Toxoplasma chitinase-like protein orchestrates cyst wall glycosylation to facilitate effector export and cyst turnover

Significance

Chronic infections often involve pathogens in energy-conserving and immune-evading states. Toxoplasma gondii remains semidormant as bradyzoites in tissue cysts within muscles and neurons. The turnover of bradyzoite cysts, including cyst wall remodeling, is crucial for immune evasion, maintaining structural integrity, and enabling cyst reactivation, ultimately supporting long-term survival and persistence within the host. However, the mechanism underlying the remodeling of the cyst wall is largely unknown. Here, we identify a noncanonical chitinase TgCLP1, which is secreted to modulate glycoproteins within the cyst wall, thereby affecting effector export and cyst turnover to assure persistence.

Abstract

Toxoplasma bradyzoites reside in tissue cysts that undergo cycles of expansion, rupture, and release to foster chronic infection. The glycosylated cyst wall acts as a protective barrier, although the processes responsible for formation, remodeling, and turnover are not understood. Herein, we identify a noncanonical chitinase-like enzyme TgCLP1 that localizes to micronemes and is targeted to the cyst wall after secretion. Genetic deletion of TgCLP1 resulted in a thickened cyst wall that decreased cyst turnover, blocked the export of virulence effectors into host cells, and resulted in failure to persist during chronic infection. Genetic complementation with a series of mutants revealed that the GH19 glycosidase domain was crucial for regulating glycosylation of several glycoproteins in the cyst wall. Overall, our findings reveal that TgCLP1 is a multifunctional survival factor that modifies glycoproteins within the cyst wall to modulate export of virulence effectors and regulate turnover of tissue cysts.

Toxoplasma gondii is a significant cause of human and animal morbidity worldwide. Although infections are normally controlled by the immune response, they persist in a long-term chronic state to avoid clearance, leading to latency. T. gondii can invade any nucleated cell within its warm-blooded host leading to acute infections, characterized by fast-replicating tachyzoites (1). Following dissemination to tissues, tachyzoites can differentiate into bradyzoites residing within the tissue cysts commonly found in the brain and muscle cells (1). Current treatments can suppress the growth of tachyzoites but do not eliminate chronic infection since they are not effective at inhibiting bradyzoites (2), which divide infrequently and asynchronously (3, 4).

During chronic infection, tissue cysts can rupture releasing bradyzoites that can infect new host cells and either differentiate to tachyzoites or remain as bradyzoites that form daughter cysts, which assure persistent chronic infection (4–6). Tissue cysts feature a central matrix that surrounds bradyzoites, and they are encased in a membrane- and protein-rich cyst wall (7), which includes numerous secretory dense granule proteins (GRAs) (8), some of which are specifically expressed by bradyzoites (9). During maturation of cysts, the distribution of GRAs forming the wall is reorganized (10), implying that the cyst wall is actively remodeled during growth.

Tachyzoites utilize a kinase cascade to activate calcium-mediated microneme secretion and motility to drive egress (11). However, bradyzoites are resistant to calcium-induced egress (12), likely because the structural rigidity of the cyst wall poses a barrier for egress. The abundant cyst wall glycoprotein CST1 is heavily glycosylated by O-linked sugars in its mucin-like domain (13), and as such it is recognized by Dolichos biflorus agglutinin (DBA) that binds to GalNAc, synthesized by a family of α-GalNAc transferases (14, 15). Deletion of CST1 leads to fragile cysts that turn over more frequently, implying that it plays a role in cell wall rigidity and that this feature is important for cyst longevity (13). Although Toxoplasma tachyzoites harbor abundant N-glycosylated and O-glycosylated proteins (16–18), the specific glycan composition of the bradyzoite cyst wall is largely unknown.

Glycosyl hydrolases (GH), also referred to as glycosidases, are a diverse group of multifunctional enzymes, capable of breaking glycosidic bonds within sugars or glycoproteins. Toxoplasma has seven putative glycosidases, among which chitinase-like protein 1 (CLP1) and GRA56 were previously identified as secretory microneme and dense granule proteins, respectively (19, 20). Previous studies showed that treatment of cysts with either chitinase or AMCase-secreting macrophages resulted in the rupture of cyst walls, suggesting that the cyst wall contains a chitin-like polysaccharide (21, 22). However, the genome lacks a chitin synthase, suggesting that a chitin-like glycosidic linkage, (i.e., GlcNAcβ-1,4-GlcNAc), may be present in the cyst wall. Therefore, investigating TgCLP1 is crucial for understanding a mechanism(s) underlying glycosylation remodeling in the cyst wall, thereby regulating cyst turnover.

Here, we combined live-cell imaging and GH treatment to demonstrate the role of carbohydrates in comprising the cyst wall. We further examined the role of CLP1 and show that it contributes to daughter cyst formation and persistence in vivo, phenotypes that are linked to alterations in cyst wall turnover, and export of virulence factors. Altogether, our work demonstrates that CLP1 modifies cyst wall glycoproteins to maintain chronic infection, thus deepening our understanding of bradyzoite biology.

Results

Essential Role of Carbohydrate Linkages in Maintaining the Stability of the Cyst Wall.

