C10 developed faster than ITO4; this difference was also found in the timing of protein expression seen by immunofluorescence. Light microscopic data were combined with transmission electron microscopic analysis using serial sectioning of ITO4 schizonts to determine nuclear number and organellar development. Thus a timetable of schizont structural maturation was established. Generally, the timing of organelle-specific antigen expression correlates well with the ultrastructural data. Rhoptries are formed mainly between second and fourth nuclear divisions, micronemes between the end of the fourth nuclear division and merozoite separation from the residual body, while dense granules are generated mainly after the micronemes. PfAMA-1 appears in micronemes before EBA-175, suggesting micronemal heterogeneity.

The first gene characterizing the clag (cytoadherence linked asexual gene) family of Plasmodium falciparum was identified on chromosome 9. The protein product (Clag9) was implicated in cytoadhesion, the binding of infected erythrocytes to host endothelial cells, but little information on the biochemical characteristics of this protein is available. Other genes related to clag9 have been identified on different chromosomes. These genes encode similar amino acid sequences, but clag9 shows least conservation. Clag9 was detected in schizonts, merozoites and ring-stage parasites after protease digestion and peptide analysis by mass spectrometry. Using antisera raised against unique regions of Clag9 and against RhopH2, a component of the RhopH high-molecular-mass protein complex of merozoites, immunofluorescence co-localized the two proteins to the apical region of merozoites. Immunoelectron microscopy co-localized Clag9 and RhopH2 exclusively to the basal bulb region of rhoptries rather than to their apical ducts. The same Clag9-specific antibodies bound the RhopH complex, and the protein was detected in the complex purified by antibodies to RhopH2. Clag9 protein was also shown to be present in ring-stage parasites, carried through from the previous cycle with the RhopH complex, in a location identical to that of RhopH2. Transcription of the clag9 gene was shown to occur at the same time as the genes for other members of the RhopH complex, rhoph2 and 3. The results indicate that Clag9 is part of the RhopH complex and suggest that, within this complex, the protein previously designated RhopH1 is composed of more than one protein product of the clag gene family. The results cast doubt on a direct role for Clag9 in cytoadhesion; we suggest that the primary role of the RhopH complex is in remodelling the infected red blood cell after invasion by the merozoite. The complex may have multiple functions dependent on its exact composition, which may include, with respect to Clag9, a contribution to the mechanism of cytoadhesion.

Apical membrane antigen 1 (AMA-1) of Plasmodium merozoites is established as a candidate molecule for inclusion in a human malaria vaccine and is strongly conserved in the genus. We have investigated its function in merozoite invasion by incubating Plasmodium knowlesi merozoites with red cells in the presence of a previously described rat monoclonal antibody (MAb R31C2) raised against an invasion-inhibitory epitope of P. knowlesi AMA-1 and then fixing the material for ultrastructural analysis. We have found that the random, initial, long-range (12 nm) contact between merozoites and red cells occurs normally in the presence of the antibody, showing that AMA-1 plays no part in this stage of attachment. Instead, inhibited merozoites fail to reorientate, so they do not bring their apices to bear on the red cell surface and do not make close junctional apical contact. We conclude that AMA-1 may be directly responsible for reorientation or that the molecule may initiate the junctional contact, which is then presumably dependent on Duffy binding proteins for its completion.

Malarial merozoites invade erythrocytes; and as an essential step in this invasion process, the 42-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP142) is further cleaved to a 33-kDa N-terminal polypeptide (MSP133) and an 19-kDa C-terminal fragment (MSP119) in a secondary processing step. Suramin was shown to inhibit both merozoite invasion and MSP142 proteolytic cleavage. This polysulfonated naphthylurea bound directly to recombinant P. falciparum MSP142 (Kd = 0.2 microM) and to Plasmodium vivax MSP142 (Kd = 0.3 microM) as measured by fluorescence enhancement in the presence of the protein and by isothermal titration calorimetry. Suramin bound only slightly less tightly to the P. vivax MSP133 (Kd = 1.5 microM) secondary processing product (fluorescence measurements), but very weakly to MSP119 (Kd approximately 15 mM) (NMR measurements). Several residues in MSP119 were implicated in the interaction with suramin using NMR measurements. A series of symmetrical suramin analogues that differ in the number of aromatic rings and substitution patterns of the terminal naphthylamine groups was examined in invasion and processing assays. Two classes of analogue with either two or four bridging rings were found to be active in both assays, whereas two other classes without bridging rings were inactive. We propose that suramin and related compounds inhibit erythrocyte invasion by binding to MSP1 and by preventing its cleavage by the secondary processing protease. The results indicate that enzymatic events during invasion are suitable targets for drug development and validate the novel concept of an inhibitor binding to a macromolecular substrate to prevent its proteolysis by a protease.

