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Shigella evades pyroptosis by arginine ADP-riboxanation of caspase-11

Nature volume 599, pages 290–295 (2021)Cite this article

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Abstract

Mouse caspase-11 and human caspase-4 and caspase-5 recognize cytosolic lipopolysaccharide (LPS) to induce pyroptosis by cleaving the pore-forming protein GSDMD1,2,3,4,5. This non-canonical inflammasome defends against Gram-negative bacteria6,7. Shigella flexneri, which causes bacillary dysentery, lives freely within the host cytosol where these caspases reside. However, the role of caspase-11-mediated pyroptosis in S. flexneri infection is unknown. Here we show that caspase-11 did not protect mice from S. flexneri infection, in contrast to infection with another cytosolic bacterium, Burkholderia thailandensis8. S. flexneri evaded pyroptosis mediated by caspase-11 or caspase 4 (hereafter referred to as caspase-11/4) using a type III secretion system (T3SS) effector, OspC3. OspC3, but not its paralogues OspC1 and 2, covalently modified caspase-11/4; although it used the NAD+ donor, this modification was not ADP-ribosylation. Biochemical dissections uncovered an ADP-riboxanation modification on Arg314 and Arg310 in caspase-4 and caspase-11, respectively. The enzymatic activity was shared by OspC1 and 2, whose ankyrin-repeat domains, unlike that of OspC3, could not recognize caspase-11/4. ADP-riboxanation of the arginine blocked autoprocessing of caspase-4/11 as well as their recognition and cleavage of GSDMD. ADP-riboxanation of caspase-11 paralysed pyroptosis-mediated defence in Shigella-infected mice and mutation of ospC3 stimulated caspase-11- and GSDMD-dependent anti-Shigella humoral immunity, generating a vaccine-like protective effect. Our study establishes ADP-riboxanation of arginine as a bacterial virulence mechanism that prevents LPS-induced pyroptosis.

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Fig. 1: S. flexneri blocks cytosolic LPS-induced pyroptosis through OspC3.
Fig. 2: OspC3 catalyses an NAD+-dependent modification of caspase-4/11.
Fig. 3: OspC3 catalyses ADP-riboxanation on an arginine in caspase-4/11.
Fig. 4: Analyses of the OspC family and mechanisms of OspC3 inactivation of caspase-4/11.
Fig. 5: OspC3 underlies evasion by Shigella of pyroptosis-mediated defence that promotes anti-Shigella humoral immunity.

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Data availability

All data supporting the findings of this study are included in this manuscript and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank L. Li and S. Chen for mass spectrometry, P. Li and H. He for technical assistance and C. Li for advice. The work was supported by National Key Research and Development Programs of China (2017YFA0505900 and 2016YFA0501500), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB37030202 and XDB29020202), the National Natural Science Foundation of China (81788104, 81922043, 21622501 and 21974002) and the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2019-I2M-5-084).

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Authors and Affiliations

  1. Research Unit of Pyroptosis and Immunity, Chinese Academy of Medical Sciences and National Institute of Biological Sciences, Beijing, China

    Zilin Li, Wang Liu, Yue Xu & Feng Shao

  2. National Institute of Biological Sciences, Beijing, China

    Zilin Li, Wang Liu, Sen Cheng, Yue Xu, Zhiqiang Wang, Xiaofan Liu, Xuyan Shi, Yaxin Liu, Xiangbing Qi, Jingjin Ding & Feng Shao

  3. Institute of Analytical Chemistry & Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Jiaqi Fu, Sen Cheng & Xiaoyun Liu

  4. Department of Microbiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China

    Xiaoyun Liu

  5. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Jingjin Ding & Feng Shao

  6. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China

    Feng Shao

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  1. Zilin Li
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Contributions

Z.L., W.L. and F.S. conceived the study. Z.L. performed most of the biochemical and functional experiments. W.L. made the initial observation of a possible new PTM on caspase-11 by OspC3. J.F., S.C. and X.L. were responsible for mass spectrometry and data analyses. Z.W. and X.Q. synthesized 2′-H-NAD+. Y.X., Y.L., X.L., X.S. and J.D. provided technical assistance and valuable suggestions. Z.L. and F.S. analysed the data and wrote the manuscript, with input from all authors. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Xiaoyun Liu or Feng Shao.

