Jocelyne Cornette

Identification and isolation of Trypanosoma cruzi trypomastigote cell surface protein with properties expected of a fibronectin receptor

The fibronectin receptor of Trypanosoma cruzi trypomastigotes was identified using immunoprecipitation procedure. Parasite radioiodinated surface material was incubated with fibronectin followed by rabbit IgG anti-fibronectin and protein A-Sepharose. The precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two radioactive bands were seen. One of 220 kDa corresponded to a subunit of fibronectin molecule present on the parasite surface at the time of isolation. The major radioactive band of 85 kDa corresponded to the fibronectin receptor. Fibronectin receptor was purified using affinity chromatography on human fibronectin coupled to Sepharose. Analysis of fibronectin receptor by sodium dodecyl sulfate-polyacrylamide gels demonstrated one major band of 85 kDa. The purified fibronectin receptor was active since 40-60% of labeled receptor could rebind to fibronectin-Sepharose. In addition, fibronectin receptor could interact with cells bearing fibronectin molecules as shown by the binding of 125I-labeled fibronectin receptor to human monocytes and neutrophils as well as cloned 3T3 fibroblasts. The binding could be inhibited by treatment of cells with anti-fibronectin antibodies. Finally, we showed that the affinity-purified fibronectin receptor and antibodies to the receptor exerted an inhibitory effect on the infection of 3T3 fibroblasts by T. cruzi trypomastigotes in an in vitro culture system.

Identification and isolation of Trypanosoma cruzi trypomastigote collagen-binding proteins: possible role in cell-parasite interaction.

We have shown here that collagen type I bound efficiently to the trypomastigote surface. In addition, monoclonal and polyclonal antibodies against collagen types I and III inhibited the infection of fibroblasts by the parasite. These results suggested the presence of collagen-binding protein(s) on the parasite surface. This protein was identified from trypomastigote surface antigens using affinity chromatography on a Gelatin Ultrogel column (denatured form of collagen). These collagen-binding proteins were revealed as a low-affinity gelatin binding protein (LAG Bp) of 98 kDa, and a high-affinity binding protein (HAG Bp) of 58 and 68 kDa under non-reducing and reducing conditions respectively. In addition, HAG Bp and LAG Bp bound to collagen type I. The 58/68 kDa protein was purified to homogeneity on a wheat germ agglutinin Sepharose column. A polyclonal antibody to this glycoprotein, as well as a monoclonal antibody (McAb) 155D3 produced against the HAG Bp, immunoprecipitated two parasite surface antigens of 160 and 58 kDa under non-reducing conditions which migrated at a position of 80-85 and 68 kDa when reduced. However, only the 80-85 kDa component could be precipitated from [35S] methionine-labelled trypomastigote antigens under reducing conditions. The antibodies to the 58/68 kDa glycoprotein as well as McAb 155D3 diminished the invasion of fibroblasts by parasites. Taken together these results suggest that the same receptor binds fibronectin and/or collagen and that both the 80-85 and 58/68 kDa glycoproteins form part of the same receptor. These trypomastigote surface molecules may interact with the host cell fibronectin and/or collagen during the initial phase of parasite-cell recognition.

The class I histone deacetylases of the platyhelminth parasite Schistosoma mansoni

Histone deacetylases (HDAC) form a conserved enzyme family that control gene expression via the removal of acetyl residues from histones and other proteins and are under increasing investigation as therapeutic targets, notably in cancer and parasitic diseases. To investigate the conservation of these enzymes in the platyhelminth parasite Schistosoma mansoni, we cloned and characterized three class I HDACs, orthologues of mammalian HDAC1, 3 and 8, and confirmed their identities by phylogenetic analysis. The identification of an HDAC8 orthologue showed that it is not vertebrate-specific as previously thought and insertions in its catalytic domain suggest specific enzymatic properties. SmHDAC1, 3, and 8 mRNAs are expressed at all schistosome life-cycle stages. SmHDAC1 repressed transcriptional activity in a mammalian cell line and this activity was dependent on its catalytic activity since transcription was partially restored by treatment with trichostatin A and a catalytic site mutant failed to repress transcription.

Occurrence of fibronectin antigenic determinants on Schistosoma mansoni lung schistosomula and adult worms

Fibronectin determinants were revealed in a soluble extract of Schistosoma mansoni adult worms by standard immunodiffusion and immunoelectrophoresis techniques. The same target molecules were demonstrated on the parasites surface using a binding assay with [125I] anti-fibronectin. The use of fluorescein- and peroxidase-conjugated-antibodies confirmed the above observations and provided a fairly precise means for locating the cross-reacting antigen on the worm surface. An in vitro cytotoxic assay using inactivated (56 degrees C, 2 h) anti-fibronectin rabbit immune serum and guinea-pig serum as a source of complement was developed. In these conditions, anti-fibronectin exerted cytotoxic activity against lung schistosomula and adult worms but not skin schistosomula. The ultrastructural damage induced by anti-fibronectin in the presence of complement was studied using transmission electron microscopy. The results suggest a close association between fibronectin determinants and S. mansoni membrane.

