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Type A cholesterol-dependent cytolysins translocate to the trans-Golgi network for NLRP3 inflammasome activation

Nature Immunology volume 26, pages 1673–1685 (2025)Cite this article

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Abstract

Cholesterol-dependent cytolysins (CDCs) constitute the largest group of pore-forming toxins and serve as critical virulence factors for diverse pathogenic bacteria. Several CDCs are known to activate the NLRP3 inflammasome, although the mechanisms are unclear. Here we discovered that multiple CDCs, which we referred to as type A CDCs, were internalized and translocated to the trans-Golgi network (TGN) to remodel it into a platform for NLRP3 activation through a unique peeling membrane mechanism. Potassium efflux was dispensable for CDC-mediated TGN remodeling and NLRP3 recruitment, but was required for the recruitment of the downstream adaptor ASC. In contrast, desulfolysin, which we referred to as type B CDC, was not internalized or translocated to the TGN due to its distinct C-terminal domain 4, despite potent pore formation on the plasma membrane, and hence could not activate NLRP3. Our discoveries uncovered the ability of CDCs to directly remodel an intracellular organelle for inflammatory response.

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Fig. 1: PFO-induced TGN dispersion and NLRP3 recruitment.
Fig. 2: C. perfringens secretion induced TGN dispersion and NLRP3 recruitment through PFO.
Fig. 3: Multiple CDCs induced TGN remodeling and NLRP3 recruitment.
Fig. 4: CDCs represent a third type of NLRP3 stimuli.
Fig. 5: A SunTag imaging system revealed that PFO translocates to the TGN.
Fig. 6: Membrane repair negatively regulated PFO-induced TGN remodeling.
Fig. 7: CDCs peeled away TGN membrane to expose PtdIns4P-enriched region for NLRP3 recruitment.
Fig. 8: DLY was not internalized and did not translocate to the TGN for TGN remodeling and NLRP3 inflammasome activation.

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Acknowledgements

We thank J. E. Vidal and A. G. Vidal (University of Mississippi Medical Center) for sharing reagents, R. Tweten, S. Melville, D. Portnoy, M. Federle, D. Missiakas, L. Comstock, T. Golovkina, A. Chervonsky, S. Light, M. Mimee and J. Lee for expertise, Y. Chen at UChicago Advanced Electron Microscopy Facility and L. Degenstein at UChicago Transgenic Mouse Facility for technical assistance, and all Chen Laboratory members for their help and support. This work was supported by the National Institute of General Medical Sciences (R35GM151390 to J.C.) and the National Institute of Allergy and Infectious Diseases (R01AI182143 to J.C.). Part of the work was performed at the Howard Taylor Ricketts Laboratory, a Regional Biocontainment Laboratory supported by the National Institute of Allergy and Infectious Diseases (UC7AI180312).

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Authors and Affiliations

  1. Department of Microbiology, University of Chicago, Chicago, IL, USA

    Nanyang Xiao, Airi Kogishi, Lisa Radochonski, Yuchong Lei & Jueqi Chen

  2. Howard Taylor Ricketts Laboratory, University of Chicago, Lemont, IL, USA

    Lisa Radochonski & Jueqi Chen

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  1. Nanyang Xiao
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  2. Airi Kogishi
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  3. Lisa Radochonski
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N.X. designed the study under the guidance of J.C. N.X., A.K., L.R., Y.L. and J.C. performed the experiments and analysis. N.X. and J.C. wrote and edited the paper.

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Correspondence to Jueqi Chen.

