Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes.

Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Human-adapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as species-specific features of an emerging pathogen.

Conformational flexibility determines selectivity and antibacterial, antiplasmodial, and anticancer potency of cationic α-helical peptides.

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.

Identification of chlorophyll a-b binding protein AB96 as a novel TGFß1 neutralizing agent.

The discovery of compounds and proteins from plants has greatly contributed to modern medicine. Vernonia amygdalina Del. (Compositae) is used by humans and primates for a variety of conditions including parasitic infection. This paper describes the serendipitous discovery that V. amygdalina extract was able to bind to, and functionally inhibit, active TGFß1. The binding agent was isolated and identified as chlorophyll a-b binding protein AB96. Given that active TGFß1 contributes to the pathology of many infectious diseases, inhibiting these processes may explain some of the benefits associated with the ingestion of this species. This is the first plant-derived cytokine-neutralizing protein to be described and paves the way for further such discoveries.

Extensive proteolytic processing of the malaria parasite merozoite surface protein 7 during biosynthesis and parasite release from erythrocytes.

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.

The cytoskeleton and motility in apicomplexan invasion.

We consider the cytoskeletal structure, function, and motility of the invasive zoites of the Apicomplexa. This monophyletic group possess a prominent microtubular cytoskeleton, with a very distinct polarity. It is associated with a non-actin based filamentous system, and with a cisternal double membrane assembly beneath the plasma membrane. The origin of the microtubular cytoskeleton is a set of apical rings. Its role in motility is still unclear, but the present knowledge of apicomplexan tubulins' molecular biology and chemistry is outlined. Actin and accessory proteins are present, and it is apparent that actin polymerisation is tightly controlled in zoites. It does not contribute to the cytoskeleton ordinarily, but is crucial in the acto-myosin linear motor which drives gliding, capping, and invasion, the best understood aspects of zoite motility. Several myosins distinct from the primary linear motor myosin are also found, but not yet well understood functionally. Many of the myosins fall into a class of the superfamily so far seen only in this phylum. The possible relationships of the actin, myosin, cytoskeletal linkage proteins, and external force-transducing adherent proteins are discussed.

Formation of the food vacuole in Plasmodium falciparum: a potential role for the 19 kDa fragment of merozoite surface protein 1 (MSP1(19)).

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.

Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes.

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.

Molecular identification of a malaria merozoite surface sheddase.

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.

Plasmodium falciparum myosins: transcription and translation during asexual parasite development.

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.

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 organelle's boundary membrane.