Trypanosoma cruzi: Differential expression and distribution of an 85-kDa polypeptide epitope by in vitro developmental stages

The expression by Trypanosoma cruzi developmental stages of an 85-kDa polypeptide epitope defined by the 155D3 monoclonal antibody (mAb) has been investigated. Immunoprecipitation revealed the presence of an 85-kDa antigen in the NP-40 soluble extract of parasites freshly released from infected fibroblasts; this antigen was not found in epimastigote and Leishmania infantum promastigote. Indirect immunofluorescence revealed that the mAb 155D3 failed to react with trypomastigotes, whereas extracellular amastigotes were heavily stained. Positive organisms displayed either surface or polar fluorescence. Since the same mAb immunoprecipitated the 85-kDa antigen in both radioactive iodine- and methionine-labeled trypomastigote detergent soluble extracts, the reactive epitope is likely to be hidden in a cryptic site in trypomastigotes. An alternative explanation for the negative immunofluorescence on trypomastigotes and the positive immunoprecipitation is the presence, in the extracts, of a small population of parasites already expressing the 155D3 epitope. Immunoelectron microscopy revealed that the target epitope is heterogenously distributed among the populations of differentiating parasites. Two types of immunogold labeling were observed: (a) mAb revealed a high amount of reactive material associated with the periphery of the parasites and (b) a label was observed on the inner surface of peripheral vacuoles that might correspond to cross sections of inflated flagellar pockets and in association with vesicles which were released by the parasites. The surface expression of the epitope recognized by the 155D3 mAb was followed by fluorescence-activated cell-sorting analysis. The results showed that the epitope is increasingly accessible during trypomastigote differentiation in vitro. Taken together, these results suggest that the epitope reacting with the 155D3 mAb is heavily expressed on extracellular amastigotes after the transformation process and, thus, appears to be developmentally regulated.

Unique functional properties of a member of the Fushi Tarazu-Factor 1 family from Schistosoma mansoni

SmFtz-F1 (Schistosoma mansoni Fushi Tarazu-Factor 1) belongs to the Ftz-F1 subfamily of nuclear receptors, but displays marked structural differences compared with its mammalian homologues SF-1 (steroidogenic factor-1) or liver receptor homologue-1. These include a long F domain (104 amino acids), an unusually large hinge region (133 amino acids) and a poorly conserved E-domain. Here, using Gal4 constructs and a mammalian two-hybrid assay, we have characterized the roles of these specific regions both in the transcriptional activity of the receptor and in its interactions with cofactors. Our results have shown that, although the AF-2 (activation function-2) region is the major activation function of the receptor, both the F and D domains are essential for AF-2-dependent activity. Modelling of SmFtz-F1 LBD (ligand-binding domain) and structure-guided mutagenesis allowed us to show the important role of helix H1 in maintaining the structural conformation of the LBD, and suggested that its autonomous transactivation activity, also observed with SF-1, is fortuitous. This strategy also allowed us to study an eventual ligand-dependence for this orphan receptor, the predicted three-dimensional models suggesting that the SmFtz-F1 LBD contains a large and well-defined ligand-binding pocket sealed by two arginine residues orientated towards the interior of the cavity. Mutation of these two residues provoked a loss of transcriptional activity of the receptor, and strongly reduced its interaction with SRC1 (steroid receptor cofactor-1), suggesting a ligand-dependent activity for SmFtz-F1. Taken together, our results argue for original and specific functional activities for this platyhelminth nuclear receptor.

Detection of IgE antibodies in onchocerciasis using a semi-purified fraction from Dipetalonema viteae total antigen.

A Dipetalonema viteae extract was separated by gel filtration on ACA 34 Ultrogel into four fractions (A, B, C and D). The allergenic activity of the D. viteae extract and its various fractions was assayed by the passive cutaneous anaphylaxis (PCA) test in rats using mouse sera obtained from Balb/c mice transplanted with D. viteae. The PCA reaction showed that fraction B was the most potent allergenic fraction of the D. viteae extract. By the radioallergosorbent test (RAST) the use of fraction B coupled to CNBr-activated paper discs showed elevated binding of IgE antibodies in onchocerciasis human sera. A comparative study demonstrated the efficiency of the above fraction in the RAST technique in distinguishing between Onchocerca volvulus-infected patients and those infected with other human filarial worms or other helminth parasites. The binding of IgE to fraction B was confirmed by the radioimmunoelectrophoresis and radio-double diffusion methods using an 125I anti-human IgE. Since D. viteae antigens are more readily obtainable than those of O. volvulus, a further purification of fraction B to improve its specificity for the detection of IgE antibodies in human onchocerciasis is warranted.