Biophysical and functional insights into YchP, a putative invasin outer membrane protein of Salmonella Typhimurium, and its role in virulence.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen responsible for gastrointestinal infections in humans and animals. Its ability to adhere to and invade intestinal epithelial cells is mediated by several outer membrane proteins (OMPs), including OmpC, OmpD, OmpA, and OmpF. YchP, a lesser-known OMP, has emerged as a putative invasin targeting β-integrins on host cells. In silico analysis revealed YchP as a 12-stranded β-barrel protein with a 41-residue signal peptide and a pore diameter that expands from 4 Å to 10 Å along an SU-shaped cavity, suggesting a potential role in selective molecular transport or gating. Biophysical studies using Size Exclusion Chromatography (SEC) and ATR-FTIR spectroscopy confirmed its monomeric state and β-sheet-rich structure, consistent with β-barrel architecture. Refolding experiments in the presence of urea, lipids, and detergents, coupled with tryptophan fluorescence and CD spectroscopy, provided insights into optimal in vitro folding conditions, with LDAO emerging as the most suitable agent for refolding and stabilizing the β-sheet conformation. Functional characterization through computational docking and pull-down assay demonstrated YchP's specific interaction with α5β1 integrin, supporting its role as a host-targeting invasin. Additionally, deletion of ychP significantly reduced adhesion (~31 %) and invasion (~50 %) of S. enterica in Caco-2 cells, highlighting its importance in host epithelial interaction. These findings collectively underscore YchP's structural and functional roles, contributing to Salmonella pathogenesis.