Intracellular S. flexneri infection causes shigellosis in humans. Although most intracellular bacteria reside in vacuoles, S. flexneri, like Burkholderia spp., live freely in the host cytosol, inevitably exposing their LPS to caspase-11/4. Wild-type (WT) mice, unlike Casp11−/− mice, survived B. thailandensis infection8 (Fig. 1a, Extended Data Fig. 1a). Mice are increasingly being used as a surrogate host for S. flexneri. Unexpectedly, both WT and Casp11−/− mice succumbed to lethal S. flexneri infection (Fig. 1a) and tolerated similarly the low-dose challenge (Extended Data Fig. 1a). Given the absence of caspase-11-mediated protection, we assayed non-canonical inflammasome activation upon S. flexneri infection. Casp1−/− immortalized bone marrow-derived macrophages (iBMDMs) were used to avoid interference by the canonical inflammasome. Although B. thailandensis and S. Typhimurium ΔsifA induced Casp11-dependent GSDMD cleavage and pyroptosis8, S. flexneri triggered little pyroptosis (Fig. 1b) despite a higher infection efficiency (Extended Data Fig. 1b). In epithelium-derived human SiHa and A431 cells, S. flexneri, unlike S. Typhimurium ΔsifA, also did not activate the caspase-4–GSDMD pyroptosis pathway (Fig. 1b, Extended Data Fig. 1c, d). Purified LPS from S. flexneri was highly pro-pyroptotic (Extended Data Fig. 1e). Thus, S. flexneri evaded caspase-11/4-mediated pyroptosis.

Fig. 1: S. flexneri blocks cytosolic LPS-induced pyroptosis through OspC3.
figure 1

a, Survival curves of WT or Casp11−/− mice intraperitoneally infected with S. flexneri or B. thailandensis (2 × 107 CFU per mouse); two-tailed log-rank (Mantel–Cox) test. b, c, Indicated SiHa cells or iBMDMs were infected with S. flexneri (S.f., WT or an ospC3 deletion/complementation strain), B. thailandensis (B.t.) or S. Typhimurium (S.T.) ΔsifA. LDH release-based cell death data are means (bars) of three individual replicates (circles). Cell supernatants were blotted with anti-cleaved GSDMD-C antibody. Data are representative of two (a) or three (b, c) independent experiments. For gel source data, see Supplementary Fig. 1.

Source data

The guanylate-binding protein (GBP) family promotes the release of LPS from intracellular bacteria and its presentation to caspase-11/4 (refs. 9,10). IpaH9.8, a Shigella ubiquitin-ligase T3SS effector, targets multiple GBPs for degradation11,12,13,14. A 2013 report proposed that S. flexneri uses the T3SS effector OspC3 to target caspase-4 but, notably, not caspase-11 (ref. 15). We examined whether IpaH9.8, OspC3 or another factor underlies evasion of pyroptosis by S. flexneri using SiHa, A431 and iBMDM cells (Fig. 1c, Extended Data Fig. 1d, f, g). Infection with ΔipaH9.8, compared to WT bacteria, caused negligibly increased pyroptosis. By contrast, ΔospC3 induced extsensive pyroptosis with evident GSDMD cleavage, which was diminished by re-expression of OspC3 in the bacteria or deletion of CASP4/11 deletion in the host cells. Deletion of all seven GBPs from A431 cells affected pyroptosis during early but not late infection (Extended Data Fig. 1h). This is consistent with the notion that GBPs, having little LPS-binding activity (Extended Data Fig. 1i), are not absolutely required for bacteria-induced caspase-4 activation. Thus, S. flexneri requires OspC3 to evade LPS-stimulated pyroptosis.

OspC3 expression in host cells blocked the induction of pyroptosis by S. flexneri ΔospC3, S. Typhimurium and even LPS alone (Extended Data Fig. 1j), suggesting that it has a bacteria-independent function. OspC3 co-immunoprecipitated with the p20/p10 form of caspase-4(C258A) (protease-deficient; C/A hereafter) in 293T cells (Extended Data Fig. 2a). The interaction did not cause p20–p10 dissociation, in contrast to earlier findings15. OspC3 also co-immunoprecipitated with inactive p20- and-p10-unprocessed caspase-4/11 (Fig. 2a, Extended Data Fig. 2b). OspC3 did not affect the proteolytic activity of caspase-4/11-p20/p10 (Extended Data Fig. 2c–e). Purified OspC3 also did not inhibit LPS-induced activation of pro-caspase-4 to cleave GSDMD, but it blocked pyroptosis when electroporated into cells (Extended Data Fig. 2f, g). Thus, hijacking of caspase-4/11 by OspC3 involves a cell-dependent mechanism.

Fig. 2: OspC3 catalyses an NAD+-dependent modification of caspase-4/11.
figure 2

a, b, Co-immunoprecipitation of caspase-4/11-p30-C/A with OspC3 and modification of caspase-4-p20/p10 by OspC3 in 293T cells. ce, Caspase-4/11-p30-C/A, expressed alone or with OspC3 in bacteria (c, d) or reacted with OspC3 with or without NAD+ in vitro (e), was analysed by native/SDS-PAGE (c, e) or ESI–MS (d). Control, OspC3-modified caspase-4-p30-C/A. f, CASP4−/− HeLa cells expressing Flag–caspase-4-p30-C/A were infected as indicated. Anti-Flag immunoprecipitates were analysed as shown. Data are representative of three (ae) or two (f) independent experiments. For gel source data, see Supplementary Fig. 1.