During the assembly of Plasmodium falciparum merozoites within the schizont stage, the parasite synthesizes and positions three sets of secretory vesicles (rhoptries, micronemes and dense granules) that are active during red cell invasion. There are up to 40 micronemes per merozoite, shaped like long-necked bottles, about 160 nm long and 65 nm at their widest diameter. On their external surfaces, they bear bristle-like filaments, each 3-4 nm thick and 25 nm long. Micronemes are translocated from a single Golgi-like cisterna near the nucleus along a band of two or three subpellicular microtubules to the merozoite apex, where they dock with the rhoptry tips. Dense granules are also formed around the periphery of the Golgi cisternae but their distribution is unrelated to microtubules. Three polyclonal antibodies raised against the recombinant PfAMA-1 ectodomain sequence recognizing both the 83 kDa and processed 66 kDa molecules label the peripheries of translocating and mature micronemes but do not label rhoptries significantly at any stage of merozoite development within schizonts. This result confirms that PfAMA-1 is a micronemal protein, and indicates that within the microneme it is located near or inserted into this organelle's boundary membrane.

Using degenerate primers, three putative myosin sequences were amplified from Australian isolates of Babesa bovis and confirmed as myosins (termed Bbmyo-A, Bbmyo-B, and Bbmyo-C) from in vitro cultures of the W strain of B. bovis. Comprehensive analysis of 15 apicomplexan myosins suggests that members of Class XIV be defined as those with greater than 35% myosin head sequence identity and that these be further subclassed into groups bearing above 50-60% identity. Bbmyo-A protein bears a strong similarity with other apicomplexan myosin-A type proteins (subclass XIVa), the Bbmyo-B myosin head protein sequence exhibits low identity (35-39%) with all members of Class XIV, and 5'-sequence of Bbmyo-C shows strong identity (60%) with P. falciparum myosin-C protein. Domain analysis revealed five divergent IQ domains within the neck of Pfmyo-C, and a myosin-N terminal domain as well as a classical IQ sequence unusually located within the head converter domain of Bbmyo-B. A cross-reacting antibody directed against P. falciparum myosin-A (Pfmyo-A) revealed a zone of approximately 85 kDa in immunoblots prepared with B. bovis total protein, and immunofluorescence inferred stage-specific myosin-A expression since only 25% of infected erythrocytes with mostly paired B. bovis were immuno-positive. Multiplication of B. bovis in in vitro culture was inhibited by myosin- and actin-binding drugs at concentrations lower than those that inhibit P. falciparum. This study identifies and classifies three myosin genes and an actin gene in B. bovis, and provides the first evidence for the participation of an actomyosin-based motor in erythrocyte invasion in this species of apicomplexan parasite., (Copyright 2002 Wiley-Liss, Inc.)

The object of this paper is to review briefly the studies on the interaction of red blood cell membrane skeletal proteins and their non-erythroid analogues with lipids in model systems as well as in natural membranes. An important question to be addressed is the physiological significance and possible regulatory molecular mechanisms in which these interactions are engaged.

