Fig. 3. Δgra45 parasites have enhanced susceptibility to IFNγ-mediated growth inhibition in rat BMDMs and human THP-1 macrophages.

a, b Murine BMDMs prestimulated with 5 ng/mL of IFNγ for 24 h were infected with luciferase-expressing WT, Δgra45, Δgra22, ΔTGGT1_263560, or ΔTGGT1_269950 parasites (MOI of 0.5) for 1 h and subsequently fixed, permeabilized, and stained for IRGA6 (a) or IRGB6 (b). A representative image from three independent experiments is shown on the left (Scale bar = 5 μm). Quantification of IRGA6/IRGB6 loading on at least 200 vacuoles is presented on the right panel. Data are displayed as mean ± SEM with independent experiments indicated by the same color dots. The significant difference between WT and knockout was analyzed with two-tailed paired t-test. c, d Brown Norway rat BMDMs prestimulated with or without IFNγ (1 ng/mL or 5 ng/mL) were infected with luciferase-expressing WT, Δgra45 (c), Δgra22 (c), ΔTGGT1_263560 (d), or ΔTGGT1_269950 (d) parasites (MOI of 0.25) for 24 h. Parasite growth for each strain was measured by luciferase assay and the growth in IFNγ-activated BMDMs is expressed relative to growth in naïve BMDMs. Data are displayed as mean ± SEM with independent experiments (n = 3) indicated by the same color dots. The significant difference between WT and knockout was analyzed with two-tailed paired t-test. e–h PMA-differentiated THP-1 macrophages prestimulated with or without IFNγ (2.5 or 5 ng/mL) were infected with luciferase-expressing WT, Δgra45 (e, n = 4), Δgra22 (f, n = 3), ΔTGGT1_263560 (g, n = 3), or ΔTGGT1_269950 (h, n = 3) parasites (MOI of 0.25) for 24 h. Parasite growth for each strain was measured by luciferase assay and the growth in IFNγ-activated THP-1 macrophages is expressed relative to growth in naïve THP-1 macrophages. Data are displayed as mean ± SEM with independent experiments indicated by the same color dots. The significant difference between WT and knockout analyzed with two-tailed paired t-test.