In 293T cells, OspC3 induced slower migration of caspase-4-p10 on an SDS gel (Fig. 2b, Extended Data Fig. 2a). Caspase-4/11-p30, co-expressed with OspC3 in Escherichia coli, exhibited a marked shift on a native gel (Fig. 2c), indicating a post-translational modification (PTM). Electrospray ionization–mass spectrometry (ESI–MS) identified a 524-Da modification, which was located to 314RDSTMGSIF322 within caspase-4-p10 by collision-induced dissociation (CID)–MS (Fig. 2d, Extended Data Fig. 2h, i). MS/MS detected fragment ions with mass-to-charge ratios of 136.06, 348.07 and 428.04, matching the mass of adenine, AMP and ADP, respectively (Extended Data Fig. 2j). This reminded us of ADP-ribosylation, in which ADP-ribose (ADPR) from nicotinamide adenine dinucleotide (NAD+) is usually transferred to serine, arginine, asparagine, aspartate, glutamate or glutamine. Although the OspC3-induced PTM is 17 Da smaller than ADP-ribosylation, NAD+ enabled recombinant OspC3 to modify caspase-4/11-p30 by 524 Da (Fig. 2e, Extended Data Fig. 2k, l). In S. flexneri infection, the 314RDSTMGSIF322 peptide and the corresponding caspase-11 peptide showed the 524-Da modification in an ospC3-dependent manner (Extended Data Fig. 2m). Consistently, caspase-4-p30-C/A from cells infected with WT S. flexneri but not ΔospC3 had a mobility shift similar to that in the in vitro assay (Fig. 2f). Thus, OspC3 catalyses an NAD+-mediated PTM on caspase-4/11.

Electron-transfer/higher-energy collision dissociation (EThcD)–MS showed that Arg314 and Arg310 in caspase-4 and -11, respectively, harboured the modification (Fig. 3a, Extended Data Fig. 3a). Replacing these residues with lysine or asparagine abolished the modification (Fig. 3b, Extended Data Fig. 3b–e). Quantitative high-performance liquid chromatography (HPLC)–MS analyses of the reaction (Extended Data Fig. 4a) revealed that one molecule of free nicotinamide (Nam) was released upon modification of one molecule of caspase-4 by one molecule of NAD+. Thus, the OspC3-catalysed PTM may contain an initial ADP-ribosylation and an additional 17-Da mass reduction reaction.

Fig. 3: OspC3 catalyses ADP-riboxanation on an arginine in caspase-4/11.
figure 3

a, EThcD–tandem mass spectrum of the Arg314-containing peptide from OspC3-modified caspase-4-p30-C/A in bacteria. b, Caspase-4-p30-C/A was reacted with OspC3 with or without NAD+, followed by native/SDS-PAGE analyses. c, Mass changes of OspC3-modified caspase-4-p30 by NAD+ analogues. d, OspC3-induced mass changes on caspase-4 Arg314-containing peptide from normal or 13C6,15N4-l-arginine–labelled 293T cells. e, Quantification of release of ammonia/ammonium from the OspC3-modification reaction; data are means (bars) of three individual replicates (circles). f, Caspase-4-p30-C/A was reacted with OspC3 and a ribosyl 2′-substituted NAD+ analogue. Control, OspC3-modified caspase-4-p30-C/A. g, Chemical structures of ADP-riboxanated and ADP-ribosylated arginine. Data are representative of three (ac, e, f) or two (d) independent experiments. For gel source data, see Supplementary Fig. 1.

Source data

Fourteen NAD+ analogues or derivatives were assayed in OspC3 modification of caspase-4 (Extended Data Fig. 4b). NAD+ fragments (ADPR, cyclic-ADPR (cADPR) and nicotinamide mononucleotide (NMN)), α-NAD+, nicotinic acid adenine dinucleotide (NAAD+), nicotinamide adenine dinucleotide phosphate (NADP+) and NADPH were inactive. By contrast, NADH, thio-NAD+ and thio-NADH, altered in the Nam part, supported the 524-Da modification (Fig. 3c, Extended Data Fig. 4b, c). Deamino-NAD+, biotin-NAD+, ε-NAD+ or nicotinamide guanine dinucleotide (NGD+) allowed modifications that preserved the mass difference between the cognate analogue and NAD+. These confirm the transfer of ADPR to caspase-4/11 with Nam being the leaving group. Indeed, OspC3-modified caspase-4 was recognized by an anti-ADP-ribose antibody16 (Fig. 2f, Extended Data Fig. 4d).

NGD+-mediated modification also had a 17-Da mass reduction from the ‘GDP-ribosylation’. A non-specific pyrophosphohydrolase, NUDT16 (ref. 17), removed an AMP from OspC3-modified caspase-4 (Extended Data Fig. 4d, e). These data suggest that the 17-Da loss occurs on the phosphoribosylated arginine. We performed stable isotope labelling by amino acids in cell culture (SILAC), using 13C6,15N4-l-arginine to label Flagcaspase-4-p20/p10 expressed alone or with OspC3 in 293T cells. MS detected a 523-Da (not 524-Da) increase on a caspase-4 Arg314-containing peptide (Fig. 3d, Extended Data Fig. 5a). The 1-Da change suggests that one Nω atom in ADP-ribosylated arginine is removed via internal deamination, explaining the 17-Da reduction. Consistent with this, free NH3/NH4+ was detected in the modification reaction (Fig. 3e).

