Diphtheria toxin activates ribotoxic stress and NLRP1 inflammasome-driven pyroptosis

Abstract

The ZAKα-driven ribotoxic stress response (RSR) is activated by ribosome stalling and/or collisions. Recent work demonstrates that RSR also plays a role in innate immunity by activating the human NLRP1 inflammasome. Here, we report that ZAKα and NLRP1 sense bacterial exotoxins that target ribosome elongation factors. One such toxin, diphtheria toxin (DT), the causative agent for human diphtheria, triggers RSR-dependent inflammasome activation in primary human keratinocytes. This process requires iron-mediated DT production in the bacteria, as well as diphthamide synthesis and ZAKα/p38-driven NLRP1 phosphorylation in host cells. NLRP1 deletion abrogates IL-1β and IL-18 secretion by DT-intoxicated keratinocytes, while ZAKα deletion or inhibition additionally limits both pyroptotic and inflammasome-independent non-pyroptotic cell death. Consequently, pharmacologic inhibition of ZAKα is more effective than caspase-1 inhibition at protecting the epidermal barrier in a 3D skin model of cutaneous diphtheria. In summary, these findings implicate ZAKα-driven RSR and the NLRP1 inflammasome in antibacterial immunity and might explain certain aspects of diphtheria pathogenesis.

Figure 1.

C. diphtheriae, DT, and exoA activate pyroptosis in primary keratinocytes. (A) Simplified cartoon representation of eukaryotic ribosome translocation and EEF1/EEF2-targeting toxins. E, exit; P, peptidyl; A, aminoacyl. (B) Live cell imaging of primary keratinocytes treated with BHI media, sterile filtrates of C. diphtheriae grown in iron-restricted media or recombinant DT (150 ng/ml). The concentration of DT in C. diphtheriae media was normalized to ∼150 ng/ml. Brightfield images were overlaid with PI fluorescence (red). Images were taken at 20× magnification. Images are representative of three independent experiments. Distinct cell death morphology is marked by yellow arrows (pyroptosis, PI+ve cells with membrane swelling) and white arrows (apoptosis, cell shriveling). Only select representative cells are marked. Scale bar represents 100 μm. (C) Quantification of the percent of PI+ve cells over time in B. Error bars represent three biological replicates, with each drug treatment considered as one replicate. Significance values were calculated from Student’s t test at the 7-h time point. *, P ≤ 0.05. **, P ≤ 0.01. (D) Immunoblot of inflammasome activation markers in primary keratinocytes 24 h after treatment. ANS (1 µM); VbP (1 µM); DT (150 ng/ml), exoA (1 µg/ml). Cells in lane 6 were incubated with BHI broth, while lanes 7 and 8 were incubated with sterile-filtered C. diphtheriae or C. striatum conditioned BHI media. Immunoblot is representative of three replicate experiments. (E) IL-1β ELISA from TNFα-primed wild-type or DPH1 KO N/TERT culture media. Media were harvested 18 h after treatment. Significance values were calculated from one-way ANOVA with multiple group comparisons, from three biological replicates. ns, not significant. ****, P ≤ 0.0001. Source data are available for this figure: SourceData F1.

Figure S1.

Additional data on the effect of EEF2 targeting bacterial toxins, including DT. (A) Anti-puromycin immunoblot of primary keratinocytes subjected to the harringtonine run-off assay. Cells were treated with BHI media, sterile filtrates of C. diphtheriae, C. striatum for 6 h, or purified DT (150 ng/ml) for 3 h before harringtonine addition (2 µg/ml). Nascent peptides were then labeled with 10 µg/ml puromycin for 10 min at different intervals. (B) Immunoblot of inflammasome components NLRP1, GSDMD, and IL-1β in N/TERT with and without TNFα priming (25 ng/ml, 8 h). Streptolysin O (SLO, 1 µg/ml) was included as additional negative control. (C) IL-1β ELISA of culture media from N/TERT cells treated with the indicated recombinant proteins or compounds. LFn (150 ng/ml): Lethal Factor from B. anthracis (aa 34–288). PA (300 ng/ml). MxiH: Shigella flexneri type 3 secretion needle protein. All cells were primed with TNFα. Significance values were calculated from one-way ANOVA with multiple group comparisons. Error bars derived from data from three technical replicates. The graph represents one of two biological replicates. (D) Kinetics of PI uptake in unprimed N/TERT cells treated with DDB/Aplidine. Significance values were calculated from Student’s t test at 4 h. Error bars are derived from data from three technical replicates. Data represent one of two biological replicates. (E) The percentage of cells with ASC-GFP specks among 293T-ASC-GFP-NLRP1 cells transfected with the indicated plasmids. Cells were fixed 24 h after transfection. ASC-GFP specks were visualized using GFP epifluorescence and normalized to the total number of cells per field of view using DAPI nuclear counterstain. Error bars are derived from data from three technical replicates. Data represent one of two biological replicates. (F) Immunoblot of overexpressed 3xFLAG-sidI and R453P glycolysase-defective mutant. Note that the level of wild-type sidI is much lower than the R453P mutant due to its strong inhibitory effect on translation, as reported previously by Subramanian et al. (2022) Preprint. (G) Immunoblot of inflammasome activator markers GSDMD and IL-1β in primary keratinocytes treated with the indicated bacterial filtrate. C. diphtheriae was cultured in BHI broth and PGT media (see Materials and methods). ***, P ≤ 0.001; ****, P ≤ 0.0001. Source data are available for this figure: SourceData FS1.

