Date Published: January 22, 2019
Publisher: Public Library of Science
Author(s): Maria Lara-Tejero, Zhuan Qin, Bo Hu, Carmen Butan, Jun Liu, Jorge E. Galán, Kelly T. Hughes.
Many bacterial pathogens and symbionts use type III secretion machines to interact with their hosts by injecting bacterial effector proteins into host target cells. A central component of this complex machine is the cytoplasmic sorting platform, which orchestrates the engagement and preparation of type III secreted proteins for their delivery to the needle complex, the substructure of the type III secretion system that mediates their passage through the bacterial envelope. The sorting platform is thought to be a dynamic structure whose components alternate between assembled and disassembled states. However, how this dynamic behavior is controlled is not understood. In S. Typhimurium a core component of the sorting platform is SpaO, which is synthesized in two tandemly translated products, a full length (SpaOL) and a short form (SpaOS) composed of the C-terminal 101 amino acids. Here we show that in the absence of SpaOS the assembly of the needle substructure of the needle complex, which requires a functional sorting platform, can still occur although with reduced efficiency. Consistent with this observation, in the absence of SpaOS secretion of effectors proteins, which requires a fully assembled injectisome, is only slightly compromised. In the absence of SpaOS we detect a significant number of fully assembled needle complexes that are not associated with fully assembled sorting platforms. We also find that although binding of SpaOL to SpaOS can be detected in the absence of other components of the sorting platform, this interaction is not detected in the context of a fully assembled sorting platform suggesting that SpaOS may not be a core structural component of the sorting platform. Consistent with this observation we find that SpaOS and OrgB, a component of the sorting platform, share the same binding surface on SpaOL. We conclude that SpaOS regulates the assembly of the sorting platform during type III secretion.
Type III protein secretion systems (T3SSs) are highly specialized multiprotein molecular machines with the capacity to inject bacterially-encoded proteins into target eukaryotic cells. Encoded by a large variety of gram-negative bacteria, T3SSs are central to the interactions of many pathogens and symbionts with their respective hosts[1–3]. The type III secretion machine is made up of several substructures that come together to form the injectisome[1, 4–7].The core component of the injectisome is the needle complex (NC), which is composed of a multi-ring base anchored in the bacterial envelope, and a filament-like extension that protrudes several nanometers from the bacterial surface[4, 6, 8, 9]. The needle filament is traversed by a narrow, ~2 nm channel and is capped at its terminal end by the tip complex, which is thought to be involved in sensing target cells and deploying the translocation pore that mediates the passage of effectors through the target cell plasma membrane[10–15]. The NC is associated to a very large cytoplasmic complex known as the sorting platform, which is responsible for selecting the type III secretion substrates and initiating them into the secretion pathway in the appropriate order. Recent cryo electron tomography (cryo-ET) studies in Salmonella Typhimurium and Shigella flexneri have provided a high-resolution view of this substructure of the injectisome[5, 17]. The sorting platform exhibits a unique cage-like architecture, enclosed by 6 pods that emerge from the NC and converge into a 6-spoke wheel-like structure that caps it at its cytoplasmic side. In the S. Typhimurium T3SS encoded within its pathogenicity island 1 (SPI-1), the cage-like structure is made up of the OrgA, SpaO, and OrgB proteins, which serve as scaffold to place the associated ATPase InvC in close apposition to the export apparatus. The ATPase plays an essential role in initiating substrates in the secretion pathway by removing their associated chaperones and unfolding the effectors prior to their threading through the narrow secretion channel.
The sorting platform is an essential substructure of the T3SS injectisome, which is critical for the recruitment and sorting of protein substrates destined to travel the type III secretion pathway. Recent cryo-ET studies have shown, in S. Typhimurium, that the sorting platform is made of a multi-protein cage-like scaffold, integrated by OrgA, SpaO, and OrgB, which serves as support for another component of this substructure, the ATPase InvC, which is involved in the initiation of substrates into the secretion pathway. Unlike other substructures of the T3SS injectisome, the sorting platform is thought to exhibit a behavior that may involve cycles of assembly and disassembly[19, 20]. However, very little information is available about the mechanisms and functional significance of this dynamic behavior. Furthermore, little is known about the mechanisms of assembly of this critical component of the T3SS machine. In this paper we have used a multidisciplinary approach to gain insight into the role of SpaO in the assembly of the sorting platform.