The above analyses predict that a nucleophile adjacent to the ADP-ribosylated arginine guanidino performs the deamination. Studies of ADP-ribosylation-based elimination18 suggest that the ribosyl-2′-OH could be a candidate nucleophile. β-2′-Deoxy-2′-H-NAD+ (2′-H-NAD+) and 2′-fluoro-NAD+ were assayed in the OspC3-catalysed modification (Extended Data Fig. 5b). 2′-Fluoro-NAD+, which is incompetent for canonical ADP-ribosylation19, could not support OpsC3 modification of caspase-4. OspC3 could use 2′-H-NAD+; notably, the modification was merely the transfer of 2′-deoxy-ADPR without further deamination (Fig. 3f, Extended Data Fig. 5c). The remaining puzzle is the atom on which the initial ADP-ribosylation occurs. Both Nω and Nδ in arginine can accept ADPR from NAD+. Ninhydrin could bond simultaneously with the two Nω in native arginine, as noted with Arg314 in unmodified caspase-4 (Extended Data Fig. 5d, e). For canonical arginine Nω-ADP-ribosylation (Rab4a by ExoS20), the modified arginine resisted conjugation by ninhydrin (Extended Data Fig. 5f). For 2′-H-NAD+-mediated modification, the 2′-deoxy-ADP-ribosylated Arg314 could react with ninhydrin (Extended Data Fig. 5e). We propose that OspC3 modifies Arg314/Arg310 of caspase-4/11 by two steps (Extended Data Fig. 5g). First, the arginine Nδ (rather than Nω) performs nucleophilic substitution of the Nam in NAD+. Second, the ribosyl-2′-OH of ADPR initiates a deamination to remove one Nω, forming an oxazolidine ring. We designate this arginine ADP-2′-imine-ribofurano[1′,2′:4,5]oxazolidination modification as ADP-riboxanation (Fig. 3g), catalysed by arginine ADP-riboxanase activity in OspC3.

S. flexneri harbours ospC1, ospC2 and a pseudogene ospC4 in the ospC3 locus. OspC1, OspC2 and OspC3 (>60% sequence identity; Extended Data Fig. 6a) share a C-terminal ankyrin-repeat domain (ARD) and an N-terminal (N) domain (Fig. 4a). ΔospC1 or ΔospC2 caused no increase in pyroptosis in infected cells (Extended Data Fig. 1f). OspC1/2 could not block cytosolic LPS-induced pyroptosis (Extended Data Fig. 6b). Purified OspC1/2 barely modified caspase-4 (Fig. 4b). Notably, the ARD of OspC3, but neither OspC1/2 nor their ARDs, readily co-immunoprecipitated with caspase-4-p30 (Fig. 4c, Extended Data Fig. 6c). Replacing the ARD in OspC3 with that of OspC1/2 diminished its caspase-4-modification and pyroptosis-blocking activity (Fig. 4b, Extended Data Fig. 6b). Conversely, chimeric proteins with the OspC1/2 N-domain and OspC3 ARD were highly active. Thus, the ARD of OspC3 determines caspase-4/11 recognition; OspC1/2 use their ARDs to target other host proteins for ADP-riboxanation.

Fig. 4: Analyses of the OspC family and mechanisms of OspC3 inactivation of caspase-4/11.
figure 4

a, Domain structure of OspC3 (red, residues essential for its ADP-riboxanase activity). b, Caspase-4-p30-C/A was reacted with OspC or a chimeric OspC protein, followed by native/SDS-PAGE analyses. c, Co-immunoprecipitation of caspase-4-p30-C/A with the ARD of an OspC in 293T cells. d, e, Caspase-4-p30-C/A modified by OspC3 (WT or D177A) in vitro (d) or in E. coli (e) was analysed by native/SDS-PAGE or MS, respectively. e, Extracted ion chromatograms of the Arg314-containing peptide. f, Indicated SiHa or A431 cells expressing OspC3 or caspase-4 (WT or R314A) were electroporated with LPS or muramyl dipeptide (MDP). g, h, GSDMD was subjected to cleavage by an indicated form of caspase-4/11-p20/p10. Control (b, d), OspC3-modified caspase-4-p30-C/A. Data are representative of two (e, f) or three (bd, g, h) independent experiments. For gel source data, see Supplementary Fig. 1.

Random mutagenesis identified Phe141, Phe186, Glu192, Glu326 and His328 in OspC3 (Fig. 4a, Extended Data Fig. 6a) as essential for ADP-riboxanating caspase-4/11 and blocking pyroptosis (Extended Data Fig. 6d–g). Another D177A mutation supported ADP-ribosylation but blocked subsequent deamination (Fig. 4d, e). Although OspC3(D177A)-modified caspase-4 was sensitive to ADP-ribosylarginine hydrolase (ADPRH), WT OspC3-catalysed ADP-riboxanation resisted demodification by ADPRH and other known host ADP-ribosylhydrolases (Extended Data Fig. 6h, i). Thus, hijacking of caspase-4/11 by ADP-riboxanation is more advantageous to bacterial virulence.

