The parasitic protozoan Tritrichomonasfoetus is the causative agent of bovine trichomoniasis, an infection of substantial agricultural importance that results in abortion and sterility. The objective of this project is to study the T foetus cell surface glycoconjugates and their involvement in adhesion of parasites to host target tissues, which will help to define the pathobiochernistry of T foetus parasite. The parasites possess novel lipophosphoglycan (LPG)-like glycoconjugates (2-3x 106 copies/parasite) anchored on the cell surface via an inositol phosphoceramide distinct from any other GPI anchor reported so far. Recent results from competition experiments using LPG suggest the involvement of glycoconjugates'in the adhesion of T foetus to HeLa cells. Anti-T foetus mAb that inhibits the adhesion of T foetus to HeLa cells recognizes T foetus LPG, as well as the glycoprotein GP 180-200 kDa. These two glycoconjugates may represent adhesion molecules. We have also identified a unique glycolipid, TF,from T foetus and because of its structural similarities to LPG, it is probable that TF, is an intermediate in the biosynthesis of LPG molecules. The specific objectives of the current project are I) to structurally characterize the LPG and clarify its relationships to GP 180-200 kDa; H) to study inununological structure/function(s) of LPG using mAbs directed against T foetus developed for this purpose; III) to study the involvement of LPG in adhesion of T foetus to HeLa cells- IV) to characterize the involvement of a unique fucosyl-inositol (alpha 1--->4) transferase in 'u,synthesis; and V) to study the biosynthesis of TF, glycolipid utilizing isotopic incorporation. Trichomonads are cultured and exposed to radio- and stable-isotopic tracers and harvested. Cells are extracted and glycoconjugate-analyzed by chemical and enzymatic digestion in combination with HPLC, GCIMS, MS, MALDI-TOF MS and NMR. The mAbs against T foetus LPGare to be generated by standard methodology to study the structure/function(s) of LPG. Experimental conditions are to be optimized to examine the involvement of LPG in adhesion of parasites to monolayers of HeLa cells. Knowledge acquired from these unique glycan structures that appear to be parasite-specific could be exploited as targets for chemotherapy by selectively inhibiting the transferases involved in their synthesis. Furthermore, these studies should provide essential knowledge of the structure/function relationships of LPG and thus lead to a better understanding of the molecular mechanisms of pathogenesis involved in host-parasite interactions. In addition, these findings will contribute to the development of effective diagnosis and therapies forbovine trichomoniasis.
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