Institute of Research, Development, and Innovation in Healthcare Biotechnology in Elche (IDiBE), Miguel Hernández University, Avda Universidad s/n, Elche-03202, Spain.
Department of Vegetal Production and Microbiology, Miguel Hernández University, Avda Universidad s/n, Elche-03202, Spain.
ACS Infect Dis. 2020 May 8;6(5):954-974. doi: 10.1021/acsinfecdis.9b00344. Epub 2020 Mar 17.
Choline-binding proteins (CBPs) from comprise a family of modular polypeptides involved in essential events of this pathogen. They recognize the choline residues present in the teichoic and lipoteichoic acids of the cell wall using the so-called choline-binding modules (CBMs). The importance of CBPs in pneumococcal physiology points to them as novel targets to combat antimicrobial resistances shown by this organism. In this work we have tested the ability of exogenously added CBMs to act as CBP inhibitors by competing with the latter for the binding to the choline molecules in the bacterial surface. First, we carried out a thorough physicochemical characterization of three native CBMs, namely C-LytA, C-Cpl1, and C-CbpD, and assessed their affinity for choline and macromolecular, pneumococcal cell-wall mimics. The interaction with these substrates was evaluated by molecular modeling, analytical ultracentrifugation, surface plasmon resonance, and fluorescence and circular dichroism spectroscopies. Van't Hoff thermal analyses unveiled the existence of one noncanonical choline binding site in each of the C-Cpl1 and C-CbpD proteins, leading in total to 5 ligand-binding sites per dimer and 4 sites per monomer, respectively. Remarkably, the binding affinities of the CBMs do not directly correlate with their native oligomeric state or with the number of choline-binding sites, suggesting that choline recognition by these modules is a complex phenomenon. On the other hand, the exogenous addition of CBMs to pneumococcal planktonic cultures caused extensive cell-chaining probably as a consequence of the inhibition of CBP attachment to the cell wall. This was accompanied by bacterial aggregation and sedimentation, causing an enhancement of bacterial phagocytosis by peritoneal macrophages. In addition, the rational design of an oligomeric variant of a native CBM led to a substantial increase in its antibacterial activity by multivalency effects. These results suggest that CBMs might constitute promising nonlytic antimicrobial candidates based on the natural induction of the host defense system.
胆碱结合蛋白（CBPs）来自肺炎链球菌，构成了一个涉及该病原体重要事件的模块多肽家族。它们使用所谓的胆碱结合模块（CBMs）识别细胞壁中磷壁酸和脂磷壁酸中存在的胆碱残基。CBPs 在肺炎链球菌生理学中的重要性表明它们是对抗该生物体表现出的抗微生物耐药性的新型靶标。在这项工作中，我们测试了外源添加的 CBM 作为 CBP 抑制剂的能力，通过与后者竞争与细菌表面的胆碱分子结合来实现。首先，我们对三种天然 CBM 进行了全面的物理化学特性分析，即 C-LytA、C-Cpl1 和 C-CbpD，并评估了它们对胆碱和大分子、肺炎链球菌细胞壁模拟物的亲和力。通过分子建模、分析超速离心、表面等离子体共振、荧光和圆二色性光谱评估了与这些底物的相互作用。范特霍夫热分析表明，在 C-Cpl1 和 C-CbpD 蛋白中，每个蛋白都存在一个非典型的胆碱结合位点，分别导致每个二聚体有 5 个配体结合位点和每个单体有 4 个结合位点。值得注意的是，CBM 的结合亲和力与它们的天然寡聚状态或与胆碱结合位点的数量不直接相关，这表明这些模块对胆碱的识别是一个复杂的现象。另一方面，将 CBM 添加到肺炎链球菌浮游培养物中会导致广泛的细胞链形成，这可能是由于 CBP 与细胞壁的附着受到抑制所致。这伴随着细菌聚集和沉淀，导致腹腔巨噬细胞对细菌的吞噬作用增强。此外，天然 CBM 寡聚变体的合理设计通过多价效应导致其抗菌活性显著增强。这些结果表明，CBM 可能构成有前途的非溶菌性抗菌候选物，基于天然诱导宿主防御系统。
