Wittlinger, J.-P.; Weninger, S.; Seneca Cardoso da Silva, J.; Tu, A.; Grey, L.; Heber, S.; Fischer, M.; Schneider, S.; Eckl-Dorna, J.; Stary, G.; Böttcher, T.; Berry, D.

Atopic dermatitis (AD) is frequently accompanied by pruritus, which has predominantly been attributed to skin colonization by Staphylococcus aureus, particularly through cleavage of protease-activated receptor 1 (PAR1) by the glutamyl endopeptidase (GEP) V8 protease. Whether related GEPs from other skin-associated staphylococci contribute to this process remains unclear. Here, we analyzed 273 staphylococcal isolates from skin swabs of 10 AD patients with pruritus, dominated by S. aureus and Staphylococcus epidermidis. Genome mining using a custom hidden Markov model identified 678 candidate GEPs, which were clustered and resolved into five structurally distinct protease architectures. Representative proteases (V8, Esp, SplB, Csp, and Hsp) were expressed and characterized. Esp displayed GEP activity and PAR1 tethered-ligand cleavage comparable to V8, generating noncanonical cleavage products, while Csp cleaved PAR1 with reduced but substantial efficiency. All representative proteases significantly disrupted barrier integrity in an epithelial barrier model. Analysis of isolate genomes and publicly available Staphylococcus genomes showed that V8 and Esp are highly conserved and largely species-restricted, whereas Csp is more broadly distributed across species. These findings identify Esp and Csp as functional GEP virulence factors in S. epidermidis and S. capitis, capable of activating itch signaling and compromising barrier function. Our study suggests that GEP-mediated pruritus and barrier dysfunction in AD may arise not only from pathogens like S. aureus, but also from commensals or opportunistic pathogens such as S. epidermidis and S. capitis.