Homology Model Reveals Novel Structural Features and an Immunodominant Surface Loop/Opsonic Target in the Treponema pallidum BamA Ortholog TP_0326

• Published in: Journal of bacteriology Vol. 197; no. 11; pp. 1906 - 1920
• Main Authors: Luthra, Amit, Anand, Arvind, Hawley, Kelly L, LeDoyt, Morgan, La Vake, Carson J, Caimano, Melissa J, Cruz, Adriana R, Salazar, Juan C, Radolf, Justin D
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.06.2015
• Subjects: Amino Acid Sequence; Animals; antibodies; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - immunology; Bacterial proteins; Bacteriology; Binding sites; biogenesis; E coli; enzyme-linked immunosorbent assay; Escherichia coli; fluorescent antibody technique; Gram-negative bacteria; human population; Humans; Immunoassay; immunoblotting; immunodominant epitopes; Immunodominant Epitopes - chemistry; Immunodominant Epitopes - genetics; Immunodominant Epitopes - immunology; Immunology; Molecular Sequence Data; Neisseria gonorrhoeae; Opsonin Proteins - immunology; outer membrane proteins; Pathogenesis; patients; prediction; Protein expression; Protein Structure, Secondary; Protein Structure, Tertiary; Rabbits; Sexually transmitted diseases; STD; Syphilis; Syphilis - immunology; Syphilis - microbiology; Topology; Treponema pallidum; Treponema pallidum - chemistry; Treponema pallidum - genetics; Treponema pallidum - immunology
• ISSN: 0021-9193; 1098-5530; 1098-5530
• DOI: 10.1128/JB.00086-15

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. IMPORTANCE Previously, we reported that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses the bipartite topology characteristic of a BamA ortholog. Using a homology model as a guide, we found that TP_0326 displays unique features which presumably relate to its function(s) in the biogenesis of T. pallidum 's unorthodox OM. The model also enabled us to identify an immunodominant epitope in a large extracellular loop that is both an opsonic target and subject to immune pressure in a human population. Ours is the first study to follow a structure-to-pathogenesis approach to map the surface topology of a T. pallidum rare OMP within the context of syphilitic infection.