The malaria merozoite, forty years on.

The invasive blood stage of malaria parasites, merozoites, are complex entities specialized for the capture and entry of red blood cells. Their potential for vaccination and other anti-malaria strategies have attracted much research attention over the last 40 years, and there is now a considerable body of data relating to their biology. In this article some of the major advances over this period and remaining challenges are reviewed.

Vesicle trafficking during sporozoite development in Plasmodium berghei: ultrastructural evidence for a novel trafficking mechanism.

Oocysts from Anopheles stephensi mosquitoes fed on murine blood infected with Plasmodium berghei berghei, were fixed for electron microscopy 6-12 days post-feeding. Ultrastructural analysis focused on Golgi-related trafficking pathways for rhoptry and microneme formation during sporogony. A small Golgi complex of 1-3 cisternae is formed close to the spindle pole body from coated vesicles budded from the nuclear envelope which is confluent with the endoplasmic reticulum. Rhoptries begin as small spheroidal bodies apparently formed by fusion of Golgi-derived vesicles, lengthening to 3-4 microm, and increasing in number to 4 per sporozoite. Ultrastructural data indicate the presence of a novel mechanism for vesicle transport between the Golgi complex and rhoptries along a longitudinal 30 nm - thick fibre (rootlet fibre or tigelle). Filamentous links between vesicles and rootlet indicate that this is a previously undescribed vesicle transport organelle. Genesis of micronemes occurs late in bud maturation and starts as spheroidal dense-cored vesicles (pro-micronemes), transforming to their mature bottle-like shape as they move apically. Filamentous links also occur between micronemes and subpellicular microtubules, indicating that as in merozoites, micronemes are trafficked actively along these structures.

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.

Microtubule associated motor proteins of Plasmodium falciparum merozoites.

We have studied the occurrence, stage specificity and cellular location of key molecules associated with microtubules in Plasmodium falciparum merozoites. Antibodies to gamma tubulin, conventional kinesin and cytoplasmic dynein were used to determine the polarity of merozoite microtubules (mt), the stage specificity of the motor proteins and their location during merozoite development. We conclude that the minus ends of the mts are located at their apical pole. Kinesin was present throughout the lifecycle, appearing as a distinct crescent at the apex of developing merozoites. The vast majority of cytoplasmic dynein reactivity occurred in late merogony, also appearing at the merozoite apex. Destruction of mt with dinitroanilines did not affect the cellular location of kinesin or dynein. In invasion assays, dynein inhibitors reduced the number of ring stage parasites. Our results show that both conventional kinesin and cytoplasmic dynein are abundant, located at the negative pole of the merozoite mt and, intriguingly, appear there only in very late merogony, prior to merozoite release and invasion.

Apical organelles of Apicomplexa: biology and isolation by subcellular fractionation.

The apical organelles are characteristic secretory vesicles of Plasmodium, Toxoplasma, Cryptosporidium and other apicomplexan organisms. They consist of rhoptries, micronemes and dense granules. Recent research has provided much new data concerning their structure, contents, functions and development. All of these organelles contain complex mixtures of proteins, with broad homologies as well as differences in molecular structure between species and genera. Many of the proteins interact with host cell membranes, and are thought to mediate selective adhesion to host cells as well as membrane modification during intracellular invasion. Micronemal proteins are important in the initial selection of host cells, and in enabling gliding motility of the parasites, while rhoptries appear to be more important in parasitophorous vacuole formation. Dense granules are involved predominantly in modifying the host cell after invasion. Research into apical organellar composition and function depends on accurate assignment of molecular identity. This requires the simultaneous application of several complementary approaches including immunolocalisation by light- and electron-microscopy, subcellular fractionation, and transgene expression. The merits and limitations of these different types of approach are discussed, and the importance of cell fractionation methods in characterising apical organelle proteins is stressed.

Ultrastructure of rhoptry development in Plasmodium falciparum erythrocytic schizonts.

