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Group A Streptococcus induces GSDMA-dependent pyroptosis in keratinocytes

Abstract

Gasdermins (GSDMs) are a family of pore-forming effectors that permeabilize the cell membrane during the cell death program pyroptosis1. GSDMs are activated by proteolytic removal of autoinhibitory carboxy-terminal domains, typically by caspase regulators1,2,3,4,5,6,7,8,9. However, no activator is known for one member of this family, GSDMA. Here we show that the major human pathogen group A Streptococcus (GAS) secretes a protease virulence factor, SpeB, that induces GSDMA-dependent pyroptosis. SpeB cleavage of GSDMA releases an active amino-terminal fragment that can insert into membranes to form lytic pores. GSDMA is primarily expressed in the skin10, and keratinocytes infected with SpeB-expressing GAS die of GSDMA-dependent pyroptosis. Mice have three homologues of human GSDMA, and triple-knockout mice are more susceptible to invasive infection by a pandemic hypervirulent M1T1 clone of GAS. These results indicate that GSDMA is critical in the immune defence against invasive skin infections by GAS. Furthermore, they show that GSDMs can act independently of host regulators as direct sensors of exogenous proteases. As SpeB is essential for tissue invasion and survival within skin cells, these results suggest that GSDMA can act akin to a guard protein that directly detects concerning virulence activities of microorganisms that present a severe infectious threat.

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Fig. 1: SpeB cleaves GSDMA.
Fig. 2: GSDMA protects mice against severe GAS skin infection.
Fig. 3: GAS induce SpeB-dependent keratinocyte pyroptosis.
Fig. 4: GSDMA activation requires cell contact and restricts iGAS.

Data availability

All data generated or analysed during this study are included in this article and its Supplementary Information. Source data are provided with this paper.

References

  1. Broz, P., Pelegrín, P. & Shao, F. The gasdermins, a protein family executing cell death and inflammation. Nat. Rev. Immunol. 20, 143–157 (2019).

    Article  Google Scholar 

  2. Zhou, Z. et al. Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells. Science 368, eaaz7548 (2020).

    CAS  Article  Google Scholar 

  3. Aglietti, R. A. et al. GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes. Proc. Natl Acad. Sci. USA 113, 7858–7863 (2016).

    CAS  Article  Google Scholar 

  4. Ding, J. et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 538, 111–116 (2016).

    ADS  Article  Google Scholar 

  5. Liu, X. et al. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 535, 153–158 (2016).

    ADS  CAS  Article  Google Scholar 

  6. Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signaling. Nature 526, 666–671 (2015).

    ADS  CAS  Article  Google Scholar 

  7. Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015).

    ADS  CAS  Article  Google Scholar 

  8. Wang, Y. et al. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 547, 99–103 (2017).

    ADS  CAS  Article  Google Scholar 

  9. Sborgi, L. et al. GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death. EMBO J. 35, 1766–1778 (2016).

    CAS  Article  Google Scholar 

  10. Tamura, M. et al. Members of a novel gene family, Gsdm, are expressed exclusively in the epithelium of the skin and gastrointestinal tract in a highly tissue-specific manner. Genomics 89, 618–629 (2007).

    CAS  Article  Google Scholar 

  11. Ralph, A. P. & Carapetis, J. R. Group A streptococcal diseases and their global burden. Curr. Top. Microbiol. Immunol. 368, 1–27 (2012).

    Google Scholar 

  12. Okada, N., Liszewski, M. K., Atkinson, J. P. & Caparon, M. Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A Streptococcus. Proc. Natl Acad. Sci. USA 92, 2489–2493 (1995).

    ADS  CAS  Article  Google Scholar 

  13. Nakagawa, I. et al. Autophagy defends cells against invading group A Streptococcus. Science 306, 1037–1040 (2004).

    ADS  CAS  Article  Google Scholar 

  14. Sil, P., Wong, S.-W. & Martinez, J. More than skin deep: autophagy is vital for skin barrier function. Front. Immunol. 9, 1376 (2018).

