Institute of Biotechnology, Life Sciences Center, Vilnius University, Sauletekio av. 7, LT-10257 Vilnius, Lithuania.
Biomolecules. 2020 Jul 8;10(7):1009. doi: 10.3390/biom10071009.
The pathogenicity of many bacteria, including depends on pore-forming toxins (PFTs) that cause host cell lysis by forming large pores in cholesterol-containing cell membranes. Therefore, PFTs-neutralising antibodies may provide useful tools for reducing pathogenic effects. This study aimed at the development and characterisation of monoclonal antibodies (MAbs) with neutralising activity to PFT pneumolysin (PLY). Five out of 10 produced MAbs were able to neutralise the cytolytic activity of PLY on a lung epithelial cell line. Epitope mapping with a series of recombinant overlapping PLY fragments revealed that neutralising MAbs are directed against PLY loops L1 and L3 within domain 4. The epitopes of MAbs 3A9, 6E5 and 12F11 located at L1 loop (aa 454-471) were crucial for PLY binding to the immobilised cholesterol. In contrast, the MAb 12D10 recognising L3 (aa 403-423) and the MAb 3F3 against the conformational epitope did not interfere with PLY-cholesterol interaction. Due to conformation-dependent binding, the approach to use overlapping peptides for fine epitope mapping of the neutralising MAbs was unsuccessful. Therefore, the epitopes recognised by the MAbs were analysed using computational methods. This study provides new data on PLY sites involved in functional activity.
许多细菌的致病性取决于形成孔的毒素（PFTs），这些毒素通过在含有胆固醇的细胞膜中形成大孔来导致宿主细胞裂解。因此，中和 PFT 的抗体可能为减少 的致病作用提供有用的工具。本研究旨在开发和表征具有中和活性的单克隆抗体（MAbs）以对抗 PFT 肺炎球菌溶血素（PLY）。在产生的 10 种 MAbs 中，有 5 种能够中和 PLY 在肺上皮细胞系上的细胞溶解活性。用一系列重组重叠 PLY 片段进行表位作图表明，中和 MAbs 针对结构域 4 内的 PLY 环 L1 和 L3。MAbs 3A9、6E5 和 12F11 的表位位于 L1 环（aa 454-471），对于 PLY 与固定化胆固醇的结合至关重要。相比之下，识别 L3（aa 403-423）的 MAb 12D10 和针对构象表位的 MAb 3F3 不干扰 PLY-胆固醇相互作用。由于结合具有构象依赖性，因此使用重叠肽来精细分析中和 MAbs 的表位映射的方法不成功。因此，使用计算方法分析了 MAb 识别的表位。本研究提供了有关参与功能活性的 PLY 位点的新数据。