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Nature Immunology thanks Seth Masters and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Ioana Staicu, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 PFO remodeled the TGN but not other organelles.

a, Coomassie blue staining for rPFO and rPFOY181A purified from E. coli. Arrows indicate rPFO and rPFOY181A proteins. Representative from at least 3 independent experiments. b, Representative LDH release assay of HeLa cells incubated with rPFO or rPFOY181A for 80 min (n = 3 wells per condition; mean ± s.d.; two-sided t-test). Representative from 3 independent experiments. c, Quantification of TGN dispersion (left) and NLRP3 recruitment (right) in HeLa cells stably expressing NLRP3-GFP incubated with rPFO or rPFOY181A for 80 min. Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition; mean ± s.d.; two-sided t-test). NLRP3 recruitment was quantified from 100 cells (n = 3). Representative from 3 independent experiments. d, Immunoblots of HEK293T cells stably expressing NLRP3, ASC, and caspase-1 (Casp1) incubated with 10 μM nigericin, 0.18/0.54/0.90/1.8 nM rPFO or 0.18/0.54/0.90/1.8 nM rPFOY181A for 40 min. pro-Casp1 has two bands because it was expressed as zeocinr-F2A-Casp1 (upper band) before ribosomal skipping to release pro-Casp1 (lower band). SE, short exposure; LE, long exposure. Representative from 3 independent experiments. e, Representative fluorescence and phase contrast images in HeLa cells stably expressing TGN46-mScarlet-I incubated with 0.90 nM rPFO for 30 min. mScarlet-I was pseudocolored to green. The TGN46 vesicles in yellow frames were highlighted in the zoom-in images. Scale bar, 5 μm. Representative from 3 independent experiments. f, Representative immunofluorescence images (left) and quantification of GM130- or giantin-containing structure areas (right) in HeLa cells incubated with 0.90 nM rPFO or not (Mock) for 80 min. Scale bar, 10 μm. Areas containing the indicated organelle markers were measured with ImageJ (n = 40 cells per condition; NS, not significant). Representative from 3 independent experiments. g, Representative immunofluorescence images (left) and quantification of colocalization between rPFO-induced NLRP3 puncta and organelle markers (right) in HeLa cells stably expressing NLRP3-GFP incubated with 0.90 nM rPFO or not (Mock) for 80 min. Scale bar, 10 μm. Colocalization of NLRP3 puncta with the indicated organelle markers after rPFO treatment was analyzed with Pearson correlation coefficient using Coloc 2 plugin of ImageJ (n = 20 cells/sample; threshold regression: Costes). Representative from 3 independent experiments.

Source data

Extended Data Fig. 2 Multiple CDCs induced TGN remodeling and NLRP3 recruitment.

a, Coomassie blue staining for CDCs purified from E. coli. Arrows indicate the CDC proteins. Representative from at least 3 independent experiments. b, Representative LDH release assay of HeLa cells incubated with the indicated CDCs for 80 min (n = 3 wells per condition; mean ± s.d.). Representative from 2 independent experiments. c, Immunoblots of HEK293T cells stably expressing NLRP3, ASC, and caspase-1 (Casp1) incubated with 10 μM nigericin, 0.18/0.54/1.8/5.4 nM rALO, or 1.8/5.4/10.8/14.4 nM rSLO for 40 min. pro-Casp1 has two bands in this cell line because it was expressed as zeocinr-F2A-Casp1 (upper band) before ribosomal skipping to release pro-Casp1 (lower band). SE, short exposure; LE, long exposure. Representative from 3 independent experiments. d, Representative immunofluorescence images (left) and quantification of TGN dispersion and NLRP3 recruitment (right) in HeLa cells stably expressing NLRP3-GFP incubated with 0.18/0.54//0.90/1.8 nM rALO or not (Mock) for 80 min. Representative images for mock treatment and 1.8 nM rALO treatment are shown. Scale bar, 10 μm. Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition; two-sided t-test). NLRP3 recruitment was quantified from 100 cells (n = 3; N.D., not detectable). Representative from at least 3 independent experiments. e, As in d, except cells were incubated with 5.4/10.8/14.4/18 nM rSLO or not (Mock). Representative images for mock treatment and 14.4 nM rSLO treatment are shown. Scale bar, 10 μm. Representative from at least 3 independent experiments.

