Unraveling the refolding dynamics, TLR2 interaction, and immunomodulatory insights of OmpA from Salmonella Typhimurium

Abstract

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium that causes gastrointestinal infection and rising antibiotic resistance worldwide. This study examines the structural-functional relationship of OmpA, a major outer membrane protein (OMP) implicated in Salmonella pathogenesis. Computational analysis revealed a high conservation of OmpA among the Enterobacteriaceae family. Due to its β-sheet-rich structure, OmpA forms inclusion bodies during overexpression. To mitigate this, solubilization under high-pH conditions and refolding with Lauryl dimethylamine N-oxide (LDAO) effectively preserves its native-like conformation. Semi-native SDS-PAGE and size exclusion chromatography suggest that OmpA exists as a dimer. Circular dichroism and heat modifiability analyses further confirmed its β-sheet-rich secondary structure, consistent with a β-barrel fold. However, high-resolution studies are required to confirm its oligomeric state and barrel architecture. These insights facilitate the selection of refolding processes and support investigations into the structure and function of OmpA. Functionally, in silico docking and in vitro assays revealed OmpA interaction with TLR2 from HEp-2 and Raji human B-cells. OmpA stimulation activates NFkB signaling in B-cells, leading to increased expression of proinflammatory cytokines such as TNF-α and upregulation of activation-induced cytidine deaminase (AID), a key enzyme involved in enhancing antibody diversity. Moreover, an immunoinformatic analysis has identified B-cell and T-cell epitopes within OmpA's extracellular domain, highlighting its potential as a vaccine candidate. Notably, immune simulations demonstrate that OmpA enhances both innate and adaptive immune responses. This response promotes memory cell generation and may contribute to bacterial clearance. Collectively, this study provides a foundation for future therapeutics against S. Typhimurium pathogenesis. © 2026 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.