Phosphoglucomutase 1 contributes to optimal cyst development in Toxoplasma gondii

Toxoplasma gondii is a ubiquitous parasite of medical and veterinary importance; however, there exists no cure for chronic toxoplasmosis. Metabolic enzymes required for the production and maintenance of tissue cysts represent promising targets for novel therapies. Here, we use reverse genetics to investigate the role of Toxoplasma phosphoglucomutase 1, PGM1, in Toxoplasma growth and cystogenesis. We found that disruption of pgm1 did not significantly affect Toxoplasma intracellular growth and the lytic cycle. pgm1-defective parasites could differentiate into bradyzoites and produced cysts containing amylopectin in vitro. However, cysts produced in the absence of pgm1 were significantly smaller than wildtype. Together, our findings suggest that PGM1 is dispensable for in vitro growth but contributes to optimal Toxoplasma cyst development in vitro, thereby necessitating further investigation into the function of this enzyme in Toxoplasma persistence in its host.


Introduction
Toxoplasma gondii is an obligate intracellular protozoan responsible for toxoplasmosis in humans and other warm-blooded animals. Infections occur mostly from consuming contaminated water, food, or undercooked meat from chronically infected animals [1]. Bradyzoites inside tissue cysts are released into the gastrointestinal tract where they invade enterocytes and convert to tachyzoites inside a parasitophorous vacuole (PV). Tachyzoites replicate rapidly, eventually lysing out of the host cell to disseminate throughout the body. In response to stressful stimuli, they convert back to bradyzoites which remain encysted in the brain and skeletal muscles for life [2].
Chronic toxoplasmosis is incurable and parasite reactivation life-threatening, particularly for the immunocompromised [3].
Here, we used the CRISPR/Cas9 gene-editing system [16] to disrupt pgm1 in a cyst-forming Toxoplasma Open Access BMC Research Notes *Correspondence: pascale.guiton@csueastbay.edu strain. Our data show that this mutation did not prevent intracellular replication or the completion of the lytic cycle. While both strains could produce amylopectincontaining cysts, we found that pgm1-defective cysts are significantly smaller than the parental cysts. Together, our findings corroborate previous reports that PGM1 is dispensable for Toxoplasma viability and demonstrate that the enzyme contributes to optimal cyst development in vitro.

Replication assay
Freshly released parasites were centrifuged at 1500 rpm for 10 min and washed once with 1XPBS. Confluent HFFs on glass coverslips were infected with 1.2 × 10 5 parasites in cDMEM for 24 h. The number of parasites per vacuole was determined by immunofluorescence microscopy, as previously described [18], following staining with mouse α-SAG1 and rabbit α-GRA7 obtained from the Boothroyd lab. Immunostaining and visualization are further described below.

Plaque assay
WT and Δpgm1 tachyzoites were syringe-lysed through a 27G needle and passed through a 5 µm filter. Confluent HFFs were infected with 250 parasites in cDMEM and incubated at 37 ºC with 5% CO 2 for 10 days undisturbed. Following methanol fixation and crystal violet staining, plaque numbers and sizes were determined using a stereoscope (Leica EZ4) and ImageJ version 1.52A (National Institutes of Health) [19,20].

Tachyzoite-to-bradyzoite differentiation
Tachyzoites were induced to differentiate into bradyzoites in HFFs as previously described [21]. Briefly, confluent HFFs on glass coverslips were infected with 4.8 × 10 4 parasites for 3 h in cDMEM before replacing the medium with Switch Medium (RPMI 1640 supplemented with 1% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin, 10 mg/mL HEPES, pH 8.2). Parasites were incubated for 4 days at 37 ºC with ambient CO 2 and the medium was changed every 24 h to maintain alkaline conditions.

Immunostaining fluorescence assay and amylopectin staining
Infected monolayers were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min at room temperature (RT). Cells were permeabilized with 0.2% or 0.4% Triton X-100 for 20 min and incubated for 1 h in 3% Bovine Serum Albumin (BSA; Fisher Scientific) in PBS. Primary antibodies diluted in 3% BSA/PBS (mouse α-SAG1 1:10,000, rabbit α-GRA7 1:1000) were added to the monolayers, when indicated, and incubated overnight at 4 ºC. Unbound antibodies were washed away with three 5 min washes in 1XPBS. The cells were then stained with secondary antibodies in 3% BSA/PBS (Goat α -Mouse 546 or Goat α-Rabbit 488 at 1:5000) for 45 min at RT. Dolichos biflorus Agglutinin (DBA; Vector Laboratories) was used at 1:100 to detect the cyst wall. After washing as described above, the coverslips were mounted with VECTASHIELD Mounting Medium containing DAPI (Vector Laboratories). Amylopectin was stained with Periodic Acid Schiff (PAS; Fisher Scientific) according to the manufacturer's guidelines.
Immunofluorescence images were obtained using an inverted microscope (Leica DM IL LED) with 100 × oil immersion objective. The number of parasites (SAG1positive) inside individual vacuole (GRA7 +) from randomly selected fields was determined from direct count under the microscope. The areas of plaques and cysts, both selected from random fields of view, were determined using ImageJ version 1.52A and 1.53, respectively [19,20].

