Pyroptosis is an inflammatory form of death mediated by gasdermin that recruits immune cells to the site of infection and promotes protective immunity. The gasdermin is a family of pore-forming proteins, including GSDMA (Gsdma1, Gsdma2, Gsdma3), GSDMB, GSDMC (Gsdmc1, Gsdmc2, Gsdmc3, Gsdmc4), GSDMD (Gsdmd), GSDME/DFNA5 (Dfna5) and GSDMF/DFNB59/PJVK (Dfnb59) [
1,
2]. Though activation of GSDMB/C/D/E has been gradually illustrated [
3], the details of GSDMA activation, particular in epithelial cells such as skin and upper gastrointestinal tract, still remain so far largely elusive.
The
Streptococcus pyogenes, also known as group A
Streptococcus (GAS), causes various acute inflammatory diseases. Among those component of GAS, Streptococcal pyrogenic exotoxin B (SpeB) is a key virulence factor [
4], contributing to GAS colonizing the epidermis and invading infection [
5], however, the specific roles need to be clarified. Recently, two studies published in
Nature by Deng
et al. [
6] and LaRock
et al. [
7] showed the crosstalk between microbial pathogens infection and pyroptosis, and reported that GSDMA was both a sensor of GAS SpeB and an effector of pyroptosis (Fig.1), which revealed a novel insight with host immune recognition and responding to pathogenic microbial infections.
First, Deng and colleagues focused on studying the skin lesions during GAS infection and observed that the skin of mice with GAS infection exhibited aberrant pyogenic and necrotic lesions signatures. Interestingly, GAS with SpeB deletion was less likely to induce cell death and neutrophil infiltration by skin local infection than GAS with wild type or other mutants, but resulted in more severe systemic inflammation, more bacterial loads in the spleen and liver and higher mortality in mice. To clarify the role of SpeB during GAS infection, Deng et al. infected mouse epidermal keratinocytes with GAS and observed pyroptotic morphology and LDH release, while GAS with SpeB mutant has no such effect. Remarkably, further electroporation of recombinant SpeB to mouse primary keratinocytes or human epidermal cell line A431 could directly lead to pyroptosis and LDH release, suggesting that SpeB directly triggers pyroptosis during GAS infection and facilitates the elimination of pathogens by the host.
To characterize the mechanism of SpeB triggering pyroptosis, Deng
et al. utilized the genome-wide CRISPR-Cas9 screen system to investigate potential SpeB-dependent host genes in A431 cells. Among those genes, only GSDMA ablation antagonized SpeB-induced pyroptosis in A431 cells. Notably, the cysteine protease inhibitor E64 completely eliminated pyroptosis caused by co-expression of GSDMA with SpeB, while the caspase inhibitors, including Z-WEHD-fmk, Z-DEVD-fmk, and Z-VAD-fmk, had no such effect. Interestingly, LaRock
et al. also found that substantial SpeB had been activated during keratinocyte infection, however, did not depend on caspase-1 activation [
7]. When the host is stimulated by a variety of exogenous or endogenous factors, gasdermins are cleaved, and the N-terminal pore-forming domain oligomerizes and forms pores in the cell membrane, causing the release of inflammatory molecules and pyroptosis [
8]. To investigate whether SpeB induces pyroptosis by direct cleavage of GSDMA, Deng
et al. performed an
in vitro cleavage assay, and demonstrated that recombinant GSDMA has been cleaved into the N-terminal P27 and C-terminal P23 fragments by SpeB in a dose-dependent manner, which could be suppressed by E64. Intriguingly, SpeB specifically cleaved GSDMA, but not other Gasdermins family members, while, LaRock
et al. held a different view, and they found that after co-incubation of the lysate of HEK293T cells overexpressing GSDMA, GSDMB, GSDMC, GSDMD, and GSDME with purified SpeB, GSDMA, GSDMC, and GSDMD rather than just GSDMA were significantly sheared. Additionally,
Mycobacterium tuberculosis,
Pseudomonas aeruginosa,
Staphylococcus aureus proteases and lysosomal cathepsins were unable to cut GSDMA, suggesting that GSDMA is a specific sensor of SpeB.