To test the effect of glycosidases on the integrity of the cyst wall, we used a transgenic line of the type II ME49 strain expressing mCherry under the regulation of bradyzoite-specific promoter bag1 (Fig. 1A). In vitro–derived cysts, produced by culture in alkaline (pH 8.2) medium for 7 d, were incubated with trypsin or a GH mixture, chitinase (TriChi), or endo-β-glucanase (TriEG) from Trichoderma and rupture of the cyst was recorded using time-lapse microscopy. Treatment with GH mixture or TriChi resulted in cyst rupture in a dose-dependent manner, whereas TriEG had no effect (Fig. 1B and SI Appendix, Fig. S1A). Incubation with GH mixture and chitinase still led to the rupture of cysts in the presence of protease inhibitors, indicating their activities are likely not due to contaminating proteases, while the digestion of the cyst wall by trypsin was completely blocked (SI Appendix, Fig. S1 B and C). Collectively, these studies indicate that cyst wall integrity depends both on proteins and carbohydrate linkages.

Fig. 1.

TgCLP1 is a microneme protein that is secreted and targeted to the cyst wall. (A) Live imaging for the treatment of in vitro–derived reporter cysts with GH mixture, chitinase (TriChi) and endoglucanase (TriEG) from Trichoderma, or trypsin for 8 min. (Scale bar, 5 μm.) (B) Quantification of the time to cyst rupture following GH treatment (N = 3 biological experiments, n = 9 technical replicates, means ± SD). (C) Intracellular localization of TgCLP1-HA in bradyzoites within in vitro–derived cysts. Stained with mouse anti-HA (green), rabbit anti-MIC2 (magenta), and biotinylated DBA (orange). (Scale bar, 5 µm.) (D) Gaussia luciferase assay of secreted TgCLP1-Gluc-HA by in vitro–derived bradyzoites pretreated with 2.5 µM Compound 1 or 5 µM 3-MB-PP1 for 5 min, followed by stimulation with either DMSO (vehicle) or 3 µM A23187 in extracellular (EC) buffer for 15 min (N = 3, n = 9). Data were represented as means ± SD and analyzed by one-way ANOVA with Dunn’s multiple comparison correction test. ***P < 0.001. (E) Ultrastructure expansion microscopy (U-ExM) of TgCLP1-HA in vitro–derived cysts treated with DMSO (vehicle) or 3 µM A23187 for 15 min. Stained with mouse anti-HA (green) and biotinylated DBA (magenta). (Scale bar, 5 µm.) (F) Quantification of relative fluorescence intensity of TgCLP1-HA distributed in the cyst wall based on U-ExM images from (E) (N = 3, n = 18). Data were represented as means (different experiments are denoted with different symbols) ± SD and analyzed by two-tailed unpaired Student’s t test. ***P < 0.001.

TgCLP1 Is a Microneme Protein that Is Secreted and Targeted to the Cyst Wall.

There are seven glycosidases in the genome of T. gondii based on conserved GH family homology domains (SI Appendix, Fig. S1D). HyperLOPIT analysis identified the glycosidase CLP1 (TGME49_293770) as a putative microneme protein. Phylogenetic analysis of proteins with a GH19 domain demonstrated that TgCLP1 is most related to the chitinase-like proteins in Neospora caninum and Hammondia hammondi, which have similar life cycles containing both tachyzoites (acute stage) and bradyzoites (chronic stage) (23) (SI Appendix, Fig. S1E).To confirm the location of TgCLP1, we introduced a 3xHA tag to the C terminus of TgCLP1 using CRISPR/Cas9 (SI Appendix, Fig. S1F). An immunofluorescence assay (IFA) revealed that TgCLP1 is colocalized with the microneme marker MIC2 in bradyzoites (Fig. 1C). To investigate whether TgCLP1 can be secreted from bradyzoites, we fused the Gaussia luciferase reporter at the C terminus of TgCLP1 together with a 3xHA tag to generate CLP1-Gluc using CRISPR/Cas9 (SI Appendix, Fig. S1 G–I). Stimulation of in vitro–derived cysts with A23187 revealed that CLP1-Gluc was secreted in a calcium-dependent manner that was inhibited by the CDPK1 inhibitor 3-methylbenzyl pyrazolopyrimidine (3-MB-PP1) and the PKG inhibitor Compound 1 (Fig. 1D). To examine the distribution of TgCLP1 after secretion, we utilized ultrastructure expansion microscopy (U-ExM) to localize TgCLP1 released from bradyzoites within the cysts. Following A23187 treatment, TgCLP1 was discharged from micronemes and displayed a punctuate distribution on the cyst wall compared to the apical micronemal pattern seen in vehicle-treated cysts (Fig. 1E). Quantitative analysis of the distribution of signal revealed that A23187-stimulated samples had a significantly higher proportion of TgCLP1 on the cyst wall compared with vehicle-treated control (Fig. 1F). This finding suggests that TgCLP1 is a microneme protein that is targeted to the cyst wall after secretion by bradyzoites.

TgCLP1 Is a Noncanonical Endochitinase.

