Antibodies inhibit the protease-mediated processing of a malaria merozoite surface protein.

When merozoites of the malaria parasite Plasmodium falciparum are released from infected erythrocytes and invade new red cells, a component of a protein complex derived from the merozoite surface protein 1 (MSP-1) precursor undergoes a single proteolytic cleavage known as secondary processing. This releases the complex from the parasite surface, except for a small membrane-bound fragment consisting of two epidermal growth factor (EGF)-like domains, which is the only part of MSP-1 to be carried into invaded erythrocytes. We report that, a group of monoclonal antibodies specific for epitopes within the EGF-like domains, some interfere with secondary processing whereas others do not. Those that most effectively inhibit processing have previously been shown to prevent invasion. Other antibodies, some of which can block this inhibition, not only do not prevent invasion but are carried into the host cell bound to the merozoite surface. These observations unequivocally demonstrate that the binding of antibody to the COOH-terminal region of MSP-1 on the merozoite surface may not be sufficient to prevent erythrocyte invasion, and show that the interaction of different antibodies with adjacent epitopes within the EGF-like domains of MSP-1 can have distinct biochemical effects on the molecule. Inhibition of MSP-1 processing on merozoites may be a mechanism by which protective antibodies interrupt the asexual cycle of the malaria parasite.

A single fragment of a malaria merozoite surface protein remains on the parasite during red cell invasion and is the target of invasion-inhibiting antibodies.

A complex of polypeptides derived from a precursor is present on the surface of the malaria merozoite. During erythrocyte invasion only a small fragment from this complex is retained on the parasite surface and carried into the newly infected red cell. Antibodies to this fragment will interrupt invasion.

Antibodies that inhibit malaria merozoite surface protein-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies.

Merozoite surface protein-1 (MSP-1) of the human malaria parasite Plasmodium falciparum undergoes at least two endoproteolytic cleavage events during merozoite maturation and release, and erythrocyte invasion. We have previously demonstrated that mAbs which inhibit erythrocyte invasion and are specific for epitopes within a membrane-proximal, COOH-terminal domain of MSP-1 (MSP-119) prevent the critical secondary processing step which occurs on the surface of the extracellular merozoite at around the time of erythrocyte invasion. Certain other anti-MSP-119 mAbs, which themselves inhibit neither erythrocyte invasion nor MSP-1 secondary processing, block the processing-inhibitory activity of the first group of antibodies and are termed blocking antibodies. We have now directly quantitated antibody-mediated inhibition of MSP-1 secondary processing and invasion, and the effects on this of blocking antibodies. We show that blocking antibodies function by competing with the binding of processing-inhibitory antibodies to their epitopes on the merozoite. Polyclonal rabbit antibodies specific for certain MSP-1 sequences outside of MSP-119 also act as blocking antibodies. Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH2-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-119 mAb 12.8. The presence of these blocking antibodies also completely abrogates the inhibitory effect of mAb 12.8 on erythrocyte invasion by the parasite in vitro. Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-119 target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti-MSP-119 antibodies. Our results suggest that an effective MSP-119-based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.

Signalling in malaria parasites. The MALSIG consortium.

Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the vertebrate liver and blood circulation, or the insect midgut and hemocoel. Cellular and molecular mechanisms enabling the parasite to sense and respond to the intra- and the extra-cellular environments are therefore key elements for the proliferation and transmission of Plasmodium, and therefore are, from a public health perspective, strategic targets in the fight against this deadly disease. The MALSIG consortium, which was initiated in February 2009, was designed with the primary objective to integrate research ongoing in Europe and India on i) the properties of Plasmodium signalling molecules, and ii) developmental processes occurring at various points of the parasite life cycle. On one hand, functional studies of individual genes and their products in Plasmodium falciparum (and in the technically more manageable rodent model Plasmodium berghei) are providing information on parasite protein kinases and phosphatases, and of the molecules governing cyclic nucleotide metabolism and calcium signalling. On the other hand, cellular and molecular studies are elucidating key steps of parasite development such as merozoite invasion and egress in blood and liver parasite stages, control of DNA replication in asexual and sexual development, membrane dynamics and trafficking, production of gametocytes in the vertebrate host and further parasite development in the mosquito. This article, which synthetically reviews such signalling molecules and cellular processes, aims to provide a glimpse of the global frame in which the activities of the MALSIG consortium will develop over the next three years.

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.

Secondary processing of the Plasmodium falciparum merozoite surface protein-1 (MSP1) by a calcium-dependent membrane-bound serine protease: shedding of MSP133 as a noncovalently associated complex with other fragments of the MSP1.

