Pfsbp1, a Maurer's cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton.

Antibodies from hyperimmune monkey sera, selected by absorption to Plasmodium falciparum-infected erythrocytes, and elution at acidic pH, allowed us to characterize a novel parasite protein, Pfsbp1 (P. falciparum skeleton binding protein 1). Pfsbp1 is an integral membrane protein of parasite-induced membranous structures associated with the erythrocyte plasma membrane and referred to as Maurer's clefts. The carboxy-terminal domain of Pfsbp1, exposed within the cytoplasm of the host cell, interacts with a 35 kDa erythrocyte skeletal protein and might participate in the binding of the Maurer's clefts to the erythrocyte submembrane skeleton. Antibodies to the carboxy- and amino-terminal domains of Pfsbp1 labelled similar vesicular structures in the cytoplasm of Plasmodium chabaudi and Plasmodium berghei-infected murine erythrocytes, suggesting that the protein is conserved among malaria species, consistent with an important role of Maurer's cleft-like structures in the intraerythrocytic development of malaria parasites.

Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extracted from normal or Plasmodium falciparum-infected erythrocyte ghosts.

Parasite-encoded membrane proteins translocated to the surface of infected erythrocytes or in specialized vesicles underneath (Maurer's clefts) play a key role in the asexual life cycle of Plasmodium falciparum (a malaria-causing protozoan), by mediating key steps such as red blood cell invasion, sequestration of infected cells in microcapillaries, and red blood cell rupture. A large-scale analysis of these membrane proteins would therefore be of great help to gain knowledge of the different stages of the Plasmodium falciparum life cycle. In order to be able to detect and identify parasite-encoded proteins directed to the red blood cell membrane, we first defined the conditions required for optimal extraction and separation of normal red blood cell ghost proteins by two-dimensional gel electrophoresis. These conditions included the use of urea, thiourea and new zwitterionic detergents in the extraction and isoelectric focusing media. The optimized conditions were then applied to analyze normal and P. falciparum-infected red blood cell ghosts. Several protein spots were found only in infected ghosts and are expected to represent parasite-encoded proteins. These proteins are currently under investigation.

Plasmodium falciparum subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1-42 maturase.

The process of human erythrocyte invasion by Plasmodium falciparum parasites involves a calcium-dependent serine protease with properties consistent with a subtilisin-like activity. This enzyme achieves the last crucial maturation step of merozoite surface protein 1 (MSP1) necessary for parasite entry into the host erythrocyte. In eukaryotic cells, such processing steps are performed by subtilisin-like maturases, known as proprotein convertases. In an attempt to characterize the MSP1 maturase, we have identified a gene that encodes a P. falciparum subtilisin-like protease (PfSUB2) whose deduced active site sequence resembles more bacterial subtilisins. Therefore, we propose that PfSUB2 belongs to a subclass of eukaryotic subtilisins different from proprotein convertases. Pfsub2 is expressed during merozoite differentiation and encodes an integral membrane protein localized in the merozoite dense granules, a secretory organelle whose contents are believed to participate in a late step of the erythrocyte invasion. PfSUB2's subcellular localization, together with its predicted enzymatic properties, leads us to propose that PfSUB2 could be responsible for the late MSP1 maturation step and thus is an attractive target for the development of new antimalarial drugs.

Non-detergent sulphobetaines enhance the recovery of membrane and/or cytoskeleton-associated proteins and active proteases from erythrocytes infected by Plasmodium falciparum.

A better understanding of the causative agent's biology and the definition of new targets for the development of drugs and/or specific immune responses is necessary to face the spred of drug-resistant malaria in developing countries and the absence of an efficient vaccine against this most important infectious disease. Non-detergent sulphobetaines enhance the recovery and isoelectric focussing of active Plasmodium falciparum proteases, cytoskeleton-associated proteins and Maurer's cleft-associated proteins. This is a significant advantage for the purification of such proteins and might help pinpoint their role for red blood cell rupture and merozoite release.

Host urokinase-type plasminogen activator participates in the release of malaria merozoites from infected erythrocytes.

Malaria infection of red blood cells is associated with plasminogen activation. Surface immunofluorescence and immunoprecipitation experiments, using specific polyclonal and monoclonal antibodies raised against human urokinase, demonstrate that this activity is due to the binding of host urokinase-type plasminogen activator to the surface of erythrocytes infected by mature forms of Plasmodium falciparum malaria parasites. Depletion of urokinase from the culture medium leads to the inhibition of merozoite release and the accumulation of segmenter-infected erythrocytes; this inhibition is reversed by the addition of human single-chain or two-chain urokinase. These findings are consistent with host urokinase being involved in the process of merozoite release from the red blood cell.