The genome of the malaria parasite, Plasmodium falciparum, contains a myosin gene sequence, which bears a close homology to one of the myosin genes found in another apicomplexan parasite, Toxoplasma gondii. A polyclonal antibody was generated against an expressed polypeptide of molecular mass 27,000, based on part of the deduced sequence of this myosin. The antibody reacted with the cognate antigen and with a component of the total parasite protein on immunoblots, but not with vertebrate striated or smooth muscle myosins. It did, however, recognise two components in the cellular protein of Toxoplasma gondii. The antibody was used to investigate stage-specificity of expression of the myosin (here designated Pf-myo1) in P. falciparum. The results showed that the protein is synthesised in mature schizonts and is present in merozoites, but vanishes after the parasite enters the red cell. Pf-myo1 was found to be largely, though not entirely, associated with the particulate parasite cell fraction and is thus presumably mainly membrane bound. It was not solubilised by media that would be expected to dissociate actomyosin or myosin filaments, or by non-ionic detergent. Immunofluorescence revealed that in the merozoite and mature schizont Pf-myo1 is predominantly located around the periphery of the cell. Immuno-gold electron microscopy also showed the presence of the myosin around almost the entire parasite periphery, and especially in the region surrounding the apical prominence. Labelling was concentrated under the plasma membrane but was not seen in the apical prominence itself. This suggests that Pf-myo1 is associated with the plasma membrane or with the outer membrane of the subplasmalemmal cisterna, which forms a lining to the plasma membrane, with a gap at the apical prominence. The results lead to a conjectural model of the invasion mechanism.

Calcium uptake by permeabilized P. chabaudi malaria parasites was measured at the trophozoite stage to assess calcium accumulation by the parasite organelles. As determined with 45Ca2+, the total calcium in the parasite was found to be 11 pmoles/10(7) cells. When the K+/H+ uncoupling agent, nigericin was present, this level fell to 6.5 pmoles/10(7) cells. A similar regulatory mechanism operates in P. falciparum, since addition of nigericin to intact parasites in calcium free-medium resulted in a transient elevation of free calcium in the parasite cytosol, as judged by fluorescent imaging of single cells loaded with the calcium indicator fluo-3,AM. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) and monensin, inhibitors of H+ ATPases and K+/H+ ionophore respectively, induced calcium elevation in fluo-3, AM-labeled intact P. chabaudi parasites. We conclude that malaria parasites utilize acidic intracellular compartments to regulate their cytosolic free calcium concentration.

The fluorescent indicator, fura-2, AM, was used to measure free calcium concentrations in the intraerythrocytic malaria parasites of Plasmodium chabaudi and Plasmodium falciparum. In both species the free cytosolic calcium concentration was maintained at low levels (between 40 and 100 nM throughout the maturation process. Digital image analysis of the indicator fluorescence was performed on parasites and evaluated with the aid of a calibration of the calcium response, based on permeabilized parasites, exposed to calcium buffers. This again revealed that free calcium concentrations in the intact parasite are maintained at a predetermined level, regardless of the free calcium in the surrounding milieu. Both species of parasites are thus capable of regulating their internal free calcium levels with high precision, presumably by means of calcium pump ATPases. A small but significant elevation of the cytosolic free calcium concentration by the tumor promoter, thapsigargin, may be taken to reflect the presence of calcium stores in the endoplasmic reticulum in P. falciparum.

We have examined the effects of seven protein kinase inhibitors (staurosporine, genistein, methyl 2,5-dihydroxycinnamate, tyrphostins B44 and B46, lavendustin A and R03) on the erythrocytic cycle of the malaria parasite, Plasmodium falciparum. One (staurosporine) strongly inhibits serine/threonine kinases, but the remainder all exhibit a strong preference for tyrosine kinases. We have been able to discriminate between effects on invasion and on intraerythrocytic development. All reagents impeded development of intraerythrocytic parasites, though at widely differing concentrations, from the sub-micromolar to the millimolar. Several inhibitors, including staurosporine, also reduced invasion. The phosphatase inhibitor, okadaic acid, had a strong inhibitory effect both on invasion and development. The regulation of malaria development by phosphorylation or dephosphorylation reactions at several points in the blood-stage cycle is implied.

