Protein O-fucosyltransferase 2–mediated O-glycosylation of the adhesin MIC2 is dispensable for Toxoplasma gondii tachyzoite infection

Abstract

Edited by Gerald W. HartToxoplasma gondii is a ubiquitous, obligate intracellular eukaryotic parasite that causes congenital birth defects, disease in immunocompromised individuals, and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune responses of many eukaryotic parasites and is also of great relevance to vaccine design. Here we demonstrate that micronemal protein 2 (MIC2), a motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat (TSR) domains. Using affinity-purified MIC2 and MS/MS analysis along with enzymatic digestion assays, we observed that at least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at these O-glycosylation sites. We found that addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homolog (TgPOFUT2) encoded by the TGGT1_273550 gene. Even though POFUT2 homologs are important for stabilizing motility-associated adhesins and for host infection in other apicomplexan parasites, loss of TgPOFUT2 in T. gondii had only a modest impact on MIC2 levels and the wider parasite proteome. Consistent with this, both plaque formation and tachyzoite invasion were broadly similar in the presence or absence of TgPOFUT2. These findings indicate that TgPOFUT2 O-glycosylates MIC2 and that this glycan, in contrast to previous findings in another study, is dispensable in T. gondii tachyzoites and for T. gondii infectivity.

Protein O-Fucosyltransferase 2 Is Not Essential for Plasmodium berghei Development

“…Recently, studies are casting new light on this issue (Bushkin et al, 2010; Sanz et al, 2013, 2016; Swearingen et al, 2016; López-Gutiérrez et al, 2017). Additionally, recent work exploring the function of PoFUT2 in Toxoplasma gondii , a Plasmodium related parasite, also revealed discrepancies in the effect of PoFUT2 null mutants on microneme protein 2 secretion and host cell attachment and invasion (Bandini et al, 2019; Gas-Pascual et al, 2019; Khurana et al, 2019). Our results suggest that within Plasmodia , nuanced differences with respect to developmental biology and host preference can also result in the observation of diverse phenotypes, which in this case is specific to O -fucosylation of sporozoite TSR domain-containing proteins.…”

Protein O- and C-Glycosylation pathways in Toxoplasma gondii and Plasmodium falciparum

“…This modification was abolished by deletion of the gene encoding POFUT2 (TGME49_273550) thereby proving the function of this glycosyltransferase in T. gondii (Fig. 3B) (Bandini et al ., 2019; Gas-Pascual et al ., 2019; Khurana et al ., 2019).…”

“…3B). Toxoplasma gondii pofut2 was knocked out in two additional studies (Gas-Pascual et al ., 2019; Khurana et al ., 2019). Gas-Pascual et al ., reported a defect in parasite replication upon its disruption, but did not address invasion or attachment as it was beyond the scope of the screen performed in this study.…”

“…Finally, in Khurana et al ., knock-out of pofut2 had no effect on cellular levels of MIC2, parasite proliferation, or its ability to attach and invade host cells. Discrepant phenotypes between the two reported pofut2 knockouts likely results from the different methods used for generating these cell lines and/or for assaying phenotype (Bandini et al ., 2019; Khurana et al ., 2019).…”

Sweet and CRISP(R)y parasite engineering

“…Despite this simplicity, there is even current controversy whether O-fucosylation of secreted T. gondii proteins (e.g. MIC2) is or is not important for invasion (5,6), but a relevant small plaque pofut2 phenotype was observed by Gas-Pascual et al (1). The authors also developed a new method to analyze glycophosphatidylinositol-type glycolipid anchors, but could not isolate any mutants affecting their core structure.…”