OspC3 blocked LPS-induced caspase-4 autoprocessing (Fig. 4f). This was recapitulated by mutations of Arg314 that also inhibited infection or LPS-induced pyroptosis (Fig. 4f, Extended Data Fig. 7a, b). Mutations of Arg310 in caspase-11 had the same effect (Extended Data Fig. 7c–e). OspC3 could also ADP-riboxanate already activated caspase-4/11 (Fig. 2b); the modified caspase-4/11-p20/p10, like their Arg314/Arg310 mutants, failed to target GSDMD (Fig. 4g, h, Extended Data Fig. 7f, g) owing to structural interference with the GSDMD-binding exosite21. Arg314/Arg310, which are conserved in caspases (Extended Data Fig. 7h), coordinate substrate P1 aspartate; caspase-4/11-p20/p10 R314/R310 mutants could not cleave the peptide substrate (Extended Data Fig. 7i). Thus, ADP-riboxanation blocked caspase-4/11 activation and cleavage of their substrate.

We used the inactive OspC3 E192A/H328A mutant (EH/AA) (Extended Data Fig. 6d–g) and assessed the function of caspase-11 ADP-riboxanation in Shigella infection. WT mice survived S. flexneri ΔospC3 infection, and this effect was reversed by complementation with OspC3 WT but not the EH/AA mutant (Fig. 5a). Accordingly, mice infected with ΔospC3 alone or ΔospC3 expressing OspC3 EH/AA had lower bacterial burdens than mice infected with WT OspC3-expressing strain (Fig. 5b). Unlike WT mice, Casp11−/− mice succumbed equally to S. flexneri WT and ΔospC3, which replicated to a similarly high level. Notably, S. flexneri ΔospC3-infected mice produced much more anti-Shigella IgG than WT bacteria-infected mice at the 10% LD50-normalized dose (the burden of ΔospC3 was not higher than WT bacteria at 24 h after infection), but this effect was abolished in Casp11−/− and Gsdmd−/− mice (Fig. 5c, Extended Data Fig. 8a). ΔospC3-infected mice were more resistant to lethal S. flexneri re-infection, and this increased resistance was also absent in Casp11−/− mice (Fig. 5d). Such effects occurred at multiple inoculation doses (Extended Data Fig. 8b, c). These findings suggest that caspase-11-mediated pyroptosis has an intrinsic function of activating humoral immunity and also highlight the importance of OspC3-catalysed ADP-riboxanation for evasion of caspase-11-mediated pyroptosis by Shigella.

Fig. 5: OspC3 underlies evasion by Shigella of pyroptosis-mediated defence that promotes anti-Shigella humoral immunity.
figure 5

a, b, WT and Casp11−/− mice were infected intraperitoneally with S. flexneri WT or an ospC3 deletion/complementation strain (2 × 107 CFU per mouse). a, Survival curves. Top, n = 16 for WT/WT and WT/ΔospC3 + pOspC3, n = 17 for WT/ΔospC3 and n = 15 for WT/ΔospC3 + pOspC3-EH/AA. Bottom, n = 6 for all groups. b, Bacterial loads. n = 5 for S. flexneri WT-infected Casp11−/− mice, n = 7 for S. flexneri ΔospC3 + pOspC3-infected WT mice and n = 6 for all other groups. cf, Indicated mice were immunized with S. flexneri WT or ΔospC3 (1.2 × 106 and 4 × 106 CFU per mouse in WT mice, respectively (both 10% LD50); both 2 × 106 CFU per mouse in Casp11−/− and Gsdmd−/− mice) (c, d) or other deletion strains (2 × 106 CFU per mouse) (e, f). c, e, Anti-Shigella antibody in the sera of immunized mice. d, f, Indicated immunized mice were re-challenged with WT S. flexneri (1.5 × 108 CFU per mouse in d and the upper panel of f and 1 × 108 CFU per mouse in the lower panel of f). a, d, f, Two-tailed log-rank (Mantel–Cox) test (****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01). b, c, e, Median values, two-tailed Mann–Whitney U-test. All data are representative of two independent experiments.

Source data

Development of a Shigella vaccine has been challenging. ΔicsA and ΔguaBA mutants are being developed as live-attenuated vaccines22. Compared to ΔicsA and ΔguaBA, S. flexneri ΔospC3 induced a higher level of anti-Shigella IgG (Extended Data Fig. 8d). Deletion of ospC3 on either ΔicsA or ΔguaBA background further boosted antibody production and conferred better protection from WT S. flexneri re-challenge (Fig. 5e, f). The better immunization of ΔicsAΔospC3 over ΔicsA also occurred at lower or higher inoculation doses in both C57BL/6 and BALB/c mice (Extended Data Fig. 8e–h). These findings are valuable for Shigella vaccine development, although with the limitation of the mouse model.