F-actin was detected in asexual-stage Plasmodium falciparum parasites by fluorescence microscopy of blood films stained with fluorescent phalloidin derivatives. F-actin was present at all stages of development and appeared diffusely distributed in trophic parasites, but merozoites stained strongly at the poles and peripheries. No filament bundles could be discerned. A similar distribution was obtained by immunofluorescence with 2 polyclonal anti-actin antibodies, one of which was directed against a peptide sequence present only in parasite actin (as inferred from the DNA sequence of the gene). A monoclonal anti-actin antibody stained very mature or rupturing schizonts but not immature parasites. Myosin was identified in immunoblots of parasite protein extracts by several monoclonal anti-skeletal muscle myosin antibodies, as well as by a polyclonal antiserum directed against a consensus conserved myosin sequence (IQ motif). The identity of the polypeptides recognised by these antibodies was confirmed by overlaying blots with biotinylated F-actin. The antiserum and one of the monoclonal antibodies were used in immunofluorescence studies and were found to stain all blood-stage parasites, with maximal intensity towards the poles of merozoites. Our results are consistent with the presence of an actomyosin motor system in the blood-stage malaria parasite.

There is conflicting evidence on whether the parasitophorous vacuole membrane, in which the malaria parasite becomes encapsulated when it enters the red cell, represents a part of the host cell membrane or is derived, at least in part, from the parasite. We have measured the surface area of populations of red cells before and after invasion by up to four merozoites of the malaria parasite, Plasmodium falciparum. The dimensions of the merozoite are such that, if it enveloped itself entirely in host cell membrane during entry, the loss of surface area would amount to some 4 square microns 2 or 3% of the total for each parasite internalized. Our measurements show that within the 99% level of confidence any loss of surface area is less than 1 square micron 2 per parasite internalized. Area measurements on red cells that have been allowed to lose known proportions of their membrane by metabolically induced vesiculation reveal, moreover, that diminutions in surface area in the range of interest are readily detectable. Our observations on recently invaded (young ring-stage) parasites appear to exclude any significant change in surface area of the host cell following invasion. This implies that, if indeed there is internalization of host cell membrane lipid on invasion, as the best evidence shows, it is compensated by parasite-derived lipid, and conversely the parasitophorous vacuole membrane probably contains a contribution of parasite-derived material, presumably that seen to be discharged by the apical organelles, the rhoptries, at the time of invasion.

The ring-infected erythrocyte surface antigen (RESA) associates with spectrin in the erythrocyte membrane (Foley, M., Tilley, L., Sawyer, W. H. and Anders, R. F. (1991) Mol. Biochem. Parasitol., 46, 137-148). A fragment of the RESA protein, which was expressed in Escherichia coli, was found to bind to inside-out vesicles of erythrocyte membranes in an apparently saturable manner. Upon extraction of inside-out vesicles with Triton X-100, the RESA fragment remained associated with the erythrocyte cytoskeleton. Using the technique of steady-state fluorescence polarisation, we have studied the thermal denaturation of fluorescein-labelled spectrin in the presence of recombinant RESA. We found that the RESA fragment partially protected spectrin against heat-induced conformational changes. Furthermore, erythrocytes infected with a RESA (-) laboratory strain (FCR3) were shown to be more susceptible to heat-induced fragmentation than erythrocytes infected with a RESA (+) strain of the parasite. RESA does not, however, appear to play an essential role in the invasion process per se as erythrocytes resealed to contain anti-RESA antibodies were efficiently invaded.

Microaggregation of band 3 proteins in hereditary ovalocytic membranes was investigated by rotational diffusion measurements and by electron microscopy. It was previously shown that band 3 in ovalocytic membranes has decreased rotational mobility compared with band 3 in normal cells (Tilley, L., Nash, G.B., Jones, G.L. and Sawyer, W.L. (1991) J. Membr. Biol. 121, 59-66). This result could arise from either altered interactions with cytoskeletal proteins or from band 3 microaggregation. In the present study it was found that removal of spectrin and actin from the membrane had no effect on the rotational mobility of ovalocytic band 3. Additional removal of ankyrin and band 4.1, as well as cleavage of the cytoplasmic domain of band 3 with trypsin, did enhance band 3 mobility, as is the case in the membranes from normal cells. However, the rotational mobility of ovalocytic band 3 was always considerably less than that of normal band 3 under the same conditions. Scanning electron microscopy and low power electron micrographs of freeze-fracture replicas revealed that the surfaces of ovalocytes were more irregular than those of normal erythrocytes. At higher magnification, numerous linearly arranged intramembranous particles were observed on the P-faces of freeze-fractured ovalocytes but not on normal cells. These clusters consist of straight or slightly curved lines of 10-15 particles in single rows. From these results it is deduced that the reduced rotational mobility of band 3 in ovalocytes is a consequence of the formation of microaggregates, which are very probably induced by the mutation in the membrane-bound domain of ovalocytic band 3.