To further understand the mechanism of SpeB activation of GSDMA, Deng et al. adopted Edman sequencing and mass spectrometry to demonstrate that SpeB cleaved GSDMA after Q246 in the GSDMA linker region, resulting in the release of two N-terminal and C-terminal fragments. Consistent with the study of Deng et al., LaRock et al. also utilized Edman sequencing to find several sites in GSDMA that could be cleaved by SpeB, and these sites were mainly located at amino acids 240–247. Deletion of amino acids 240–247 could destroy the cleavage effect of SpeB on GSDMA and prevent pyroptosis. Further studies of recombinant SpeB transfected into 293T cells by electroporation showed that SpeB regulated the cleavage of GSDMA, resulting in increased LDH release and pyroptosis. Besides, purified GSDMA-NT induced pyroptosis only when electroporated into cells but failed to exert killing effects in the culture medium. Intriguingly, LaRock et al. found that extracellular SpeB did not effectively activate GSDMA, and purified Speb induced pyroptosis only when packaged in liposomes, and cytoplasmic translocation of Speb required bacterial-cell binding, a process that may involve bacterial adhesins and envelopes.
To explore whether GSDMA induces pyroptosis by drilling holes in lipid membranes, Deng et al. conducted in vitro experiments based on protein–lipid overlay assay, and found that SpeB cleaved GSDMA in a concentration-dependent manner, leading to destruction of certain acidic lipid-containing membranes. Interestingly, GSDMA-N could bind directly to phosphatidylserine (PS), 3-O-sulfogalactosylceramide and cardiolipin (CL) depending on its 215–246 amino acid to form oligomers, thus promoting liposome leakage. These results demonstrate that GSDMA-N may promote pyroptosis through the formation of oligomers on cell membrane.
Deng
et al. also demonstrated the role of GSDMA in host resistance to GAS infection. It should be mentioned that among the three mouse Gsdma genes (Gsdma1/2/3), Gsdma1 and Gsdma3 are selectively expressed on the skin [
9], but only the Gsdma1-encoded protein contains SpeB cleavage sites. After GAS infection, mice with Gsdma1 knockout showed symptoms similar to those of wild-type mice infected with SpeB-deficient GAS, that is, reduced local neutrophil infiltration, increased levels of multi-organ inflammation, and showed high mortality. Similarly, LaRock
et al. also demonstrated that Gsdma1-3-knockout mouse primary keratinocytes blocked SpeB-mediated pyroptosis, as did GSDMA knockout human primary keratinocytes. These results indicated that Gsdma/GSDMA was a substrate of SpeB leading to pyroptosis in mouse.
In summary, Deng et al. and LaRock et al. unveiled the unique contributing role of GSDMA in sensing the GAS virulence factor SpeB and causing pyroptosis, adding novel mechanisms for host immune recognition and response to pathogenic microbial infections. Given the essential role of GSDMA in systemic infection, it is promising to investigate whether people with a variant of the GSDMA gene are more susceptible to GAS infection. Notably, SpeB has been identified in previous studies as an IL-1β processor distinct from caspase-1, and more research is needed to understand how SpeB enters the cytosol of host cells. In addition, Deng et al. found that SpeB is specific for GSDMA cleavage, and only GSDMA is the substrate of SpeB among the gasdermins family members, which limits the application of this study. However, LaRock et al. found that Speb not only cleaved GSDMA, but also activated GSDMC and GSDMD. Unfortunately, the authors did not further explore the specific sites where SpeB cleaved GSDMC and GSDMD, which may also be an interesting research field. These studies still complement the mechanism of pathogen-induced cell death. Although the mechanism of SpeB cleavage of GSDMA has enriched our understanding of pyroptosis, other upstream molecules of GSDMA need to be further discovered, and perhaps other gasdermin members also play important roles in microbial infection. Taken together, these two studies have improved our understanding of the functional diversity and specificity of virulence factors of pathogens.
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