TgCLP1 harbors two conserved glutamic acid residues E158 and E177 in the catalytic loop, while one potential catalytic glutamic acid residue that is found in most GH19 chitinases (24), is instead histidine H169 (Fig. 2A and SI Appendix, Fig. S2A). To investigate the enzymatic activity of TgCLP1, we utilized five potential substrates containing different units of β-1,4-HexNAc linkages (Fig. 2B). Compared with TriChi, recombinant full-length TgCLP1 (rTgCLP1) purified from Escherichia coli (SI Appendix, Fig. S2 B and C) displayed modest activity against chitin using the dinitrosalicylic acid (DNS) assay that measures free reducing sugars released by hydrolysis of glycosidic bonds (Fig. 2C). Additionally, rTgCLP1 exhibited greater activity against the fluorescent substrate 4-MU-(GlcNAc)₃ compared with 4-MU-(GlcNAc)₂ (Fig. 2D), which quantifies hydrolysis of the internal linkage to 4-methyl umbelliferol (4-MU), indicating a preference to act as an endochitinase. Consistent with this activity, coincubation with chitopentaose, but not cellopentaose, dampened the catalytic degradation of 4-ΜU-(GlcNAc)₃ by rTgCLP1 (Fig. 2E). To investigate the residues essential for the enzymatic activity, we expressed a truncated GH19 domain (SI Appendix, Fig. S2 B and D), which had slightly lower activity than full-length enzyme (SI Appendix, Fig. S2E), and generated a series of point mutations (SI Appendix, Fig. S2D). Compared with wild-type rTgCLP1^GH19, E158A, H169A, and E177A mutants all impeded activity on 4-MU-(GlcNAc)₃ (Fig. 2F). In addition, mutation of H169 to glutamic acid (E) impaired the enzymatic activity to 4-MU-(GlcNAc)₂ and 4-MU-(GlcNAc)₃ (Fig. 2F and SI Appendix, Fig. S2F), indicating that the enzymatic mechanism of TgCLP1 differs from classical chitinases requiring two glutamic acids for activity. Taken together, these findings indicate that TgCLP1 displays activity on short oligosaccharides composed of β-1,4-linked GlcNAc, with relatively weak activity on chitin, consistent with an active site that is distinct from canonical chitinases.

Fig. 2.

TgCLP1 is a noncanonical endochitinase. (A) Multiple sequence alignment of CLP orthologs. Conserved residues in gray, catalytic residues in green boxes. (B) Diagram of chitinase substrates. (C) Enzyme activity of full-length recombinant TgCLP1 (rTgCLP1) acting on 1% colloidal chitin. TriEG and TriChi serve as controls. One-way ANOVA with Dunn’s multiple comparison correction test (N = 3, n = 9, means ± SD), *P < 0.05, ***P < 0.001. (D) Activities of rTgCLP1 on fluorescent oligosaccharides 4-MU-(GlcNAc)₂ and 4-MU-(GlcNAc)₃. rTgCLP1, TriEG, or TriChi (each at 10 µM), were incubated with 10 µM fluorescent substrates for 1 h. Two-way ANOVA with Geisser–Greenhouse correction test (N = 3, n = 9), n.s. = not significant, ***P < 0.001. (E) Substrate competition assay using 50 µM cellopentaose or 50 µM chitopentaose in the presence of 10 µM of 4-MU-(GlcNAc)₃ incubated with 10 µM of rTgCLP1. Kruskal–Wallis test (N = 3, n = 9, means ± SD), n.s. = not significant, ***P < 0.001. (F) Activities of wild-type rTgCLP1^GH19 and point mutants on 10 µM 4-MU-(GlcNAc)₃. Kruskal–Wallis test (N = 3, n = 9, means ± SD), n.s. = not significant, ***P < 0.001.

TgCLP1 Contributes to Cyst Turnover.

During chronic infection, tissues cysts undergo successive waves of slow expansion, followed by rupture and generation of daughter cysts (5, 6, 25, 26) (Fig. 3A). To monitor this process in vitro, we examined the formation of daughter cysts, defined by their small size, using in vitro–derived cysts (Fig. 3B). Deletion of clp1 using CRISPR/Cas9 (SI Appendix, Fig. S3 A and B) led to a decrease in daughter cell number (Fig. 3C) and increase in cyst size (SI Appendix, Fig. S3D), but did not affect the growth of tachyzoites (SI Appendix, Fig. S3C). In contrast, knockout of cst1, a wall glycoprotein needed for structural integrity of the cyst (13), led to dramatically increased daughter cyst formation (Fig. 3 B and C). Despite decreased ability of ∆clp1 parasites to generate daughter cysts, there was no difference in the percentage of DBA-positive cysts initially formed under alkaline growth conditions, indicating that TgCLP1 is not required for tachyzoite to bradyzoite stage conversion (SI Appendix, Fig. S3E).

Fig. 3.

TgCLP1 contributes to the cyst turnover. (A) Schematic of daughter cyst formation. (B) IFA of in vitro–derived cysts from parental and mutant lines. White arrows indicate daughter cysts stained with biotinylated DBA to mark the cyst wall or rabbit anti-GRA7 to mark the parasitophorous vacuole membrane (green), mouse anti-BAG1 (red), rabbit anti-SAG1 (magenta), and DAPI (blue). (Scale bars, 10 µm.) (C) Percentage of daughter cysts after cultured at alkaline pH for 7 d. Statistics were analyzed by two-tailed unpaired Student’s t test (N = 3, n = 9, means ± SD), ***P < 0.001. (D) Electron microscopy (EM) of in vitro–derived cysts. (Scale bar, 500 nm.) White arrows indicate the cyst wall. (E) Cyst wall thickness measured from electron graphs of in vitro–derived cysts, two-tailed unpaired Student’s t test (N = 2, n = 16, means ± SD), ***P < 0.001. (F) Percentage of in vitro–derived cysts showing partial egress in response to A23187, two-tailed unpaired Student’s t test (N = 3, n = 9, means ± SD), ***P < 0.001. (G) IFA of spontaneous rupture of ex vivo cysts treated with EC buffer for 0 or 48 h. Stained with DBA (white). (Scale bar, 5 µm.) (H) Comparison of spontaneous rupture of in vitro–derived or ex vivo cysts. rTgCLP1 (50 µM) was incubated with ∆clp1 cysts in EC buffer containing 0.25% octyl glucoside. Data from 48 h were normalized to 0 h, multiple t test with false discovery rate (N = 3, n = 9, means ± SD), n.s., not significant, ***P < 0.001. (I) Examining the role of TgCLP1 in the reactivation of bradyzoites using bradyzoites released from ex vivo cysts by pepsin treatment. HFFs were infected with bradyzoites and cultured for 24 h in normal medium to allow for the reactivation, followed by quantification of vacuoles positive for tachyzoite stage–specific SAG1 and bradyzoite stage–specific BAG1 in IFA. Data were represented as mean ± SD pooled from three biological replicates (N = 3, n = 9). Two-way ANOVA, n.s. not significant.

