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 restricted to some trypanosome species. By expressing developmentally regulated trans-sialidases, these protozoan parasites cleave sialic acids from host glycoconjugates and transfer them to acceptors on their own cell surfaces. The biological function of this process is not understood, but trans-sialylation is expected to be important in the invasion of mammalian cells by Trypanosoma cruzi and the survival of Trypanosoma brucei within its insect vector. Since a conventional gene knockout approach was precluded, we developed a dominant-negative strategy, in which fusion proteins consisting of a bacterial sialidase and trypanosome proteins were expressed in T.brucei and T.cruzi. The strong recombinant sialidase activity shifted the reaction equilibrium from sialic acid transfer to hydrolysis, in this way creating a sialic-acid-negative phenotype. Taking advantage of a recently introduced inducible expression system, we were able to control the expression of sialidase fusion proteins in T.brucei. Reversion of the sialic-acid-negative state to wild-type sialylation was accomplished by selective inhibition of the foreign sialidase, leaving the parasite trans-sialidase unaffected. Both desialylation and resialylation of trypanosomes was rapidly achieved. Our results show that neither T.brucei nor T.cruzi require sialic acids for survival in vitro, ruling out the involvement of sialylation in cell surface integrity. The versatile system introduced here will allow a detailed in vivo study of the role of trans-sialylation during the trypanosome infection cycle. Furthermore, cell-surface sialic acids are implicated in a multitude of (patho-) biochemical processes in other organisms. The quantitative and qualitative manipulation of cell surface sialic acids, by expressing of counteracting enzymes, constitutes a novel approach with potentially broad applications in glycobiology.
Engstler
M
M
Laboratory of Molecular Parasitology, Rockefeller University, New York, NY 10021, USA.
Wirtz
E
E
Cross
G A
GA
D000818:Animals; D003001:Cloning, Molecular; D003013:Clostridium / Q000235:genetics; D004912:Erythrocytes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005799:Genes, Dominant; D009439:Neuraminidase / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012794:Sialic Acids / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000235:genetics / Q000378:metabolism*
D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D012794:Sialic Acids; D009439:Neuraminidase