O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites

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.

Keywords: MIC2; O-fucosylation; Toxoplasma gondii; apicomplexa parasite; fucosyltransferase; glycoprotein secretion; glycosylation; invasion; protein glycosylation; thrombospondin-like repeat.

Figure 1.

MIC2 TSR1, -3, -4, and -5 are modified by dHexHex and TSR3, -4, and -5 are also C-mannosylated. A, amino acid sequences of the six TSR domains of MIC2. Cys resides are labeled in green, the O-fucosylation acceptor Ser/Thr is marked in red. C-Mannosylation sites are shown in blue. B, ETD of a semi-tryptic peptide from TSR1. The presence of the three product ions z9, z10, and y10 containing dHexHex identifies Ser-285, the predicted POFUT2 acceptor, as the glycosylated residue. C, HCD fragmentation of a TSR3 semi-tryptic peptide is consistent with modification by dHexHex disaccharide plus two C-mannoses. Collision energy of 10 eV (top) shows ion species corresponding to the precursor minus a Hex, a dHexHex, or minus dHexHex and 120.04 Da (broken green circle). Increasing the collision energy to 15 eV (bottom) allows detection of the precursor minus Hex, dHexHex, and two Hex −120.04 Da fragments. D, ion trap ETD of a semi-tryptic peptide from TSR4 identified the z7 product ion carrying both Hex and dHex, indicating Ser-485, the predicted POFUT2 acceptor site, as the residue modified by a dHexHex disaccharide. E, 10-eV HCD fragmentation of the Hex₂dHex glycoform, m/z 1120.7058 (top), and the Hex₃dHex ion species, m/z 1161.2106 (bottom), is shown. The most abundant ion, corresponding to the precursor minus the O-linked sugar, was the same for both species, consistent with the difference between the glycoforms being the Hex elongating the O-fucose and not one of the C-Man. Red triangle, fucose; green circle, mannose; broken green circle, C-Man cross-ring fragment; white circle, Hex; *, cysteine carbamidomethylation.

Figure 2.

HCD MS/MS of an O-fucosylated and C-mannosylated glycopeptide from TSR3 of MIC2. An HCD MS/MS spectrum was recorded with collision energy of 25 eV. Detection of the singly charged b11 and doubly charged y13, y13-NH₃ ions allows positioning of C-Man residues to the first and second Trp residues of the WXXWXXC motif, respectively. Other ions of interest are the doubly charged y19 (two Hex residues) and y22 (two intact Hex(s) or one intact Hex plus one Hex −120.04 Da fragment). Red triangle, fucose; green circle, mannose; white circle, Hex; *, cysteine carbamidomethylation.

Figure 3.

HCD MS/MS of an O-fucosylated and C-mannosylated glycopeptide from TSR4 of MIC2. HCD MS/MS spectrum was obtained with a collision energy of 20 eV. Detection of the Hex in the y-series ions only from y13 onward, both on singly and doubly charged species, confirms positioning of the Hex on the Trp in the WXXC motif, consistent with C-mannosylation. The y7 + dHexHex ion supports the presence of the disaccharide on Ser-485, the predicted POFUT2 site. Red triangle, fucose; green circle, mannose; white circle, Hex; *, cysteine carbamidomethylation.

Figure 4.

Localization of POFUT2 and generation of Δpofut2 and Δnst2 mutants. A, schematic representation of the strategy used to in situ tag pofut2 and the regions recognized by the primers used in B. B, PCR to show integration of the 3xMYC tag in the correct locus (P35 + P40). A control reaction amplifying a fragment of the pofut2 ORF was also performed (P37 + P38). C, IFA of RH ΔKu80 tachyzoites expressing pofut2 tagged at the C terminus with a 3xMYC tag and an ER marker (p30HDEL-YFP) shows partial co-localization. Scale bar, 2 μm. D, schematic representation of the strategy used to generate the pofut2 and nst2 gene disruptions. Cas9 was directed to excise each gene in the first and last exon. An mGFP-expressing cassette was inserted in each locus. The primers used in E and F are marked by arrows. E and F, genomic DNA was extracted after a first enrichment for mGFP-positive cells (mixed population) and after the final cloning step (KO). PCR analyses show substitution of the WT ORF with the mGFP cassette in both strains. Mixed pop, mixed population. All primer sequences can be found in Table S2. G, Δpofut2 parasites were complemented by expression of T. gondii POFUT2 fused to a 3xMYC C-terminal tag. MYC staining by IFA is consistent with ER localization. Scale bar, 2 μm.

Figure 5.