Merozoites of the human malaria parasite, Plasmodium falciparum, when treated with cytochalasin B, will attach irreversibly to red cells with formation of a vestigial internal (parasitophorous) vacuole, but they are inhibited from moving into the cell. The existence of an actin-based motile mechanism is implied. Immunoblotting, peptide mapping and the DNase inhibition assay have been used to show that the merozoite contains actin. It makes up an estimated 0.3% of the total parasite protein and is partitioned in the ratio of about 1:2 between the cytosolic and particulate protein fractions. In the former it is unpolymerised and in the latter filamentous. Most of the anti-actin-reactive protein in the soluble fraction and about 20% of that in the pellet has an apparent molecular weight of 55,000 and reacts with an anti-ubiquitin antibody; it is thus evidently ubiquitinyl actin, or arthrin, which has so far been detected only in insect flight muscle.

Hereditary ovalocytes (stomatocytic ovalocytes), when examined within 1-2 days from the time that the blood sample is drawn, are invaded by Plasmodium falciparum in culture to the extent of at least 55% of normal control cells. The ovalocytes have extremely rigid membranes, characterised by a shear elastic modulus some 3-4 times greater than that of normal cells. The extent of invasion falls off very much more rapidly than that into normal cells on storage, and we surmise that this is the reason for earlier reports of resistance of ovalocytes to malarial invasion in vitro. The initial loss of susceptibility to invasion with time is not accompanied by any change in membrane rigidity, but is primarily a consequence of a rapid decline in intracellular ATP concentration: this falls to below the threshold level required for invasion (approx. 0.1 mM) over a period in which the ATP in normal cells remains almost constant. Incubation in a metabolic regenerating medium leads to a rise in the intracellular ATP concentration and invasion by P. falciparum is recovered, though to a much lower extent than in normal cells. The resistance of ovalocytes to invasion becomes irreversible, due possibly to degradative processes in the membrane, on further storage. The developing parasites in ovalocytes have a reduced number of merozoites and show distinct morphological abnormalities.

Exclusion of magnesium ions from resealed ghosts or their extraction from intact human red cells by means of an ionophore results in a reversible drop in susceptibility to invasion by Plasmodium falciparum merozoites in vitro. Resealed ghosts, containing magnesium-ATP and diluted cytosol, are invaded with high efficiency only when the original hypotonic lysis is carried out in the presence of magnesium ions. This effect is not related to the loss of membrane-associated constituents when magnesium ions are absent. Ghosts containing calcium ions, together with the protective agent, flunarizine, were essentially resistant to invasion; this effect is again at least partially reversible. A possible explanation of these phenomena is that entry of the merozoite may be inhibited by breakdown of the host cell phospholipid asymmetry, with the appearance of aminophospholipids at the outer cell surface.

We have attempted to determine whether the parasitophorous vacuole membrane, in which the malaria parasite (merozoite) encapsulates itself when it enters a red blood cell, is derived from the host cell plasma membrane, as the appearance of the invasion process in the electron microscope has been taken to suggest, or from lipid material stored in the merozoite. We have incorporated into the red cell membrane a haptenic phospholipid, phosphatidylethanolamine, containing an NBD (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) group, substituted in the acyl chain, and allowed it to translocate into the inner bilayer leaflet. After invasion of these labelled cells by the parasite, Plasmodium falciparum, immuno-gold electron microscopy was used to follow the distribution of the labelled lipid; this was found to be overwhelmingly in favour of the host cell membrane relative to the parasitophorous vacuole. Merozoites of P. knowlesi were allowed to attach irreversibly to red cells without invasion, using the method of pretreatment with cytochalasin. The region of contact between the merozoite and the host cell membrane was in all cases devoid of the labelled phosphatidylethanolamine. These results lead us to infer that the parasitophorous vacuole membrane is derived wholly or partly from lipid preexisting in the merozoite.

