Prevalent glutamyl-endopeptidases in the commensal skin microbiome have itch-relevant activity

Technical Summary: Prevalent Glutamyl-Endopeptidases in the Commensal Skin Microbiome Have Itch-Relevant Activity

Problem Statement
Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by intense pruritus (itch) and barrier dysfunction. While Staphylococcus aureus colonization is strongly linked to AD severity and itch, a significant subset of patients experiences active disease and pruritus without detectable S. aureus. The known mechanism involves the S. aureus V8 protease (a glutamyl-endopeptidase, or GEP), which cleaves protease-activated receptor 1 (PAR1) on sensory neurons to trigger itch signaling and degrades epithelial barrier proteins. However, the contribution of other skin-colonizing staphylococci, particularly coagulase-negative staphylococci (CoNS) like Staphylococcus epidermidis, to this GEP-mediated pathogenesis remains unclear.

Methodology
The study employed a multi-omics and functional characterization approach:

1. Isolation and Sequencing: The authors cultured 349 bacterial isolates from skin swabs of 10 AD patients with pruritus. Whole-genome sequencing (WGS) of 273 staphylococcal isolates (dominated by S. aureus and S. epidermidis) was performed using Oxford Nanopore technology.
2. Genome Mining: A custom Hidden Markov Model (HMM) was constructed using known GEP sequences to screen the 273 genomes. This identified 678 candidate GEPs.
3. Structural and Functional Classification: Candidate sequences were clustered (CD-HIT) and analyzed for signal peptides. AlphaFold2 was used to predict protein structures, resolving the candidates into five distinct structural architectures based on active site conservation. Representative proteases from each group (V8, Esp, SplB, Csp, and Hsp) were selected.
4. In Vitro Characterization:
   • Expression: Proteases were heterologously expressed in E. coli and enzymatically activated using thermolysin.
   • Enzymatic Activity: GEP activity was measured using a chromogenic substrate (Z-FLE-pNA) on both culture supernatants and purified recombinant proteins.
   • PAR1 Cleavage: A PAR1 tethered-ligand construct (PAR1A22-T102) was incubated with proteases. Cleavage sites were mapped and quantified using mass spectrometry (LC-MS/MS).
   • Barrier Integrity: Primary human nasal epithelial cells were treated with activated proteases, and barrier integrity was monitored over time using the xCELLigence impedance-based system.
5. Genomic Distribution Analysis: The prevalence of these GEP homologs was assessed in the patient isolate collection and a large public database of 12,588 Staphylococcus genomes using BLAST.

Key Results

• Diversity of GEPs: Genome mining revealed a diverse array of GEPs across 13 staphylococcal species. Structural analysis resolved these into five groups, with S. aureus V8 and S. epidermidis Esp showing the highest structural similarity (TM-score ~0.99).
• Functional Activity of Esp and Csp:
  • Esp (S. epidermidis): Displayed GEP activity comparable to V8. Crucially, Esp cleaved the PAR1 tethered ligand at glutamyl residues (specifically E43), generating non-canonical cleavage products similar to those produced by V8.
  • Csp (S. capitis): Showed moderate GEP activity and cleaved PAR1, though with reduced efficiency compared to V8 and Esp.
  • SplB, Hsp: Showed minimal enzymatic activity against synthetic substrates and PAR1 constructs in this assay context.
• Barrier Disruption: All activated proteases (V8, Esp, SplB, Csp, Hsp) significantly reduced epithelial barrier integrity in the cell model, indicating that GEP-mediated barrier breakdown is a shared trait among these homologs, regardless of their specific PAR1 cleavage efficiency.
• Species Distribution:
  • V8: Highly conserved in S. aureus (98.9% of genomes).
  • Esp: Highly conserved in S. epidermidis (98.3% of genomes) and present in S. caprae.
  • Csp: Broadly distributed across S. capitis, S. caprae, S. haemolyticus, and S. condimenti.
  • Hsp: The most phylogenetically widespread, found across nine species.

Significance and Claims
The paper identifies Esp (from S. epidermidis) and Csp (from S. capitis) as functional GEP virulence factors capable of activating the PAR1 itch signaling pathway and disrupting epithelial barrier integrity.

The authors claim that their findings suggest:

1. Functional Redundancy: Itch and barrier dysfunction in AD may not be restricted to S. aureus. Commensal or opportunistic pathogens like S. epidermidis and S. capitis can sustain GEP-driven pathology even in the absence of S. aureus.
2. Therapeutic Implications: Current therapeutic strategies targeting only S. aureus (e.g., specific anti-V8 approaches) may overlook alternative drivers of disease. The collective proteolytic load of multiple staphylococcal species likely contributes to the AD phenotype.
3. Mechanistic Insight: The study provides evidence that non-canonical cleavage of PAR1 by Esp can still generate agonistic peptides, linking S. epidermidis directly to the itch signaling axis previously attributed solely to S. aureus.

The authors maintain a modest tone regarding future steps, noting that confirming these roles in complex biological systems (e.g., mouse AD models or direct clinical quantification of Esp transcription) is a necessary next step, but the current data establishes a strong biochemical and genomic basis for the involvement of non-aureus staphylococci in AD pathogenesis.