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The authors declare no competing interests.

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Peer review information Nature thanks Klaus Aktories and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 S. flexneri suppresses cytosolic LPS-induced host defense through the OspC3 effector.

a, Survival curves of WT or Casp11−/− mice infected intraperitoneally with S. flexneri or B. thailandensis (5 × 106 CFU per mouse, n = 10 for each group; two-tailed log-rank (Mantel-Cox) test). b, Casp1−/−Casp11−/− iBMDMs were infected with S. flexneri (S.f.) or B. thailandensis (B.t.) at the indicated MOIs for 1.5 h. Extracellular bacteria were killed by gentamycin. Intracellular bacteria were stained by anti-Shigella or anti-Burkholderia antibodies (scale bar, 20 μm). The numbers of bacteria/cell (mean values) were calculated from 5 randomly selected images. c, g, WT or CASP4−/− A431 cells were infected with S. flexneri (WT, an ospC3 deletion or complementation (pOspC3) strain) or S. Typhimurium ΔsifA. d, WT or CASP4−/− SiHa cells were infected with S. flexneri WT or ΔospC3 at the indicated MOIs. e, Indicated cells were electroporated with LPS purified from S. flexneri, B. thailandensis or E. coli (E.c.). f, WT or CASP4−/− SiHa cells and Casp1−/− or Casp1−/−Casp11−/− iBMDMs were infected with S. flexneri WT or an indicated mutant. h, WT or CASP4−/− or GBPs−/− (lacking all seven human GBPs) A431 cells were infected with indicated bacteria strains; cell death was assayed at the indicated time points post-infection. i, Streptavidin pull-down assay of the binding of biotin-conjugated LPS or Pam3CSK4 to Flag-tagged caspase-4/11-C/A and GBP1 in transfected 293T cell lysates. j, WT, CASP4−/−, or OspC3-expressing SiHa cells were infected with S. flexneri ΔospC3 or S. Typhimurium ΔsifA, or electroporated with LPS. c, g, j, Cell lysates were immunoblotted as shown and supernatants were blotted with the anti-cleaved GSDMD-C antibody. f, h, Cells were primed with 100 ng ml−1 IFNγ overnight prior to infection. Cell death (c, d, f–h, j) was quantified by LDH release at 3.5 h post-infection unless noted and ATP-based cell viability (e) was measured 2 h post-electroporation; data are means (bars) of three individual replicates (circles). Data are pooled from two experiments (a) and representative of three independent experiments (b–i). For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 2 OspC3 catalyzes an NAD+-dependent modification of caspase-4/11.

a, b, Co-immunoprecipitation of OspC3 with caspase-4-p20/p10 (a) or full-length caspase-4/11 (b). C/A, the catalytic-cysteine mutant. c–e, GSDMD (c, d) or the fluorescent peptide substrate Ac-WEHD-AFC (e) were assayed for in vitro cleavage by caspase-4/11-p20/p10 in the presence of OspC3 at indicated concentrations. f, Cleavage of GSDMD by LPS-activated caspase-4 in the presence or absence of OspC3. g, LPS alone or mixed with purified OspC3 or EGFP was electroporated into HeLa cells or iBMDMs. ATP-based cell viability was measured. h, k, Caspase-11-p30-C/A, expressed alone/with OspC3 in bacteria (h) or reacted with OspC3 ± NAD+ in vitro (k), was analysed by ESI–MS (h) or native/SDS-PAGE (k). Control, OspC3-modified caspase-11-p30-C/A. i, j, CID-tandem mass spectrum of a chymotryptic caspase-4 peptide bearing the 524-Da modification. Purified caspase-4-p30-C/A, modified by OspC3 in E. coli, was subjected to mass spectrometry analyses. j, The upper and lower panels show the tandem mass spectra of a fragment ion (m/z = 428.04) from the modified peptide in i and the ADP standard, respectively. *, OspC3-modified; Mox, oxidized methionine. l, ESI–MS determination of the molecular mass of caspase-4/11-p30-C/A that had been reacted with OspC3 in the presence or absence of NAD+. m, EThcD–MS of purified caspase-4/11; extracted ion chromatograms of the R314/R310-containing peptides or a control peptide. *, OspC3-modified; Mox, oxidized methionine. e, g, Data are means (bars) of three individual replicates (circles). Data are representative of three (a–k) or two (l, m) independent experiments. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 3 OspC3 modifies Arg314 in caspase-4 and the equivalent Arg310 in caspase-11.

