Abstract
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.
Highlights
T3SS are found in numerous Gram-negative bacteria and share strong homologies among different invasive pathogens
Induction of pyroptosis was measured by LDH released from murine bone marrow-derived macrophages that were treated with increasing concentrations of IpaB-knot in its C-terminal region (Knot) (Fig. 1B)
We observed a similar dose-dependent release of LDH with IpaB-Knot as has been demonstrated for IpaB alone [25]. 50 mg/ml of recombinant fusion protein caused LDH release as efficiently as 1% detergent (Triton X-100). These results demonstrate that IpaB is still functional when fused to the Knot
Summary
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 [1][2]. One pathogen depending on T3SS-mediated virulence is Shigella flexneri, a human pathogen of the intestine that depends on effector protein delivery to establish an infection. In S. flexneri serovar 5a M90T, the T3SS is encoded on a 210 kb-extrachromosomal plasmid [5], where genes encoding the NC are clustered in distinct operons. Together with the invasion plasmid antigen (ipa)-operon, this region is referred to as the entry region which is necessary and sufficient for invasion of host cells [6][7]. The translocators IpaB and IpaD regulate secretion by forming a complex at the tip of the needle [8] and deletion of either ipaB or ipaD causes hypersecretion of effectors [9]
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