Fig 1.
Glycans and UDP-GlcNAc synthesis in T. gondii.
A) Proteins of the secretory pathway of Toxoplasma gondii are commonly modified as N-glycans, O-glycans or GPI-anchored. The typical glycan structures in T. gondii are shown, as well as the modification of Skp1, which harbours a specific O-glycosylation [73]. Free, non-protein bound GPIs (glycoinositolphospholipids, GIPLs) are distinguished from GPI anchors by a Glc residue linked to GalNAc (see dashed line). B) The activated sugar nucleotide UDP-GlcNAc is synthesised from glucose in six conserved enzymatic reactions, with the glucosamine-phosphate N-acetyltransferase (GNA1) converting glucosamine-6-phosphate (GlcN6P) to N-acetylglucosamine-6-phosphate (GlcN6P) C) Overview of T. gondii enzymes in UDP-GlcNAc synthesis, listing their names, accession number (ID) [25], fitness score (FS) [23] and putative localisation (hLOPIT) [74], as well as the accession number [75] and mutagenesis fitness score (MFS) [76] in the related Plasmodium falciparum parasite. Abbreviations: PI: phosphatidylinositol; EtN: ethanolamine; GT1: glucose transporter 1; PM: plasma membrane; Fru, fructose; PI, phosphatidylinositol; GPI, glycosylphosphatidyl inositol; UDPGlcNAc, uridine diphosphate N-acetylglucosamine. Other abbreviations, see panel A and C.
Fig 2.
GNA1 is essential for T. gondii.
A) Cartoon schematic depicting the DNA segment and transcripts encoding GNA1 as well as the resulting protein(s). The approximate sites targeted by the single guide RNAs (sgRNAs) in the genome-wide fitness screen are shown. Dotted vertical lines illustrate where the sgRNAs impact on the resulting transcripts and protein. Positions of the varying in frame start codons and the resulting GNA1 isoforms are depicted. B) Immunofluorescence assay (IFA) showing the pellicle marker GAP45 and Ty signal in GNA1-mAID-Ty parasite line and its parental line (TIR1). C) Western blot revealing the signal of Ty-tagged GNA1 and TIR1 at different time points of auxin (IAA) treatment. D) IFA showing Ty signal in GNA1-mAID-Ty parasites in the absence of IAA and 18 hours after IAA treatment. E) Lysis plaques formed over one week of TIR1 and GNA1-mAID-Ty parasite cultivation in the presence or absence of IAA. F) Growth assay of TIR1 and GNA1-mAID-Ty parasites showing the number of parasites per vacuole after 24 hours of growth and varying durations of IAA treatment. B-E show representative images of three independent experiments. F shows the means and standard deviation (error bars) from representative data of one of three independent biological replicates averaging technical triplicates. p-values are given comparing the average number of parasites by two-sided Student’s t-test, between the indicated conditions. Abbreviations: MW, molecular weight; CAT, catalase.
Fig 3.
UDP-GlcNAc synthesis is disrupted in T. gondii that lack GNA1.
A) Percent 13C-labelling in T. gondii (TIR1) derived N-acetylglucosamine-6-phosphate (GlcNAc6P) in unlabelled parasites (natural abundance) or after incubation of intracellular or extracellular parasites in medium containing U-13C6-glucose for 24 or three hours, respectively. B) Relative abundance and fractional 13C-labelling in TIR1 and GNA1-mAID-Ty parasite metabolite extracts, following incubation of extracellular parasites in medium containing U-13C6-glucose for five hours in the absence of auxin (−IAA) or following 18 hours pre-treatment (+IAA). TIR1 −IAA parasites were incubated in medium with natural abundance glucose as an unlabelled control. Note that metabolites for which the abundance of labelled and unlabelled ions was too low to obtain reliable labelling data were deemed below limit of detection (<LOD, sum of ion intensity <1000 arbitrary units). C) Relative metabolite levels in TIR1 and GNA1-mAID-Ty, following no treatment (−IAA) or treatment with IAA for 18 hours during intracellular growth (+IAA). Data plotted show the means and standard deviation of three (A, B) or four (C) independent biological replicates. p-values from two-sided Student’s t-tests are given in A and C, comparing the indicated conditions. Abbreviations: Glc6P, glucose-6-phosphate; GlcN6P, glucosamine-6-phosphate; GlcNAc6P, N-acetylglucosamine-6-phosphate; Fru6P, fructose-6-phosphate; GlcNAc1P, N-acetylglucosamine-1-phosphate; UDPGlcNAc, uridine diphosphate N-acetylglucosamine.
