A Substrate-Fusion Protein Is Trapped inside the Type III Secretion System Channel in Shigella flexneri

The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.

三型分泌系统（Type III Secretion System, T3SS）是革兰氏阴性菌用于单步跨细菌被膜、从细胞质分泌效应蛋白的大分子复合物。对于多数病原菌而言，T3SS是关键毒力因子，可介导细菌与对应宿主产生相互作用并操控宿主细胞。
T3SS的典型结构特征为针状复合物（needle complex, NC）。该复合物形似注射器：其基体横跨细菌双膜，带有一条从细菌表面向外延伸的长针状结构。
基于这一注射器样作用范式，学界普遍认为效应蛋白与转位因子需先解折叠，再经由基体与针状通道从细菌细胞质中被分泌出去。尽管针对T3SS已有广泛研究，但这一假说仍缺乏实验证据支撑，其具体分泌机制尚未完全明晰。
为阐明T3SS分泌机制的细节，我们构建了由T3SS底物与携带打结基序的大体积蛋白融合而成的重组蛋白。由于打结结构无法被解折叠，这类融合蛋白可被T3SS识别为底物，但会滞留在NC通道内并阻塞该分泌系统。据我们所知，本研究首次实现了底物融合蛋白与分离纯化的NC的共可视化，并证实底物蛋白会以N端先行的方式直接经由通道完成分泌。该通道会物理包裹融合蛋白，使其免受蛋白酶降解与化学修饰。
本研究结果夯实了学界对T3SS作用机制的基础认知，同时为未来开展原位结构研究提供了一种高效工具。该方法或同样可推广应用于其他需先解折叠底物才能完成分泌的蛋白分泌系统。