Statistical methods
Statistical analyses were performed using GraphPad Prism version 8.4.3. A p-value ≤ 0.05 was considered a statistically significant difference between groups.

Toxoplasma phosphoglucomutases are upregulated during chronic infection in mice
Comparative transcriptomic and proteomic analyses [22] revealed that Toxoplasma expresses stage-specific proteins which enable the parasite to survive and to be efficiently transmitted between hosts. We mined the transcriptional data from Pittman et al. [12] available on the commonly used Toxoplasma Informatics Resources database (ToxoDB) [10,23] to specifically identify metabolic enzymes involved in gluconeogenesis and glycolysis that are significantly upregulated at least 2 folds in chronic vs. acute infection. Of the 422 genes upregulated in chronic infection, our analysis revealed 21 that are specifically associated with carbohydrate metabolism (Fig. 1A, B, Additional file 1). As expected, these genes include well-known glycolytic isoenzymes involved in tissue cyst formation, such as lactate dehydrogenase 2 (ldh2) [24] and enolase 1 (eno1) [25]. Interestingly, unlike ldh1/ldh2 and eno1/eno2 which are expressed in a stage-dependent manner, both PGM isoforms (pgm1 and pgm2) were upregulated 6.4 and 3.1 folds, respectively, in the chronic stage, 28 days post-infection (dpi) [12]. Transcriptional analyses of gene expression at 28, 90, and 120 dpi from Garfoot et al. [26] indicate that unlike pgm2 whose expression remained similar up to 120 dpi, pgm1 transcripts further increased from 28 to 120 dpi. Together, this analysis strongly suggests that transcriptional regulation of pgm1/pgm2 may be critical for the development and/or maintenance of tissue cysts in mice. Furthermore, the increased expression of PGM1 during chronic infection and its enzymatic activity at the intersection of energy storage and production pathways, namely glycolysis and amylopectin metabolism, warrant determining the role of this enzyme during Toxoplasma growth and differentiation.

Disruption of pgm1 does not hinder parasite growth in vitro
To determine the contribution of PGM1 to Toxoplasma growth, we used the CRISPR-Cas9 gene-editing system to create an insertional mutant Me49ΔhxgprtΔpgm1 (Δpgm1) by introducing a hxgprt selection cassette at the pgm1 locus [16] (Fig. 2A, B, Additional file 1: Figure S1).
We assessed the intracellular growth of Δpgm1 parasites vs. WT 24 h after infection of HFFs in glucose replete growth medium. SAG1-positive parasites inside GRA7positive vacuoles were enumerated. We found similar numbers of Δpgm1 vacuoles with either 2, 4, or ≥ 8 parasites as WT (Fig. 2C). Likewise, no significant differences in plaque numbers and sizes were observed 10 days after infection (Fig. 2 D, E). Thus, as previously reported for Toxoplasma RH strain [15,27], our data indicate that PGM1 is dispensable for Toxoplasma intracellular growth and lytic cycle in vitro, albeit in glucose-rich conditions.

pgm1-defective parasites produced smaller amylopectin-containing cysts in vitro
Given the upregulation of pgm1 in chronic infection, we tested whether disruption of pgm1 would impede tissue cyst formation. We induced tachyzoites to differentiate into bradyzoites in nutrient-poor, alkaline conditions in ambient CO 2 [21]. After 4 days, we stained the monolayers with Dolichos biflorus agglutinin (DBA) to detect the cyst wall and Periodic Acid Schiff (PAS) to visualize amylopectin [8]. Both WT and Δpgm1 parasites produced PAS-positive cysts (Fig. 3A), suggesting that PGM1 is not essential for amylopectin accumulation during stage conversion in vitro. However, further studies are required to determine any differences in the relative amount of this polysaccharide between WT and Δpgm1 cysts. Interestingly, Δpgm1 cysts were on average ~ 4060 pixels 2 smaller than WT (p = 0.0362 by Mann-Whitney test, Fig. 3C). Together, our results indicate that although PGM1 is not required for stage conversion and amylopectin storage, the enzyme contributes to optimal cyst development in vitro.