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.

Time-resolved phosphorescence anisotropy was used to study the molecular organisation of band 3 in the erythrocyte membrane. Three different rotational relaxation regimes of mobile band 3 were resolved. These populations may represent different aggregation states of band 3 within the membrane, or they may result from association of band 3 with other proteins at the cytoplasmic surface. The polycation spermine decreases the apparent mobility of band 3 by a mechanism that does not involve the underlying cytoskeleton. A monoclonal antibody directed against the cytoplasmic portion of band 3 can also cause an increase in the immobile fraction of band 3 molecules. This monoclonal antibody will inhibit invasion of erythrocytes by malaria parasites. Membranes prepared from erythrocytes infected with mature stages of the malaria parasite, Plasmodium falciparum, show altered dynamic properties corresponding to a marked restriction of band 3 mobility.

Within the circulation, the invasive stage of Plasmodium is the merozoite, a small elliptical cell. Electron microscopy shows that the merozoite can attach reversibly to erythrocytes by its adhesive coat, then form a close, irreversible contact by its apical end, triggering secretion from membranous vesicles (rhoptries and micronemes) on to the erythrocyte membrane. This causes the erythrocyte membrane to invaginate and the merozoite then becomes enclosed within a cavity lined by interiorized membrane. In uninfected erythrocytes, the surface membrane consists of a lipid bilayer in which lie various integral membrane proteins and glycoproteins, associated at their cytoplasmic ends with a network of other proteins constituting the membrane skeleton. There is much evidence that during invasion the membrane proteins and skeleton are removed from the invaginated membrane. There are also ultrastructural data suggesting that the rhoptries are able to generate membrane-like materials, which are inserted into the erythrocyte membrane to cause its inward expansion. Further expansion may be induced by the liberation of parasite secretions from another set of organelles (microspheres) released after the first stage of invasion.

Human red cells, when lysed by dialysis at high haematocrit against a medium of low ionic strength and then dialysed back to physiological saline at 37 degrees C, give rise to resealed ghosts that are invaded with high efficiency by Plasmodium falciparum parasites. When the haematocrit is reduced, a critical concentration is reached, such that the resealed ghosts no longer support invasion. This indicates that a constituent of the cytoplasm becomes diluted to a concentration below a critical level. This constituent is evidently ATP, for when extraneous ATP is added to the diluent and the dialysate, the susceptibility to invasion is fully restored. This does not occur when the non-hydrolysable analogue, adenylyl-imidodiphosphate (AMP-PNP) is substituted for ATP, whereas the hydrolysable ATP analogue, adenosine-5'-O-(3-thiotriphosphate) (ATP-gamma-S), which can be utilised by kinases, can partly replace ATP. Stimulation of invasion by the addition of 2,3-diphosphoglycerate was also associated with a perceptible rise in ATP concentration. The invasion process does not appear to involve intracellular calcium, for EGTA introduced into the resealed ghost has no detectable effect. Moreover, vanadate in the medium does not appreciably inhibit invasion, and it is thus unlikely that the requirement for ATP is linked to the activity of membrane ion-pump enzymes. An inhibitor of phosphorylation, adenosine, introduced into the cells at high concentration, causes significant inhibition of invasion. The results suggest that ATP is required for maintaining the turnover of phosphoryl groups of membrane-associated proteins, such as spectrin. A basic scheme for the mechanism of the invasion process is suggested. In addition to the effect of ATP, it is also shown that with greater dilution, and in the presence of ATP, there is an abrupt loss of susceptibility to invasion. It is inferred that this is due to the dilution of another essential cytoplasmic constituent to below a critical concentration. This second constituent has not yet been identified.

