Anton R. Dluzewski

Apical Membrane Antigen 1, a Major Malaria Vaccine Candidate, Mediates the Close Attachment of Invasive Merozoites to Host Red Blood Cells

ABSTRACT 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.

Plasmodium falciparum apical membrane antigen 1 (PfAMA-1) is translocated within micronemes along subpellicular microtubules during merozoite development

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 organelles boundary membrane.

The Malaria Parasite Cyclic GMP-Dependent Protein Kinase Plays a Central Role in Blood-Stage Schizogony

ABSTRACT 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 ≥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.

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.

The Plasmodium falciparum clag9 gene encodes a rhoptry protein that is transferred to the host erythrocyte upon invasion.

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.

Motile Systems in Malaria Merozoites: How is the Red Blood Cell Invaded?

The ability of the malaria parasite to invade erythrocytes is central to the disease process, but is not thoroughly understood. In particular, little attention has been paid to the motor systems driving invasion. Here, Jennifer Pinder, Ruth Fowler and colleagues review motility in the merozoite. The components of an actomyosin motor are present, including a novel unconventional class XIV myosin, now called Pfmyo-A, which, because of its time of synthesis and location, is likely to generate the force required for invasion. In addition, there is a subpellicular microtubule assemblage in falciparum merozoites, the f-MAST, the integrity of which is necessary for invasion.

Formation of the Food Vacuole in Plasmodium falciparum: A Potential Role for the 19 kDa Fragment of Merozoite Surface Protein 1 (MSP119)

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 (MSP119), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP119 during the parasites subsequent intracellular development using immunochemical analysis of metabolically labeled MSP119, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP119 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 MSP119 and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP119 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.

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.

Suramin and suramin analogues inhibit merozoite surface protein-1 secondary processing and erythrocyte invasion by the malaria parasite Plasmodium falciparum

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 μm) and to Plasmodium vivax MSP142 (Kd = 0.3 μm) 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 μm) secondary processing product (fluorescence measurements), but very weakly to MSP119 (Kd ∼ 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.

Three-dimensional ultrastructure of the ring stage of Plasmodium falciparum: Evidence for export pathways

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.