Figure 2.

DT and C. diphtheriae trigger RSR downstream of EEF2 inactivation. (A) Immunoblot of ZAKα, p38 NLRP1 in primary keratinocytes treated with the indicated triggers in the presence of DMSO or M443 (1 µM). M443 was added 10 min before DT or bacterial filtrate. Lysates were harvested 3 h after treatment. Immunoblot representative of three replicate experiments. (B) Immunoblot of RSR kinases and GFP-NLRP1^linker in control and ZAKα KO N/TERT cells. ANS-treated cells were harvested 2 h after treatment. Indicated cells were primed with 25 ng/ml TNFα overnight and then treated with 150 ng/ml DT. Immunoblot representative of three replicate experiments. Source data are available for this figure: SourceData F2.

Figure 3.

DT and C. diphtheriae cause pyroptosis in a ZAKα and NLRP1-dependent manner but also cause other forms of cell death. (A) Comparison of PI uptake kinetics between control and NLRP1 KO primary keratinocytes in response to purified DT. Error bars represent three biological replicates, with each drug treatment considered as one replicate. Significance values were calculated from Student’s t test at the 7-h time point. **, P ≤ 0.01. (B) Immunoblot of GSDMD, cleaved caspase-3, and IL-1β in WT and NLRP1 KO keratinocyte lysates or media 24 h after the indicated treatment. Immunoblot is representative of three replicate experiments. (C) Comparison of PI uptake kinetics between control and ZAKα KO primary keratinocytes in response to purified DT. Error bars represent three biological replicates, with each drug treatment considered as one replicate. Significance values were calculated from Student’s t test at the 7-h time point. *, P ≤ 0.05. (D) Immunoblot of ZAKα, GSDMD, MCL-1, cleaved caspase-3 and IL-1β in WT and ZAKα KO keratinocyte lysates or media 24 h after the indicated treatment. Salinomycin (10 µM) does not activate the NLRP1 inflammasome and was used as a negative control. Immunoblot is representative of three replicate experiments. Source data are available for this figure: SourceData F3.

Figure S2.

Additional characterization of NLRP1 phosphorylation, ZAKα, and ATF3 in DT-induced pyroptosis and apoptosis. (A) IL-1β ELISA from control and NLRP1 KO human primary keratinocytes. Media were harvested 18 h after treatment. Purified DT (150 ng/ml). ANS (1 µM). VbP (3 µM). (B) IL-1β ELISA from NLRP1 KO N/TERT cells rescued with wild-type NLRP1 or NLRP1 3A mutant primed or treated with the indicated conditions. TNFa priming 18 h, media harvested 18 h later after treatment. (C) Kinetics of PI uptake for control and ZAKα KO primary keratinocytes. Error bars are derived from data from three technical replicates. Data represent one of two biological replicates. Significance values were calculated from Student’s t test at the 7-h time point. (D) Immunoblot of apoptotic markers (cleaved caspase-3 and PARP1) from the lysates of TNFα-primed N/TERT cells treated with C. diphtheriae BHI media filtrate and the indicated inhibitors. Beln: caspase-1 inhibitor belnacasan (5 µM). Emri: pan-caspase inhibitor emricasan (5 µM). 6p (0.5 µM). (E) Immunoblot of apoptotic markers from the lysates of TNFα-primed WT and ZAKα KO N/TERT cells treated with C. diphtheriae BHI media filtrate and the indicated inhibitors. Beln: caspase-1 inhibitor belnacasan (5 µM). Emri: pan-caspase inhibitor emricasan (5 µM). 6p (0.5 µM). Nefla: p38 inhibitor neflammapimod (0.5 µM). (F) ATF3 immunoblot in control and ATF3 KO primary keratinocytes. Lysates were harvested 3 h after ANS. (G) Immunoblot of GSDMD and IL-1β comparing inflammasome activation of Cas9 control and ATF3 KO keratinocytes treated with C. diphtheriae media (normalized to ∼150 ng/ml DT). ns, not significant; **, P ≤ 0.01; ****, P ≤ 0.0001. Source data are available for this figure: SourceData FS2.

Figure 4.

