O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites

Giulia Bandini, Deborah R. Leon, Carolin M. Hoppe, Yue Zhang, Carolina Agop-Nersesian, Melanie J. Shears, Lara K. Mahal, Françoise H. Routier, Catherine E. Costello, John Samuelson

Research output: Contribution to journalArticle

Abstract

Toxoplasma gondii is an intracellular parasite that causes disseminated infections that can produce neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2), a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for T. gondii motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C-mannose and O-fucose in Plasmodium spp. and mammals. Here, using MS analysis, we found that the four TSRs in T. gondii MIC2 with protein O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, whereas Trp residues within three TSRs are also modified with C-mannose. Disruption of genes encoding either POFUT2 or the putative GDP–fucose transporter (NST2) resulted in loss of MIC2 O-fucosylation, as detected by an antibody against the GlcFuc disaccharide, and in markedly reduced cellular levels of MIC2. Furthermore, in 10 –15% of the pofut2 or nst2 vacuoles, MIC2 accumulated earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts was reduced for these knockouts, which both exhibited defects in attachment to and invasion of host cells comparable with the mic2 phenotype. These results, indicating that O-fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that Plasmodium falciparum pofut2 strain has decreased virulence and also support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes.

Original languageEnglish (US)
Pages (from-to)1967-1983
Number of pages17
JournalJournal of Biological Chemistry
Volume294
Issue number6
DOIs
StatePublished - Feb 8 2019

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Bandini, G., Leon, D. R., Hoppe, C. M., Zhang, Y., Agop-Nersesian, C., Shears, M. J., Mahal, L. K., Routier, F. H., Costello, C. E., & Samuelson, J. (2019). O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites. Journal of Biological Chemistry, 294(6), 1967-1983. https://doi.org/10.1074/jbc.RA118.005179