Prior to the separation of merozoites from the Plasmodium falciparum schizont, various stage-specific organelles are synthesized and assembled within each merozoite bud. The apical ends of the merozoites are initiated close to the ends of endomitotic spindles. At each of these sites, the nuclear membrane forms coated vesicles, and a single discoidal or cup-like Golgi cisterna appears. Reconstruction from serial sections indicates that this structure receives vesicles from the nuclear envelope and in turn gives off coated vesicles to generate the apical secretory organelles. Rhoptries first form as spheroidal structures and grow by progressive fusion of small vesicles around their margins. As each rhoptry develops, 2 distinctive regions separate within it, an apical reticular zone with electron-lucent areas separated by cords of granular material, and a more homogenously granular basal region. The apical part elongates into the duct, with evidence for further vesicular fusion at the duct apex. The rounded rhoptry base becomes progressively more densely packed to form a spheroidal mass, and compaction also occurs in the duct. Typically, one rhoptry matures before the other. Cryofractured rhoptry membranes show asymmetry in the sizes and numbers of intramembranous particles at the internally- and externally-directed fracture faces.

A brief illustrated guide to the ultrastructure of Plasmodium falciparum asexual blood stages.

Interpretation of the new information arising from the Plasmodium falciparum Genome Project requires a good working knowledge of the ultrastructure of the parasite; however many aspects of the morphology of this species remain obscure. Lawrence Bannister, John Hopkins and colleagues here give an illustrated overview of the three-dimensional (3-D) organization of the merozoite, ring, trophozoite and schizont stages of the parasite, based on available data that include 3-D reconstruc-tion from serial electron microscope sections. The review describes the chief organelles present in these stages, emphasizing the continuity of structure in addition to specialized, stage-specific features developed during the asexual erythrocytic cycle.

Microtubules in Plasmodium falciparum merozoites and their importance for invasion of erythrocytes.

Plasmodium falciparum merozoites have an array of 2-3 subpellicular microtubules, designated f-MAST. We have previously shown that colchicine inhibits merozoite invasion of erythrocytes, indicating a microtubular involvement in this process. Colchicine inhibition of invasion was reduced by the Taxol-stabilization of merozoite microtubules prior to colchicine exposure. Immunofluorescence assays showed that the number and length of f-MASTs were reduced in colchicine-treated merozoites, confirming that microtubules were the target of colchicine inhibition. The dinitroaniline drugs, trifluralin and pendimethalin, were shown by immunofluorescence to depolymerize the f-MAST and both drugs were inhibitory in invasion assays. These results demonstrate that the integrity of the f-MAST is important for successful invasion. Fluorescence imaging demonstrated the alignment of mitochondria to f-MAST, suggesting that mitochondrial transport might be perturbed in merozoites with disorganized f-MAST. Depolymerizing mt in late-stage schizonts did not affect the allocation of mitochondria to merozoites.

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.

Precise timing of expression of a Plasmodium falciparum-derived transgene in Plasmodium berghei is a critical determinant of subsequent subcellular localization.

The development of transfection technology for malaria parasites holds significant promise for a more detailed characterization of molecules targeted by vaccines or drugs. One asexual blood stage vaccine candidate, apical membrane antigen-1 (AMA-1) of merozoite rhoptries has been shown to be the target of inhibitory, protective antibodies in both in vitro and in vivo studies. We have investigated heterologous (trans-species) expression of the human malaria Plasmodium falciparum AMA-1 (PF83/AMA-1) in the rodent parasite Plasmodium berghei. Transfected P. berghei expressed correctly folded and processed PF83/AMA-1 under control of both pb66/ama-1 and dhfr-ts promoters. Timing of expression was highly promoter-dependent and was critical for subsequent subcellular localization. Under control of pb66/ama-1, PF83/AMA-1 expression and localization in P. berghei was limited to the rhoptries of mature schizonts, similar to that observed for PF83/AMA-1 in P. falciparum. In contrast the dhfr-ts promoter permitted PF83/AMA-1 expression throughout schizogony as well as in gametocytes and gametes. Localization was aberrant and included direct expression at the merozoite and gamete surface. Processing from the full-length 83-kDa protein to a 66-kDa protein was observed not only in schizonts but also in gametocytes, indicating that processing could be mediated outside of rhoptries by a common protease. Trans-species expressed PF83/AMA-1 was highly immunogenic in mice, resulting in a response against a functionally critical domain of the molecule.

A role for microtubules in Plasmodium falciparum merozoite invasion.

