Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands.
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CH, Utrecht, The Netherlands.
Sci Rep. 2023 Nov 1;13(1):18836. doi: 10.1038/s41598-023-46026-x.
Antibodies play a key role in the immune defence against Gram-negative bacteria. After binding to bacterial surface antigens, IgG and IgM can activate the complement system and trigger formation of lytic membrane attack complex (MAC) pores. Molecular studies to compare functional activity of antibodies on bacteria are hampered by the limited availability of well-defined antibodies against bacterial surface antigens. Therefore, we genetically engineered E. coli by expressing the StrepTagII antigen into outer membrane protein X (OmpX) and validated that these engineered bacteria were recognised by anti-StrepTagII antibodies. We then combined this antigen-antibody system with a purified complement assay to avoid interference of serum components and directly compare MAC-mediated bacterial killing via IgG1 and pentameric IgM. While both IgG1 and IgM could induce MAC-mediated killing, we show that IgM has an increased capacity to induce complement-mediated killing of E. coli compared to IgG1. While Fc mutations that enhance IgG clustering after target binding could not improve MAC formation, mutations that cause formation of pre-assembled IgG hexamers enhanced the complement activating capacity of IgG1. Altogether, we here present a system to study antibody-dependent complement activation on E. coli and show IgM's enhanced capacity over IgG to induce complement-mediated lysis of E. coli.
抗体在针对革兰氏阴性菌的免疫防御中发挥着关键作用。与细菌表面抗原结合后，IgG 和 IgM 可以激活补体系统并触发形成溶膜攻击复合物（MAC）孔。由于针对细菌表面抗原的定义明确的抗体的有限可用性，因此分子研究比较抗体在细菌上的功能活性受到阻碍。因此，我们通过在外膜蛋白 X（OmpX）中表达 StrepTagII 抗原来对大肠杆菌进行基因工程改造，并验证这些经过工程改造的细菌被抗-StrepTagII 抗体识别。然后，我们将该抗原-抗体系统与纯化的补体测定法相结合，以避免血清成分的干扰，并直接比较 IgG1 和五聚体 IgM 介导的 MAC 诱导的细菌杀伤。虽然 IgG1 和 IgM 都可以诱导 MAC 介导的杀伤，但我们表明，与 IgG1 相比，IgM 具有诱导补体介导的大肠杆菌杀伤的更大能力。虽然增强靶标结合后 IgG 聚集的 Fc 突变不能改善 MAC 形成，但导致预组装 IgG 六聚体形成的突变增强了 IgG1 的补体激活能力。总之，我们在此提出了一种研究抗体依赖性补体激活大肠杆菌的系统，并显示 IgM 诱导补体介导的大肠杆菌裂解的能力强于 IgG1。
