Host-derived glucose and its transporter in the obligate intracellular pathogen Toxoplasma gondii are dispensable by glutaminolysis

Abstract

Toxoplasma gondii, as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgST1-3), of which TgST2 is expressed at its surface, whereas TgST1 and TgST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike Deltatgst2, the modest impairment in Deltatggt1 and Deltatggt1/Deltatgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The Deltatggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1-complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in Deltatggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of Deltatggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells.

Fig. 1.

TgGT1 can mediate the import of glucose, mannose, fructose, and galactose in L. mexicana null mutant (Δlmgt). (A) TgGT1-complemented promastigotes were assayed for their ability to transport 100 μM d-[³H]glucose, d-[³H]mannose, d-[¹⁴C]fructose, or d-[¹⁴C]galactose. The pX63NeoRI vector was used as the control. (B) Substrate saturation curve for d-[³H]glucose. Uptake was determined over a 30-s period for a range of substrate concentrations (0.01–2 mM). (C) Inhibition of 100 μM d-[³H]glucose uptake in the presence of sugar inhibitors (0–50 mM). The K_m and K_i were determined by Michaelis–Menten and nonlinear regression analyses.

Fig. 2.

TgGT1 and TgST2 localize in the plasma membrane of T. gondii, whereas TgST1 and TgST3 reside in intracellular vesicles. (A) The parental or Δtggt1 strains were transfected with TgGT1-HA-pNTP3 and TgGT1-HA-pGT1 constructs, respectively. (B) Transiently transfected parasites expressing N-terminally HA-tagged TgST1, TgST2, or TgST3 in pNTP3 vector. (C) C-terminally Ty1-fused and TUB8-regulated T. gondii sugar permeases in stably transfected parasites. (D) TgST1 and TgST3 were tagged with Ty1 between sixth and seventh transmembrane domains in pTUB8.

Fig. 3.

The Δtggt1 but not Δtgst2 strain of T. gondii demonstrates a protracted in vitro growth. (A) Representative images of the parental, Δtggt1, Δtgst2, and Δtggt1/Δtgst2 strains as generated by plaque assays. (B) The images were digitized and plaques were manually encircled to calculate the area of 80 plaques formed by the individual strains by using the ImageJ program. (C) Replication rates of T. gondii tachyzoites in human foreskin fibroblasts

Fig. 4.

The Δtggt1 but not Δtgst2 strain of T. gondii is compromised in using host-derived glucose and exhibits a delayed replication. (A) Glucose utilization assays were performed with parasite-infected human foreskin fibroblasts (HFF) monolayers (multiplicity of infection = 4) at 24-h postinfection. (B) HFFs were infected for 40 h in glucose-free or normal media, and the inoculum-normalized parasite yield was calculated.

Fig. 5.

Glutamine fulfills the metabolic needs of the Δtggt1 mutant. Freshly harvested tachyzoites were used to perform the motility assays. (A) Representative images of the parental and Δtggt1 strains in Hanks's balanced salt solution supplemented with the indicated reagents. (B) Quantitative diagram of the motile fraction in three independent experiments. (C) Glutamine (0.5 μCi/mmol of [³H]glutamine) incorporation assays were executed with parasite-infected HFF monolayers (multipliciy of infection = 4) at 24 h postinfection.