Starvation of low-density lipoprotein-derived cholesterol induces bradyzoite conversion in Toxoplasma gondii

Abstract

Background: Lacking enzymes for sterol synthesis, the intracellular protozoan Toxoplasma gondii scavenges cholesterol from host cells to multiply. T. gondii has a complex life cycle consisting of two asexual stages; the proliferative stage (tachyzoite), and the latent stage characterized by tissue cysts (bradyzoite). In vitro, bradyzoite development can be induced by mimicking host immune response stressors through treatment with IFN-γ, heat shock, nitric oxide, and high pH. However, the extent to which host nutrients contribute to stage conversion in T. gondii is unknown. In this study, we examined the impact of host cholesterol levels on stage conversion in this parasite.

Methods: Growth of T. gondii tachyzoites (ME49 strain) was investigated in Chinese hamster ovary (CHO) cells using various concentrations of low-density lipoprotein (LDL), oleic acid, or glucose. Squalestatin, which is an inhibitor of squalene synthase and is, therefore, an inhibitor of sterol synthesis, was used to treat the CHO cells. Tachyzoite to bradyzoite conversion rates were analyzed by indirect fluorescent antibody tests.

Results: Parasite growth was significantly enhanced by addition of exogenous LDL, whereas no such enhancement occurred with oleic acids or glucose. In ME49, growth inhibition from squalestatin treatment was not obvious. Although growth of the RH strain was unaffected by squalestatin in the presence of lipoprotein, in its absence growth of this strain was suppressed. The frequency of BAG1-positive vacuoles in ME49 increased under lipoprotein-free conditions. However, addition of exogenous LDL did not increase tachyzoite to bradyzoite conversion in this strain. Furthermore, treatment with squalestatin did not enhance stage conversion.

Conclusion: Our results suggest that LDL-derived cholesterol levels play a crucial role in bradyzoite conversion in T. gondii.

Figure 1

Parasite growth in the presence of LDL, fatty acids, or glucose. Uracil incorporation was assayed using CHO cells infected with the ME49 strain of T. gondii for 20 h in medium containing 5% LPDS treated with LDL or oleic acid at the concentrations indicated (A), or in glucose-free medium containing 10% FBS treated with glucose at the concentrations indicated (B). Data are expressed as a percentage relative to the control (incubation without lipid or glucose), which was taken as 100% ± the standard deviation (n = 4).

Figure 2

Squalestatin treatment and parasite growth. Uracil incorporation was assayed using CHO cells infected with the ME49 strain (A) or RH strain (B) of T. gondii for 44 h in medium containing 5% LPDS or 5% FBS treated with squalestatin at the concentrations indicated. Data are expressed as a percentage relative to that of the control (incubation in the presence of 5% LPDS without squalestatin), which was taken as 100% ± the standard deviation of triplicate samples. Statistical analysis of the data was conducted using a one-way ANOVA followed by Tukey’s multiple comparison tests. (*) Values of P < 0.01 were considered statistically significant when compared with incubation without squalestatin.

Figure 3

IFAT analysis of BAG1 expression in parasite vacuoles. CHO cells were infected with T. gondii ME49. After 72 h, the parasite-infected cells were subjected to IFAT analyses. (A) Infected CHO cells were stained with a SAG1 antibody (tachyzoite specific marker) and a BAG1 antibody (bradyzoite specific marker). Scale bar: 10 μm. (B) Percentage of vacuoles expressing BAG1. Data represent the mean percentage of BAG-positive vacuoles from one hundred vacuoles (from triplicate samples). Statistical analysis of the data was conducted using a one-way ANOVA followed by Tukey’s multiple comparison tests. Different symbols indicate statistically significant differences (P < 0.01). SS, 50 μM squalestatin.