Toxoplasma gondii Is Dependent on Glutamine and Alters Migratory Profile of Infected Host Bone Marrow Derived Immune Cells through SNAT2 and CXCR4 Pathways
Figure 1
T. gondii is dependent on glutamine for optimal infection, replication and viability in vitro.
Experiments were conducted in human foreskin fibroblast (HFF) monolayers. (A) Representative confocal images on the top row show the intracellular tachyzoites expressing GFP (green) and one extracellular parasite staining with SAG1 (red, also indicated by the white arrow) in the presence of L-glutamine. A 63× oil objective was used to examine the SAG1-positive parasite at higher magnification (bottom left corner). Pictures on the second row represent the SAG1 staining of extracellular T. gondii in the absence of L-glutamine. The nuclei of HFFs were stained with DAPI (blue), and the merged images of the fluorescence and the bright field shows that the HFF monolayer was confluent when the experiment was carried out. (B) Infection efficiency is defined as the ratio of intracellular parasites to total parasites in the examined field on the coverslip after 6 hours of infection. Infection efficiency was reduced ∼65% by omitting L-glutamine (Student's t-test, P<0.001). (C) Examples of the confocal images showing T. gondii replication in HFFs after 22 hours in the presence of glutamine. The replication cycle numbers were indicated in white. (D) In the presence of L-glutamine, T. gondii tachyzoites were able to duplicate up to 4 times in 22 hours, but without L-glutamine, over 50% of parasites only divided twice. (E) In the absence of L-glutamine, mean replication cycle number declined approximately 25% in 22 hours (Student's t-test, P<0.05). (F) MTT assay was used to evaluate parasite viability. Glutamine starvation significantly decreased the viability of T. gondii by 29%, and D-glutamine was unable to substitute for L-glutamine (one-way ANOVA, F(2,89) = 45.036, P<0.001). Asterisks indicate significant difference from medium containing 2 mM L-glutamine (post hoc Dunnett's test, P<0.001). Data show mean value ± SEM from three independent experiments.