Source data

Extended Data Fig. 3 K+ efflux was essential for ASC recruitment induced by multiple CDCs.

a–c, Representative immunofluorescence images (left) and quantification of TGN dispersion and NLRP3 recruitment (right) in HeLa cells stably expressing NLRP3-GFP incubated with 1.8 nM rALO (a), 14.4 nM rSLO (b), or 5.4 nM rPLY (c) for 80 min in the presence of KCl at the indicated concentrations. Scale bar, 10 μm. Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition, mean ± s.d.; two-sided t-test; NS, not significant). NLRP3 recruitment was quantified from 100 cells (n = 3; N.D., not detectable). Representative from 3 independent experiments. d, Representative immunofluorescence images (left) and quantification of ASC speck formation (right) in HeLa cells stably expressing NLRP3-GFP and ASC incubated with 1.8 nM rALO, 14.4 nM rSLO or 5.4 nM rPLY for 60 min in the presence of KCl at the indicated concentrations. Scale bar, 25 μm. The percentage of cells with ASC speck formation was quantified from 100 cells (n = 3). Representative from 3 independent experiments. e, Model: CDCs represent a third type of NLRP3 stimuli. The canonical K+ efflux-dependent stimuli (for example, nigericin) does not require K+ efflux for TGN dispersion but requires K+ efflux for NLRP3 recruitment to the dispersed TGN. The K+ efflux-independent stimuli (for example, imiquimod) does not require K+ efflux for any step. CDCs do not require K+ efflux for either TGN dispersion or NLRP3 recruitment but require K+ efflux for ASC recruitment.

Source data

Extended Data Fig. 4 Neither retrograde trafficking nor endosomal maturation was required for PFO-mediated TGN remodeling or NLRP3 inflammasome activation.

a–c, Representative immunofluorescence images (a), quantification of TGN dispersion (b) and NLRP3 recruitment (c) in HeLa cells stably expressing NLRP3-GFP pre-treated with DMSO (solvent control), 25 µM retro-2cycl or 20 µM EGA for 1 h before addition of 0.90 nM rPFO or not (Mock) for 80 min. Scale bar, 10 μm. Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition; mean ± s.d.; two-sided t-test; NS, not significant). NLRP3 recruitment was quantified from 100 cells (n = 3; N.D., not detectable). Representative from 3 independent experiments. d, Immunoblots of HEK293T cells stably expressing NLRP3, ASC, and caspase-1 (Casp1) pre-treated with DMSO (solvent control), 25 µM retro-2cycl, or 20 µM EGA for 1 h before addition of 0.90 nM rPFO for 40 min. pro-Casp1 has two bands in this cell line because it was expressed as zeocinr-F2A-Casp1 (upper band) before ribosomal skipping to release pro-Casp1 (lower band). Representative from 2 independent experiments.

Source data

Extended Data Fig. 5 Endocytosis was not required for PFO trafficking or PFO-mediated NLRP3 inflammasome activation.

a–c, Representative immunofluorescence images (a), quantification of rPFO colocalization with the TGN (b) and TGN dispersion (c) in HeLa cells stably expressing scFv-sfGFP pre-treated with DMSO (solvent control), 25 µM retro-2cycl or pitstop 2 (20 µM) for 1 h before addition of 0.90 nM 10xGCN4-rPFO. Scale bar, 5 μm. Areas of rPFO foci colocalizing with the TGN were measured with ImageJ (n = 40 cells per condition; mean ± s.d.; two-sided t-test; N.D., not detectable, NS, not significant). Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition). Representative from 2 independent experiments. d, Immunoblots of HEK293T cells stably expressing NLRP3, ASC, and caspase-1 (Casp1) pre-treated with DMSO (solvent control) or 20 µM pitstop 2 for 1 h before addition of 0.90 nM rPFO for 40 min. pro-Casp1 has two bands in this cell line because it was expressed as zeocinr-F2A-Casp1 (upper band) before ribosomal skipping to release pro-Casp1 (lower band). Representative from 2 independent experiments.