MIC2 is not O-fucosylated in either Δpofut2 and Δnst2 strains. A, structure of Glc-β-1,3-Fuc-α-KLH antigen. B, ELISA of the purified anti-GlcFuc antibody (1 μg/ml) shows the specificity of the polyclonal IgY for BSA-GlcFuc versus BSA-Fuc. C, Western blot testing of purified anti-GlcFuc antibody confirms its specificity for BSA modified with the GlcFuc disaccharide. No reactivity is observed probing with the preimmune IgY fraction. D, Western blot analysis with anti-FucGlc antibody identifies a β-elimination–sensitive band in WT corresponding to MIC2. No reactivity is shown in all three knockouts analyzed. MIC2 and tubulin controls are also shown. E, Western blotting of tachyzoites cell lysate with monoclonal anti-MIC2 shows reduced levels of cellular MIC2 in both knockouts. F, MIC2 levels were normalized to tubulin, and the average of three biological repeats is shown. G, Western blot analysis shows that complementation with POFUT2 restores reactivity to the anti-GlcFuc antibody, consistent with restored O-fucosylation. Additionally, MIC2 levels are again comparable with WT. Student's t test was used to compare the samples, and significant differences are marked: *, 0.05 < p < 0.01; **, p = 0.001. Error bars, S.D.

Figure 6.

MIC2 is mislocalized in both Δpofut2 and Δnst2 strains. A, MIC2 and, consequently, M2AP localization is affected in the KO, with both proteins accumulating in the early secretory pathway in a fraction of the vacuoles (white arrows). This is not a general microneme defect, as AMA4 location is unaffected. The white squares indicate the areas magnified on the right. Merge, antibody staining (red), DAPI (blue), and mGFP (green) for the O-fucosylation mutants. B, quantification of the percentage of vacuoles with abnormal MIC2, M2AP, and AMA4 localization. The average of three biological repeats ± S.D. (error bars) is shown. Student's t test was used to compare the samples, and significant differences are marked: *, 0.05 < p < 0.01; **, 0.01 < p < 0.0001; ***, p < 0.0001. n.s., not significant.

Figure 7.

Accumulated MIC2 partially co-localizes with a cis-Golgi marker. Parasites were transiently electroporated with GRASP55-mRFP (A) or P30HDEL-mCherry (B) and analyzed by structured illumination microscopy. Mislocalized MIC2 partially co-localizes with the cis-Golgi marker (GRASP55), but not with an ER-resident protein (P30HDEL). Additionally, GRASP55 localization itself is aberrant (A, arrowheads). White arrows, accumulation of MIC2 in the early/mid-secretory pathway. White squares, areas magnified below. Scale bars, 2 μm.

Figure 8.

Growth, attachment, and invasion are affected in Δpofut2 and Δnst2. A, representative images from the plaque assay. Scale bars, 2 mm. B, quantification of the number of plaques shows a 40% reduction plaque formation in the KOs compared with the parental strain. The average of four biological repeats ± S.D. (error bars) is shown. ***, p < 0.0005. C, representative images from the red/green invasion assays. Attached parasites look yellow (red + green), and invaded parasites are green. D, box plot for the red/green assay, comparing the invasion rate between parental lines and mutants (ratio of invaded to total parasites). E, box plot for the red/green assay, comparing the invasion rates between parental strain, Δpofut2, and the complemented parasites (Δpofut2 + POFUT2). Student's t test was used to compare the samples. and significant differences are marked: *, 0.05 < p < 0.01; **, p < 0.001; ***, p < 0.0005; ****, p < 0.00001. All p values are reported in Table S3. Scale bars, 5 μm.

Figure 9.

Egress defect in Δpofut2 and Δnst2 is not as severe as in the Δmic2. A, representative images showing the different phenotypes observed in the assay. Extracellular parasites were labeled with anti-SAG1 (in red). After permeabilization, all parasites were labeled with anti-β-tubulin (in green). As a result, parasites in intact vacuoles are green, whereas parasites in permeabilized vacuoles or that have egressed are labeled in green and red. B, quantification of the egress phenotypes. Quantifications are given as the average of at least three biological repeats ± S.D. (error bars). Student's t test was used to compare the samples, and all p values are reported in Table 2. Scale bars, 5 μm.

Figure 10.

Comparison of mic2 and O-fucosylation KOs. Shown is a schematic representation of differences and similarities between Δmic2 and Δpofut2 and Δnst2. Disruption of O-fucosylation is sufficient to reproduce the attachment and invasion defects observed when MIC2 is not being synthesized. Conversely, the parasite ability to egress is much more compromised in the Δmic2 versus Δpofut2 and Δnst2. Dark red arrows indicate a stronger defect, whereas small light red arrows indicate a milder phenotype. N/A, not applicable.