EM demonstrated that ∆clp1 in vitro–derived cysts had thicker cyst walls compared to parental strain cysts (Fig. 3 D and E), indicating that TgCLP1 may play a role in modification of the cyst wall. We have previously shown that bradyzoites undergo limited egress following stimulation with A23187 resulting in selective release of a few bradyzoites without cyst rupture (12). Cysts containing ∆clp1 parasites showed lower stimulated egress compared to parental cysts in response to A23187 (Fig. 3F and SI Appendix, Fig. S3F), suggesting that the thicker wall of ∆clp1 cysts is more rigid. To further confirm the role of TgCLP1 in cyst wall rigidity, we developed a spontaneous excystation assay by incubating cysts with EC buffer to allow the spontaneous rupture of cyst and release of bradyzoites, as described previously (27). We observed a significant reduction in the spontaneous rupture of in vitro–derived or ex vivo ∆clp1 cysts compared to parental control (Fig. 3 G and H), a defect that was restored by addition of rTgCLP1 in the presence of octyl glucoside for permeabilization of the vacuole membrane surrounding the cyst wall (Fig. 3H and SI Appendix, Fig. S3G). A previous study suggested that TgCLP1 was involved in the reactivation of in vitro–induced bradyzoites (19). We further evaluated whether disruption of clp1 affected in vivo–derived bradyzoite reactivation following cyst wall rupture. Once liberated from ex vivo cysts by pepsin treatment, ∆clp1 bradyzoites were inoculated onto HFFs in normal medium for 24 h to allow for the reactivation. Quantification of the vacuoles positive for tachyzoite-specific marker SAG1 and/or bradyzoite-specific marker BAG1 showed that disruption of clp1 did not affect reactivation (Fig. 3I). Taken together, these findings suggest that TgCLP1 participates in cyst wall maturation and/or turnover, and in its absence, the cyst wall is thicker and more rigid.

Lectin Pulldown Identifies Several Secretory Glycoproteins Modulated by TgCLP1.

To explore the glycan composition of the cyst wall, we developed a method to enrich cyst walls from in vitro–derived cysts (Fig. 4A), resolved glycoproteins by SDS-PAGE, and probed them with a panel of lectins with different sugar specificities (SI Appendix, Fig. S4 A and B). We found that DBA, HPA (Helix pomatia agglutinin), WGA (wheat germ agglutinin) and Jacalin recognized glycoproteins in the cyst wall, indicative of the presence of α-linked GalNAc, β-GlcNAc and Gal β-1,3-GalNAc (SI Appendix, Fig. S4B). To investigate whether TgCLP1 is involved in modulating these sugar linkages, we performed lectin blotting using both tachyzoites and cyst wall fractions of parental vs ∆clp1 mutants. We only observed significant differences in the glycoproteins profiled by WGA in cyst wall fractions (Fig. 4B), while the other lectin blotting assays did not reveal obvious differences (SI Appendix, Fig. S4 C and D). To identify glycoproteins differentially affected by TgCLP1, we performed tandem mass spectrometry (MS/MS) analysis of the WGA pulldown products from in vitro–derived cysts. We observed abundant glycoproteins in the cyst wall including CST1, MAG1, BPK1, and GRA7 (9) (Fig. 4C and Dataset S5). To identify potential substrates of TgCLP1, we used these criteria to search the MS/MS data including 1) secretory proteins that localize to the cyst wall; 2) ≥ 4.91 of Log₂fold changes; 3) the presence of predicted N-glycosylation or O-glycosylation sequences. We identified two up-regulated proteins KRUF4 and TGME49_250955, and two down-regulated proteins GRA56 and TGME49_291630 (Fig. 4 C and D). To validate whether disruption of TgCLP1 affects glycosylation of these candidates, we epitope-tagged these proteins using CRISPR/Cas9 (SI Appendix, Fig. S4 F–H). KRUF4 and TGME49_250955 share the same gene sequence in their 3′ ends (SI Appendix, Fig. S4E), and peptides from the MS/MS analyses could potentially match either; however, since TGME49_250955 is not expressed in bradyzoites (SI Appendix, Fig. S4H), we focused on KRUF4. Loss of TgCLP1 resulted in reduced staining of GRA56-HA on the cyst wall, while KRUF4 redistributed from the matrix to aggregate on the cyst wall (Fig. 4E). In contrast, knockout of clp1 did not affect the localization of all candidates in tachyzoites (SI Appendix, Fig. S4H).

Fig. 4.