Identification of transcripts that are induced by multiple RSR agents across multiple cell types. (A) Design of RNAseq experiment using primary keratinocytes. Compound 6p is a ZAKα inhibitor. (B) Venn diagram showing the identification of 285 ZAKα-dependent, DT-induced upregulated transcripts in primary keratinocytes. (C) Gene Ontology (GO) analysis of ZAKα-dependent DT-induced upregulated transcripts. (D) Venn diagram demonstrating the overlap of transcriptional changes elicited by the indicated RSR agents. The source cell types are indicated in brackets. Fold change (FC) > 2 used a cutoff for datasets published in Subramanian et al. (2022) (Preprint; DDB, ANS, and L. pneumoniae). FC > 1.5 was used as the cutoff for UVB (GEO accession: GSE116968) in accordance with the depositing authors’ analysis. (E) Transcripts per kilobase million (TPM) transcript levels of ATF3, PMAIP1/NOXA, and GADD45A in mock and DT-treated control or 6p-treated primary keratinocytes. Significance values were calculated from -way ANOVA from three biological replicates shown. ns, not significant; ****, P ≤ 0.0001. (F) Kinetics of PI uptake of primed wild-type and ATF3 KO N/TERT cells treated with DT. Significance values were calculated from Student’s t test at the 7-h time point. n.s., not significant. (G) IL-1β ELISA of control and ATF3 KO primed N/TERT cells after the drug/toxin treatments. Significance values were calculated from one way ANOVA from two biological replicates. n.s., not significant.

Figure 5.

ZAKα inhibition rescues epidermal integrity by limiting pyroptosis in a model of cutaneous diphtheria. (A) Outline of the 3D human skin model of cutaneous diphtheria. (B) H&E staining demonstrating the histological changes caused by DT and VbP. Yellow arrows indicate dyskeratotic keratinocytes with vacuolated cytoplasm and condensed nuclei. Images representative of three biological replicates. Tissues were fixed 24 h after treatment. (C) Correlation between the fold change (FC) of 65 cytokines/chemokines after 24 h of DT or VbP treatment. (D) H&E staining of 3D skin treated with the indicated bacteria filtrate or compound. Yellow arrows indicate dyskeratotic keratinocytes with vacuolated cytoplasm and condensed nuclei. Gray arrows mark putative apoptotic cells with eosin-rich cytosol and condensed nuclei. Compound 6p is a ZAKα inhibitor. Images representative of three biological replicates. Tissues were fixed 24 h after treatment. (E) Quantification of the extent of the detachment between the dermal–epidermal layer based on the H&E staining of 3D skin in D. Significance values were calculated from one-way ANOVA. **, P ≤ 0.01. ****, P ≤ 0.0001. (F) GSDMD p30 staining of 3D skin treated with C. diphtheriae filtrate with and without compound 6p. Black arrows indicate membranous staining around foci of epithelial barrier damage. (G) Western blot of IL-18 and IL-1β p17 in the cultured media of 3D skin after the indicated treatment. (H) Significantly upregulated cytokines/chemokines in DT-treated 3D skin culture relative to untreated 3D skin from D. Log₁₀(adjusted P values) and log₂(fold change) were calculated from a multiparametric t test. Fold change cutoff is set at 5. (I) Significantly upregulated cytokines/chemokines in DT + 6p 3D skin culture relative to 6p only 3D skin from D. Log₁₀(adjusted P values) and log2(fold change) were calculated from a multiparametric t test. Fold change cutoff is set at 5. Source data are available for this figure: SourceData F5.

Figure S3.

Further data on C. diphtheriae–induced skin damage in 3D skin cultures. (A) Quantification of the percentage of cells with condensed nuclei and the detachment between the dermal–epidermal layer, based on the H&E staining of 3D skin. (B) Quantification of the percentage of cells with condensed nuclei based on the H&E staining of 3D skin sections shown in Fig. 5. (C) Immunohistochemistry (IHC) staining of plakoglobin (desmosome and adherens junction marker) and plectin (hemidesmosome marker) in 3D skin sections treated with the indicated conditions. Black arrows indicate foci of epidermal damage with loss of plakoglobin staining. Brackets indicate areas of DEJs with disorganized plectin staining. Scale bar = 50 µm. (D) H&E staining of 3D skin treated with DT and neflamapimod (p38i). Tissues were fixed 24 h after treatment. Scale bar = 50 µm. (E) H&E staining of 3D skin treated with DT and belnacasan (CASP1i). Tissues were fixed 24 h after treatment. Scale bar = 50 µm. (F) Quantification of the extent of the detachment between the dermal–epidermal layer based on the H&E staining of p38i-treated organotypic skin samples. (G) Quantification of the extent of the detachment between the dermal–epidermal layer based on the H&E staining of CASP1i-treated organotypic skin samples. (H) Significantly upregulated cytokines/chemokines in the indicated organotypic skin samples. Log₁₀(adjusted P value) and log₂(fold change) were calculated from a multiparametric t test. The samples correspond to D and F. Media from organotypic skin collected 24 h after treatment with DT and the inhibitors. (I) Significantly upregulated cytokines/chemokines in the indicated organotypic skin samples. Log₁₀(adjusted P value) and log₂(fold change) were calculated from a multiparametric t test. The samples correspond to E and G. Media from organotypic skin were collected 24 h after treatment with DT and the inhibitors. ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.