Source data

Extended Data Fig. 6 Endocytosis was not required for PFO-mediated TGN remodeling or NLRP3 inflammasome activation.

a,b, Representative immunofluorescence images (a) and quantification of TGN dispersion (b) in HeLa cells transfected with V5-tagged dynamin 1 (DNM1) (WT or dominant-negative mutant K44A), dynamin 2 (DNM2) (WT or dominant-negative mutant K44A) or not (Original) before incubated with 0.90 nM rPFO or not (Mock) for 80 min. Scale bar, 5 μm. b, Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition; mean ± s.d.; two-sided t-test; NS, not significant). Representative from 2 independent experiments. c, Immunoblots of HEK293T cells stably expressing NLRP3, ASC, and caspase-1 (Casp1) before transfected as in a (top) and the same cell lines incubated with 0.90 nM rPFO for 40 min (bottom). pro-Casp1 has two bands in this cell line because it was expressed as zeocinr-F2A-Casp1 (upper band) before ribosomal skipping to release pro-Casp1 (lower band). Representative from 3 independent experiments.

Source data

Extended Data Fig. 7 Membrane repair negatively regulated PFO translocation to the TGN.

a, Schematic of Ca2+ influx-driven membrane repair. Left: in Ca2+-containing medium, PFO induces Ca2+ influx, which leads to membrane repair. Right: in Ca2+-free medium, PFO cannot induce Ca2+ influx, and thus is unable to trigger membrane repair. b,c, Representative fluorescence and phase contrast images (b) and quantification of propidium iodide-positive cells (c) in HeLa cells incubated with rPFO in Ca2+-containing medium or Ca2+-free medium supplemented with 50 μg/mL propidium iodide for 80 min. Scale bar, 25 μm. c, The percentage of cells with propidium iodide signal was quantified from 100 cells (n = 3; mean ± s.d.; two-sided t-test; NS, not significant). Representative from 3 independent experiments. d, Representative immunofluorescence images (left) and quantification of rPFO colocalization with the TGN (right) in HeLa cells stably expressing scFv-sfGFP incubated with 10xGCN4-rPFO in Ca2+-containing medium or Ca2+-free medium for 80 min. Areas of rPFO foci colocalizing with the TGN were measured with ImageJ (n = 40 cells per condition; N.D., not detectable). Scale bar, 5 μm. Representative from 3 independent experiments.

Source data

Extended Data Fig. 8 Membrane repair inhibitors enhanced PFO-mediated TGN remodeling and NLRP3 recruitment.

a, Representative LDH release assay of HeLa cells pre-treated with DMSO (solvent control), 20 µM U0126, or 20 µM mirdametinib for 30 min before addition of rPFO for 80 min (n = 3 wells per condition; mean ± s.d.; two-sided t-test; NS, not significant). Representative from 3 independent experiments. b–d, Representative immunofluorescence images (b), quantification of TGN dispersion (c) and NLRP3 recruitment (d) in HeLa cells stably expressing NLRP3-GFP pre-treated with DMSO (solvent control), 20 µM U0126 or 20 µM mirdametinib for 30 min before addition of rPFO or not (Mock) for 80 min. Scale bar, 10 μm. Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition). NLRP3 recruitment was quantified from 100 cells (n = 3; N.D., not detectable). Representative from 3 independent experiments.

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Extended Data Fig. 9 DLY did not activate the NLRP3 inflammasome.

