We recently demonstrated that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses characteristic BamA bipartite topology. Herein， we used immunofluorescence analysis (IFA) to show that only the β-barrel domain of TP_0326 contains surface-exposed epitopes in intact T. pallidum. Using the solved structure of Neisseria gonorrhoeae BamA， we generated a homology model of full-length TP_0326. Although the model predicts a typical BamA fold， the β-barrel harbors features not described in other BamAs. Structural modeling predicted that a dome comprised of three large extracellular loops， loop 4 (L4)， L6， and L7， covers the barrel's extracellular opening. ... More
We recently demonstrated that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses characteristic BamA bipartite topology. Herein， we used immunofluorescence analysis (IFA) to show that only the β-barrel domain of TP_0326 contains surface-exposed epitopes in intact T. pallidum. Using the solved structure of Neisseria gonorrhoeae BamA， we generated a homology model of full-length TP_0326. Although the model predicts a typical BamA fold， the β-barrel harbors features not described in other BamAs. Structural modeling predicted that a dome comprised of three large extracellular loops， loop 4 (L4)， L6， and L7， covers the barrel's extracellular opening. L4， the dome's major surface-accessible loop， contains mainly charged residues， while L7 is largely neutral and contains a polyserine tract in a two-tiered conformation. L6 projects into the β-barrel but lacks the VRGF/Y motif that anchors L6 within other BamAs. IFA and opsonophagocytosis assay revealed that L4 is surface exposed and an opsonic target. Consistent with B cell epitope predictions， immunoblotting and enzyme-linked immunosorbent assay (ELISA) confirmed that L4 is an immunodominant loop in T. pallidum-infected rabbits and humans with secondary syphilis. Antibody capture experiments using Escherichia coli expressing OM-localized TP_0326 as a T. pallidum surrogate further established the surface accessibility of L4. Lastly， we found that a naturally occurring substitution (Leu(593) → Gln(593)) in the L4 sequences of T. pallidum strains affects antibody binding in sera from syphilitic patients. Ours is the first study to employ a "structure-to-pathogenesis" approach to map the surface topology of a T. pallidum OMP within the context of syphilitic infection.