Discussion
PGM1 is one of two PGM isoforms differentially expressed in Toxoplasma [10,12,28]. In this study, we showed that disruption of pgm1 in a cyst-forming Toxoplasma strain did not prevent intracellular growth or completion of the lytic cycle in glucose-replete conditions, corroborating previous studies in non-cyst forming Type I tachyzoites [15,27]. Our observation that tachyzoites lacking pgm1 could differentiate into bradyzoites in the absence of glucose further supports the nonessential role of PGM1 and PGM1-dependent glucose-6-phosphate production in tachyzoites as suggested by Imada et al. [29]. Interestingly, PGM1 has been implicated in Ca 2+ -dependent microneme secretion in tachyzoites [11,13,15], and thus, like functionally characterized PGMs in other organisms [9,30], it may play an unconventional role during Toxoplasma development.
Additionally, the absence of pgm1 did not abrogate amylopectin biosynthesis and storage, probably due to functional compensation with PGM2. While both pgm1 and pgm2 transcripts are higher in bradyzoites than tachyzoites [28], the proteins share only 25% homology. PGM2 has a significantly lower enzymatic activity than PGM1 [29]. Interestingly, Saha et al. [15] demonstrated that PGM2 didn't compensate for the deletion of PGM1 in the context of Ca 2+ -regulated microneme secretion in tachyzoites.
Although glycolysis is not required for tachyzoite viability, it is critical for tissue cyst formation and pathogenesis in mice [31]. Parasites lacking hexokinase, the first enzyme in glycolysis that catalyzes the phosphorylation of glucose to glucose-6-phosphate, produce smaller cysts in vitro [31]. This phenotype was recapitulated in pgm1-defective parasites, further supporting the importance of glycolytic intermediates during cystogenesis. While the bradyzoite burden of PGM1-deficient cysts and their infectivity remain to be determined, it is plausible that the parasites inside these mutant cysts have decreased resistance to proteases and are less infectious following oral infection, as previously shown for Bradyzoite Pseudokinase 1 (BPK1) mutants [32]. Because the absence of PGM1 does not significantly alter the replication rate of tachyzoites, it is conceivable that the bradyzoite burdens in the mutant and wildtype cysts be comparable. This assertion is supported by Watts et al. who showed that cyst size is not a strong predictor bradyzoite burden [33].
Overall, this study suggests that PGM1 is not critical for Toxoplasma growth and differentiation; however, it is required for optimal cyst maturation, which is critical for the establishment of chronic Toxoplasma infections. Future studies are needed to parse out the interplay and diverse activities of Toxoplasma PGMs Fig. 2 Disruption of pgm1 and growth assays. A Schematic representation of disruption of pgm1 using CRISPR-Cas9 gene-editing system for nonhomologous insertion of the hxgprt selectable marker cassette. The dotted line represents the region in the first exon of pgm1 targeted by the small guide RNA (sgPGM1). B Image of DNA gel electrophoresis of PCR1-3 performed using DNA from wildtype (WT) and mutant (Δpgm1) to demonstrate integration of the hxgprt expression cassette at the pgm1 locus. The expected product for PCR1 (212 bp) was obtained only for WT while products for PCR2 (813 bp) and PCR3 (1185 bp) were amplified only with Δpgm1 DNA. C Intracellular growth. HFFs were infected with 1.2 × 10 5 WT or Δpgm1 parasites for 24 h in cDMEM. Monolayers were fixed and stained with antibodies raised against SAG1 (tachyzoite surface marker) and GRA7 (PV marker). Intracellular parasites were enumerated in at least 20 vacuoles/strain/experiment, N = 3 independent experiments; error bars = standard error of the mean; p-value was determined by Chi-square test. D Total numbers of plaques counted 10 days after infection of HFFs with 250 WT or Δpgm1 parasites. E Plaque areas were determined for 85 WT and 109 Δpgm1 plaques using Fiji/ImageJ in pixels 2 , N = 3 replicates/strain in a single experiment, error bar = standard deviation; ns: p-value > 0.05 by nonparametric Mann-Whitney test and understand how they affect central carbon metabolism and developmental differentiation in this ubiquitous parasite. PGMs are among several metabolic enzymes whose transcripts are significantly upregulated during chronic infection with Toxoplasma. While PGM1 was our initial focus, future work will evaluate the contributions of other poorly characterized glycolytic enzymes identified in our bioinformatic search. Similar to PGM1, these enzymes may be critical to Toxoplasma biology and serve as potential therapeutic targets against chronic toxoplasmosis.