WGA pulldown identifies glycoproteins that are modulated by TgCLP1. (A) Purification of in vitro–derived cysts for lectin profiling. (B) Biotinylated succinylated WGA was used to probe glycoproteins in the lysates of in vitro–derived cysts, followed by detection with IRDye^® 800CW Streptavidin. ALD1 was used as loading control. (C) Relative enrichment (fold change of Log₂) based on total spectral counts from LC–MS/MS analysis of WGA pulldown products from in vitro–derived cysts of ∆clp1 vs parental (n = 2). (D) Summary of top hits identified by LC–MS/MS analysis of WGA pulldown. (E) Localization of GRA56 and KRUF4 in in vitro–derived cysts. Top panel, domain architecture of GRA56 and KRUF4 with added HA or Ty tag. Stained with mouse anti-Ty or anti-HA (green), rabbit anti-GRA7 (red), biotinylated DBA (magenta). (Scale bar, 10 µm.) The percentage of GRA56-HA and Ty-KRUF4 in the cyst wall was shown on the Right panel. Two-tailed unpaired Student’s t test (N = 3, n = 18, means ± SD), ***P < 0.001. (F) WGA pulldown products from in vitro–derived cysts were resolved on SDS-PAGE gels, blotted with mouse anti-Ty or anti-HA, rabbit anti-ALD1, rabbit anti-GRA7, and imaged with LI-COR-specific secondary antibodies. ALD1 and GRA7 serve as loading controls. White arrows indicate target bands. Quantification of the ratio of gray intensities from Western blotting of GRA56-HA and Ty-KRUF4 normalized to GRA7 was shown on the bottom (N = 3, means ± SD). (G) Model showing the proposed modulation of GRA56 and KRUF4 by TgCLP1. The gray hexagon represents unknown monosaccharide. The blue square represents the GlcNAc unit.

To further confirm the differential glycosylation of GRA56 and KRUF4, we performed WGA lectin pulldowns of in vitro–derived cyst walls using HA or Ty-tagged parasites. GRA56 was readily detected in the pulldown from parental cysts but absent from ∆clp1 samples, suggesting that TgCLP1 modifies GRA56, enabling its recognition by WGA (Fig. 4F). Also, we observed significant enrichment of KRUF4 in the pulldown from cyst wall fraction of the ∆clp1 sample, suggesting that KRUF4 is modified by TgCLP1 to prevent WGA recognition (Fig. 4F). In contrast, TGME49_291630 was not affected in the lectin pulldown assay (SI Appendix, Fig. S4I), ruling out its possibility of modulation by TgCLP1. Collectively, these findings suggest TgCLP1 contributes to the cleavage of glycosidic bonds within GRA56 and KRUF4 that result in their recognition or masking from WGA, respectively (Fig. 4G).

Mutant Complementation of TgCLP1 Identifies Its Key Functional Domains.

To dissect functional domains of TgCLP1, we complemented the ∆clp1 mutant parasite with constructs designed to test targeting and glycosidase function (Fig. 5A and SI Appendix, Fig. S5 A–C). Deletion of the N terminus (CLP1^ΔN-ter) or replacement with the N terminus containing a carbohydrate-binding domain from rice chitinase OsChi (CLP1^OsCBD) resulted in loss of apical targeting in tachyzoites, while deletion of the C terminus (CLP1^ΔC-ter) had no effect on targeting (Fig. 5B). Consistent with this finding, fusion of the N terminus of TgCLP1 with a YFP reporter resulted in apical targeting, while a C-terminal fusion remained trapped in the ER and loss of the signal peptide resulted in accumulation in the cytosol (Fig. 5C).

Fig. 5.

Functional analysis of TgCLP1 domains. (A) Schematic of wild-type versus mutant TgCLP1 complementation constructs. (B) IFA of EC TgCLP1 complementation tachyzoites. Stained with mouse anti-HA (green) and rabbit anti-MIC2 (magenta). (Scale bar, 2 µm.) (C) IFA of fluorescent reporter YFP constructs fused with TgCLP1 terminal domains. Parasites fixed at 48 h after transient expression and stained with mouse anti-HA (green), rabbit anti-MIC2 (magenta), and DAPI (blue). (Scale bar, 5 µm.) (D and E) Daughter cyst formation assay (D) and Spontaneous excystation (E) of TgCLP1 complementation constructs expressed by in vitro–derived cysts. Data in (D and E) were both pooled from three independent experiments containing three technical replicates each (N = 3, n = 9) and represented as means ± SD. Statistics were analyzed by one-way ANOVA with Dunn’s multiple comparison correction test, ***P < 0.001.

To investigate whether complementation restored functions of TgCLP1 in cyst turnover, we determined the percentage of daughter cyst formation among complemented parasite lines. As expected, complementation with constructs that fail to target correctly (i.e., CLP1^OsCBD, CLP1^ΔN-ter) led to a lower percentage of daughter cysts, as did deletion of the C terminus (i.e., CLP1^∆C-ter) (Fig. 5D). Consistent with deletion of GH19 catalytic domain, mutation of conserved catalytic residues H169 or E177 to alanine or glutamic acid failed to restore daughter cyst formation (Fig. 5D), implying a functional role of the GH19 domain in cyst turnover. Furthermore, replacement of the GH19 domain of TgCLP1 with that of rice chitinase OsChi (CLP1^OsGH19) fully rescued the defective daughter cyst formation in ∆clp1 parasites (Fig. 5D). Next, we also checked whether these complemented lines restored the phenotype of spontaneous cyst rupture. Consistent with the data of daughter cyst formation, only CLP1^WT and CLP1^OsGH19 restored cyst rupture (Fig. 5E), while the other complementation lines failed to rescue the defect. In summary, our data suggest that the N terminus of TgCLP1 is required for targeting to micronemes. Moreover, the GH19 domain contributes to the spontaneous cyst rupture and turnover, which does not appear to depend on a unique specificity of the GH19 domain of TgCLP1.