Merozoites of the malaria parasite Plasmodium falciparum possess on their surface proteolytically processed fragments of the merozoite surface protein-1 (MSP1). Secondary processing of one of these fragments, MSP1(42), always occurs prior to, or at the point of successful erythrocyte reinvasion. It is shown that a product of this secondary processing, MSP1(33), is shed in the form of a noncovalently-associated complex with a number of other proteins, including the MSP1-derived species MSP1(38) and MSP1(83). Secondary processing of MSP1(42) is inhibited by the chelating agents ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(beta-aminoethyl ether)-tetraacetic acid (EGTA), and this inhibition is reversible by addition of excess calcium. Secondary processing occurs in preparations of washed, disrupted merozoites, and is inhibited by the protease inhibitors phenylmethylsulphonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), indicating that the protease responsible is a membrane-associated serine protease.

Processing of the Plasmodium falciparum major merozoite surface protein-1: identification of a 33-kilodalton secondary processing product which is shed prior to erythrocyte invasion.

We have previously shown that only a single 19-kDa fragment of the Plasmodium falciparum major merozoite surface protein (MSP1) is carried with an invading merozoite into the infected red cell. This fragment (MSP1(19] is derived from the C-terminal membrane-bound end of a major product, MSP1(42), of the primary stage of MSP1 proteolytic processing. Using a monoclonal antibody mapped to an epitope within the N-terminal region of MSP1(42), we have shown that a soluble 33-kDa polypeptide (MSP1(33) corresponding to the N-terminal region of MSP1(42) is shed into culture supernatants during merozoite release and erythrocyte invasion. These observations provide further evidence that the secondary processing of MSP1(42) involves a highly site-specific proteolytic activity.

A conserved parasite serine protease processes the Plasmodium falciparum merozoite surface protein-1.

The merozoite surface protein-1 of the human malaria parasite Plasmodium falciparum undergoes an extracellular proteolytic cleavage (secondary processing) intrinsic to successful erythrocyte invasion. In the T9/96 clone of P. falciparum the protease responsible has been characterised as a membrane-associated, calcium-dependent activity, sensitive to irreversible inhibitors of serine proteases. Here we extend these studies and show that secondary processing activity in intact merozoites of P. falciparum strains expressing the alternative dimorphic type of merozoite surface protein-1 has identical characteristics, and that the cleavage site is close to or identical to that in the protein from T9/96. The protease responsible is shown to be parasite-derived, and able to catalyse processing of native substrate only when present in the same membrane. Cleavage of the substrate follows apparent first order kinetics for at least 2 half-lives. It is concluded that secondary processing of both dimorphic forms of the P. falciparum merozoite surface protein-1 is a conserved event, mediated by a mechanistically conserved protease located on the merozoite surface. These observations provide clues to the identity of the protease and show that, irrespective of the dimorphic type, secondary processing results in the same, highly conserved region of the merozoite surface protein-1 remaining on the surface of the invading merozoite.

Proteolytic processing of the Plasmodium falciparum merozoite surface protein-1 produces a membrane-bound fragment containing two epidermal growth factor-like domains.

The amino-terminal sequence has been obtained for 2 fragments of the Plasmodium falciparum T9/94 merozoite surface protein precursor (PfMSP1) and these have been compared with the sequence predicted from the gene. These data define the position of these fragments in the precursor and indicate that the C-terminal sequence which is carried into the red cell during invasion consists of 2 epidermal growth factor (EGF)-like domains. A homologous cleavage sequence and domain structure can be identified in the MSP1 molecules of other malarial species. In addition the results suggest that the smaller fragment is not N-glycosylated.

A 22 kDa protein associated with the Plasmodium falciparum merozoite surface protein-1 complex.

The Plasmodium falciparum merozoite surface protein-1 (MSP-1) is synthesized as a precursor of approximately 195 kDa and is processed to form a complex of polypeptides on the surface of free merozoites. As a result of a second processing event, the entire MSP-1 complex is shed from the surface, apart from a C-terminal fragment that remains anchored to the merozoite membrane. We have identified a 22 kDa protein (p22) on the surface of merozoites by cell surface radioiodination and indirect immunofluorescence assay on unfixed free merozoites. p22 is also a component of the shed MSP-1 complex where it is present in part as a 19 kDa form (p22(19)) as shown by immunochemical and peptide mapping analyses. The soluble complex contains MSP-1-derived polypeptides and p22 in approximately stoichiometrically equal amounts. N-terminal amino acid sequence analyses of p22/p22(19) showed that the protein is not derived from the MSP-1 precursor.

Structural analysis of the glycosyl-phosphatidylinositol membrane anchor of the merozoite surface proteins-1 and -2 of Plasmodium falciparum.

Plasmodium falciparum accumulates the two merozoite surface proteins-1 and -2 during schizogony. Both proteins are proposed to be anchored in membranes by glycosyl-phosphatidylinositol membrane anchors. In this report the identity of these GPI-anchors is confirmed by labelling with tritiated precursors and additionally by specific enzymatic and chemical treatments. Detailed structural analysis of the core-glycans showed that the GPI-anchors of both proteins possess an extra alpha 1-2 linked mannose at the conserved trimannosyl-core-glycan. MSP-1 and MSP-2 labelled with tritiated myristic acid possess primarily radioactive myristic acid at inositol rings in both GPI-anchors. Additionally the hydrophobic fragments released from [3H]myristic acid labelled GPI-anchors were identified as diacyl-glycerols, carrying preferentially [3H]palmitic acid in an ester-linkage.