A BLAST search identified 27 OspC homologues in diverse bacteria, including Vibrio, Salmonella, Erwinia and Chromobacterium (Extended Data Fig. 9a). Homology of their catalytic domains and ARDs to those of OspC3 ranges from 99% to 56% and from 100% to 27%, respectively. Certain homologues readily ADP-riboxanated caspase-4/11 and blocked LPS-induced pyroptosis, and this effect was abolished by the corresponding EH/AA mutations (Extended Data Fig. 9b, c). For homologues that could not modify caspase-4/11, replacing their ARDs with that of OspC3 enabled the modification (Extended Data Fig. 9c). CopC from Chromobacterium violaceum, a deadly bacterium that causes hepatic abscesses in humans, could ADP-riboxanate caspase-4 and inhibit LPS-induced pyroptosis, less potently than OspC3 (Extended Data Fig. 10a–c). CopC could also modify caspase-4/11 during infections, and C. violaceum ΔcopC showed decreased replication in infected mouse liver (Extended Data Fig. 10d–f). Thus, OspC-like ADP-riboxanases are widely used by bacteria for various functions, including blocking pyroptosis.

In summary, Shigella uses OspC3 to modify caspase-11/4 and thereby thwart the inflammasome/pyroptosis-mediated defence. This differs from known bacterial inflammasome-modulating strategies that are self-alterations or indirect, such as inhibition of the Pyrin inflammasome by Yersinia YopM23. Future studies will uncover other inflammasome/pyroptosis-targeting effectors. OspC3 catalyses arginine ADP-riboxanation; the activity is shared by the OspC family in bacteria. Arginine ADP-riboxanation might also exist in eukaryotes, and could be identified by mining ADP-ribosylome proteomic data.


No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment.

Plasmids, antibodies and reagents

DNAs for ospC1, ospC2 and ospC3 were amplified from S. flexneri 2a strain 2457T. DNA for exoS was amplified from Pseudomonas aeruginosa strain PAO1. DNA for sdeA was amplified from Legionella pneumophila strain Lp02. DNA for copC was amplified from the genomic DNA of C. violaceum strain 12472. Complementary DNA (cDNA) for NUDT16 was from an ORF library of Invitrogen (clone ID: IOH61424). cDNAs for human caspase-4, GSDMD, Rab4A, YWHAB and caspase-11 were described previously2,24,25. The DNAs were ligated into pCS2-Flag, pCS2-3×Flag, pCS2-3×Flag-GST, pCS2-3×HA, pLVX-3×Flag or FUIGW-Flag vectors for transient or stable expression in mammalian cells. For recombinant protein expression in E. coli, the DNAs were inserted into pGEX-6p-2, pET28a-6×His, pET28a-6×His-SUMO, pET21a, pACYCDuet-1, pQE-30 or pAC-SUMO (generated by replacing the replication origin of pET28a-6×His-SUMO with the p15A replicon derived from the pACYC vector). For complementation in the S. flexneri ΔospC3 strain, C-terminal Flag-tagged DNAs for ospC3 and its mutants, together with its native promoter sequence, were cloned into the pET28a vector. The pSpCas9(BB)-2A-GFP plasmid (PX458) used for generating knockout cells was obtained from Addgene (48138). All truncations and point mutations were generated by the standard polymerase chain reaction (PCR) cloning method. All plasmids were verified by DNA sequencing.

Monoclonal antibodies against human GSDMD (ab210070/EPR19829), mouse GSDMD (ab219800/EPR20859), human cleaved GSDMD-C domain (ab227821/EPR20885-203)21 and mouse cleaved GSDMD-C domain (ab255603/EPR20859-147)21 were from Abcam. Antibodies against Flag (F3165/M2 and F7425/polyclonal) and tubulin (T5168/B-5-1-2) were from Sigma-Aldrich. The anti-HA antibody (3724/C29F4) was from Cell Signaling Technology, and rabbit Fc-fused mono-ADP-ribose binding reagent (MABE1076) was from Sigma-Aldrich. The monoclonal antibody for caspase-4 and polyclonal antibody for caspase-11 were generated by the in-house facility at the National Institute of Biological Sciences (NIBS) in Beijing, China. For western blot, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (NA931/polyclonal) and HRP-conjugated anti-rabbit IgG (NA934/polyclonal) were purchased from GE Healthcare Life Sciences. For enzyme-linked immunosorbent assay (ELISA), HRP-conjugated goat anti-mouse IgG (1036-05/polyclonal) was purchased from SouthernBiotech (lot no. D4913-WJ86H). Antibodies for Flag and tubulin were used at 1:5,000 dilution, and all other primary antibodies were used at 1:1,000 dilution for western blot. Secondary antibodies were used at 1:5,000 and 1:6,000 dilutions for western blot and ELISA, respectively.

The following chemical reagents were purchased from Sigma-Aldrich: NAD+ (N1636), NADH (N8129), NADPH (N7505), NGD+ (N5131), ε-NAD+ (N2630), Thio-NAD+ (T7375), Deamino-NAD+ (N6506), NAAD+ (N4256), α-NAD+ (N6754), α-NADH (N6879), NMN+ (N3501), ADPR (A0752) and ninhydrin (N4876). The ammonia assay kit (MAK310) was also from Sigma-Aldrich. 2′-F-NAD+ (D148) was from the BIOLOG Life Science Institute. Biotin-NAD+ (4670-500-01) was from Trevigen. Thio-NADH (BIB5005) was from Apollo Scientific. NADP+ (432216) was from J&K Scientific. Cyclic-ADPR (21417) was from Cayman Chemical. 2′-H-NAD+ was synthesized at the chemical centre of our institute. All other chemical reagents used were from Sigma-Aldrich unless otherwise noted.

