Division of Biological Science and Technology, Yonsei University, Wonju, 26493, Republic of Korea.
Department of Biomedical Laboratory Science, Yonsei University, Wonju, 26493, Republic of Korea.
Sci Rep. 2018 Apr 11;8(1):5814. doi: 10.1038/s41598-018-24254-w.
Flagellin is a major component of the flagellar filament. Flagellin also functions as a specific ligand that stimulates innate immunity through direct interaction with Toll-like receptor 5 (TLR5) in the host. Because flagellin activates the immune response, it has been of interest to develop as a vaccine adjuvant in subunit vaccines or antigen fusion vaccines. Despite the widespread application of flagellin fusion in preventing infectious diseases, flagellin-antigen fusion designs have never been biophysically and structurally characterized. Moreover, flagellin from Salmonella species has been used extensively despite containing hypervariable regions not required for TLR5 that can cause an unexpected immune response. In this study, flagellin from Bacillus cereus (BcFlg) was identified as the smallest flagellin molecule containing only the conserved TLR5-activating D0 and D1 domains. The crystal structure of BcFlg was determined to provide a scheme for fusion designs. Through homology-based modeling and comparative structural analyses, diverse fusion strategies were proposed. Moreover, cellular and biophysical analysis of an array of fusion constructs indicated that insertion fusion at BcFlg residues 178-180 does not interfere with the protein stability or TLR5-stimulating capacity of flagellin, suggesting its usefulness in the development and optimization of flagellin fusion vaccines.
鞭毛蛋白是鞭毛丝的主要组成部分。鞭毛蛋白还作为一种特定的配体，通过与宿主中的 Toll 样受体 5（TLR5）直接相互作用，刺激固有免疫。由于鞭毛蛋白激活免疫反应，因此它已被开发为亚单位疫苗或抗原融合疫苗中的疫苗佐剂而受到关注。尽管鞭毛蛋白融合在预防传染病方面得到了广泛应用，但鞭毛蛋白-抗原融合设计从未在生物物理和结构上进行过特征描述。此外，尽管沙门氏菌属的鞭毛蛋白含有不需要 TLR5 的超变区，可能会引起意外的免疫反应，但仍被广泛使用。在这项研究中，鉴定出来自蜡状芽孢杆菌（BcFlg）的鞭毛蛋白是仅含有保守的 TLR5 激活结构域 D0 和 D1 的最小鞭毛蛋白分子。确定了 BcFlg 的晶体结构，为融合设计提供了方案。通过同源建模和比较结构分析，提出了多种融合策略。此外，对一系列融合构建体的细胞和生物物理分析表明，在 BcFlg 残基 178-180 处进行插入融合不会干扰鞭毛蛋白的蛋白稳定性或 TLR5 刺激能力，这表明它在开发和优化鞭毛蛋白融合疫苗方面具有实用性。
