Shigella ubiquitin ligase IpaH7.8 targets gasdermin D for degradation to prevent pyroptosis and enable infection

Abstract

The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors pathogens that circumvent this host defense mechanism. Here, we show that the Shigella ubiquitin ligase IpaH7.8 functions as an inhibitor of GSDMD. Shigella is an enteroinvasive bacterium that causes hemorrhagic gastroenteritis in primates, but not rodents. IpaH7.8 contributes to species specificity by ubiquitinating human, but not mouse, GSDMD and targeting it for proteasomal degradation. Accordingly, infection of human epithelial cells with IpaH7.8-deficient Shigella flexneri results in increased GSDMD-dependent cell death compared with wild type. Consistent with pyroptosis contributing to murine disease resistance, eliminating GSDMD from NLRC4-deficient mice, which are already sensitized to oral infection with Shigella flexneri, leads to further enhanced bacterial replication and increased disease severity. This work highlights a species-specific pathogen arms race focused on maintenance of host cell viability.

Keywords: GSDMD; Shigella; inflammasome; proteasome; pyroptosis; ubiquitin ligase; virulence.

Figure 1.. IpaH7.8 ubiquitin ligase activity blocks pyroptosis in human cells

(A) Shigella effector positive selection screen strategy (NGS, next generation sequencing). (B) The non-canonical inflammasome pathway. (C) Volcano plot depicting changes in effector abundance in the LPS- and dox-treated sample relative to the dox-only control. The x-axis corresponds to the Log₂ fold change in gene expression, and the y-axis indicates the adjusted p-value. Wald test, FDR adjustment by Benjamini-Hochberg method (n = 3 per group). (D, E, G and H) Graphs indicate the percentage of LDH released from Ea.hy926 cells (D, G, and H) or iMacs (E) after LPS electroporation (D, E and G) or treatment with 25 μM Valboro-Pro (H, VbP). (F) Domain architecture of IpaH7.8. Where indicated, doxycycline (dox) was used to induce expression of wild-type (WT) IpaH7.8 or mutant IpaH7.8(C357A). Bars represent the mean of 3–4 biological replicates, each plotted as a single data point. See also figure S1.

Figure 2.. IpaH7.8 targets GSDMD for proteasomal degradation

(A) Proteomics strategy to identify IpaH7.8 substrate(s) in Ea.hy926 cells. (B) Scatter plot depicting doxycycline (dox)-induced IpaH7.8-dependent changes in global protein abundance. Results represent 2 biological replicates. The x-axis corresponds to the Log₂ ratio of 6 h dox / 0 h no dox. The Log₂ ratio of 6 h dox / 6 h no dox is on the y-axis. P-values determined by ANOVA. (C, D and E) GSDMD (C), GLMN (D), and NLRP1, (E) (red circles) and associated ubiquitinated lysines (blue circles) are quantified at different times in Ea.hy926 cells after doxycycline (dox)-induced expression of IpaH7.8. Each circle represents the Log₂ ratio of dox treated / no dox (2 biological replicates each). (F) Immunoblots of 293T cells co-transfected with myc-tagged gasdermins and Flag-IpaH7.8 (WT, wild-type or CA, mutant C357A). Results representative of 3 independent experiments. (G) The mammalian ubiquitin pathway. Specific inhibitors of UAE/E1 (MLN-7243), NAE1 (MLN-4924), and the proteasome (Btz) are annotated at the relevant steps. Human cullin-RING family E3 ligases require the co-factor NEDD8 (NED) for activity. (H) Immunoblots of Ea.hy926 cells with dox-inducible Flag-IpaH7.8 after treatment for 4 h with DMSO vehicle or 1 μM Btz, MLN-7243 (UAE/E1), or MLN-4924 (NAE1). (I) Immunoblots of in vitro ubiquitination reactions using GSDMD and IpaH7.8 (WT or C357A). Results representative of 3 independent experiments. See also figure S2 and data S1.