Bacterial strains and infection

S. flexneri 2a strain 2457T, B. thailandensis E264, C. violaceum 12472 and S. Typhimurium SL1344 were used in cell culture infections, and the first three were used to infect mice. S. flexneri ΔospC1, ΔospC2 and ΔospC3 mutants were generated by using the λ Red recombineering method26. C. violaceum ΔcopC was generated by homologous recombination using the suicide vector pDM4-SacB. S. flexneri ΔipaH9.8 and S. Typhimurium ΔsifA have been described previously1,11. For infection in epithelial cells, S. flexneri strains were transformed with an afimbrial adhesin (Afa) locus to achieve high infection efficiency. For in vivo infection, the mouse-passaged B. thailandensis strain was used8.

Cultured cells were seeded in 24-well plates 12–16 h before infection. iBMDMs were primed with 0.1 µg ml−1 of LPS for 16 h to stimulate caspase-11 expression. The bacteria were cultured overnight at 37 ºC in 2× YT medium with shaking at 220 rpm. Overnight bacterial cultures were diluted 1:50 (1:20 for B. thailandensis) in fresh 2× YT medium and cultured at 37 ºC with shaking for about 3.5 h (4.5 h for B. thailandensis) when OD600 reached about 1.7. Bacteria were diluted in serum-free Dulbecco’s modified Eagle’s medium (DMEM) to achieve the desired multiplicity of infection (MOI: 25 for S. flexneri; 50 for S. Typhimurium; 100 for B. thailandensis; and 10 for C. violaceum). Infection was started by centrifugation at 800g for 5 min at room temperature followed by incubation at 37 ºC in a 5% CO2 incubator. After 1 h of infection (0.5 h for S. Typhimurium in iBMDMs, 1.5 h for B. thailandensis), cell culture media were replaced with fresh serum-free DMEM supplemented with appropriate antibiotics (100 μg ml−1 of gentamycin and 34 μg ml−1 of chloramphenicol for S. flexneri and S. Typhimurium, 100 μg ml−1 of gentamycin for B. thailandensis). Infected cells were further incubated for different time durations dependent upon the context of infection. Specifically, iBMDMs were incubated for 1 h (S. Typhimurium), 2.5 h (S. flexneri) and 6.5 h (B. thailandensis); SiHa and A431 cells were incubated for 3.5 h. The cell culture media were collected and subjected to LDH release assay or trichloroacetic acid precipitation to obtain released protein samples. Infection of A431 cells with S. flexneri was performed in the presence of 4 μM human α-defensin 5 (kindly provided by Dr. Dan Xu, Xi'an Jiaotong University, China). The LDH assay was performed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega).

WT C57BL/6 mice, purchased from Beijing Vital River Laboratory Animal Technology, and Casp11−/− C57BL/6 mice6, were used for infection in animals. All mice were maintained in the specific pathogen-free facility at NIBS under standard housing conditions (12-h dark/light cycle, 20–26 ºC, 40–70% humidity, noise ≤ 60 dB) in accordance with the national guidelines for housing and care of laboratory animals (National Health Commission, China). Mice were transferred to a Biosafety Level 2 facility with the same housing conditions to conduct infections. The protocols for mouse experiments are in accordance with institutional regulations after review and approval by the Institutional Animal Care and Use Committee of NIBS. The bacteria were cultured overnight at 37 ºC in 2× YT medium with shaking at 220 rpm. Overnight cultures were diluted 1:100 in fresh 2× YT medium and grown until OD600 reached 1.5. The bacteria were collected by centrifugation (4,000 rpm. for 5 min) and re-suspended in PBS. Next, 8–10-week-old female mice were infected intraperitoneally with 200 μl of bacteria suspension (2 × 107 colony-forming units (CFU) for each mouse). For infection of C. violaceum, 7-week-old female mice were infected intravenously with 5 × 103 C. violaceum (in 200 μl of PBS). Survival of mice was checked daily for 10 d. To measure bacterial burden, livers and spleens of infected mice were collected 13–16 h after infection and homogenized in sterile PBS. The CFU numbers in the tissue were measured by plating serial-diluted homogenates on 2× YT agar plates.

