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
Department of Medicine, University of Connecticut Health, Farmington, Connecticut, USA.
Department of Pediatrics, University of Connecticut Health, Farmington, Connecticut, USA.
J Bacteriol. 2015 Jun;197(11):1906-20. doi: 10.1128/JB.00086-15. Epub 2015 Mar 30.
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.
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.
我们最近证明，TP_0326是梅毒螺旋体中一种真正的罕见外膜蛋白（OMP），并且它具有典型的BamA二分拓扑结构。在此，我们使用免疫荧光分析（IFA）表明，在完整的梅毒螺旋体中，只有TP_0326的β-桶状结构域含有表面暴露的表位。利用淋病奈瑟菌BamA的已解析结构，我们生成了全长TP_0326的同源模型。尽管该模型预测了典型的BamA折叠，但β-桶状结构具有其他BamA中未描述的特征。结构建模预测，由三个大的细胞外环，即环4（L4）、L6和L7组成的穹顶覆盖了桶状结构的细胞外开口。L4是穹顶主要的可表面接触环，主要包含带电荷的残基，而L7基本呈中性，并且在两层构象中包含一个多聚丝氨酸序列。L6伸入β-桶状结构，但缺乏将L6锚定在其他BamA中的VRGF/Y基序。IFA和调理吞噬作用试验表明，L4暴露于表面且是调理吞噬的靶点。与B细胞表位预测一致，免疫印迹和酶联免疫吸附测定（ELISA）证实，L4是梅毒螺旋体感染的兔子和二期梅毒患者体内的免疫显性环。使用表达定位于外膜的TP_0326的大肠杆菌作为梅毒螺旋体替代物的抗体捕获实验进一步证实了L4的表面可及性。最后，我们发现梅毒螺旋体菌株L4序列中一个天然存在的替换（Leu(593)→Gln(593)）影响梅毒患者血清中的抗体结合。我们的研究是首次采用“结构到发病机制”的方法，在梅毒感染的背景下绘制梅毒螺旋体OMP的表面拓扑结构。
此前，我们报道TP_0326是梅毒螺旋体中一种真正的罕见外膜蛋白（OMP），并且它具有BamA直系同源物的二分拓扑结构特征。以同源模型为指导，我们发现TP_0326表现出独特的特征，这些特征可能与其在梅毒螺旋体非传统外膜生物合成中的功能有关。该模型还使我们能够在一个大的细胞外环中鉴定出一个免疫显性表位，该表位既是调理吞噬的靶点，又在人群中受到免疫压力的影响。我们的研究是首次采用结构到发病机制的方法，在梅毒感染的背景下绘制梅毒螺旋体罕见OMP的表面拓扑结构。
