Phosphoglucomutase 1 contributes to optimal cyst development in Toxoplasma gondii

Abstract

Objective: 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.

Results: 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.

Keywords: Amylopectin; Gluconeogenesis; Glycolysis; Phosphoglucomutase; Stage conversion; Tissue cysts; Toxoplasma.

Fig. 1

Identification of upregulated metabolic genes during Toxoplasma chronic infection. A Workflow for identification of genes associated with glycolysis and gluconeogenesis with higher expression in chronic vs. acute infection in dataset from Pittman et al. [12]; the analysis was performed on ToxoDB [10]. B Word cloud of enriched pathways among the 422 genes upregulated during chronic infection in mice. The image was generated on ToxoDB. C Transcript levels of differentially regulated glycolytic and gluconeogenic enzymes in Toxoplasma. Values were obtained from Pittman et al. dataset available on ToxoDB version 54

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 × 10⁵ 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 pixels², N = 3 replicates/strain in a single experiment, error bar = standard deviation; ns: p-value > 0.05 by nonparametric Mann–Whitney test

Fig. 3

In vitro stage conversion assay. A Representative fluorescence images of amylopectin-containing WT and Δpgm1 cysts at 4 days post-induction. Infected monolayers were stained with PAS to detect amylopectin (red), DBA to label the cyst wall (green), and DAPI for nuclei (blue); Scale bar = 10 microns. B Representative images of WT and Δpgm1 cysts 4 days post-induction in vitro. The images are representative of the mean value of cyst areas for each strain. Cysts were stained with DBA (red), anti-GRA7 (green), and DAPI (blue); Scale bar = 10 microns. C Quantification of cyst sizes. The areas of 176 WT and 185 Δpgm1 cysts were determined in pixels² at 4 days post-induction from 3 independent experiments; *p = 0.0362 by nonparametric Mann–Whitney test