DataSheet_1_Multifunctional interaction of CihC/FbpC orthologs of relapsing fever spirochetes with host-derived proteins involved in adhesion, fibrinolysis, and complement evasion.pdf

IntroductionRelapsing fever (RF) remains a neglected human disease that is caused by a number of diverse pathogenic Borrelia (B.) species. Characterized by high cell densities in human blood, relapsing fever spirochetes have developed plentiful strategies to avoid recognition by the host defense mechanisms. In this scenario, spirochetal lipoproteins exhibiting multifunctional binding properties in the interaction with host-derived molecules are known to play a key role in adhesion, fibrinolysis and complement activation.
MethodsBinding of CihC/FbpC orthologs to different human proteins and conversion of protein-bound plasminogen to proteolytic active plasmin were examined by ELISA. To analyze the inhibitory capacity of CihC/FbpC orthologs on complement activation, a microtiter-based approach was performed. Finally, AlphaFold predictions were utilized to identified the complement-interacting residues.
Results and discussionHere, we elucidate the binding properties of CihC/FbpC-orthologs from distinct RF spirochetes including B. parkeri, B. hermsii, B. turicatae, and B. recurrentis to human fibronectin, plasminogen, and complement component C1r. All CihC/FbpC-orthologs displayed similar binding properties to fibronectin, plasminogen, and C1r, respectively. Functional studies revealed a dose dependent binding of plasminogen to all borrelial proteins and conversion to active plasmin. The proteolytic activity of plasmin was almost completely abrogated by tranexamic acid, indicating that lysine residues are involved in the interaction with this serine protease. In addition, a strong inactivation capacity toward the classical pathway could be demonstrated for the wild-type CihC/FbpC-orthologs as well as for the C-terminal CihC fragment of B. recurrentis. Pre-incubation of human serum with borrelial molecules except CihC/FbpC variants lacking the C-terminal region protected serum-susceptible Borrelia cells from complement-mediated lysis. Utilizing AlphaFold2 predictions and existing crystal structures, we mapped the putative key residues involved in C1r binding on the CihC/FbpC orthologs attempting to explain the relatively small differences in C1r binding affinity despite the substitutions of key residues. Collectively, our data advance the understanding of the multiple binding properties of structural and functional highly similar molecules of relapsing fever spirochetes proposed to be involved in pathogenesis and virulence.

引言：回归热（Relapsing fever, RF）仍是一种未得到充分关注的人类疾病，其由多种致病性疏螺旋体属（Borrelia, B.）的不同物种引发。回归热螺旋体以人体血液中极高的菌体密度为典型特征，已演化出多种策略以逃避免疫宿主的防御识别机制。在此背景下，研究证实，在与宿主源性分子互作过程中展现多功能结合特性的螺旋体脂蛋白，在黏附、纤维蛋白溶解及补体激活过程中发挥关键作用。
方法：本研究通过酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assay, ELISA），检测了CihC/FbpC同源蛋白与不同人类蛋白质的结合活性，以及蛋白结合型纤溶酶原向具有蛋白水解活性的纤溶酶的转化过程。为分析CihC/FbpC同源蛋白对补体激活的抑制能力，本研究采用了基于微孔板的实验体系。最后，本研究借助AlphaFold预测工具，鉴定出与补体互作的氨基酸残基。
结果与讨论：本研究阐明了来自不同回归热螺旋体的CihC/FbpC同源蛋白结合特性，涉及的菌株包括帕克疏螺旋体（B. parkeri）、赫姆斯疏螺旋体（B. hermsii）、图里卡塔疏螺旋体（B. turicatae）以及回归热疏螺旋体（B. recurrentis），其靶分子包括人类纤连蛋白、纤溶酶原及补体成分C1r。所有CihC/FbpC同源蛋白均分别对上述三类分子展现出相似的结合活性。功能实验结果显示，纤溶酶原与所有疏螺旋体蛋白的结合呈剂量依赖性，并可转化为活性纤溶酶。氨甲环酸可几乎完全抑制纤溶酶的蛋白水解活性，表明赖氨酸残基参与了该丝氨酸蛋白酶的互作过程。此外，野生型CihC/FbpC同源蛋白以及回归热疏螺旋体的C端CihC片段，均展现出对补体经典途径的强效灭活能力。将人类血清与除缺失C端区域的CihC/FbpC变体之外的疏螺旋体分子预孵育，可使血清敏感型疏螺旋体细胞免受补体介导的裂解。本研究结合AlphaFold2预测结果与已公开的晶体结构，在CihC/FbpC同源蛋白上定位了参与C1r结合的潜在关键残基，试图解释尽管存在关键残基的替换，C1r结合亲和力仍仅存在较小差异的现象。综上，本研究的数据加深了我们对回归热螺旋体结构与功能高度相似的多功能结合分子的理解，这类分子被认为与致病过程及毒力调控密切相关。