Figure 3.. IpaH7.8 targets human GSDMD, but not mouse GSDMD

(A) Immunoblots of in vitro ubiquitination reactions using human (hs) or mouse (mm) GSDMD and IpaH7.8. Results representative of 3 independent experiments. (B) Immunoblots of 293T cells transfected with the GSDMD and IpaH7.8 constructs indicated. Results representative of 3 independent experiments. (C and D) Immunoblots of 293T cells co-transfected with IpaH7.8 and GSDMD (wild-type, WT, or the mutants indicated). 3KR, K55R/K62R/K205R. 15KR, pan KR mutant. mm, mouse. hs, human. mm-K, introduces the 6 non-conserved lysine residues from human GSDMD into mouse GSDMD. (E) Immunoblots showing co-immunoprecipitation of IpaH7.8(C357A) and GSDMD from co-transfected 293T cells. (F) Binding isotherms of hsGSDMD (black circles) and mmGSDMD (turquoise circles) with IpaH7.8 measured by microscale thermophoresis (MST). The y-axis indicates the fraction bound, the x-axis is the Log₁₀ of the IpaH7.8 concentration in molar. Each circle represents the mean ± SD of 3 technical replicates. Results representative of 3 independent experiments. See also figure S3.

Figure 4.. The GSDMD Pore-forming domain is the target of IpaH7.8

(A) Domain swapping strategy depicts PFD-boundaries for human and mouse GSDMD. (B, C and E) Immunoblots of 293T cells co-transfected with IpaH7.8 (wild-type, WT, unless mutant C357A, CA is indicated) and GSDMD (wild-type, WT, or the mutants indicated). mm, mouse. hs, human. 5XA, the five residues indicated were replaced with five alanines. PFD, pore-forming domain; CTD, C-terminal domain. hs↔mm aa swap, human-mouse GSDMD chimera with indicated residues swapped in. (D) Alignment of mouse (mm) and human (hs) GSDMD N-terminal protein sequences highlighting human residues 11–25 (red bar). (F) Structural overlay of human (hs) GSDMD (6N9O, magenta, aa 11–25) and mouse (mm) GSDMD (6N9N, cyan, aa 11–26). N, N-terminal. C, C-terminal. mm18G is colored red. (G) Truncation mutants of IpaH7.8(C357A) used in (H) and (I). (H and I) Immunoblots showing co-immunoprecipitation of the IpaH7.8 LRR and GSDMD from co-transfected 293T cells. Results representative of 3 independent experiments. See also figure S4.

Figure 5.. GSDMD restricts S. flexneri replication in Nlrc4^−/− mice

(A) Immunoblots of HeLa cells infected by S. flexneri 5a strain M90T wild-type (WT) or ΔipaH7.8. h.p.i, hours post-infection. Results representative of 3 independent experiments. (B) Immunoblots of HeLa cells infected with S. flexneri 5a strain M90T for 2 h in the presence of DMSO vehicle or 1 μM Bortezomib (Btz), or MLN-7243 (UAE1). Results representative of 3 independent experiments. (C) Immunoblots of iMacs infected by S. flexneri 5a strain M90T WT or ΔipaH7.8. Results representative of 3 independent experiments. (D) Percentage of infected Caco-2 cells killed by GFP-expressing S. flexneri 5a strain M90T wild-type (WT, grey circles), ipaH7.8 deficient (ΔipaH7.8, green circles), or un-infected control (black circles) based on their uptake of propidium iodide (GFP/PI++ cells over total GFP+ cells). Circles represent the mean ± SD calculated from 6 infected wells. Data representative of 3 independent experiments. (E to L) Characterization of control (black circles, n = 6), Gsdmd^−/− (grey circles, n = 5), Nlrc4^−/− (green circles, n = 9) and Nlrc4^−/−Gsdmd^−/−(dKO, white circles, n = 6) female mice at 48 h.p.i. challenged with 10⁷ CFU (colony forming units) of S. flexneri 2a strain 2457T. Endpoint harvests were performed 48 h.p.i. (E) Representative images of cecum and colon. Note the cecum tissue thickening (size reduction), macroscopic edema, and lack of stool pellets (blue arrows) in dKO tissue. (F) Percentage change in body weight. (G) Feces weights before and after dehydration. (H) Fecal blood scores (1 = occult blood, 2 = macroscopic blood). (I) Representative images of H&E stained cecum and colon tissue from infected mice (50 μm scale bar). (J) Blinded quantification of histology score (cumulative) for tissues in I. (K) Tissue KC levels measured by ELISA. (L) CFUs in the IEC-enriched fraction of gentamicin-treated cecum and colon (combined). (G and H, J to L) Each symbol represents one mouse, lines represent mean ± SD. (F) Each symbol represents the mean of all animals within the indicated group ± SD. Mann-Whitney test, * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant (p > 0.05). See also figure S5.