TgCLP1 Depletion Impairs the Export of Effectors.

Given its role in modulating glycoproteins in the cyst wall in vitro, we next investigated the role of TgCLP1 during infection in mice (Fig. 6A). No difference was observed in the dissemination of ∆clp1 vs. parental parasites following i.p. inoculation of CD-1 mice that were monitored by bioluminescent imaging of luciferase expression (Fig. 6B and SI Appendix, Fig. S6A). We quantified the cyst burden in brains of chronically infected mice by DBA staining (SI Appendix, Fig. S6B), and observed that the parental strain underwent a slight decrease after 6 wk, that rebounded later in infection, consistent with the production of daughter cysts (Fig. 6C). In contrast, ∆clp1 infected mice initially established chronic infection in the brain, but then underwent a progressive decline that showed no recovery (Fig. 6C and SI Appendix, Fig. S6C). Quantification of the cyst size demonstrated that ∆clp1 cysts were significantly bigger than parental cysts (Fig. 6D and SI Appendix, Fig. S6D), consistent with in vitro–derived cysts (SI Appendix, Fig. S3D). To explore the mechanism underlying the clearance of ∆clp1 cysts in vivo, we investigated two major pathways that have been associated with the control of cyst burden: alternatively activated macrophages (AAMØ) that secrete chitinase (AMCase) (22) and interferon-γ responses modulated by parasite effectors (28–30).

Fig. 6.

TgCLP1 mediates effector export to facilitate immune evasion. (A) Schematic for chronic infection in vivo. (B) Bioluminescence monitoring of acute infection in CD-1 mice (N = 3, n = 9, means ± SD). (C and D) Quantification of cyst numbers/brain (C) and size (D) at weeks postinfection. Data in (C) were combined from 3 to 5 mice for each group (each dot represents a single animal). Cyst size in (D) was from n = 20 cysts for each time-point (pooled from 3 to 5 animals). The dotted line represents the limit of detection (=50 cysts). Data from (D) were represented as means ± SD. Statistics were analyzed by the multiple t test with FDR approach, n.s., not significant, *P < 0.05, ***P < 0.001. (E) Percentage of ruptured ex vivo purified cysts after incubation with BMDMs for 6 h (N = 3, n = 9, means ± SD). One-way ANOVA with Dunn’s multiple comparison correction test, n.s., not significant, ***P < 0.001. (F) IRF1 induction in host cell nucleus after 100 U/mL IFN-γ stimulation for 24 h, following 6 d of culturing at alkaline pH. Stained with biotinylated DBA (green), IRF1 (magenta), and cell nucleus (blue). Dotted white cycles indicate the area for quantification. (Scale bar, 10 µm.) Quantification of the relative fluorescence intensities of IRF1 was shown on the Right panel (N = 3, n = 30). Values were corrected using parasite-infected host cells without IFN-γ stimulation. One-way ANOVA with Dunn’s multiple comparison correction test, n.s., not significant, ***P < 0.001. (G) Monitoring the export of effectors TgNSM-Ty and TgIST-Ty from in vitro–derived bradyzoites. Parental, ∆clp1, and ∆clp1/CLP1 complemented strains were stably transformed with Ty-epitope tagged copies of TgNSM and TgIST to evaluate export. Stained with biotinylated DBA (magenta), mouse anti-Ty (green), and DAPI (blue). Dotted white outlines indicate the area for quantification. (Scale bar, 5 µm.) Quantification of relative fluorescence intensities of effectors in the host nucleus was shown on the Right panel (N = 3, n = 30). Values were corrected using noninfected host cells. Data are represented as means ± SD. Statistics were analyzed by two-way ANOVA with Dunn’s multiple comparison correction test, n.s., not significant, ***P < 0.001.

To test the first mechanism, we isolated bone marrow–derived macrophages (BMDMs) from C57BL/6 mice and induced M2 polarization using Interleukin-4 (IL-4), leading to high chitinase-like activity against 4-MU-(GlcNAc)₂ (SI Appendix, Fig. S6E). Incubation of parental, ∆clp1, and clp1 complemented cysts with M2 cells showed that mutant cysts were more resistant to the BMDMs digestion (Fig. 6E and SI Appendix, Fig. S6F), suggesting that macrophages are not responsible for the clearance of ∆clp1 cysts in vivo.

We next explored the role of interferon-γ responses by monitoring the expression of host cell interferon regulatory factor 1 (IRF1) in the nucleus, a process that is blocked by prior infection (31). Previous studies showed that TgIST and TgNSM are trafficked to the host cell nucleus where they disrupt IFN-γ-induced gene expression and cell death pathways (28–30). Here, we utilized Pru∆ist and ME49∆nsm as controls for the IRF1 staining in the nuclei of infected host cells. Infection with parental bradyzoites led to undetectable IRF1 in the nucleus, while ∆clp1 bradyzoites lost the ability to suppress this pathway in comparison to the complemented parasites (Fig. 6F). In contrast, we found that clp1 knockout in tachyzoites did not affect the IRF1 induction in host cells after IFN-γ stimulation (SI Appendix, Fig. S6 G and H). To define the molecular basis for this phenotype, we further examined the secretion of IST and NSM. Loss of TgCLP1 did not affect the export of NSM and IST in tachyzoites (SI Appendix, Fig. S6 I–K) but resulted in significant reduction in secretion by bradyzoites into the host nucleus, while complementation of clp1 successfully restored the secretion of TgNSM and TgIST in bradyzoites (Fig. 6G). Collectively, these findings indicate that the clearance of cysts in mice infected with ∆clp1 parasites may be secondary to inhibition of effector export to the host nucleus once parasites have converted to bradyzoites/become encysted.