    Article  Google Scholar 

  15. Barnett, T. C. et al. The globally disseminated M1T1 clone of Group A Streptococcus evades autophagy for intracellular replication. Cell Host Microbe 14, 675–682 (2013).

    CAS  Article  Google Scholar 

  16. Nelson, D. C., Garbe, J. & Collin, M. The cysteine proteinase SpeB from Streptococcus pyogenes– a potent modifier of immunologically important host and bacterial proteins. Biol. Chem. 392, 1077–1088 (2011).

    CAS  Article  Google Scholar 

  17. LaRock, D. L., Russell, R., Johnson, A. F., Wilde, S. & LaRock, C. N. Group A Streptococcus infection of the nasopharynx requires proinflammatory signaling through the interleukin-1 receptor. Infect. Immun. 88, e00356–20 (2020).

    CAS  Article  Google Scholar 

  18. LaRock, C. N. et al. IL-1β is an innate immune sensor of microbial proteolysis. Sci. Immunol. 1, eaah3539 (2016).

    Article  Google Scholar 

  19. Cookson, B. T. & Brennan, M. Pro-inflammatory programmed cell death. Trends Microbiol. 3, 113–114 (2001).

    Article  Google Scholar 

  20. Uhlen, M. et al. A genome-wide transcriptomic analysis of protein-coding genes in human blood cells. Science 366, eaax9198 (2019).

    CAS  Article  Google Scholar 

  21. Nizet, V. et al. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 414, 454–457 (2001).

    ADS  CAS  Article  Google Scholar 

  22. Walker, M. J. et al. DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection. Nat. Med. 13, 981–985 (2007).

    CAS  Article  Google Scholar 

  23. Schrager, H. M., Rheinwald, J. G. & Wessels, M. R. Hyaluronic acid capsule and the role of streptococcal entry into keratinocytes in invasive skin infection. J. Clin. Invest. 98, 1954–1958 (1996).

    CAS  Article  Google Scholar 

  24. O’Seaghdha, M. & Wessels, M. R. Streptolysin O and its co-toxin NAD-glycohydrolase protect Group A Streptococcus from xenophagic killing. PLoS Pathog. 9, e1003394 (2013).

    Article  Google Scholar 

  25. Barnett, T. C., Bowen, A. C. & Carapetis, J. R. The fall and rise of Group A Streptococcus diseases. Epidemiol. Infect. 147, e4 (2019).

    Article  Google Scholar 

  26. Svensson, M. D. et al. Role for a secreted cysteine proteinase in the establishment of host tissue tropism by group A streptococci. Mol. Microbiol. 38, 242–253 (2000).

    CAS  Article  Google Scholar 

  27. Cywes, C. & Wessels, M. R. Group A Streptococcus tissue invasion by CD44-mediated cell signalling. Nature 414, 648–652 (2001).

    ADS  CAS  Article  Google Scholar 

  28. Molinari, G. & Chhatwal, G. S. Invasion and survival of Streptococcus pyogenes in eukaryotic cells correlates with the source of the clinical isolates. J. Infect. Dis. 177, 1600–1607 (1998).

    CAS  Article  Google Scholar 

  29. Wang, J. R. & Stinson, M. W. M protein mediates streptococcal adhesion to HEp-2 cells. Infect. Immun. 62, 442–448 (1994).

    CAS  Article  Google Scholar 

  30. Sakurai, A. et al. Specific behavior of intracellular Streptococcus pyogenes that has undergone autophagic degradation is associated with bacterial streptolysin O and host small G proteins Rab5 and Rab7. J. Biol. Chem. 285, 22666–22675 (2010).

    CAS  Article  Google Scholar 

  31. Ruan, J., Xia, S., Liu, X., Lieberman, J. & Wu, H. Cryo-EM structure of the gasdermin A3 membrane pore. Nature 557, 62–67 (2018).

    ADS  CAS  Article  Google Scholar 

  32. LaRock, C. N. et al. Group A Streptococcal M1 protein sequesters cathelicidin to evade innate immune killing. Cell Host Microbe 18, 471–477 (2015).

    CAS  Article  Google Scholar 

  33. Aziz, R. K. et al. Invasive M1T1 group A Streptococcus undergoes a phase-shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB. Mol. Microbiol. 51, 123–134 (2004).

