Reviewer #1 (Public Review):

Summary:
In their revised manuscript, the authors analyze the evolution of the gasdermin family and observe that the GSDMA proteins from birds, reptiles and amphibians does not form a clade with the mammalian GSDMAs. Moreover, the non-mammalian GSDMA proteins share a conserved caspase-1 cleavage motif at the predicted activation site. The authors provide several series of experiments showing that the non-mammalian GSDMA proteins can indeed be activated by caspase-1 and that this activation leads to cell death (in human cells). They also investigate the role of the caspase-1 recognition tetrapeptide for cleavage by caspase-1 and for the pathogen-derived protease SpeB.

Strengths:
The evolutionary analysis performed in this manuscript appears to use a broader data basis than what has been used in other published work. An interesting result of this analysis is the suggestion that GSDMA is evolutionary older than the main mammalian pyroptotic GSDMD, and that birds, reptiles and amphibians lack GSDMD but use GSDMA for the same purpose. The consequence that bird GSDMA should be activated by an inflammatory caspase (=caspase1) is convincingly supported by the experiments provided in the manuscript.

Weaknesses:
While the cleavability of bird/reptile GSDMA by caspase-1 is well-supported by several experiments, the role of this cleavage for pyroptotic cell killing is addressed more superficially. The experiments performed to this end all use human cells; it is likely - but not guaranteed - that the human model recapitulaes the physiological role of non-mammalian GSDMA proteins. While the data provided in this paper help to understand GSDMA evolution and the activation mechanism of bird/reptile GSDMA, it does not address the still elusive activation mechanism for mammalian GSDMA

As a consequence, the significance of this finding is mostly limited to birds and reptiles.