Elucidating protein-protein interactions between host cell receptors and outer membrane protein a (ompa) of orientia tsutsugamushi in the pathogenesis of cellular invasion

Scrub typhus, caused by Orientia tsutsugamushi, remains an underdiagnosed and re- emerging infectious disease. Its antigenic variability complicates vaccine and diagnostic development. Outer membrane protein A (OtOmpA), highly conserved across strains and detected during various stages of infection, has been proposed as a promising target. However, its specific role in the pathogenesis of scrub typhus, particularly in human endothelial cells, the primary target cells, remains poorly understood. This study aimed to investigate the interaction between OtOmpA and proteins from human pulmonary (HPAEC) and cerebral microvascular endothelial cells (HBEC-5i). DNA sequence alignment using Jalview and ligand-binding pocket prediction with AutoDockFR- AGFR, visualised by PyMOL, confirmed OtOmpA’s suitability as a study target and guided sequence selection for cloning. The Gilliam strain DNA sequence was cloned into three expression vectors (pET-14b(+), pET-20b(+), and pET-28a(+)). Positive colonies were identified by PCR and agarose gel electrophoresis, and DNA sequencing confirmed the inserts. Recombinant vectors were expressed in E. coli BL21(DE3) pLysS, and OtOmpA production was verified via SDS-PAGE, Western blotting, LC- MS/MS, and positive serum from scrub typhus a patient. The adherence and invasion capabilities of OtOmpA were assessed by immunofluorescence microscopy. Protein– protein interactions were analysed through a poly-His-tag pull-down assay and 1D-LC- MS/MS. Molecular docking and binding site prediction were performed using HDOCK and visualised in PyMOL, while MEMSAT-SVM predicted transmembrane helix topology. In this study, although all vectors contained the correctly oriented OtOmpA sequence, only pET-20b(+) successfully expressed the protein. OtOmpA enhanced E. coli association with endothelial cells, suggesting a role in adhesion and invasion. An integrated in vitro and in silico approach identified several host interacting proteins: heat shock protein β, integrin alpha-X, caspase-4, baculoviral IAP repeat-containing protein 6, serine/threonine-protein kinase ATR, glucose-6-phosphate dehydrogenase, oxysterol-binding protein-related protein 8, and phosphatidylinositol 4-kinase beta. Two OtOmpA regions, V68–D112 and R124–T157, were predicted as key binding domains. In conclusion, this study provides novel insights into OtOmpA’s role in scrub typhus pathogenesis, offering potential guidance for developing targeted diagnostics, vaccines,and therapeutic strategies.