Discussion

Chronic infection by T. gondii is maintained by tissue cysts surrounded by a wall that provides a protective niche and resists immune clearance to maintain a reservoir of infectious bradyzoites. How the cyst wall is formed and modified during growth is poorly understood. We show that Toxoplasma bradyzoites secrete a microneme chitinase-like protein TgCLP1 that is targeted to the cyst wall. Phenotypical characterization demonstrated that TgCLP1 contributes to cyst turnover and in its absence, thickening of the cyst wall impedes formation of daughter cysts. Biochemical studies demonstrated that TgCLP1 is a noncanonical endochitinase, despite its glycan substrate not being found in the cyst wall. Lectin pulldown revealed that TgCLP1 differentially affects the glycosylation of glycoproteins in the cyst wall. Finally, TgCLP1 is needed to maintain efficient export of virulence effectors from bradyzoites. Collectively our studies reveal that modification of glycoproteins is critical to proper formation of the cyst wall and for persistence in vivo.

TgCLP1 localizes to secretory micronemes and is targeted to the cyst wall after induced secretion, consistent with a role in modifying components of the cyst wall. Genetic complementation demonstrated that the TgCLP1 N terminus is involved in its trafficking to micronemes, while its C terminus likely plays a role in its targeting to the cyst wall. The majority of microneme proteins involved in host cell invasion by T. gondii are secreted onto the surface of EC tachyzoites (32). In contrast, TgCLP1 is released into the cyst matrix, similar to the perforin-like protein PLP1 that is released into the parasitophorous vacuole (PV) prior to egress by tachyzoites (33). Previous studies showed that microneme secretion is driven by calcium signaling in tachyzoites during invasion and egress (11); however, this pathway is dampened in bradyzoites (12). Hence, it remains uncertain what signaling pathway drives release of TgCLP1 within tissue cysts.

Previous literature suggests several key events in cyst turnover, including cyst rupture, bradyzoite invasion, daughter cyst formation, and cyst growth during chronic infection by T. gondii (3, 27, 34, 35). A significant gap in understanding cyst turnover is the mechanism by which bradyzoites egress from the cyst. Our findings that cyst walls are thicker and daughter cyst formation is impaired in ∆clp1 parasites, support a model where TgCLP1 contributes to the rupturing and/or remodeling of the cyst wall, facilitating the egress by bradyzoites. Recently, TgCLP1 was reported to affect the recrudescence of bradyzoites in vitro based on failure of in vitro differentiated cysts to re-emergence as tachyzoites after return to normal culture (19). In our study, we allowed tissue cysts to form in vivo and then liberated bradyzoites using pepsin. Once liberated, clp1 bradyzoites readily infected cells and reverted to tachyzoites as shown by repression of BAG1 and expression of SAG1. Collectively, we interpret these studies to indicate that TgCLP1 is not required for bradyzoite to tachyzoite reactivation but rather is involved in cyst wall modifications that are critical for escape.

Chitinases specifically target chitin, which in its occurrence in arthropod exoskeletons and fungal cell walls consists of crystalline or semicrystalline arrays of polysaccharides composed of repeated units of β-1,4-N-acetylglucosamine (GlcNAc) (36). GH19 chitinases are unified by their shared lysozyme-like tertiary structure and ability to hydrolyze internal -4GlcNAcβ1,4-GlcNAcβ1- linkages, but vary considerably in their ability to work on chitin, modified forms of the free polysaccharide, and nonreducing terminal linkages (37). TgCLP1 is unusual in that it lacks one of two acidic residues conserved in GH19 active sites (24) that is instead replaced by a histidine (H169). Nonetheless, biochemical assays demonstrate that TgCLP1 hydrolyzes short oligosaccharides containing -4GlcNAcβ1,4-GlcNAcβ1- linkages, and appears to prefer internal linkages and free oligosaccharides over chitin. In addition, complementation with the GH19 domain from rice chitinase fully recovered the phenotype of ∆clp1 cysts, suggesting that an overlapping and conserved hydrolase activity is essential for the cyst turnover. Additionally, chitinase from Trichoderma can digest the cyst wall, well supporting the idea that the cyst wall contains chitin-like linkages. Given that T. gondii and its mammalian hosts lack candidate genes for a chitin synthase, the cyst wall is likely to contain alternative chitin-like linkages for which the current substrates may serve as proxy. For example, N-glycans contain a core -4GlcNAcβ1,4-GlcNAcβ1- linkage, which endo-β-N-acetylglucosaminidases from the CAZy GH18 family of glycohydrolases, including other classes of chitinases, have evolved to cleave. N-glycans of Toxoplasma tachyzoites possess this conserved linkage (16), but little is known about linkages of O-glycans of bradyzoites as the small quantities of available material pose challenges to current analytical methods. Further studies are warranted to define the precise specificity and mode of action of TgCLP1.