PfSUB-2: a second subtilisin-like protein in Plasmodium falciparum merozoites.

Erythrocyte invasion by the malaria merozoite requires the activity of merozoite proteases. We have previously identified a Plasmodium falciparum protein belonging to the superfamily of subtilisin-like serine proteases, which is expressed in a subset of secretory organelles in free merozoites. Here we describe the identification of a second P. falciparum subtilisin-like merozoite protein. Called PfSUB-2, it is encoded by a single copy gene and is expressed as a large putative type I integral membrane protein which undergoes extensive post-translational processing. The terminal processing product is expressed in an apical location in merozoites. PfSUB-2 may mediate one or more of the serine protease activities known to be associated with erythrocyte invasion.

Proteases involved in erythrocyte invasion by the malaria parasite: function and potential as chemotherapeutic targets.

Malaria places an increasing burden on global public health resources. In the face of growing resistance of the malaria parasite to available antimalarial drugs, there is a need for new drugs and the identification of new chemotherapeutic targets. The malaria parasite has a complex life cycle which includes a number of obligate intracellular stages. Clinical malaria results from cyclic asexual replication of the blood-stage parasite in circulating erythrocytes of the human host. Erythrocyte entry and host cell rupture require the activity of parasite proteases, and these enzymes are, therefore, attractive targets for rational approaches to new drug development. Malarial proteases play a role in at least two distinct aspects of host cell invasion; modification of parasite proteins involved in host cell recognition and entry; and restructuring of the host cell itself, during and following invasion, and in order to allow parasite release from the host cell. This review details recent advances in the identification of these proteases, describes current understanding of their activation and functional role, and discusses their potential as targets for protease inhibitor-based drugs.

Use of a recombinant baculovirus product to measure naturally-acquired human antibodies to disulphide-constrained epitopes on the P. falciparum merozoite surface protein-1 (MSP1).

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure antibody levels in human sera to a candidate vaccine antigen, merozoite surface protein-1 (MSP1), of the malaria parasite Plasmodium falciparum. To ensure the detection of antibodies reactive with important conformational epitopes, antigens used in the ELISA were obtained from either in vitro parasite cultures, or from a baculovirus expression system in which correct folding of recombinant MSP1-derived polypeptides has been previously demonstrated. The specificity of the ELISA was confirmed using a novel antibody affinity select method. The assay was used to investigate the pattern of acquisition of anti-MSP1 antibodies in a cross-sectional survey of 387 3-8 year old residents of a malaria endemic area of The Gambia. A significant positive correlation between anti-MSP1 antibody levels and age was evident, though individual responses to two antigens corresponding to two distinct domains of the MSP1 varied widely.

Merozoite surface protein 1, immune evasion, and vaccines against asexual blood stage malaria.

There is an urgent need for a vaccine against malaria and proteins on the surface of the merozoite are good targets for development as vaccine candidates because they are exposed to antibody. However, it is possible that the parasite has evolved mechanisms to evade a protective immune response to these proteins. Merozoite surface protein 1 (MSP-1) is a candidate for vaccine development and its C-terminal sequence is the target of protective antibody. MSP-1 is cleaved by proteases in two processing steps, the second step releases the bulk of the protein from the surface and goes to completion during successful red blood cell invasion. Antibodies binding to the C-terminus of Plasmodium falciparum MSP-1 can inhibit both the processing and erythrocyte invasion. Other antibodies that bind to either the C-terminal sequence or elsewhere in the molecule are 'blocking' antibodies, which on binding prevent the binding of the inhibitory antibodies. Blocking antibodies are a mechanism of immune evasion, which may be based on antigenic conservation rather than diversity. This mechanism has a number of implications for the study of protective immunity and the development of malaria vaccines, emphasising the need for appropriate functional assays and careful design of the antigen.

Glycosyl-phosphatidylinositols in protozoa structure, biosynthesis and intracellular localisation.

We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and biosynthesis data should lead us to common and individual features of the GPI-biosynthesis and transport in different organisms.

What is the function of MSP-I on the malaria merozoite?

In the absence o f any clear enzymatic activity, attempts to define the role of merozoite surface protein-I have focused mainly on analysis of its structure, on its interaction with the immune system and on binding assays. But how does our knowledge of the structure o f this protein contribute to functional studies? Are there data to suggest a role in the evasion of effective host immune responses? Binding studies have used the intact protein or various fragments and peptides, but do such approaches provide a reliable indicator of function? In this article, Tony Holder and Mike Blackman review these areas.