Direct evidence of O-GlcNAcylation in the apicomplexan Toxoplasma gondii: a biochemical and bioinformatic study.

Toxoplasma gondii and Plasmodium falciparum are apicomplexan parasites responsible for serious diseases in humans. Many studies have focused on the post-translational modifications (PTMs) found in the two protists including phosphorylation, acetylation or SUMOylation but only a few of these are concerned with the nuclear and cytosolic-specific glycosylation O-GlcNAcylation. O-GlcNAcylation is a highly dynamic PTM-regulated by the ON and OFF enzymes: O-GlcNAc transferase and O-GlcNAcase-that can compete with phosphorylation but its function remains unclear. In this work, we directly prove the O-GlcNAcylation in T. gondii using antibodies specifically directed against the modification and we strongly suggest its occurrence in P. falciparum. We found that the inducible 70 kDa-Heat Shock Protein is O-GlcNAcylated, or associated with an O-GlcNAc-partner, in T. gondii. Using anti-OGT antibodies we were able to detect the expression of the glycosyltransferase in T. gondii cultured both in human foreskin fibroblast and in Vero cells and report its putative sequence. For the first time the presence of O-GlcNAcylation is unequivocally shown in T. gondii and suspected in P. falciparum. Since the O-GlcNAcylation is implicated in many biological fundamental processes this study opens a new research track in the knowledge of apicomplexans' life cycle and pathogenic potential.

The dual origin of Toxoplasma gondii N-glycans.

N-Linked glycosylation is the most frequent modification of secreted proteins in eukaryotic cells that plays a crucial role in protein folding and trafficking. Mature N-glycans are sequentially processed in the endoplasmic reticulum and Golgi apparatus through a pathway highly conserved in most eukaryotic organisms. Here, we demonstrate that the obligate intracellular protozoan parasite Toxoplasma gondii independently transfers endogenous truncated as well as host-derived N-glycans onto its own proteins.Therefore, we propose that the apicomplexan parasite scavenges N-glycosylation intermediates from the host cells to compensate for the rapid evolution of its biosynthetic pathway, which is primarily devoted to modification of proteins with glycosylphosphatidylinositols rather than N-glycans.

Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii.

GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-alpha production by macrophages exposed to T. gondii GPIs. Importantly, TNF-alpha response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88(-/-) mice were more susceptible to death than wild-type (WT), TLR2(-/-), TLR4(-/-), TLR2/4(-/-), and CD14(-/-) mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4(-/-), but not TLR2(-/-), TLR4(-/-), and CD14(-/-), mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-gamma production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2(-/-) and TLR2/4(-/-) mice produced more IFN-gamma than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.

Toxoplasma gondii grown in human cells uses GalNAc-containing glycosylphosphatidylinositol precursors to anchor surface antigens while the immunogenic Glc-GalNAc-containing precursors remain free at the parasite cell surface.

Toxoplasma gondii is a ubiquitous parasite that infects nearly all warm-blooded animals. Developmental switching in T. gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for the disease propagation after alteration of the immune status of the carrier. The redifferentiation event is characterized by an over expression of a tachyzoite specific set of glycosylphosphatidylinositol anchored surface antigens and free GPIs. T. gondii grown in animal cells uses two glycosylphosphatidylinositol precursors to anchor the parasite surface proteins. The first form has an N-acetylgalactosamine residue bound to a conserved three-mannosyl core glycan, while the second structure contains an additional terminal glucose linked to the N-acetylgalactosamine side branch. Sera from persons infected with T. gondii reacted only with the glucose-N-acetylgalactosamine-containing structure. Here we report that T. gondii cultured in human cells uses predominantly the N-acetylgalactosamine-containing structure to anchor the parasite surface antigens. On the other hand, glycosylphosphatidylinositol structures having an additional terminal glucose are found exclusively on the parasite cell surface as free glycolipids participating in the production of cytokines that are implicated in the pathogenesis of T. gondii. We also provide evidence that such free glycosylphosphatidylinositols are restricted mainly to the lipid microdomains in the parasite cell surface membrane and mostly associated with proteins involved in the parasite motility as well as invasion of the host cell.