a, EThcD-tandem mass spectrum of the Arg310-containing chymotryptic peptide from modified caspase-11-p30-C/A obtained by co-expression with OspC3 in bacteria. Fragmentation patterns generating the observed c/z and b/y ions are indicated along the peptide sequence. b, d, Caspase-4/11-p30-C/A or an Arg314/R310 mutant were expressed alone or together with OspC3 in 293T cells. Immunopurified caspase-4/11-p30 were analysed by ESI–MS to determine the total molecular mass.♦, a contamination ion that only appeared in the particular experiment. c, e, Caspase-4/11-p30-C/A or an R314/R310 mutant protein were reacted with OspC3 in the presence or absence of NAD+. The reactions were analysed by native/SDS-PAGE (c). Chymotrypsin-digested caspase-4/11-p30 proteins were analysed by mass spectrometry (e). Shown are the extracted ion chromatograms of the R314/R310-containing peptides. a, e, Mox, oxidized methionine. Data are representative of three (a, c, e) or two (b, d) independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 4 OspC3 modification of caspase-4/11 contains a step of ADP-ribosylation.

a, HPLC–MS quantification of small-molecule products present in indicated in vitro caspase-4 modification reactions. Mean values ± s.d., n = 3 (independent experiments), two-tailed unpaired Student’s t-test (***P < 0.001, **P < 0.01, ns, non-significant). b, c, Assessing the ability of various NAD+ analogues or derivatives to support in vitro modification of caspase-4-p30-C/A by OspC3. b, The reactions were subjected to native/SDS-PAGE analyses. Control, OspC3-modified caspase-4-p30-C/A in bacteria. c, Following the modification, caspase-4-p30 was digested with chymotrypsin and analysed by mass spectrometry. Shown are the extracted ion chromatograms of the R314-containing peptides. Mass changes of each analogue from NAD+ are illustrated underneath the corresponding chromatograms. A, adenine; N, nicotinamide; P, phosphate; R, ribose. d, e, Caspase-4/11-p30-C/A modified by OspC3 in bacteria was treated with SdeA or NUDT16 overnight. d, The caspase-4 samples were then immunoblotted with an anti-ADP-ribosylation antibody. e, Caspase-4/11 after NUDT16 treatment was digested with chymotrypsin and analysed by mass spectrometry. Shown are extracted ion chromatograms of the R314/R310-containing peptides. All data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 5 OspC3-catalyzed modification involves ADP-ribosylation-dependent arginine deamination.

a, Caspase-4-p20/p10-C/A was expressed alone or co-expressed with OspC3 in 293T cells metabolically labeled with 13C6, 15N4-L arginine. Immunopurified caspase-4 was analysed by mass spectrometry. b, Structural illustration of 2’-H-NAD+ and 2’-F-NAD+. c, Caspase-4-p30-C/A was reacted with OspC3 in the absence or presence of 2’-H-NAD+, and then analysed by mass spectrometry. d, Chemical scheme of ninhydrin modification of an arginine. e, Unmodified caspase-4-p30-C/A or OspC3-modified caspase-4-p30-C/A using NAD+ or 2’-H-NAD+ were incubated with 5 mM ninhydrin for 8 h at room temperature, and then digested with chymotrypsin and analysed by mass spectrometry. f, Purified Rab4a was ADP-ribosylated by ExoS (activated by 14-3-3 protein). ADP-ribosylated and native Rab4a was incubated with 5 mM ninhydrin, followed by mass spectrometry analyses. g, A proposed scheme of OspC3-catalyzed arginine ADP-riboxanation. a, c, e, f, Extracted ion chromatograms of caspase-4 R314-containing peptides (a, c, e) or Rab4a R84-containing peptide (f). Data are representative of two (a) or three (c, e, f) independent experiments.

