Correlation of structural development and differential expression of invasion-related molecules in schizonts of Plasmodium falciparum.

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.

Apical membrane antigen 1, a major malaria vaccine candidate, mediates the close attachment of invasive merozoites to host red blood cells.

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.

Myosins of Babesia bovis: molecular characterisation, erythrocyte invasion, and phylogeny.

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.

Interaction of membrane skeletal proteins with membrane lipid domain.

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.

Acidic calcium pools in intraerythrocytic malaria parasites.

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.

Actomyosin motor in the merozoite of the malaria parasite, Plasmodium falciparum: implications for red cell invasion.

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 homeostasis in intraerythrocytic malaria parasites.

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.

Inhibition of invasion and intraerythrocytic development of Plasmodium falciparum by kinase inhibitors.

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.

Contractile protein system in the asexual stages of the malaria parasite Plasmodium falciparum.

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.

Origins of the parasitophorous vacuole membrane of the malaria parasite: surface area of the parasitized red cell.

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 Plasmodium falciparum protein RESA interacts with the erythrocyte cytoskeleton and modifies erythrocyte thermal stability.

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.

Aggregation of band 3 in hereditary ovalocytic red blood cell membranes. Electron microscopy and protein rotational diffusion studies.

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.

Actin in the merozoite of the malaria parasite, Plasmodium falciparum.

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.

Invasion of hereditary ovalocytes by Plasmodium falciparum in vitro and its relation to intracellular ATP concentration.

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.

Effect of intra-erythrocytic magnesium ions on invasion by Plasmodium falciparum.

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.

Origins of the parasitophorous vacuole membrane of the malaria parasite, Plasmodium falciparum, in human red blood cells.

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.

Basis of unique red cell membrane properties in hereditary ovalocytosis.

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.

Rotational dynamics of the integral membrane protein, band 3, as a probe of the membrane events associated with Plasmodium falciparum infections of human erythrocytes.

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.

The ultrastructure of red cell invasion in malaria infections: a review.

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.