title: A Homology Model Reveals Novel Structural Features and an Immunodominant Surface Loop/Opsonic Target in the Treponema pallidum BamA Ortholog TP_0326.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Department of Medicine, University of Connecticut Health, Farmington, Connecticut, USA.
authors.forename: Amit
authors.initials: A
authors.lastname: Luthra
authors.affiliation: Department of Medicine, University of Connecticut Health, Farmington, Connecticut, USA.
authors.forename: Arvind
authors.initials: A
authors.lastname: Anand
authors.affiliation: Department of Pediatrics, University of Connecticut Health, Farmington, Connecticut, USA.
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Hawley
authors.affiliation: Department of Medicine, University of Connecticut Health, Farmington, Connecticut, USA.
authors.forename: Morgan
authors.initials: M
authors.lastname: LeDoyt
authors.affiliation: Department of Pediatrics, University of Connecticut Health, Farmington, Connecticut, USA.
authors.forename: Carson J
authors.initials: CJ
authors.lastname: La Vake
authors.affiliation: Department of Medicine, University of Connecticut Health, Farmington, Connecticut, USA Department of Pediatrics, University of Connecticut Health, Farmington, Connecticut, USA Department of Molecular Biology and Biophysics, University of Connecticut Health, Farmington, Connecticut, USA Connecticut Children's Medical Center, Hartford, Connecticut, USA.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Caimano
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Cali, Colombia.
authors.forename: Adriana R
authors.initials: AR
authors.lastname: Cruz
authors.affiliation: Department of Pediatrics, University of Connecticut Health, Farmington, Connecticut, USA Connecticut Children's Medical Center, Hartford, Connecticut, USA.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Salazar
authors.affiliation: Department of Medicine, University of Connecticut Health, Farmington, Connecticut, USA Department of Pediatrics, University of Connecticut Health, Farmington, Connecticut, USA Department of Molecular Biology and Biophysics, University of Connecticut Health, Farmington, Connecticut, USA Department of Genetics and Genomic Sciences, University of Connecticut Health, Farmington, Connecticut, USA Department of Immunology, University of Connecticut Health, Farmington, Connecticut, USA Connecticut Children's Medical Center, Hartford, Connecticut, USA JRadolf@uchc.edu.
authors.forename: Justin D
authors.initials: JD
authors.lastname: Radolf
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D016056:Immunodominant Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D009895:Opsonin Proteins / Q000276:immunology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013587:Syphilis / Q000276:immunology* / Q000382:microbiology; D014210:Treponema pallidum / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D016056:Immunodominant Epitopes; D009895:Opsonin Proteins
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