Extended Data Fig. 6 Domain architecture and enzymatic properties of the OspC family.

a, Multiple sequence alignment of OspC1, OspC2 and OspC3. The alignment was performed using the ClustalW2 algorithm and illustrated using ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Identical residues are in red background and conserved residues are colored in red. The N-terminal putative ADP-riboxanase domain and the C-terminal ARD are marked along the sequence. Residues required for OspC3 ADP-riboxanase activity are highlighted by black rectangle. b, HeLa cells were electroporated with LPS together with recombinant OspC or an indicated chimeric protein. Cell viability was determined by the ATP assay. c, Co-immunoprecipitation of caspase-4-p30-C/A with an OspC-family member. d, e, Caspase-4/11-p30-C/A was reacted with OspC3 or an indicated mutant in the presence of NAD+. The reactions were subjected to native/SDS-PAGE analyses. Control, OspC3-modified caspase-11/4-p30-C/A in bacteria. EH/AA, OspC3 E326A/H328A. f, g, SiHa cells and Casp1−/− or Casp1−/−Casp11−/− iBMDMs were infected with WT S. flexneri or an ospC3 deletion/complementation strain. Cell death was measured by the LDH-release assay. h, Flag-caspase-4-p30-C/A was co-expressed with OspC3 or an indicated mutant in 293T cells. i, Caspase-4-p30-C/A was left unmodified (Unmod.) or pre-modified by OspC3 (WT or D177A) in vitro. Anti-Flag immunoprecipitates (h) or caspase-4-p30-C/A (i) were left untreated or treated with ADP-ribosylarginine hydrolase (ADPRH) or other indicated ADP-ribosylhydrolases. The samples were subjected to anti-ADPR immunoblotting. b, f, g, Data are means (bars) of three individual replicates (circles). Data are representative of three (b–h) or two (i) independent experiments. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 7 R314/R310 ADP-riboxanation of caspase-4/11 blocks their autoprocessing and also cleaving of GSDMD.

a, c, CASP4−/− A431 cells (a) or Casp1−/−Casp11−/− iBMDMs (c) stably expressing an Arg314 or Arg310 mutant caspase-4 or -11, respectively, were electroporated with LPS. Cell viability was measured 2 h post-electroporation. b, d, e, CASP4−/− A431 cells stably expressing caspase-4 (WT or an indicated R310 mutant) or Casp1−/−Casp11−/− iBMDMs stably expressing caspase-11 (WT or an indicated R314 mutant) were infected with S. Typhimurium ΔsifA (b), or S. flexneri ΔospC3 (d) or B. thailandensis (e), respectively. Pyroptosis was measured by the LDH-release assay. b, d, Cell lysates were immunoblotted as shown and supernatants were blotted with the anti-cleaved GSDMD-C antibody. f, WT or R314/R310-mutant caspase-4/11-p20/p10 proteins were purified from bacteria and analysed by SDS-PAGE. g, Gel-filtration chromatography analyses of caspase-4-p20/p10 (WT or R314A)–GSDMD-C complex formation. Caspase-4-p20/p10-A, R314-ADP-riboxanated form obtained by co-expression with OspC3 in bacteria. h, Alignment of caspase sequences around Arg314 in caspase-4. The alignment was performed using the ClustalW2 algorithm and presented using ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Identical residues are colored in red background and conserved residues are in red. The invariant arginine is highlighted by a black rectangle. Sequence numbers of the starting residues are indicated on the left. i, Cleavage of the fluorescent peptide substrate Ac-WEHD-AFC by WT or R314/R310-mutant caspase-4/11-p20/p10 proteins at the indicated concentrations. a–e, Data are means (bars) of three individual replicates (circles). All data are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 8 S. flexneri ΔospC3 promotes adaptive anti-Shigella immunity.

