Unité de Génie Enzymatique et Cellulaire (GEC), CNRS UMR 7025, Université de Technologie de Compiègne, Compiègne, 60203, France.
Robotein®, InBioS Research Unit, University of Liège, Building B6, Quartier Agora, Allée du 6 Août, 13, Sart-Tilman, Liège, 4000, Belgium.
Appl Microbiol Biotechnol. 2024 Jul 23;108(1):425. doi: 10.1007/s00253-024-13195-2.
Borrelia, spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia, including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF. KEY POINTS: • DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment • Borrelia spp. possesses immune evasion mechanisms, including human host complement • CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1.
伯氏疏螺旋体（Borrelia），通过蜱传播的螺旋体，是许多全身性疾病的病原体，如莱姆病（LB）和蜱传回归热（TBRF）。本研究关注两种参与这些疾病的两种伯氏疏螺旋体亚种的表面蛋白：CspZ，由伯氏疏螺旋体亚种严格表达（也称为补体调节蛋白 2 ），以及因子 H 结合 A（FhbA），由赫尔姆斯伯氏疏螺旋体表达。包括后者在内的许多伯氏疏螺旋体亚种能够通过多种方式逃避各种潜在脊椎动物宿主的免疫防御。在这种情况下，先前的数据表明，这两种表面蛋白通过与人类补体系统替代途径的关键调节剂因子 H（FH）和 FH 样蛋白 1（FHL-1）相互作用，在两种伯氏疏螺旋体亚种的免疫逃避中发挥作用。重组蛋白 CspZ 和 FhbA 在大肠杆菌中表达，并通过一步金属亲和层析纯化，分别从 1L 培养菌中获得 15 和 20mg 或纯蛋白。纯度通过 SDS-PAGE 和 HPLC 评估，接近 95%。CspZ 和 FhbA 的质量通过质谱（MS）检查。通过圆二色性（CD）确认 CspZ 和 FhbA 的正确折叠，并通过 SPR 表征其生物学活性，即它们与从人血清中纯化的 FH（重组 FH 和重组 FHL-1）的相互作用。这项研究为研究蛋白的生化特性及其生物分子相互作用提供了基础，这是开发改善 LB 和 TBRF 现有诊断方法的必要前提。关键点：• DLS、CD、SEC-MALS、NMR、HPLC 和 MS 是蛋白质质量评估的工具• 伯氏疏螺旋体具有免疫逃避机制，包括人类宿主补体• CspZ 和 FhbA 与人 FH 和 rFHL-1 高亲和力（pM 至 nM）相互作用。
