M.A. Ouaissi

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.

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.

Schistosoma mansoni: ultrastructural damages due to complement on schistosomula in vitro.

Abstract The ultrastructural damage induced by complement in vitro on the schistosomula of Schistosoma mansoni was studied using transmission and scanning electron microscopy. The sequence of events which leads to the killing of the schistosomula is as follows: (a) the lytic activity starts at the anterior end of the schistosomula; (b) lesions progress simultaneously in two distinct directions: from the anterior to the posterior end, and from the outer membrane to the muscle layer; (c) “bubbles” appear around parasite which might correspond to increased membrane permeability; (d) the lytic activity of the late complement components occur in the syncytium; (e) the schistosomula lose their tegument completely, exposing the muscle layer. These findings and our previous work suggest that the activation of the alternate pathway and late complement components is sufficient, without antibodies or cells, to kill schistosomula in vitro.