Immuno-gold labelling electron microscopy of thin sections was used to determine the distribution of red cell membrane and membrane skeleton proteins in the vicinity of internalized malaria parasites. When examined immediately after invasion (young ring-stage parasites), the parasitophorous vacuole membranes of both Plasmodium falciparum and P. knowlesi were found to be characterized by the essentially complete absence of spectrin, ankyrin and the most abundant transmembrane protein, band 3. P. knowlesi merozoites were trapped in the attached but not internalized state by pretreatment with cytochalasin B. In this merozoite-red cell complex antibody labelling showed that band 3 had been eliminated from the region of the host cell membrane in contact with the parasite. Internal vesicles, originating apparently from the site of attachment, were often observed in the red cell. Opposite the attached parasite a cavity was also sometimes seen in the host cell, presumably representing an incipient internal vesicle. The membrane was intact, as judged by the absence of protein (haemoglobin) in the cavity, and, like the membranes surrounding the internal vesicles, was devoid of membrane proteins. A large multilamellar body was sometimes seen in the merozoite close to its point of attachment. The lamellar spacing was about 50 nm. The electron microscope images suggest a diffusion of electron-dense material from the lamellar body into the cavity in the host cell.

The invasion of resealed human red cell ghosts by Plasmodium falciparum, and those from monkey cells by P. knowlesi, was strongly inhibited by anti-spectrin antibodies introduced into their cytoplasm. Univalent F(ab)1 fragments gave no such effect, but a combination of these fragments and goat anti-rabbit immunoglobulin, to restore bifunctionality, caused perceptible inhibition of invasion. Disulphide cross-links introduced between spectrin molecules in intact red cells by the membrane-permeant oxidizing agent, diamide, again led to inhibition of invasion. This effect was largely reversed by reduction of the cross-links. Gel electrophoresis was used to confirm that cross-linking was essentially confined to spectrin, and that extended covalent networks were not formed. It follows that local formation of bridges, whether by antibodies or oxidation of thiol groups, functions by inhibiting a local rearrangement of the cytoskeleton that forms a step in the invasion process.

Experimental procedures are described that lead to the formation of sealed red cell ghosts capable of efficient invasion by malaria parasites. Both human and monkey cells have been studied with respect to invasion by Plasmodium falciparum and P. knowlesi respectively. Resealed ghosts containing about half the normal concentration of haemoglobin are prepared by osmotic lysis and resealing at a haematocrit of 70%. When examined in the scanning electron microscope, populations of these ghosts contain few echinocytes, but have an abundance of stomatocytic forms. When undiluted haemolysate is substituted for the aqueous saline diluent, in which the cells are suspended for lysis and resealing, discocytes result, with a morphology very similar to that of the original cells. Invasion is somewhat, but not dramatically higher for this material. An investigation of the effect of intracellular potassium concentration revealed that this had no major effect on invasion. A small increase in invasion resulted from addition of 50 or 100 mg/ml of albumin to the medium in which the cells were resealed, but intra-erythrocytic protein content per se is evidently not a major factor in determining the efficiency of invasion. Augmentation of the cytoplasmic ATP concentration during lysis and resealing increased the level of parasitaemia significantly, and the parasites in these ghosts developed normally and gave rise to viable merozoites. It was found that albumin at certain concentrations passed freely into the lysed cells and reached equilibrium with that in the external medium. By contrast, the high molecular weight solute Blue Dextran 2000 was completely excluded from the lysed cells.

It has previously been shown that antibodies against the transmembrane proteins, band 3 and glycophorin A, inhibit entry of the merozoite into the red cell and, in the case of band 3, it was established that attachment of the parasite to the cell is not prevented. We have found that antibodies against the cytoplasmic domains of band 3 and of glycophorin A, when present in the interior of resealed ghosts of human red cells, also inhibit invasion by P. falciparum. It is inferred that attachment of the merozoite to the red cell causes structural effects that are transduced to the membrane cytoskeleton and the antibodies against transmembrane proteins interfere with the invasion sequence at this level.

Human erythrocytes contain 7 electrophoretically different alpha-naphthyl acetate esterases. Their solubility properties indicate that 5 are present in the erythrocyte cytoplasm and 2 are tightly bound to the stroma; the stroma-bound esterases can be inhibited by 10(-5) M physostigmine. No additional physostigmine-sensitive enzymes were detectable in Plasmodium falciparum-infected cells. Addition of physostigmine to malaria parasites in culture killed the parasites rapidly.