a–c, C57BL/6 mice were infected intraperitoneally with S. flexneri WT or ΔospC3 at the indicated doses (n = 12 for each group). 1%, 2.5%, 5%, 10% and 15% LD50 equal 1.2 × 105, 3 × 105, 6 × 105, 1.2 × 106, and 1.8 × 106 CFU, respectively, for S. flexneri WT, and 4 × 105, 1 × 106, 2 × 106, 4 × 106, and 6 × 106 CFU, respectively, for S. flexneri ΔospC3. a, Bacterial loads (CFU per gram of tissue) were determined 24 h post-infection. b, c, Fourteen days after immunization, serum anti-Shigella antibodies were determined (b); two additional days later, mice were re-challenged intraperitoneally with WT S. flexneri (1.5 × 108 CFU per mouse) and survival was monitored daily (c). d–h, C57BL/6 (d, e, f) or BALB/c (d, g, h) mice were immunized with an indicated S. flexneri deletion strain at the indicated CFUs. Fourteen days after immunization, serum anti-Shigella antibodies were determined (d, e, g); two additional days later, mice immunized in e and g were re-challenged intraperitoneally with WT S. flexneri (1.5 × 108 and 8 × 107 CFU per mouse for e and g, respectively) and survival was monitored daily (f, h). d, n = 12 for ΔospC3 groups in C57BL/6 and BALB/c mice, n = 18 for ΔicsA and ΔguaBA groups in C57BL/6 mice, and n = 15 for ΔicsA and ΔguaBA groups in BALB/c mice. The data in C57BL/6 mice are representations from those in b for ΔospC3 and in Fig. 5e for ΔicsA and ΔguaBA. e–h, n = 11 for the ΔicsA (4 × 106 CFU) group in f, and n = 12 for all other groups. a, b, d, e, g, Median values; two-tailed Mann-Whitney U-test (****P ≤ 0.0001). c, f, h, Two-tailed log-rank (Mantel-Cox) test. All data are representative of two independent experiments.

Source data

Extended Data Fig. 9 The wide presence of OspC-like arginine ADP-riboxanase in bacteria.

a, Domain organization of OspC homologs identified through BLAST searches. The source of bacterial species and the accession number of the homologs are indicated on the left of the diagram. Sequence similarity of each homolog to OspC3 within the ADP-riboxanase domain and the ARD are indicated. b, CASP4−/− HeLa cells stably expressing Flag-caspase-4/11 were transfected with an indicated OspC3 homolog. LPS was electroporated into the cells and ATP-based cell viability was measured 2 h post-electroporation. Data are means (bars) of three individual replicates (circles). Cell lysates prepared prior to LPS electroporation were subjected to anti-Flag immunoprecipitation. EH/AA, mutations corresponding to the E326A/H328A double mutation in OspC3. c, Lysates of 293T cells co-transfected with Flag-caspase-4-C/A and a chimeric OspC-family protein (replacing the ARD with that of OspC3) were subjected to immunoprecipitation. The homolog from C. amalonaticus used is WP_103774980.1. b, c, Anti-Flag, anti-HA or anti-ADPR immunoblotting. Data are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 10 Functional analyses of the CopC effector from C. violaceum.

a, b, Purified caspase-4-p20/p10-C/A (a) or caspase-4-p30-C/A (b) was reacted with recombinant CopC or OspC protein (CopC ΔN50 and OspC3 ΔN52 were used in a) at the indicated molar ratio in the presence of NAD+. Reactions were analysed by SDS-PAGE (a) or native-PAGE (b). Red * indicates ADP-riboxanation of caspase-4-p10. c, HeLa cells expressing CopC or an empty vector were electroporated with LPS. Cell death was measured by the LDH-release assay. Data are means (bars) of three individual replicates (circles). d, e, 3xFlag-caspase-4/11-expressing 293T cells were infected with S. Typhimurium SL1344 harboring CopC WT or E325A (d) or C. violaceum WT, ΔcviC or ΔcopC (e). Immunoprecipitated Flag-caspase-4/11 was subjected to anti-Flag or anti-ADPR immunoblotting. f, C57BL/6 mice (n = 14 for each group) were intravenously infected with C. violaceum WT or ΔcopC (5 × 103 CFU per mouse). Seventy-two h later, bacterial burden in the mouse liver (CFU per gram) was determined (median values, two-tailed Mann-Whitney U-test). Data are representative of three (a–c) or two (d–f) independent experiments. For gel source data, see Supplementary Fig. 1.

Source data

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Supplementary Fig. 1

Figures of the uncropped immunoblots for key data are presented in the main text and Extended Data section of the manuscript.

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Li, Z., Liu, W., Fu, J. et al. Shigella evades pyroptosis by arginine ADP-riboxanation of caspase-11. Nature 599, 290–295 (2021). https://doi.org/10.1038/s41586-021-04020-1

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