Generation of constitutive and inducible trans-sialylation dominant-negative phenotypes in Trypanosoma brucei and Trypanosoma cruzi

Trans-sialylation is a unique enzymatic process that is restrictedto some trypanosome species. By expressing developmentally regulatedtrans-sialidases, these protozoan parasites cleave sialic acidsfrom host glycoconjugates and transfer them to acceptors ontheir own cell surfaces. The biological function of this processis not understood, but trans-sialylation is expected to be importantin the invasion of mammalian cells by Trypanosoma cruzi andthe survival of Trypanosoma brucei within its insect vector.Since a conventional gene knockout approach was precluded, wedeveloped a dominant-negative strategy, in which fusion proteinsconsisting of a bacterial sialidase and trypanosome proteinswere expressed in T.brucei and T.cruzi. The strong recombinantsialidase activity shifted the reaction equilibrium from sialicacid transfer to hydrolysis, in this way creating a sialic-acid-negativephenotype. Taking advantage of a recently introduced inducibleexpression system, we were able to control the expression ofsialidase fusion proteins in T.brucez. Reversion of the sialic-acid-negativestate to wild-type sialylation was accomplished by selectiveinhibition of the foreign sialidase, leaving the parasite trans-sialidaseunaffected. Both desialylation and resialylation of trypanosomeswas rapidly achieved. Our results show that neither T.bruceinor T.cruzi require sialic acids for survival in vitro, rulingout the involvement of sialylation in cell surface integrity.The versatile system introduced here will allow a detailed invivo study of the role of trans-sialylation during the trypanosomeinfection cycle. Furthermore, cell-surface sialic acids areimplicated in a multitude of (patho-) biochemical processesin other organisms. The quantitative and qualitative manipulationof cell surface sialic acids, by expressing of counteractingenzymes, constitutes a novel approach with potentially broadapplications in glycobiology. sialidase trans-sialidase sialic acids PARP procyclin dominant-negative phenotype