Fig 5. HeLa cells retard growth of Toxoplasma by an IFNγ and ubiquitin-dependent but acidification-independent mechanism.

(A) Ubiquitin is recruited to type II Toxoplasma vacuoles in IFNγ-stimulated HeLa cells. Inhibiting ubiquitination with the E1 inhibitor UBEI-41 reduces recruitment of ubiquitin to type II vacuoles at 6h p.i.. Counts are expressed as % positive vacuoles and the mean of 3 experiments is shown. Significance was determined using 2way ANOVA, ***, p ≤ 0.001, ****, p ≤ 0.0001 and ns, not significant. (B) Inhibiting ubiquitination increased the ability of type II Toxoplasma to replicate in IFNγ-stimulated HeLa at 24h and 18h p.i., but no difference in replication was observed at 6h. HeLa were pre-treated with 50μm UBEI-41 to inhibit ubiquitination prior to infecting with type I or type II Toxoplasma for 24, 18 and 6h. Replicating Toxoplasma counts with and without inhibition of ubiquitination are shown, mean of ≥4 experiments are shown Significance was determined using 2way ANOVA where * indicates p ≤ 0.05, **** indicates p ≤ 0.0001 and ns not significant. (C) LC3B is recruited to the type II vacuole. HeLa cells stimulated or not with IFNγ for 18h were infected with type I or type II parasites for the indicated times. Recruitment of LC3B was monitored by antibody staining. Graphs indicating the percentage of positive vacuoles are shown for 3 experiments. Significance was determined using 2way ANOVA, *, p ≤ 0.05, ***, p ≤ 0.001, ****, p ≤ 0.0001 and ns, not significant. (D) The type II Toxoplasma vacuole in IFNγ-stimulated HeLa cells does not acquire lysosomal markers and does not acidify. HeLa cells were infected with type I or type II Toxoplasma for the indicated times. LAMP1 staining was recorded and expressed as % positive vacuoles for 3 experiments. Significance was determined using 2way ANOVA, **, p ≤ 0.01 and ns, not significant. (E) Inhibiting acidification has no effect on the ability of type II Toxoplasma to replicate in IFNγ-stimulated HeLa, 6h p.i.. HeLa cells were treated with 10mM NH₄Cl 1h after infection to prevent acidification and infection continued until 6h. Replicating Toxoplasma counts with and without NH₄Cl are shown, mean of ≥3 experiments are shown. Significance was determined using 2way ANOVA, ns = not significant.