Toxoplasma gondii strains defective in oral transmission are also defective in developmental stage differentiation

Abstract

Toxoplasma gondii undergoes differentiation from rapidly growing tachyzoites to slowly growing bradyzoites during its life cycle in the intermediate host, and conversion can be induced in vitro by stress. Representative strains of the three clonal lineages showed equal capacity to differentiate into bradyzoites in vitro, as evidenced by induction of bradyzoite antigen 1, staining with Dolichos biflorus lectin (DBL), pepsin resistance, and oral infectivity in mice. We also examined several recently described exotic strains of T. gondii, which are genetically diverse and have a different ancestry from the clonal lineages. The exotic strain COUG was essentially like the clonal lineages and showed a high capacity to induce bradyzoites in vitro and in vivo, consistent with its ability to be efficiently transmitted by the oral route. In contrast, exotic strains MAS and FOU, which are defective in oral transmission, showed a decreased potential to develop into bradyzoites in vitro. This defect was evident from reduced staining with DBL and the cyst antigen CST1, failure to down-regulate tachyzoite antigens, such as tachyzoite surface antigens 1 and 2A, and decreased resistance to pepsin treatment. Despite normal in vitro differentiation, the exotic strains CAST and GPHT also showed decreased oral transmission, due to formation of smaller cysts and a lower tissue burden during chronic infection, traits also shared by MAS and FOU. Collectively, these findings reveal that the limited oral transmission in some strains of T. gondii is due to inefficient differentiation to the bradyzoite form, leading to defects in the formation of tissue cysts.

FIG. 1.

In vitro differentiation to bradyzoites by clonal lineages. Representative strains for type I (GT-1), type II (ME49), and type III (CTG) strains were induced to differentiate by high-pH culture for 5, 7, and 9 days. Cyst development was detected by staining with fluoresceinated DBL and by using the mAb 8.25.8 to BAG1 followed by secondary antibodies conjugated to Alexa 594. (A) Representative example of BAG1 staining (red channel) and DBL-positive staining (green channel) for in vitro-derived cysts of T. gondii. Nuclei are stained in blue (DAPI). Scale bar = 10 μm. (B) The average numbers of cysts per ×40 magnification field were similar among all three lineages at all time points. (C) Average sizes (area in μm²) of cysts formed by T. gondii strains from the three lineages. ME49 formed the largest cysts, while cysts formed by GT-1 and CTG were somewhat smaller. The differences between ME49 and CTG were statistically significant at day 7 and day 9 (*, P ≤ 0.05). Error bars show standard deviations.

FIG. 2.

Expression of cyst antigens following in vitro differentiation of T. gondii strains. The majority of strains strongly express both the cell wall epitope detected by DBL (green) and the CST-1 antigen (red). However, MAS expressed both markers poorly and FOU failed to stain with DBL, while staining positively for CST-1. At 5 days postinduction, the cells were fixed and stained with fluoresceinated DBL (green) and antibody to CST-1 followed by goat anti-mouse IgG conjugated to Alexa 594 (red). Nuclei are stained in blue. Scale bar = 10 μm. All pictures shown were recorded under similar optical conditions, imaged with the same exposure time, and processed identically.

FIG. 3.

qPCR analysis of stage-specific genes during in vitro differentiation of T. gondii. Parasites were cultured in vitro under conditions that induce differentiation. After 5 days, total RNA was harvested and analyzed by qPCR for the stage-specific genes shown. The ME49 and COUG strains showed strong repression of the tachyzoite-specific genes SAG1 and SAG2A, while strains MAS and FOU continued to express these genes at levels typical of tachyzoites. All strains showed induction of the bradyzoite-specific gene BAG1, and all strains except FOU showed induction of LDH2. The values represent the fold change based on C_T comparison (see Materials and Methods).

FIG. 4.

Electron microscopy of T. gondii cysts formed after 9 days of induction in vitro. (A) Intracellular cyst of ME49 demonstrating the convoluted cell wall (arrows) and internal granular matrix (M). Parasite nuclei (N) are located posteriorly. (B) Enlargement of ME49 cyst demonstrating posterior nuclei (N), abundant micronemes (black arrowheads), amylopectin granules (arrows), and electron-dense rhoptries (white arrowheads). (C) Cyst of MAS containing parasites that resemble tachyzoites. Although the cyst wall (arrows) and internal matrix (M) were similar, the parasites lacked features of bradyzoites. Parasite nuclei (N) were located centrally. (D) Enlargement of MAS cyst showing parasites with honeycombed rhoptries (white arrowhead), centrally located nucleus (N), fewer micronemes, and fewer amylopectin granules. (E) Cyst of FOU showing tachyzoite-like features, including centrally located nucleus (N) and honeycombed rhoptries (arrowhead). (F) Enlarged cyst of FOU showing characteristic tachyzoite-like features, including centrally located nuclei (N), fewer micronemes, and absence of amylopectin granules. Scale bars in A and C = 1 μm; scale bars in B, D, E, and F = 500 nm.

FIG. 5.

In vivo-derived cysts of T. gondii stained with DBL. All strains of T. gondii produced cysts in vivo that stained strongly with lectin (white areas). Brain homogenates from chronically infected mice or rats were fixed and stained with fluoresceinated lectin. Scale bars = 10 μm.