Toxoplasma phosphoglucomutases are upregulated during chronic infection in mice
Comparative transcriptomic and proteomic analyses [19] 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 Toxoplasma Informatics Resources database (ToxoDB) [10] 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) [20] and enolase 1 (eno1) [21]. 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. [22] 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.
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). We assessed the intracellular growth of Δpgm1 parasites vs. WT 24 hours after infection of HFFs in glucose replete growth medium. SAG1-positive parasites inside GRA7-positive 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. 2D-E). Thus, as previously reported for Toxoplasma RH strain [15, 23], 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 CO2 [18]. After four 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 pixels2 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.