Fig. 7: MDH-FAD is required for T. gondii growth.

a The growth of Δmdh-fad and DiCre tachyzoites in vitro was compared by plaque assay (7 d, 100 tachyzoites/well and 3 wells for each strain). b The relative size of plaques from (a) (means ± SEM; ****p ≤ 0.0001, Student’s t test). c The replication assays of DiCre and Δmdh-fad in vitro. After 24 h of infection with HFF cells, the number of parasites in vacuoles was determined by IFA. d Measurement of parasite burden in ICR mice. ICR mice were infected by intraperitoneal injection of DiCre and Δmdh-fad tachyzoites (10⁴ tachyzoites/mouse and 5 mice/strain). Parasite loads in peritoneal fluid were calculated by qPCR after 5 days of infection. e ICR mice were injected intraperitoneally with wild-type and Δmdh-fad tachyzoites (100 parasites/mouse and 10 mice/strain). f DiCre and Δmdh-fad tachyzoites (5 × 10⁷) were harvested and cultured for 4 h in a glucose-free medium supplemented with 8 mM ¹³C₅-glutamine. The levels of ¹³C incorporation in glycolysis and TCA cycle metabolites were determined by the LC-MS. M0-M5 represents the number of carbons in the ¹³C-labeled metabolites. Values are mean ± SEM of five independent experiments (n = 5, two-way ANOVA). g Incorporation of ¹³C into glycolysis and TCA cycle metabolites, as determined by UHPLC-HRMS. The extracellular tachyzoites of the DiCre and Δmdh-fad strains were incubated with 8 mM ¹³C₆-glucose for 4 h (n = 5 biologically independent samples, means ± SEM; Student’s t test). h Effect of MDH-FAD deletion on malate formation (data from panel (f)). The peak area represents the sum of M0-M4 peak areas (Student’s t test). i Effect of MDH-FAD deletion on malate formation (data from panel (g)). The peak area represents the sum of M0-M4 peak areas (Student’s t test). j The total of NAD⁺ and NADH in DiCre and Δmdh-fad parasites was measured using a NAD⁺/NADH assay kit. Means ± SEM from two independent experiments (n = 2), each with two replicates (****p ≤ 0.0001, Student’s t test). k Mitotracker staining was used to examine the effect of MDH-FAD depletion on the membrane potential (Δψm). l The number of Δψm positive tachyzoites in DiCre and Δmdh-fad strains was counted (****p ≤ 0.0001, Student’s t test). The parental tachyzoites showed the typical intense staining of the mitochondrion and were classified as Δψm-positive.