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

Authors:
Khurana S, Coffey MJ, John A, Uboldi AD, Huynh MH, Stewart RJ, Carruthers V, Tonkin CJ, Goddard-Borger ED, Scott NE
In:
Source: J Biol Chem
Publication Date: (2018)
Issue: Epub:
Research Area:
Parasitology
Platform:
4D-Nucleofector® X-Unit
Experiment

Disruption of TGPOFUT2 using CRISPR in T. gondii tachyzoits:

10 µg of the Cas9 plasmid was combined with 20 µg of PCR product, then precipitated using EtOH/NaAc prior to transfection. The dried DNA was resuspended in 3 µL of elution buffer (EB, Qiagen), followed by 20 µL P3 solution (Lonza). A washed parasite pellet containing approximately 10exp6 tachyzoites was then resuspended in this solution and transfected using the code FI-115 in a 16-well Nucleocuvette Strip in an Amaxa 4D Nucleofector (Lonza). Parasites containing the knockout construct were then selected as normal following addition of phleomycin and subsequently sub-cloned until a stable population was obtained.

Abstract

Toxoplasma 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 the addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homologue (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.