a, Representative LDH release assay of HeLa cells incubated with rDLY for 80 min (n = 3 wells per condition; mean ± s.d.). Representative from 2 independent experiments. b, Representative fluorescence and phase contrast images (left) and quantification of propidium iodide-positive cells (right) in HeLa cells incubated with rDLY or not (Mock) in 50 μg/mL propidium iodide-containing medium for 80 min. Scale bar, 25 μm. The percentage of cells with propidium iodide signal was quantified from 100 cells (n = 3; mean ± s.d.; two-sided t-test). Representative from 3 independent experiments. c, Immunoblots of HEK293T cells stably expressing NLRP3, ASC, and caspase-1 (Casp1) incubated with 10 μM nigericin or 0.18/0.54/1.8/5.4 nM rDLY for 40 min. pro-Casp1 has two bands in this cell line because it was expressed as zeocinr-F2A-Casp1 (upper band) before ribosomal skipping to release pro-Casp1 (lower band). SE, short exposure; LE, long exposure. Representative from 3 independent experiments. d, Schematic for rPFO and rDLY chimeric proteins. The signal peptide (residue 1–28) of rPFO was deleted. rDLY does not have a signal peptide and therefore no deletion was performed. Domain (D)1, D2, D3, and D4 in rPFO are swapped with the corresponding domains in rDLY. D1, D2, and D3 are discontinuous domains. e, Representative LDH release assay of HeLa cells incubated with rPFO_DLYD4 (rPFO with its D4 replaced by D4 of rDLY) for 80 min (n = 3 wells per condition). Representative from 2 independent experiments. f, Representative immunofluorescence images (top) and quantification of TGN dispersion and NLRP3 recruitment (bottom) in HeLa cells stably expressing NLRP3-GFP incubated with 0.90 nM rPFO, 1.8 nM rPFO_DLYD4 or not (Mock) for 80 min. Scale bar, 10 μm. Areas containing TGN structures were measured with ImageJ (n = 40 cells per condition; NS, not significant). NLRP3 recruitment was quantified from 100 cells (n = 3; N.D., not detected). Representative from 3 independent experiments. g, Model: CDCs can be grouped into two types based on whether they can be internalized by host cells to remodel the TGN. Type A CDCs form pores on the plasma membrane before being internalized inside cells and translocating to the TGN. This translocation is mediated by D4 and is negatively regulated by Ca2+ influx-driven membrane repair, as membrane repair facilitates the clearance of CDC pores. After reaching the TGN, type A CDCs peel away the PtdIns4P-negative TGN membrane into multiple vesicles, thus exposing the remodeled perinuclear PtdIns4P-positive TGN membrane for NLRP3 inflammasome assembly. CDC-induced K+ efflux is not essential for TGN dispersion or NLRP3 recruitment but required for ASC recruitment. Type B CDCs (represented by DLY), while also capable of forming pores on the plasma membrane, do not get internalized. As a result, type B CDCs do not translocate to the TGN and therefore cannot remodel the TGN to activate the NLRP3 inflammasome.

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Extended Data Table 1 Summary of pathogenic bacteria-derived CDCs examined in this study
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Supplementary information

Supplementary Information

Supplementary Figs. 1–11 and source data (uncropped blots) for supplementary figures.

Reporting Summary

Supplementary Video 1

PFO remodeled the PtdIns4P-positive TGN structure. Time-lapse imaging of HeLa cells stably expressing OSBPPH–GFP, a PtdInsp4P marker, before incubated with 0.90 nM rPFO at 37 °C with 5% (v/v) CO2 and imaged with the GFP channel. Representative from three independent experiments.

Supplementary Video 2

STED super-resolution imaging of SunTag-rPFO in HeLa scFv–sfGFP cells. STED super-resolution microscopy analysis of HeLa cells stably expressing scFv–sfGFP incubated with 0.90 nM 10xGCN4–rPFO at 37 °C for 60 min. Z-stack images were generated using a step size of 0.025 µm with 3D reconstruction applied. Representative from three independent experiments.

Supplementary Video 3

NLRP3 was recruited to the remodeled perinuclear region after rPFO treatment. Time-lapse imaging of HeLa cells stably expressing NLRP3–GFP incubated with 0.90 nM rPFO at 37 °C with 5% (v/v) CO2 before being imaged with the GFP channel. Representative from three independent experiments.

Supplementary Video 4

NLRP3 was recruited to the dispersed giant vesicles after nigericin treatment. Time-lapse imaging of HeLa cells stably expressing NLRP3–mNeonGreen incubated with 10 μM nigericin at 37 °C with 5% (v/v) CO2 before being imaged with the mNeonGreen channel. Similar results with NLRP3–GFP have been observed in our previous study (PMID 30487600). Representative from three independent experiments.

Supplementary Data 1

Numerical source data for supplementary figures.

Source data

Source Data All Figures

Numerical source data. Each tab represents one figure panel.

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Xiao, N., Kogishi, A., Radochonski, L. et al. Type A cholesterol-dependent cytolysins translocate to the trans-Golgi network for NLRP3 inflammasome activation. Nat Immunol 26, 1673–1685 (2025). https://doi.org/10.1038/s41590-025-02277-6

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