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.

Characterisation of PfSec61, a Plasmodium falciparum homologue of a component of the translocation machinery at the endoplasmic reticulum membrane of eukaryotic cells.

Plasmodium falciparum secretes several proteins that cause changes in the erythrocyte membrane enabling it to survive within red blood cells. Little is known of the mechanisms involved in the secretion and targeting of parasite polypeptides to the various cell compartments. The P. falciparum gene homologous to the mammalian Sec61alpha, gene, which encodes a component of the translocation pore in the endoplasmic reticulum of eukaryotic cells, was characterised to investigate the translocation process in the parasite. PfSec61 is present as a unique copy in the parasite genome and was mapped to chromosome 13. It encodes a 40 kDa polypeptide, as shown by immunoblotting and immunoprecipitation of [35S]methionine metabolically-labelled parasite extracts. The deduced amino acid sequence of PfSec61 is 87% similar to the mammalian polypeptide, and the two proteins give similar hydropathy plots. These results strongly suggest that PfSec61 has the same topological orientation and functional role as Sec61alpha. Anti-PfSec61 antibodies were used to investigate the cellular location and kinetics of expression of the polypeptide in the parasite. Immunofluorescence confocal microscopy showed that PfSec61 was located in the parasite cytoplasm, close to the nucleus, in a position consistent with its being in the endoplasmic reticulum.

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.

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

Immunogenicity and antigenicity of a Plasmodium falciparum protein fraction (90-110 kDa) able to protect squirrel monkeys against asexual blood stages.

A monkey vaccination trial using a Plasmodium falciparum protein fraction containing antigens of 90-110 kDa is reported. The fraction was obtained by electroelution from preparative polyacrylamide gels. Three monkeys out of five resisted a heavy challenge dose of highly virulent parasites. Using specific antisera, several components of the fraction were identified, namely heat shock protein 90 (hsp90), Ag44/RhopH3, ABRA, 96tR/GBP130 and Pf96 protease. The fraction did not contain KAHRP, nor the SERP antigen. The antibody response of the monkeys was studied on these individual antigens purified by preparative immunoprecipitation. Surprisingly, hsp90 was found in the immunoprecipitates obtained with SERP antisera. Interestingly, the response to hsp90 correlated with protection, high antibody titres being found only in the protected monkeys. In contrast, no correlation with protection could be found for the response to the other antigens.

Plasmodium chabaudi p68 serine protease activity required for merozoite entry into mouse erythrocytes.

To define the role of malaria parasite enzymes during the process of erythrocyte invasion, we have developed an in vitro serum-free invasion assay of mouse erythrocytes by purified Plasmodium chabaudi merozoites. The sensitivity of a merozoite-specific serine protease (p68) to various inhibitors and the effect of these inhibitors on invasion indicate a crucial role for p68. The substrate specificity of the purified enzyme has been partially defined using fluorogenic peptides. Consistent with this, in vitro incubation of mouse erythrocytes with the merozoite enzyme led to the cleavage of band 3 protein. The possible implication of erythrocyte band 3 truncation for the successful entry of the merozoite into the erythrocyte is discussed.

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.

Plasmodium falciparum: analysis of B epitopes of a polypeptide antigen expressed in Escherichia coli, using monoclonal antibodies.

Monoclonal antibodies were raised against a recombinant molecule corresponding to the polypeptide 72 kDa, previously described as possibly related to protection in Plasmodium falciparum infection. Selection of hybridoma cell lines was done by immunofluorescence to guarantee the reactivity of the monoclonal antibodies both against the recombinant and the native molecule of the parasite. Monoclonal antibodies were characterized by serological and immunochemical techniques. Competitive binding assays between monoclonal antibodies defined four different B epitopes. One epitope is specific for P. falciparum, a second is also present in P. vivax, while the two others seem to be ubiquitous and are also present in the rodent parasite P. chabaudi. The ubiquitous epitope 72.C is apparently the only one recognized by squirrel monkey sera presenting protective antibodies against the asexual blood infection by P. falciparum.

Structure and function of a thymic peptide is mimicked by Plasmodium falciparum peptides.

Numerous Plasmodium falciparum antigens contain repetitive amino acid sequences. Two blood stage antigens, Pf11-1 and Pf332, were characterized in our laboratories and present high cross-reactivities, defining a family of cross-reacting antigens. In this report, we show that amino acid sequence homologies might explain these cross-reactivities, but that they extend to polypeptides from the host, namely thymosin-alpha 1 (T alpha 1). An antiserum raised in chickens and Saimiri monkeys against the synthetic Pf11-1 peptide cross-reacts with synthetic T alpha 1. Synthetic Pf11-1 and Pf332 peptides share some of the biological activities of T alpha 1. These results are discussed with respect to the mechanisms devised by malaria parasites for escape from the host immune response.

Immunogenicity of Plasmodium falciparum antigenic determinants produced by Escherichia coli recombinant clones.

The immunogenicity of two parasite antigens produced by Escherichia coli as proteins fused to beta-galactosidase was investigated in three animal species: mice, rabbits and squirrel monkeys. 2L protein carries 71 amino acids of a parasite antigen and 11.1 protein carries 23 repeats of a 9-amino-acid repetitive unit. The humoral response was studied using indirect immunofluorescence and immunoprecipitation. The results indicate that immunization of mice, rabbits and squirrel monkeys using SDS-denatured 2L fusion protein induced antibodies able to bind to parasite antigen 2L in the IFA or in the immunoprecipitation assays. Immunization using the native fusion protein did not induce antibodies able to immunoprecipitate the 2L parasite antigen. The same observation was made for the animals immunized with 11.1 recombinant protein. In this case, the antibody response was also measured by ELISA using synthetic dimers of the repeat as antigen. In mice and rabbits, high titres of anti-11.1 antibodies were found by ELISA. However, when the antigen produced by the parasite itself was used to evaluate the response, low titres were found. This indicates that the animals produced high levels of antibodies to a structure which is not exposed in the parasite. In squirrel monkeys, the same observation was made, but the overall levels of the response to 11.1 antigen were considerably lower than those observed in mice or rabbits.

Plaque antibody selection: rapid immunological analysis of a large number of recombinant phage clones positive to sera raised against Plasmodium falciparum antigens.

A library of Plasmodium falciparum genomic DNA on the lambda gt11 phage vector was screened for clones positive to a rabbit serum raised against a purified fraction of P. falciparum proteins and a pool of sera from malaria patients. The positive clones were characterized with antibodies purified by the plaque antibody selection technique. This technique consist of purifying specific antibodies on a nitrocellulose filter blotted directly on a lawn of plaques of an antigen-producing phage clone. The purified antibodies are then used as a probe in a Western blot of parasite protein extract, for preliminary characterization of the clones. Using this method, two different clones coding for P. falciparum antigens were identified with the rabbit serum and about 20 with the human sera. This method can be of general use, i.e. it is not limited to parasite systems, and facilitates the immunological analysis and identification of a large number of clones.