Mutation of the bacterial transcription factor MxiE results in linear ubiquitylation of S. flexneri in IFNγ-primed epithelial cells.

(A - C) Cells were primed with 100 U/ml IFNγ or left unprimed and then infected with WT or ΔmxiE S. flexneri at an MOI of 50 - 100. Cells were fixed 3 - 4 h post infection (hpi) and stained for linear ubiquitin (M1-Ub) with anti-M1 antibody. Percentage of M1-Ub-positive bacteria was quantified in infected A549 epithelial cells at 4 hpi (B) and HT29 epithelial cells at 3 hpi (C). Graphs show the average of three independent experiments and depict means ± SEM. Two-way ANOVA with Tukey’s multiple comparison tests were performed; all statistically significant comparisons are shown. ***p<0.001; **** p<0.0001; ns, not significant.

S. flexneri ΔmxiE mutants become decorated with linear and lysine-linked ubiquitin in IFNγ-primed A549 cells.

(A) WT A549 cells expressing the indicated internally Strep-tagged Ubiquitin (INT-Ub) variants were primed with 100 U/ml IFNγ overnight and co-localization of INT-Ub with cytosolic S. flexneri (Sf) ΔmxiE was assessed at 4 hpi. Ubiquitin linkage-specific antibodies were used to determine the percentage of S. flexneri ΔmxiE staining positive for K27-linked ubiquitin (K27-Ub) (B) or K63-linked ubiquitin (C) in IFNγ-primed untransduced WT A549 cells. Co-staining of anti-M1 and anti-ubiquitin (FK2) on the surface of S. flexneri ΔmxiE in IFNγ-primed (100U/ml) A549 cells is shown in (D). Quantification of anti-M1-FK2 co-staining in untreated and IFNγ-primed A549 cells is depicted in (E). All infections were done at an MOI of 50 - 100. Data was generated from at least three independent experiments and shows mean ± SEM. One-way ANOVA followed by Dunnet’s multiple comparisons were performed of all groups against WT-ubiquitin group (A). Two-way ANOVA with Tukey’s multiple comparison tests were performed for (B) and (C). An unpaired t-test was performed between “both FK2+M1” groups (gray bars) (E). *p<0.05; **p<0.01; ***p<0.001; **** p<0.0001

Ubiquitylation of S.flexneri ΔmxiE is dependent on RNF213 but not LUBAC.

(A) Immunoblotting for HOIP, HOIL-1, and RNF213 protein expression in untreated and IFNγ-primed WT and the corresponding gene deletion (KO) A549 cells. (B) Percentage of M1-linked ubiquitin positive ΔmxiE S.flexneri in IFNγ-primed WT, HOIPKO, HOIL-1KO and RNF213KO A549 cells. (C) Percentage of WT and 7KR INT-Ub-positive ΔmxiE S. flexneri in IFNγ-primed WT, HOIPKO, and RNF213KO A549 cells. (D – F) Untreated and IFNγ-primed A549 and HT29 cells were infected with the indicated S. flexneri strains and immuno-stained for RNF213 and ubiquitin (FK2). Representative immunofluorescence microscopy images are shown for A549 infections in (E). RNF213-S. flexneri colocalization percentages were quantified in A549 (D) and HT29 (F) cells. (G) Quantification of ubiquitin and RNF213 colocalization with ΔmxiE S. flexneri in A549 cells. Percentages of ΔmxiE S. flexneri staining positive for antibodies specific for K27-linked (H) and K63-linked ubiquitin (I) are also provided. All data are represented by the mean ± SEM from at least three independent experiments. One-way ANOVA followed by Dunnet’s multiple comparisons were performed of all groups against WT A549 group in (A). Two-way ANOVA with Tukey’s multiple comparison tests were performed in (C, D and F). an unpaired t-test was performed between “both Ub+RNF213” groups (gray bars) in (G). For (H and I), an unpaired-t test was performed. **p<0.01;; **** p<0.0001.

S. flexneri virulence factors IpaH1.4 and IpaH2.5 induce proteasomal degradation of RNF213.

(A-B) All S. flexneri strains express PilT to enhance adhesion and infection rates and infections were carried out at an MOI of 5 - 25 for 3 hours (A) Untreated and IFNγ-primed (100 U/ml) A549 cells were infected with indicated S. flexneri strains and protein lysates were probed for RNF213 expression. (B) Infected and uninfected A549 cells were cultured in the presence of different concentrations of the proteasomal inhibitor MG132 and RNF213 expression was monitored by immunoblotting. (C) HEK293T cells stably expressing mCherry-RNF213 were transiently transfected with individual GFP-tagged IpaH effectors for 24 hours and RNF213 expression was assessed. (D) WT and catalytically inactive C368S mutants of IpaH1.4 and IpaH2.5 were transiently transfected into HEK293T cells expressing mCherry-RNF213 and cell lysates were subjected to immunoblotting. (C-D) Denaturation of cell lysates at lower temperature (56°C) was required for RNF213 detection but also resulted in the formation of double bands for all IpaH-GFP constructs. Images are representative of three independent experiments UI: Uninfected.

Loss of IpaH1.4 is sufficient to render S. flexneri susceptible to RNF213-driven ubiquitylation.

(A) IFNγ-primed A549 cells were infected with the indicated PilT+ S. flexneri strains for 3 hours at an MOI of 5 - 25 and RNF213 protein levels were assessed by Western blotting. (B) Representative microscopy image depicting RNF213 recruitment to cytosolic ΔIpaH1.4 bacteria in IFNγ-primed A549 cells. (C) Percentage of RNF213+ WT and ΔIpaH1.4 S. flexneri in IFNγ-primed A549 cells. (D) Representative microscopy image showing ΔIpaH1.4 S. flexneri decorated with M1-linked ubiquitin in IFNγ-primed A549 cells. Percentage of M1-linked (E), K27-linked (F), and K63-linked (G) ubiquitin-positive WT and ΔIpaH1.4 S. flexneri in IFNγ-primed WT (E-G) and RNF213KO (E) A549 cells. (H) The ilux operon was introduced into S. flexneri strains to use bioluminescence (RLU) as a proxy for bacterial growth. RLU was measured in IFNγ-primed WT A549 cells infected with the indicated S. flexneri strains at an MOI of 5. (C, F-H) Data represent the mean ± SEM from at least three independent experiments. An unpaired t-test was conducted for (C, F, G). One-way ANOVA followed by Dunnet’s multiple comparisons were performed between all groups against the group of WT A549 cells (E). *p<0.05; ***p<0.001; **** p<0.0001.