Invasion of human red blood cells by Plasmodium falciparum is inhibited by the protease inhibitors, leupeptin and chymostatin. The efficacy of chymostatin was reduced if the cells were first treated with chymotrypsin. On the other hand, exposure of fresh cells to the supernatant from a synchronous culture at the reinvasion stage showed no such effect. This suggests that a proteolytic step occurs in the course of invasion and may be confined to the region of contact between the invading parasite and the erythrocyte. To test this, leupeptin or chymostatin was introduced into lysed cells, which were then resealed. The intracellular inhibitor strongly reduced invasion. Leupeptin also caused a striking effect on the development of the trophozoite stage of the parasites: a massive vacuole, apparently containing undigested haemoglobin, developed within the parasite. This did not totally stop development and the vacuolated parasites could be recovered in relatively pure form by lysis of the parasitised host cells with saponin.

The effects of changes in red cell membrane properties on invasion by Plasmodium falciparum have been studied by varying the cholesterol content and the intracellular concentration of polyamines. Increased cholesterol content is known to cause large reductions in the internal fluidity of the phospholipid bilayer and a change in its preferred direction of bending, but does not cause changes in gross mechanical rigidity. Polyamines, on the other hand, are thought to increase the cohesion of the membrane cytoskeleton and impede translational diffusion of transmembrane particles, as well as increase the mechanical rigidity of the membrane. Cells with membranes augmented by 50% in cholesterol show no reduction in their susceptibility to parasitic invasion, whereas an increase in cytosolic polyamine (especially spermine) concentration leads to strong inhibition of invasion. In neither case is the development of the intracellular parasite affected. We conclude that it is the macroscopic, rather than the microscopic rheoelastic properties of the membrane that influence the invasion process. Depletion of membrane cholesterol leads to a substantial reduction in parasitaemia; it is suggested that this is linked to the reduced phosphorus incorporation into spectrin in these cells. Polyamines may exert a significant effect at physiological concentrations and the possibility must be considered that the elevated polyamine levels found in red cells in sickle cell disease may account for the protection against P. falciparum.

Mature asexual parasites from cultures of knobby or knobless clones of P. falciparum containing 5 to 10% late forms were harvested by layering up to 10(9) erythrocytes on 3 ml Percoll. The density of the Percoll was adjusted to between 1.081 and 1.091 g cm-3, depending on the maturity of the parasites. Centrifugation at 1500 g for 10 min produced a sharp band at the interface containing mature parasites with a purity averaging 86%. Washed parasites derived from the layer or pellet showed good viability in vitro.

Whereas "white' ghosts, conventionally prepared by hypotonic lysis and washing are resistant to invasion by malaria parasites, cells lysed at high haematocrit and resealed without separating the membranes from the cytosol are invaded with high efficiency (not less than one-third the frequency of intact cells). Similar results were obtained with human and monkey cells, exposed respectively to Plasmodium falciparum and P. knowlesi. Microspectrophotometry of individual cells was used to ascertain the the susceptibility to invasion of lysed, resealed cells was not related to its haemoglobin content.

The shape and mechanical properties of human red cells were modified in several ways and the consequences for the efficiency of invasion by Plasmodium falciparum in culture were investigated. Inhibition of invasion by depletion of ATP was shown to be unrelated to cell shape or deformability changes. Treatment of cells with N-ethylmaleimide (NEM), which dissociates some 70% of the native spectrin tetramers into the dimer, grossly reduced deformation of the cells under shear and increased by a factor of two or more the shear elastic modulus, as measured by the micropipette aspiration technique. Cells thus treated were efficiently invaded by P. falciparum (ca. 75% of control). In a population of cells pretreated with chlorpromazine, parasites were found in stomatocytic cells which were highly undeformable under shear. There was also considerable invasion into cells from subjects with hereditary pyropoikilocytosis, and two types of elliptocytosis. Cells treated with wheat germ agglutinin showed a dose-dependent increase in rigidity; a fivefold increase in elastic modulus (with total loss of deformation under shear in our conditions) still permitted invasion at a level of 50% of the control. The results suggest that gross mechanical properties of the membrane per se, at least within any physiologically relevant range, are unlikely to be the primary determinant of malarial invasion; this may instead be linked to the freedom of membrane proteins to migrate in the course of entry of the parasite.