Colchicine, a drug which poisons the polymerization of microtubules, was assayed for effects on the invasion of Plasmodium falciparum merozoites into red cells in order to investigate if merozoite microtubules have a function in invasion. Culture conditions and concentrations of colchicine were established where the maturation and rupture of schizonts was unaffected by the drug. This was judged first by light microscopy, including morphology and counts of nuclear particle numbers, then by ultrastructural studies which excluded deranged organellogenesis as a cause of merozoite failure, and finally by diachronic cultures in which both recruitment and loss of schizonts could be counted. Specific invasion inhibition was seen when 10 microM-1 mM colchicine was present. Red cells pre-incubated in colchicine and then washed showed no reduction in their extent of invasion, and neither red cell lysis, sphering nor blebbing were apparent. We conclude that intact microtubules are necessary for successful merozoite function.

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.

The role of the cytoskeleton in Plasmodium falciparum merozoite biology: an electron-microscopic view.

The biochemical, ultrastructural and experimental data concerning the organization and biological roles of the merozoite cytoskeleton are briefly reviewed. Actin is known to be expressed in the asexual erythrocytic stages, and has also been demonstrated in Plasmodium falciparum merozoites biochemically and visualized by fluorescence microscopy after appropriate labelling. Experimental evidence indicates that actin-myosin-based motility is important in merozoite locomotion during red-cell invasion. Microtubules also occur in P. falciparum merozoites in the form of a small longitudinal band of subpellicular microtubules, and experiments with anti-microtubule drugs indicate that microtubules are involved in some aspect of invasion. In the late-stage schizont, microtubules are also important in merozoite morphogenesis. The numbers and positions of the merozoite apices within the schizont are spatially related to the spindle poles of the final mitotic division, and extranuclear microtubules are probably responsible for the trafficking of vesicles from a single Golgi cisterna to form the apical organelles. In addition to these cytoskeletal structures, numerous short cytoskeletal filaments of unknown composition attach the merozoite plasma membrane to the underlying pellicular cisterna, and this process may drive the budding of merozoites from the parent schizont.

Effects of unilateral deafening on the cochlear nucleus of the guinea pig at different ages.

Developmental changes in spherical cell sizes were measured in the ventral cochlear nucleus (VCN) in normal guinea pigs aged 2 days and 2, 7 and 12 weeks to establish the time course of postnatal neuronal growth, as a baseline for our experimental work. A continued growth of spherical cell size was observed in the VCN up to 7 weeks postnatally. Animals were unilaterally deafened by cochlear perfusion with kanamycin sulphate at ages 1 and 6 weeks. After 6 weeks survival the spherical cells were measured in the VCNs of both sides. Unilateral deafferentation at both ages caused an ipsilateral reduction in spherical cell size, neurons of the younger group being smaller than in the older. On the contralateral side these cells in the older group were larger than age-matched controls while in the younger group there was no difference compared with age-matched controls. These findings suggest that the results of deafferentation are age-dependent, and may indicate an ability of the neural circuitry to adapt to the loss of sensory input on the other side.

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.

Protein gene product 9.5 in the developing cochlea of the rat: cellular distribution and relation to the cochlear cytoskeleton.

Protein gene product 9.5 was immunolocalized in the adult and early postnatal (P2-P15) rat cochlea, and its distribution compared with a 200 kDa highly phosphorylated neurofilament subunit (neurofilament 200) and alpha-tubulin. In the adult, Protein gene product 9.5 was expressed exclusively in cochlear nerve fibres and ganglion cells, a small percentage of these (Type II ganglion cells and olivocochlear bundle fibres) being intensely positive for both protein gene product and neurofilament 200. In postnatal development, pillar and Deiters' cells were at first (P2-P15) strongly positive for protein gene product 9.5, and hair cells moderately so. At P2, all nerve fibres and ganglion cells showed co-expression of protein gene product 9.5 and neurofilament 200, but at later stages, the subset of intensely co-labelled neurons appeared, nerve fibres at P7 onwards and ganglion cells from P12. There was no overt correlation between the onset of protein gene product 9.5 and alpha-tubulin expression in any cochlear component. Protein gene product 9.5 expression in ganglion cells was at first (P2 and P7) mainly nuclear, and later also cytoplasmic. It is concluded that there is a clear correlation of high levels of protein gene product 9.5 and neurofilament protein expression, and that protein gene product 9.5 is expressed in some non-neuronal cells of the cochlea during its early development, persisting until after hearing has commenced.