GSDMA roles in skin innate immune defense

PROJECT SUMMARY/ABSTRACT Streptococcus pyogenes (group A Streptococcus; GAS) causes more than half a million deaths annually through invasive infections and post-infection immune sequelae like rheumatic heart disease. The pathogenesis of these diseases is poorly understood despite their public health importance, and they carry high mortality even with treatment. The first barrier to infection is the skin. GAS frequently lives asymptomatically, but can invade further to cause impetigo and erysipelas, and deeper still to cause the “flesh-eating disease” necrotizing fasciitis. The long-term goal of this work is to understand the specific cellular processes that GAS virulence factors manipulate when infections proceed to more severe invasive diseases, in order to develop more effective therapeutic countermeasures. This proposal tests the hypothesis that keratinocytes sense invasive infection through a new cell death mechanism of gasdermin A-dependent pyroptosis. The major secretion product of GAS is a protease, SpeB, which degrades tissue to promote spread and immune factors to protect the bacterium. GAS can also invade skin keratinocytes, which usually can restrict intracellular pathogens through autophagy, but hypervirulent GAS escapes this also using SpeB. Notably, the inert cytosolic protein gasdermin A is processed by SpeB into an active pore-forming effector. Keratinocytes express high gasdermin A and die of gasdermin A-dependent pyroptosis during GAS infection. Thus, activation of gasdermin A is intrinsically linked to the same virulence factors essential for invasion and serves as a cell-autonomous sensor of pathogenicity. This proposal dissects cell death at the host-pathogen interface with three aims. In the first, a panel of defined mutant GAS and clinical isolates naturally differing in their ability to cause invasive will be used to examine the molecular requirements for cell death during infection, with specific reporters used to measure SpeB activity, gasdermin A activation, and bacterial and host cell survival. Then, the molecular effector functions of gasdermin A and how pyroptosis results in bacterial killing is examined. Lastly, in vivo models are used to separately examine inflammation, wound healing, and bacterial killing to determine the specific contribution of cell death to the establishment and progression of skin infection. These studies will lay the foundation for how gasdermin A acts as an immune sensor of skin infection, dramatically improving our fundamental understanding of GAS pathogenesis and revealing therapeutic targets to treat disease.