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

doi:10.1074/jbc.ra118.005179

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 Δpofut2 or Δnst2 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 Δmic2 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 Δpofut2 strain has decreased virulence and also support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes.

Highlights

Toxoplasma gondii is an intracellular parasite that causes disseminated infections that can produce neurological damage in fetuses and immunocompromised individuals
Using MS analysis, we found that the four TSRs in T. gondii Microneme protein 2 (MIC2) with protein O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, whereas Trp residues within three TSRs are modified with C-mannose
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 support a

Summary

To whom correspondence should be addressed

Conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes. A knockout of P. falciparum pofut showed that O-fucosylation of TSRs is required for efficient mosquito infection by ookinetes and for hepatocyte invasion by sporozoites [32] These phenotypes are due to defective folding/stabilization of the parasite TSR-containing proteins, such as TRAP and circumsporozoite protein, consistent with the observations made in metazoans and a potential role for O-fucosylation in efficient folding and/or stabilization of TSRs [23, 32]. We use MS to show that four of six TSRs of MIC2 are modified by O-fucose and that in addition to TSR5 (as shown by Hoppe et al [25]), two other TSRs are modified with C-Man. We identify the putative POFUT2 and GDP– fucose transporter (NST2) and show that knockout of either pofut or nst results in loss of O-fucosylation of MIC2, defects in the protein stability and localization, and a decrease in the ability of parasites to invade host cells

Results

Discussion

Experimental procedures