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.

In vivo time course of synthesis and processing of major schizont membrane polypeptides in Plasmodium falciparum.

A rapid method of separating membrane co-sedimentable and soluble components of Plasmodium infected erythrocytes is presented. We propose a nomenclature for major P. falciparum polypeptides, applicable to different isolates and based on their cellular location and stage specificity. For four of these polypeptides (185 kDa = Mp1; 120 kDa = Mp3; 76 kDa = Mp5; 90 kDa= Sp2) supposed to play a role in protective immunity, monospecific antibodies were available. We have studied their fate at the time of merozoïte release and reinvasion, and the possible correlations between these polypeptides, by pulse-chase experiments.

The S-antigen of Plasmodium falciparum Palo Alto represents a new S-antigen serotype.

The S-antigen from the Palo Alto isolate of Plasmodium falciparum has been characterized. The partial sequence for the gene coding for this antigen (clone 281) reveals the presence of tandem repeats of eight amino acids which defines a new S-antigen serotype. Antibodies raised against the 281 recombinant clone reacted with a 140 kDa antigen by immunoblotting with parasite extracts and culture supernatants. The 140 kDa peptide was also identified by immunoprecipitation of metabolic labelled parasites. The 281 mouse antiserum was used to localize the antigen on parasite smears by indirect immunofluorescence assay and more precisely by immunoelectron microscopy. The S-antigen is localized within the parasitophorous vacuole. Furthermore, different isolates were examined for the presence of the Palo Alto S-antigen specificity.

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.

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.

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.

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.

Screening for lamB missense mutations which alter all lambda receptor activities in Escherichia coli K12.

Previously described missense mutations in gene lamB, the structural gene for the lambda receptor in Escherichia coli K12, affected only some of the activities of this multifunctional protein. We isolated lamB mutations, some of which could be of the missense type, and which affected all of the activities of the LamB protein. Among 8 of these mutations, 5 affected the stability of the LamB protein and 3 did not markedly decrease the amount of LamB protein in the mutant strains. In these 3 cases, the mutated LamB proteins were recovered with the envelope of the mutants. We briefly discuss the nature of these mutations and their possible effects on LamB protein structure and location.

In vivo and in vitro functional alterations of the bacteriophage lambda receptor in lamB missense mutants of Escherichia coli K-12.

lamB is the structural gene for the bacteriophage lambda receptor in Escherichia coli K-12. In vivo and in vitro studies of the lambda receptor from lamB missence mutants selected as resistant to phage lambda h+ showed the following. (i) Resistance was not due to a change in the amount of lambda receptor protein present in the outer membrane but rather to a change in activity. All of the mutants were still sensitive to phage lambda hh*, a two-step host range mutant of phage lambda h+. Some (10/16) were still sensitive to phage lambda h, a one-step host range mutant. (ii) Resistance occurred either by a loss of binding ability or by a block in a later irreversible step. Among the 16 mutations, 14 affected binding of lambda h+. Two (lamB106 and lamB110) affected inactivation but not binding; they represented the first genetic evidence for a role of the lambda receptor in more than one step of phage inactivation. Similarly, among the six mutations yielding resistance to lambda h, five affected binding and one (lamB109) did not. (iii) The pattern of interactions between the mutated receptors and lambda h+ and its host range mutants were very similar, although not identical, in vivo and in vitro. Defects were usually more visible in vitro than in vivo, the only exception being lamB109. (iv) The ability to use dextrins as a carbon source was not appreciably affected in the mutants. Possible working models and the relations between phage infection and dextrins transport were briefly discussed.

Explanations accounting for transduction by bacteriophage lambda in maltose negative bacteriophage lambda resistant mutants of Escherichia coli K-12.

Entry of DNA from lambda phages particles into lambdarMAl- mutants of Escherichia coli K-12 is shown to be due to two distinguishable processes. One, residual transduction, results from a low level expression of lamB. The other one, background transduction, is independent of gene lamB. Interpretations are presented for these results. It is propos that residual transduction is due to a weak promoter pB3 located within or near the distal part of the gene preceeding lamB in the same operon. It is proposed that background transduction is due to a secondary receptor structure for phage lambda. Finally a tentative hypothesis relatin pB3 to insertion sequences is presented.

General method for fine mapping of the Escherichia coli K-12 lamB gene: localization of missense mutations affecting bacteriophage lambda adsorption.

lamB is the structural gene for the bacteriophage lambda receptor, a multifunctional protein located in the outer membrane of Escherichia coli K-12. We present a method for deletion mapping of any lamB mutations with a recognizable pheno-type. This method involves a transducing phage constructed by in vitro recombination which can also be used for complementation, deoxyribonucleic acid sequence, and in vitro protein synthesis studies with the mutated lamB gene. Using this method, we mapped 18 lamB missense mutations which confer resistance to phage lambda h+ (wild-type host range). The main results were the following. (i) None of the 18 mutations was located in the first 4 deletion intervals out of the 11 of the genetic map. (ii) These mutations were clustered according to their phenotype as follows. (a) Class I mutations, which allow growth of lambda h and lambda hh* (one-step and two-step host range mutants of lambda, respectively), were located in three regions--three in interval V, four in interval VIII-IX, and three in interval X-XI. Only the last three mutations still allowed growth of phage K10 which also uses the lambda receptor, and two of them still allowed reversible binding of lambda h+. (b) All seven class II mutations allowed only growth of lambda hh* and mapped in interval V. These results are discussed in the frame of a genetic approach to the functional topology of the lambda receptor.

Structure of the malB region in Escherichia coli K12. I. Genetic map of the malK-lamB operon.

A series of deletions, Mu insertions and point mutations affecting the malK-lamB operon have been isolated. They were used to establish a deletion map of this operon, which could be divided in 27 intervals, with 16 in malK and 11 in lamB. One interesting feature of this map is the lack of randomness in the distribution of Mu insertions in the lamB gene; by using data published elsewhere on the physical length of the deletion intervals it can be concluded that about 25% of these Mu insertions are clustered in a segment representing 2 to 8% of the gene. This map is presently being used to study the biosynthesis, structure, and function of the lamB product, which is an outer membrane protein involved in the transport of maltose and maltodextrin, and which in addition constitutes the receptor for phage lambda.

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.

Activation of a Plasmodium falciparum protease correlated with merozoite maturation and erythrocyte invasion.

Protease-dependent processes of the P. falciparum schizogonic cycle are briefly described. The P. falciparum p76 protease is the first example of a biochemically regulated protease, the activation of which is related to merozoite maturation and/or erythrocyte invasion. The main known properties of the p76 protease are reviewed and some original results concerning its biosynthesis and biological properties are described.

lamB mutations in E. coli K12: growth of lambda host range mutants and effect of nonsense suppressors.

Over sixty EMS induced mutations affecting gene lamB, presumably the structural gene for the lambda receptor in Escherichia coli K12, were examined for growth of lambda host range mutants and effect of nonsense suppressors. By the first criterion the mutations could be grouped in three classes. Bacteria with class I mutations allow growth of lambda mutants with extended host range (noted lambdah) of the type already described (Appleyard, MacGregor and Baird, 1956). Bacteria with class II mutations allow growth of lambdah mutants with still more extended host range (noted lambdahh). No host range mutants of lambda could be found which would grow on bacteria with class III mutations. Using nonsense suppressors it was found that class I and II consist of missense mutations, while class III consists of nonsense mutations. Exceptions are likely to exist (especially in class III) but were not found among the mutations tested. These observations are briefly discussed in terms of outer membrane protein integration and of phage receptor interaction.

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.

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.

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.