Mice immunisation and ELISA

Female C57BL/6 and BALB/c mice (8 weeks old) were immunized by intraperitoneal injection of bacteria (1.2 × 106 CFU for WT S. flexneri, 4 × 106 CFU for ΔospC3 mutant and 2 × 106 CFU for all candidate vaccine strains unless specifically indicated) re-suspended in 200 μl of PBS. Owing to the more efficient clearance of the ΔospC3 strain in mice, the doses of immunization for WT and the ΔospC3 mutant were normalized by their virulence, and 10% of the median lethal dose (LD50) of each strain was used. Sera samples were collected 14 d after the immunisation, and Shigella-specific antibody levels were assessed by standard ELISA assay. In brief, 96-well ELISA plates (Nunc MaxiSorp) were coated with 2 × 107 CFU of live S. flexneri re-suspended in 100 μl of carbonate-bicarbonate buffer (pH 9.6) at 4 ºC overnight. The Shigella-coated plates were washed three times with PBST buffer (0.05% Tween 20 in PBS) before blocking with 200 μl of the buffer (2% BSA in PBST) for 2 h at 37 ºC. After washing with PBST three times, twofold serial dilutions of the sera (diluted in the blocking buffer) were added to the plates (100 μl per well) and incubated for 2 h at 37 ºC. The plates were then washed four times with PBST before adding HRP-conjugated goat anti-mouse IgG antibody (1:6,000 diluted in the blocking buffer, 100 μl per well) for 1 h at 37 ºC. After washing with PBST four times, peroxidase substrate o-phenylenediamine (0.4 mg ml−1, dissolved in 0.15 M phosphate-citrate buffer supplemented with 0.03% H2O2, pH 5.0) was added to the plates (100 μl per well) and incubated for 15 min at room temperature. The reaction was then stopped by adding 2 M H2SO4 (50 μl per well), and OD492 was measured. Antibody titres were defined as the reciprocal of the last dilution having an OD492 value at least twofold higher than that obtained in the control wells; the blocking buffer was used as the mock serum sample.

Cell culture, transfection and immunoprecipitation

HeLa, SiHa, A431, 293T and iBMDM cells were grown in DMEM supplemented with 10% (v/v) FBS and 2 mM l-glutamine. All cell lines were obtained from the American Type Culture Collection except for the previously described iBMDMs1. Cells were maintained at 37 ºC in a humidified 5% CO2 incubator. All cells were tested for mycoplasma using the standard PCR method. Cell identity was checked frequently by morphological features but was not authenticated by short tandem repeat profiling. Knockout cell lines were generated by the CRISPR–Cas9 method as previously described1. In brief, PX458 plasmids containing the guide RNAs targeting Casp1, Casp11 or CASP4 were electroporated into the cells (1 μg of DNA per 1 × 106 cells). Three days later, GFP-positive cells were sorted into single clones on 96-well plates by flow cytometry. Single clones were screened and verified by sequencing of the PCR fragments and confirmed by western blot. Sequences for the guide RNAs used are 5′-GGAATTCTGGAGCTTCAATC-3′ for CASP1, 5′-GGTCCACACTGAAGAATGTC-3′ for Casp11 and 5′-CAAGAGAAGCAACGTATGGC-3′ for CASP4. The HeLa CASP4−/− cell line was described previously1. Transient transfection was performed using jetPRIME (Polyplus-transfection) following the manufacturer’s instructions. Cell lines with stable gene expression were generated by lentiviral infection as previously described1. For immunoprecipitation, 293T cells grown to 60% confluence were transfected with indicated plasmids. Twenty-four hours after transfection, cell pellets were harvested and lysed in the lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM CaCl2, 1% Triton X-100 and a protease inhibitor cocktail) for 0.5 h followed by centrifugation at 4 ºC (15,000 rpm for 10 min). The lysates were incubated with anti-Flag M2 affinity beads at 4 ºC with gentle rotation for 2 h. The beads were washed five times with the lysis buffer, and the immunoprecipitants were eluted from the beads with Flag or 3×Flag peptides.

Recombinant protein purification

The E. coli BL21 (DE3) strain was used for all recombinant protein expression. Induction of protein expression (18 ºC for 16 h) was achieved with 0.4 mM isopropyl β-d-1-thiogalactopyranoside after OD600 of bacterial culture reached 1.0. Bacterial cells were harvested and lysed by an ultrasonic homogenizer in buffer containing 20 mM HEPES (pH 7.0), 300 mM NaCl, 5 mM CaCl2 and 5 mM dithiothreitol. GST-fusion OspC or OspC chimeric proteins were purified by glutathione sepharose affinity chromatography. The GST tag was removed by overnight digestion with homemade PreScission Protease (PPase) at 4 ºC. The untagged proteins were further purified by HiTrap SP HP cation exchange chromatography and Superdex G75 gel filtration chromatography (GE Healthcare Life Sciences). The purified proteins were concentrated and stored at −80 ºC in buffer containing 20 mM HEPES (pH 7.0), 150 mM NaCl, 2 mM CaCl2 and 5 mM dithiothreitol. pAC-SUMO-CASP4-p30-C/A plasmid was used to express caspase-4-p30-C/A protein. Bacteria cells were harvested and lysed in the buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole and 10 mM 2-mercaptoethanol. The His6-SUMO-tagged protein was first purified by affinity chromatography using Ni-chelating sepharose resin, and the tag was removed by overnight digestion at 4 ºC with homemade ULP1 protease. The untagged protein was further purified by HiTrap Q HP anion exchange and Superdex G75 gel filtration chromatography. Purified caspase-4 proteins were concentrated and stored at −80 ºC in buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 5 mM dithiothreitol. A similar procedure was followed to purify other His6-SUMO-tagged proteins, including caspase-11-p30-C/A, GSDMD, NUDT16, ExoS (ADP-ribosyltransferase domain starting from Ala233), Rab4a, 14-3-3β, ADPRH, ADPRS, OARD1, MACROD2 and His6-tagged MACROD1 (starting from Thr91). Accession numbers (for the NCBI protein database, of the proteins used in this study are: OspC3 (WP_015683184.1), OspC2 (WP_000701108.1), OspC1 (WP_001026857.1), CASP4 (NP_001216.1), CASP11 (NP_031635.2), GSDMD (NP_001159709.1), NUDT16 (NP_689608.2), ExoS (WP_003113791.1), RAB4A (NP_004569.2), 14-3-3β (NP_647539.1), ADPRH (NP_001116.1), ADPRS (NP_060295.1), OARD1 (NP_001316613.1), MACROD2 (NP_542407.2) and MACROD1 (NP_054786.2).

