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. Author Summary Type III Secretion Systems (T3SS) secrete bacterial effector proteins from the cytoplasm across the cell wall, but mechanistic details of this process remain mostly elusive. We locked the T3SS of Shigella flexneri in an actively secreting state by expression of substrate fusions that consist of a functional translocator and a stably-folded knotted protein. Although recognized as T3SS substrates, the fusions are not released from secreting Shigella and impede transport of other effector proteins by obstructing the T3SS channel. We localized the fusion at isolated channels and observed that the translocator is secreted with the N-terminus first. We further demonstrate that the channel physically encloses the partially transported substrate. Our analysis elucidates important steps of the T3SS mechanism. Furthermore, we developed fusion proteins useful for advanced structural investigations of one of the most complex bacterial virulence devices known and our approach may help to also understand other protein transport mechanisms.