A role for erythrocyte band 3 degradation by the parasite gp76 serine protease in the formation of the parasitophorous vacuole during invasion of erythrocytes by Plasmodium falciparum.

A purified Plasmodium falciparum serine protease (gp76) implicated in erythrocyte invasion, degrades human erythrocyte band 3 and glycophorin A. Inhibition studies using synthetic peptides derived from the presumed band 3 enzymatic cleavage sites and the observed uptake of fluorescent phospholipids following gp76 treatment, suggest that band 3 degradation by this serine protease participates in the formation of the parasitophorous vacuole by restructuring the red cell cytoskeleton. These results provide a rationale for the elaboration of specific inhibitors to block red cell invasion by malaria parasites.

Identification of a family of Rab G-proteins in Plasmodium falciparum and a detailed characterisation of pfrab6.

As a first step towards developing a set of compartment-specific probes for studying protein trafficking in the malaria-infected erythrocyte, we describe here a family of Plasmodium falciparum Rab proteins. We characterise in detail P. falciparum Rab6 (PfRab6) a marker which in other cells is specific for the Golgi/trans Golgi network. Although PfRab6 mRNA is expressed throughout the intraerythrocytic cycle, maximal expression occurs at the trophozoite stage. Immunofluorescence microscopy shows that the distribution of PfRab6 changes during the final stages of parasite maturation, coalescing into multiple foci, each of which is associated with the nucleus of a forming daughter parasite.

Non-detergent sulphobetaines: a new class of mild solubilization agents for protein purification.

The action of non-detergent sulphobetaines (NDSBs) as new mild agents for protein purification is described. The solubilization effects of non-detergent sulphobetaines are shown in different examples; all obtained under non-denaturing conditions: (1) microsomal proteins extraction; (2) recovery after dialysis of nuclear proteins; (3) reduction of precipitation in isoelectric focusing experiments under non-denaturing conditions; and (4) purification of a membrane-bound serine protease from Plasmodium falciparum involved in erythrocyte invasion by malaria merozoites. The absence of a significant denaturation effect induced by NDSBs is demonstrated by tests on beta-galactosidase and alkaline phosphatase. A simple NDSB synthesis and some possible explanations of the action of NDSBs are also presented.

Malaria parasites: enzymes involved in red blood cell invasion.

Three enzymes have been described in malaria merozoites: a serine-protease and two phospholipases. The parasite serine-protease is necessary for parasite entry into the red blood cell. This enzyme is synthesized by intraerythrocytic schizonts as a glycolipid-anchored membrane precursor, harbouring a preformed serine-protease active site but no detectable proteolytic activity. Detection of the enzymatic activity correlates with the solubilisation of the enzyme by a parasite glycolipid-specific phospholipase C in merozoites. A third enzyme has been detected with glycolipid-degrading activity, presumably a lipase A. These activities participate in a biochemical cascade originating with the attachment of the merozoite to the red blood cell, including the translocation of the phospholipase C to the membrane-bound protease, the solubilisation/activation of the protease and its secretion at the erythrocyte/parasite junction and ending with the entry of the parasite into the host cell. Both the phospholipase C and the lipase A might generate secondary messages in the merozoite. Our current knowledge concerning these enzymes is presented.

Malaria parasites: enzymes involved in red blood cell invasion

Three enzymes have been described in malaria merozoites: a serine-protease and two phospholipases. The parasite serine-protease is necessary for parasite entry into the red blood cell. This enzyme is synthesized by intraerythrocytic schizonts as a glycolipid-anchored membrane precursor, harbouring a performed serine-protease active site but not detectable proteolytic activity. Detection of the enzymatic activity correlates with the solubilisation of the enzyme by a parasite glycolipid-specific phospholipase C in merozoites. A third enzyme has been detected with glycolipid-degrading activity, presumably a lipase A. These activities participate in a biochemical cascade originating with the attachment of the merozoite to the red blood cell, including the translocation of the phospholipase C to the membrane-bound protease, the solubilisation/activation of the protease and its secretion at the erytrocyte/parasite junction and ending with the entry of the parasite into the host cell. Both the phospholipase C and the lipase A might generate secondary messages in the merozoite. Our current knowledge concerning these enzymes is presented

Malaria proteases and red blood cell invasion.