By capturing glycoproteins in the cyst wall that bind to WGA, we demonstrate that TgCLP1 regulates the glycosylation of two GRA proteins including KRUF4 and GRA56 (20, 38), to ensure their proper location within the cyst wall. The differential recognition of these GRA proteins by WGA, which recognizes terminal GlcNAc, suggests that TgCLP1 is involved in trimming oligosaccharides to either mask terminal GlcNAc in the case of KRUF4 or to expose terminal GlcNAc as in the case GRA56. Additionally, other GRA proteins were identified in the differential MS/MS study, suggesting the action of TgCLP1 may be more widespread. The altered cyst wall thickness and increased rigidity of the cyst wall in ∆clp1 parasites suggest that these trimming actions are important in maturation of the cyst wall to assure proper plasticity needed for maturation and turnover. Previous studies on CST1, GRA2, and NST1 have also revealed that glycosylation is key for the cyst wall development (13, 39, 40).

IFN-γ immune responses (41), along with AMCase secreted by alternatively activated macrophages in the CNS (22), both play critical roles in controlling the cyst burden during chronic infection. Mutant ∆clp1 parasites were less susceptible to rupture by BMDM, suggesting that AMCase is not responsible for the clearance observed in vivo. Instead, we show that TgCLP1 is involved in the bradyzoite effector export, which is possibly associated with the regulation of host cell immune responses, indicating its role as a bradyzoite survival factor. Toxoplasma secretes several effectors, such as TgIST and TgNSM to inhibit INF-gamma signaling in host cells thus evading and co-opting the host’s immune defenses to favor its own survival (28, 29) and these have also been shown to be important for survival of bradyzoites within cysts (30, 42). TgCLP1 disruption blocked the export of TgIST and TgNSM into the host cell nucleus, which would be expected to render cysts susceptible to clearance in IFN-gamma-activated cells in vivo. The block in export of virulence effectors TgIST and TgNSM may result from the thicker cyst wall, changes in its carbohydrate components, or rearrangement of glycoproteins that form the cyst wall, as these have been shown to dynamically reorganize during development (8). Alternatively, loss of TgCLP1 may affect MYR1, the core component of the effector translocon, comprising several GRAs such as MYR1-4 that governs the translocation of effectors in both tachyzoites and bradyzoites (43–45). Such an association would have to occur in a stage-specific manner as we did not detect a loss of effector export in ∆clp1 tachyzoites. This finding is consistent with the fact that the ∆clp1 mutant has normal dissemination during acute infection but is only impaired in chronic infection.

Summary

Taken together, our findings demonstrate that TgCLP1 is an important bradyzoite survival factor that modifies cyst wall glycoproteins to modulate its maturation. Loss of TgCLP1 alters the cyst wall composition, impairs effector export, and results in loss of cysts during chronic infection.

Materials and Methods

Parasite and Host Cell Culture.

T. gondii tachyzoites were cultured on human foreskin fibroblasts (HFFs, ATCC). They were grown in D10 [Dulbecco’s modified Eagle’s medium (DMEM); Life Technologies] with a pH of 7.4, supplemented with 10% fetal bovine serum (FBS), 10 μg/mL gentamicin (Thermo Fisher Scientific), 10 mM glutamine (Thermo Fisher Scientific), at 37 °C in a 5% CO₂ incubator. For the induction of in vitro–derived cysts, HFFs were infected with tachyzoites at a multiplicity of infection (MOI) of 0.2 and were maintained in D10 for 2 h. The medium was shifted to RPMI 1640 medium without bicarbonate (MP Biomedicals) buffered to pH 8.2 using 25 mM HEPES and containing 5% FBS and replaced daily for 7 d. All strains and host cell lines were Mycoplasma-free, confirmed by the e-Myco plus kit (Intron Biotechnology). T. gondii type II strain ME49 Fluc (46) and ME49 TIR1-3FLAG (47) were used for genetic modification.

Animal Care and Ethics Statement.

Mice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-approved facility at Washington University School of Medicine. All animal studies were conducted in accordance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals, and protocols were approved by the Institutional Animal Care and Use Committee at the School of Medicine, Washington University in St. Louis. All details of animal studies were described in SI Appendix.

Daughter Cyst Formation Assay.

Freshly egressed parasites were inoculated onto HFFs on coverslips at a MOI of 0.2. Bradyzoites were induced for 7 d followed by fixation and staining for BAG1, SAG1, along with the cyst wall staining using biotinylated DBA. Daughter cysts, defined as cysts with one or two bradyzoites that are SAG1-negative, BAG1-positive, and DBA-positive, were quantified from at least 100 induced cysts. The percentage of daughter cysts was averaged and assessed for statistical significance.

Interferon Regulatory Factor 1 (IRF1) Expression in IFN-γ Stimulated HFFs.

Bradyzoites were induced for 7 d at alkaline pH. At day 6 postinduction, 100 U/mL human interferon-γ recombinant protein (R&D) was added to the stress medium and incubated for 24 h, followed by fixation and staining for SAG1, human IRF1, and the cyst wall using biotinylated DBA. Images were captured using a 63x Oil Plan-Apochromat lens (NA 1.4) on an Axioskop 2 MOT Plus Wide Field Fluorescence Microscope (Carl Zeiss, Inc.). Images were processed and quantified (N = 3, n = 30) for the relative fluorescence intensity via ZEN 3.3 (Blue edition, Carl Zeiss, Inc.).

Additional Methods and detailed protocols are provided in SI Appendix. Refer to SI Appendix for gene accession information, primers, plasmids, parasite strains, key resources, and lectin pulldown MS/MS data (Datasets S1–S6).

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Dataset S02 (XLSX)

Dataset S03 (XLSX)

Dataset S04 (XLSX)

Dataset S05 (XLSX)

Dataset S06 (XLSX)

Dataset S07 (XLSX)

Data, Materials, and Software Availability

All study data are included in the article and/or supporting information.