DELETION OF AN IMMUNODOMINANT TRYPANOSOMA-CRUZI SURFACE GLYCOPROTEIN DISRUPTS FLAGELLUM CELL-ADHESION

Null mutants of the Trypanosoma cruzi insect stage-specific glycoprote in GP72 were created by targeted gene replacement. Targeting plasmids were constructed in which the neomycin phosphotransferase and hygromyc in phosphotransferase genes were flanked by GP72 sequences. These plas mids were sequentially transfected into T. cruzi epimastigotes by elec troporation. Southern blot analyzes indicated that precise replacement of the two genes had occurred. No aberrant rearrangements occurred at the GP72 locus and no GP72 gene sequences had been translocated elsew here in the genome. Western blots confirmed that GP72 is not expressed in these null mutants. The morphology of the mutants is dramatically different from wild-type. In both mutant and wild-type parasites, the flagellum emerges from the flagellar pocket. In the null mutant the no rmal attachment of the flagellum to the cell membrane of the parasite is lost.