    CAS  Article  Google Scholar 

  34. Klock, H. E. & Lesley, S. A. in High Throughput Protein Expression and Purification: Methods and Protocols (ed. Doyle, S. A.) 91–103 (Humana, 2009).

  35. Miao, E. A. et al. Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1β via Ipaf. Nat. Immunol. 7, 569–575 (2006).

    CAS  Article  Google Scholar 

  36. Lichti, U., Anders, J. & Yuspa, S. H. Isolation and short term culture of primary keratinocytes, hair follicle populations, and dermal cells from newborn mice and keratinocytes from adult mice, for in vitro analysis and for grafting to immunodeficient mice. Nat. Protoc. 3, 799–810 (2008).

    CAS  Article  Google Scholar 

  37. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    ADS  CAS  Article  Google Scholar 

  38. LaRock, C. N. & Cookson, B. T. The Yersinia virulence effector YopM binds caspase-1 to arrest inflammasome assembly and processing. Cell Host Microbe 12, 799–805 (2012).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank V. Nizet for insights and bacterial strains. This study received materials from the Emory Investigational Clinical Microbiology Core (supported by the Department of Medicine, Division of Infectious Diseases) and the Emory Mouse Transgenic and Gene Targeting Core (subsidized by the Emory University School of Medicine and receives support from National Institutes of Health (NIH; UL1TR000454)). C.N.L. received support from NIH grants AI130223 and AI153071. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the NIH.

Author information

Authors and Affiliations

Authors

Contributions

D.L.L. performed all biochemistry and transfection experiments. A.F.J., J.S.S. and C.N.L. performed in vitro infections. S.W., M.M. and C.N.L. performed in vivo experiments and analysed results. C.N.L. conceived the study. C.N.L. and D.L.L. designed the experiments, analysed data, and wrote the manuscript with input from all co-authors.

Corresponding author

Correspondence to Christopher N. LaRock.

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Competing interests

D.L.L. and C.N.L. are named inventors on a US provisional patent application (No. 63/234,810) that describes GSDMA activities.

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Nature thanks Dario Zamboni 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 SpeB cleaves GSDMA.

a, Cytotoxicity of HEK293Ts transfected with human GSDMs ± SpeB. Data are the mean±s.d. of 4 technical replicates. P values were calculated by two-tailed Student’s t-test. b, Membranes spotted with lipids were incubated with indicated proteins and binding was assessed by immunoblot for GSDMA. c, Thermal melt analysis of GSDMA and GSDMAΔ240-247 with melting temperatures indicated. (a–c) data are representative of three independent experiments.

Extended Data Fig. 2 Alignment of human and mouse GSDMAs.

Human GSDMA (Q96QA5), mouse GSDMA_1 (Q9EST1), mouse GSDMA_2 (Q32M21), mouse GSDMA_3 (Q5Y4Y6) aligned with Clustal Omega algorithm in DNASTAR. Disordered solvent accessible loop (gray box). Inverted arrows identify cleavage sites of SpeB on hGSDMA. Residues 240–247 of hGSDMA are identified (green bar).

Extended Data Fig. 3 Lytic activity, SpeB production, and binding of GAS strains.

a, b, Keratinocytes were infected (MOI = 100) with GAS 5448, isogenic mutant controls, or clinical isolates for 4 h and (a) lysis measured by LDH release and (b) SpeB activity measured using specific substrate Mca-IFFDTWDNE-Lys-Dnp. c, GAS labeled with Bocillin was incubated with keratinocytes (MOI = 100) 1 h, washed, and adherence measured by fluorescence. Data are representative of three independent experiments with 3 technical replicates and are presented as the mean±s.d. P values were calculated by two-way ANOVA compared to 5448 (M1) control; P < 0.0001.

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LaRock, D.L., Johnson, A.F., Wilde, S. et al. Group A Streptococcus induces GSDMA-dependent pyroptosis in keratinocytes. Nature 605, 527–531 (2022). https://doi.org/10.1038/s41586-022-04717-x

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