Studies of malaria proteases have focused on two general groups, corresponding to activities specific to malaria parasites: (1) proteases involved in hemoglobin degradation which are active in the food vacuole and which exhibit optimal activity at low pH; and (2) proteases specific to schizonts and/or merozoites which are involved in merozoite maturation and red blood cell invasion and which exhibit optimal activity at neutral pH. In this paper, Catherine Braun Breton and Luis H. Pereira da Silva will focus on those activities necessary for the release of infectious merozoites and the entry of the parasite into its host cell.

Plasmodium falciparum and Plasmodium chabaudi: characterization of glycosylphosphatidylinositol-degrading activities.

Merozoites of malaria parasites have a membrane-bound serine protease whose solubilization and subsequent activity depend on a parasite-derived glycosylphosphatidylinositol-phospholipase C (GPI-PLC). The GPI-degrading activities from both Plasmodium falciparum and Plasmodium chabaudi have been characterized and partially purified by phenylboronate chromatography. They are membrane-bound, developmentally regulated, calcium-independent enzymes and as such they resemble GPI-PLC of Trypanosoma brucei. Furthermore, a T. brucei GPI-PLC-specific monoclonal antibody (mAT3) immunoprecipitates the plasmodial GPI-degrading activity. Thin-layer chromatography is suggestive of two activities: a GPI-PLC and a phospholipase A.

Identification of phosphatidylinositol-specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor?

A phospholipase C which cleaves phosphatidylinositol and glycosylphosphatidylinositol (GPI) anchors was identified in Listeria monocytogenes. This 36 kDa protein is encoded by the gene plcA, and is homologous to the Bacillus cereus, Bacillus thuringiensis and eukaryotic phosphatidylinositol-specific phospholipases C (PI-PLC). Expression of the plcA gene in Escherichia coli correlates with the appearance of PI-PLC activity in the cells. In Listeria monocytogenes, the activity is secreted to the culture medium. PI-PLC activity was only found in the two pathogenic species of the genus Listeria, namely L. monocytogenes and L. ivanovii. PI-PLC activity was lost and virulence decreased when the plcA gene was disrupted in the chromosome. This suggests that the PI-PLC of L. monocytogenes might be involved in virulence.

Glycolipid anchorage of Plasmodium falciparum surface antigens.

Human red blood cells (RBC) were infected with the malarial parasite Plasmodium falciparum, the anchoring of schizont proteins to RBC membranes by glycoinositol phospholipids was demonstrated by three criteria: (1) metabolic incorporation of 3H-ethanolamine and 3H-myristate into the protein; (2) release of 35S-methionine-labelled protein into the supernatant after incubation with phosphatidylinositol-specific phospholipase C; and (3) the exposure of a glycoinositol phosphate epitope on the methionine-labelled protein following phospholipase C cleavage. Labelled proteins were analysed by immunoprecipitation, polyacrylamide gel electrophoresis in sodium dodecylsulphate and gel fluorography. Several candidate proteins were observed when each criteria was investigated. Among these, 3 proteins which met all three criteria were identified by immunoprecipitation with monospecific sera or monoclonal antibodies. These included 3 possible vaccine candidates, the p190 major surface antigen, the p76 serine protease and the p71 protein which is thought to be a member of the family of heat-shock Hsp70 proteins.

Induction of the proteolytic activity of a membrane protein in Plasmodium falciparum by phosphatidyl inositol-specific phospholipase C.

Membrane anchoring of proteins by a covalently attached glycosyl-phosphatidylinositol moiety has been reported in many different eukaryotic cells including parasite protozoa. The diversity of proteins in which this phospholipid attachment is found suggests that it is functionally important and perhaps also functionally pleiotropic. Studies on the Thy-1 antigen of murine lymphocytes indicate that it can facilitate the lateral mobility of membrane proteins. It can also permit the rapid and specific release of the anchored proteins from the membrane following cleavage by a phosphatidylinositol-specific phospholipase C (PI-PLC). Here we show that this type of anchoring may be involved in the regulation of an enzymatic activity. PI-PLC releases a Plasmodium falciparum membrane protein of relative molecular mass (Mr) 76K (p76) from intact merozoites or isolated schizont membranes and induces a proteolytic activity associated with its soluble form. Endogenous activation of the proteolytic activity of p76 appears to occur at the end of the schizogony and could initiate a cascade of biochemical events associated with merozoite maturation.