Abstract
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.
Highlights
Type III protein secretion systems (T3SSs) are highly specialized multiprotein molecular machines with the capacity to inject bacterially-encoded proteins into target eukaryotic cells
Type III secretion systems or injectisomes are large, multiprotein complexes composed of several substructures: the needle complex, a multiring structure with a protruding needle-like appendage anchored in the bacterial envelope; the export apparatus, a set of membrane proteins that form a gate in the inner-membrane for the passage of effector proteins; and the sorting platform, a large cytosolic complex that delivers the effectors to the needle complex in an orderly fashion
We find that in the absence of SpaOS, the sorting platform still forms and functions slightly less efficiently than in the wild-type situation, and we conclude that SpaOS is most likely not a central structural component of the sorting platform and may play a regulatory role during the cycles of assembly and disassembly that the sorting platform undergoes
Summary
Type III protein secretion systems (T3SSs) are highly specialized multiprotein molecular machines with the capacity to inject bacterially-encoded proteins into target eukaryotic cells. The type III secretion machine is made up of several substructures that come together to form the injectisome[1, 4,5,6,7].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 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[16]. 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
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