OspC3-catalysed modification reaction and native-PAGE

The reaction was carried out in a buffer containing 20 mM HEPES (pH 7.0), 150 mM NaCl, 1 mM CaCl2 and 5 mM dithiothreitol. Purified caspase-4/11-p30-C/A and OspC3 proteins (molar ratio: 1:1) were incubated at 16 ºC for 12 h in the presence of 1 mM NAD+. Native-PAGE was used to analyse OspC3-catalysed modification of caspase-4/11. Samples of the in vitro reaction or the purified protein were separated on a 4–20% gradient non-denaturing polyacrylamide gel in a buffer containing 25 mM Tris (pH 8.3) and 192 mM glycine. The electrophoresis was started at a voltage of 90 V; the voltage was gradually increased to 180 V, and the electrophoresis was stopped until the dyes in the loading buffer (bromophenol blue and xylene cyanol FF) migrated out of the gel. The gel was stained with Coomassie Brilliant Blue R-250 to visualize the mobility shift of the modified caspase-4/11 protein.

Stable isotope labelling by amino acids in cell culture experiment

293T cells were first adapted to SILAC DMEM medium in which l-arginine was replaced by heavy isotope labelled arginine (l-arginine-13C6, 15N4), and regular FBS was replaced by dialysed FBS. The cells were cultured in SILAC DMEM medium and passaged at a ratio of 1:10 for five times to ensure complete incorporation, which was further checked by LC–MS analysis before the experiment. Flag–caspase-4 p20/p10 complexes were expressed alone or co-expressed with OspC3 by transient transfection in the adapted 293T cells. Thirty-six hours after transfection, cell pellets were harvested and lysed in a buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Triton X-100 and a protease inhibitor cocktail. Standard immunoprecipitation was performed to purify caspase-4 p20/p10 proteins that were subjected to subsequent LC–MS analyses.


For LC–MS/MS identification of the modified peptides, samples were separated on an SDS‐PAGE gel, and the protein bands of interest were reduced and alkylated using dithiothreitol and iodoacetamide, respectively. The protein samples were digested overnight with chymotrypsin. The resulting peptides were dried on a SpeedVac vacuum concentrator and then re-suspended in an aqueous buffer before LC–MS/MS analysis. A Hybrid Ion Trap Orbitrap mass spectrometer (LTQ Orbitrap Velos, Thermo Fisher Scientific) was used to identify and analyse the modified peptides, and profiling of the modification sites was performed on the Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific).

For CID–MS and EThcD–MS analyses, a capillary column (75 μm × 150 mm) with a laser-pulled electrospray tip (Model P-2000, Sutter Instrument) was home-packed with 4-mm, 100-Å Magic C18AQ silica-based particles. The LC mobile phase comprised solvent A (97% H2O, 3% acetonitrile and 0.1% formic acid) and solvent B (80% acetonitrile, 20% H2O and 0.1% formic acid). An EASY-nLC 1200 HPLC system (Thermo Fisher Scientific) was used to generate the following HPLC gradient: solvent B was increased from 7% to 40% in 40 min and then raised to 95% in 2 min and kept for 10 min followed by 100% solvent A for column equilibration. A data-dependent acquisition mode was enabled for peptide fragmentation with one full MS scan (m/z range 350.00–1,200.00, resolution 60,000) followed by CID. To identify the modification site, precursor ions were subjected to EThcD for peptide fragmentation. Raw MS files were processed using Mascot (version 2.3.02, Matrix Science). The following settings were used for database search: 20 ppm precursor mass error tolerance and 0.8-Da fragment mass error tolerance for LTQ Orbitrap Velos and 10 p.p.m. precursor mass error tolerance and 0.02-Da fragment mass error tolerance for Orbitrap Fusion Lumos. Carbamidomethylation of cysteine residues was set as a fixed modification, and oxidation of methionine was set as a variable modification. A maximum of two missed cleavage sites was allowed. Peptide and protein identifications were filtered at less than 1% false discovery rates. The corresponding peptide peaks were obtained from Thermo Xcalibur 2.2 (Thermo Fisher Scientific).

To measure the total molecular weight of caspase-4 or -11, the purified proteins were separated on a C18 reversed-phase column. The EASY-nLC 1200 HPLC system was used to generate the following gradient: 20–50% B in 20 min, 50–70% B in 3 min and maintained at 70% B for 20 min. MS data were processed using Thermo Scientific Protein Deconvolution software. The parameters were specified according to the mass spectrometer setting. Deconvoluted mass of the most abundant ion was selected as the mass of the target protein with a mass tolerance of 30 ppm.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.