Fig 4.
Disruption of TgGNA1 cannot be rescued by GlcNAc supplementation.
A) Lysis plaques formed by TIR1 and GNA1-mAID-Ty parasites over one week of growth when treated with auxin (+IAA) or not (−IAA) and supplemented with varying concentrations of glucosamine (GlcN) and/or N-acetylglucosamine (GlcNAc) as indicated. B-C) Relative metabolite abundance and fractional 13C-labelling in TIR1 and GNA1-mAID-Ty parasite extracts, incubated for five hours extracellularly in medium without glucose and containing U-13C6-glucosamine (B) or U-13C6-N-acetylglucosamine (C) in the absence of IAA or following IAA pre-treatment (+IAA, 18 h). TIR1 −IAA parasites were incubated in medium with natural abundance glucose as an unlabelled control. Note that metabolites for which the abundance of labelled and unlabelled ions was too low to obtain reliable labelling data were deemed below limit of detection (<LOD, sum of ion intensity <1000 arbitrary units). D) Relative metabolite levels in TIR1 and GNA1-mAID-Ty parasites, following no treatment (−IAA) or treatment with IAA (+IAA, 18 h) during intracellular growth in the presence or absence of glucose and supplemented with GlcNAc as indicated for the same duration. Overview on the left and detail on the right. E) Intracellular growth assay showing the number of parasites per vacuole after 24 hours of growth, treated for 48 hours with IAA, Glc or GlcNAc as indicated. F) Lysis plaques formed by TIR1 and GNA1-mAID-Ty parasites over one week of growth in presence or absence of auxin (IAA) and supplemented with glucose (Glc) or N-acetylglucosamine (GlcNAc) in Glc-free medium as indicated. A) shows representative images of three independent experiments. Data plotted in B-E show the means and standard deviation of three (B, C, E) and four (D) independent biological replicates, respectively. p-values from two-sided Student’s t-tests are given in D and E, comparing the indicated conditions. p-values in E compare the average number of parasites per vacuole. F) shows a representative image from three independent experiments. Abbreviations: Glc, glucose; GlcNAc, N-acetylglucosamine. Other abbreviations, see Fig 3.
Fig 5.
Lack of TgGNA1 disrupts GPI-anchored proteins causing an invasion defect.
A) Immunofluorescence assays (IFAs), showing the staining of the pellicle marker GAP45 and the GPI-anchored protein surface antigen 1 (SAG1) in TIR1 and GNA-mAID-Ty parasites after varying durations of auxin (IAA) treatment. B) Quantification of vacuoles displaying normal (even distribution) and abnormal SAG1 signal (uneven, patchy distribution with predominant accumulation inside the residual body), based on IFA images as shown in panel A. C) IFAs, showing staining of the rhoptry marker ARO and actin in TIR1 and GNA-mAID-Ty parasites after varying durations of auxin (IAA) treatment. D) Quantification of vacuoles displaying normal (club-shaped) and abnormal rhoptry morphology (shorter rhoptries with diffuse staining), based on IFA images as shown in panel C. E) Relative glycoinositolphospholipid (GIPL) abundance of untreated (−IAA) or IAA-treated (+IAA, 36 h) TIR1 and GNA1-mAID-Ty parasites. F) Percent of invaded TIR1 and GNA1-mAID-Ty parasite in the absence (–IAA) or after IAA treatment (18 hours) as determined by a red/green invasion assay. G) Quantification of glycoinositolphospholipids (GIPLs) in cells grown with the indicated treatments/supplementations for 18 hours. H) Percentage of invaded TIR1 and GNA1-mAID-Ty parasites, following the indicated treatment over 18 hours. A) and C) show representative images of three independent experiments. B, D-H show the means and standard deviation (error bars) from one of three independent biological replicates, averaging technical triplicates. For B and D, >100 vacuoles were counted and categorised, per replicate. p-values are given in B, D-H following two-sided Student’s t-tests, comparing the indicated conditions. p-values <0.05 were considered significant. Abbreviations: ARO, Armadillo repeats only protein. For other abbreviations, see Figs 3 and 4.