Guy Tronchin

Localization, biosynthesis, processing and isolation of a major 126 kDa antigen of the parasitophorous vacuole of Plasmodium falciparum.

Monoclonal antibodies prepared against a 50 kDa antigen found in Plasmodium falciparum culture supernatants identify a 126 kDa polypeptide which can be localized by immunofluorescence and immunoelectronmicroscopy at the periphery of the schizonts. This polypeptide is released from the infected erythrocytes by mild saponin lysis and is probably a component of the parasitophorous vacuole. Pulse chase kinetic analysis demonstrated its disappearance from the parasitized red blood cell from 6 to 10 h after being synthesized and the concomitant appearance of the 50 kDa molecule in the culture supernatant. Purification of metabolically labeled, schizont infected cells demonstrated that spontaneous release of merozoites is needed for the processing of the 126 to the 50 kDa whereas reinvasion is not. Polyclonal antibodies were raised in rabbit against affinity purified 126 kDa protein. These antibodies, together with another 126 kDa specific monoclonal antibody have enabled us to characterize two other cleavage products of the 126 kDa antigen in culture supernatants, namely 47 and 18 kDa polypeptides. We believe that the processing of the 126 kDa protein into low molecular weight fragments reflects a proteolytic event which may participate in merozoite release.

Characterization of a 225 kilodalton rhoptry protein of Plasmodium falciparum

A monoclonal antibody (24C6 4F12) raised against Plasmodium falciparum culture supernatant antigens gave a multiple dot picture on schizonts when assayed by immunofluorescence on P. falciparum erythrocytic stages. The corresponding antigen was localized in the peduncle of rhoptries by immunoelectronmicroscopy. On Western blots of P. falciparum schizonts, a major antigen of 225 kDa and a minor one of 240 kDa were recognized by this McAb. Pulse chase analysis of [35S]methionine biosynthetic labeling of P. falciparum culture demonstrated that the 240 kDa molecule was the precursor of the 225 kDa and that its processing occurred between 0 and 4 h after synthesis. Biosynthesis of the 240-225 kDa antigen occurred only during schizogony.

Cytochemical and ultrastructural studies of Candida albicans: II. Evidence for a cell wall coat using concanavalin A

Ultrastructural modifications of the cell wall coat of Candida albicans during adherence to host cells were investigated using various cytochemical techniques. Attachment of the fungus to buccal epithelial cells appeared to involve spatial rearrangement of their cell wall surface. In particular adhering yeast developed a fibrogranular surface layer visualized by the periodic acid — thiocarbohydrazide silver proteinate technique (a polysaccharide detectron technique); Concanavalin A binding sites detected on their cell wall coat were highly increased. Attachment of yeasts to epithelial cells appeared mediated by fibrillar structures or polysaccharidic granules distributed on the cell wall coat. But free extra-cell wall material containing mannoproteins released from the yeast surface suggested additional mechanisms.

Antigenic variations of Candida albicans in vivo and in vitro—Relationships between P antigens and serotypes

Serum samples from patients with candidosis and from rabbits experimentally infected with serotype B C. albicans strains consistently showed higher antibody titers against Candida strains with serotype A antigens than strains with serotype B antigens, in indirect fluorescent antibody tests. When sera from rabbits infected with C. albicans serotype B strains were absorbed with blastospores of the homologous strain they continued to react against strains of C. albicans serotype A and a C. tropicalis strain with serotype A antigens. Serotype A-specific antisera reacted against tissue forms of C. albicans serotype B in vivo and against serotype B germ tubes, but not their parent blastospores, in vitro. These findings suggest that C. albicans B serotype cells may sometimes express, in vitro or in vivo, one or several antigens so far considered as specific of serotype A. The results have implications for the classical concept of C. albicans serotypes and for the serological diagnosis of candidosis in relation with the previously described strain P variable antigens.

Etudes Cytochimiques et Ultrastructurales de la Paroi de Candida albicans

RésuméLa localisation ultrastructurale des polysaccharides paritétaux de Candida albicans a été réalisée par utilisation de Concanavaline A sur sections ultrafines de résine hydrosoluble (glycol méthacrylate). Les sites récepteurs de la lectine sont révélés en incubant successivement les coupes avec de la péroxydase, puis un mélange 3-3′ diaminobenzidine-H2O2. Cette méthode a permis de localiser les mannanes, qui sont les seuls polysaccharides pariétaux de C. albicans susceptibles de réagir avec la Concanavaline A. Deux couches continues réactives ont été ainsi mises en évidence à la périphérie de la paroi des blastospores. Les résultats sont discutés en fonction de ceux obtenus par une méthode différente de mise en évidence des polysaccharides (PATAg).AbstractThe ultrastructural localization of polysaccharides in the cell wall of Candida albicans was carried out by means of Concanavalin A on glycol methacrylate ultrathin sections. The sections were incubated successively with horseradish peroxydase, 3-3′ diaminobenzidine and H2O2, for revealing the binding sites of the lectin. This method allowed us to localize mannan, since Concanavalin A does not react with other polysaccharides of the C. albicans cell wall. In these conditions mannan was found to be located in two continuous reactive layers at the periphery of blastospores cell wall. The results are discussed in relation with those obtained by another method using the polysaccharide detection technique described by Thiery (PATAg).