Fig. 5: The cellular targeting of RNF213 by IpaH1.4 relies on the specific IpaH1.4 LRR/RNF213 RING interaction.

a–e Representative confocal micrographs of RNF213-knockout HeLa cells stably expressing the AcGFP-tagged wild type RNF213 or RNF213 L4036R mutant were infected by the ipaH1.4/ipaH2.5 double-knockout (ΔipaH1.4/ΔipaH2.5) S. flexneri strain 4 h after transfection of relevant different mCherry-tagged IpaH1.4 variant or the control mCherry tag and further expressed 24 h. HeLa cells were stained with the DAPI reagent for the nuclei. WT wild type; Scale bars, 10 µm. f Statistical results related to the percentage of the IpaH1.4-positive RNF213-coats on cytosolic bacteria in (a–d). The data represent the mean ± SEM of 40, 39, 41, and 43 cells for each group, presented in the same order as in (f). An unpaired ordinary one-way ANOVA analysis followed by Sidak multiple comparisons test was used to define a statistically significant difference. g Fold replication of the wild-type S. flexneri M90T and the ipaH1.4/ipaH2.5 double-knockout (ΔipaH1.4/ΔipaH2.5) S. flexneri in the infected wild type or RNF213-knockout HeLa cells calculated at 6 h time point normalized to 2 h time point after infection. Bacteria were counted by serial dilutions of cell lysate on LB agar plates. Data are presented as mean ± SEM from three independent biological repeats. A two-tailed unpaired Student’s t test analysis was used to define a statistically significant difference. h Fold replication of the ipaH1.4/ipaH2.5 double-knockout (ΔipaH1.4/ΔipaH2.5) S. flexneri rescued with the wild type IpaH1.4 or different IpaH1.4 variant in infected wide type HeLa cells calculated at 6 h time point normalized to 2 h time point after infection. Bacteria were counted by serial dilution of cell lysate on LB agar plates. Data are presented as mean ± SEM from three independent biological repeats. An unpaired ordinary one-way ANOVA analysis followed by Sidak multiple comparisons test was used to define a statistically significant difference.