Date Published: January 16, 2014
Publisher: Public Library of Science
Author(s): Kim Dohlich, Anna Brotcke Zumsteg, Christian Goosmann, Michael Kolbe, Denise M. Monack.
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.
T3SS are found in numerous Gram-negative bacteria and share strong homologies among different invasive pathogens. Using the T3SS, bacteria are able to secrete effector proteins that translocate into the host-cell where they target metabolic or signal transduction pathways for example . The T3SS is a key instrument in interactions between bacteria and eukaryotes, as it is used also by symbiotic bacteria in plants .
In this study we demonstrate that T3SS substrate proteins travel through the NC channel during type III secretion. While largely assumed, our experiments provide convincing evidence for this model. Based on the strong conservation of the NC among different bacterial species , we would predict that our findings are of general relevance for T3 secretion. We generated fusion proteins consisting of the translocator IpaB as a T3SS substrate and a protein with a trefoil-knot in its C-terminal region (Knot). We show that IpaB-Knot has a functional IpaB domain and a folded Knot domain which indicates that the fusion protein is folded prior to secretion. However, the fusion protein attenuates invasion as observed for an ipaB-deficient mutant. In order to pass through the narrow NC channel, the T3SS needs to unfold the Knot. We observed that IpaB-Knot inhibits secretion of T3SS translocators and effectors in the hypersecretor S. flexneriipaD. Obstruction may occur because the T3SS cannot unfold the Knot domain within IpaB-Knot and consequently, IpaB-Knot blocks the channel. Our results are supported by previous reports which show a direct correlation between T3SS secretion and the ability of a protein to be thoroughly inserted into the channel and its folding status .