A streptococcal M protein activates the NLRP 3 inflammasome

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. 1 Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA. 2 Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA. 3 Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA. 4 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA. *e-mail: pghosh@ucsd.edu; vnizet@ucsd.edu Group A Streptococcus (GAS) causes diseases ranging from common pharyngitis to life-threatening necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS), and is the immunologic trigger for rheumatic heart disease1. With an estimated 500,000 deaths annually, GAS ranks among the ten deadliest human pathogens2. The pathogen expresses numerous surface-bound and secreted virulence factors that subvert innate immune defences1. M protein, the most abundant protein on the GAS surface3, extends as hair-like fimbriae4, with structure, function and immunochemistry unique among known virulence molecules5. Sequence analysis of encoding emm genes suggests more than 220 variants6. However, only a few M protein/emm types are widespread and associated with systemic infections, with serotype M1/emm1 the most prevalent worldwide7. A globally disseminated serotype M1T1 subclone has been the leading cause of severe invasive GAS infections in recent decades8. M proteins mediate host epithelial cell adhesion9 and resistance to opsonophagocytosis by binding host components including fibrinogen, C4b-binding protein and immunoglobulin Fc10. M proteins also block the membrane-lytic activities of host antimicrobial peptides and histones by sequestering them away from the bacterial membrane11,12. Soluble M proteins released during infection by neutrophil-derived granule proteases13 can trigger inflammation by forming a supramolecular assembly with fibrinogen that activates neutrophils13,14. Indeed, soluble M1 protein is sufficient in animal models to trigger vascular leakage and tissue injury similar to severe GAS diseases such as NF and STSS13,15. Under physiological conditions, M1 protein is also shed naturally from the GAS surface and is detected at micromolar concentrations in the extracellular medium16, provided it is not degraded by the broad-spectrum GAS cysteine protease SpeB17. In M1T1 GAS and certain other invasive serotypes, mutations in the covR/S (aka csrR/S) two-component regulatory system can upregulate several virulence factors while suppressing expression of SpeB18,19. Loss of SpeB expression spares M protein from proteolytic degradation and increases the levels of its soluble form. Effects of soluble M1 on macrophages have not previously been investigated. Macrophages play a central role in pathogen recognition and the activation of innate immune and inflammatory responses. Inflammasomes are multimeric cytosolic protein complexes that integrate pathogen-triggered signalling cascades in macrophages, activating caspase-1 and proteolytic processing of pro-IL-1β and pro-IL-18 to their mature cytokine forms, IL-1β and IL-18 (ref. 20). Inflammasomes can activate a rapid pro-inflammatory form of macrophage cell death termed ‘pyroptosis’, characterized by plasma membrane rupture, release of cytosolic contents and DNA fragmentation21. Pyroptosis restricts replication of intracellular bacterial pathogens22 and IL-1β signalling promotes immune resistance to several microbes, including GAS23. Among the various inflammasomes, those containing intracellular pattern recognition molecule NACHT, LRR and pyrin domains-containing protein 3 (NLRP3) are best studied, as their deregulation contributes to inherited autoinflammatory disorders24 and chronic metabolic diseases, including atherosclerosis, type 2 diabetes, gout and obesity25. So far, two GAS-secreted toxins are known to activate macrophage NLRP3 inflammasome-mediated IL-1β signalling and pyroptosis: pore-forming cytolysin streptolysin O (SLO)26 and C3 ADP-ribosyltransferase SpyA27. The consequences of GAS toxin-mediated NLRP3 inflammasome activation range from a protective immune response to exaggerated inflammatory host cell damage. Here, we report that the classical GAS virulence factor M1 protein triggers macrophage cell death in a manner requiring its B-repeat region. M1 is a second signal for caspase-1-dependent NLRP3 inflammasome activation, leading to IL-1β processing and pyroptotic macrophage cell death. M1 uptake occurs through clathrin-mediated endocytosis accompanied by K+ efflux, both essential events in NLRP3 activation. M1-induced pyroptosis and IL-1β signalling may exert pro-immune and pro-inflammatory effects of Group A streptococcal M protein activates the NLRP3 inflammasome