Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System

Abstract

Shigella flexneri, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisome’s cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 “pods” that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.

Distinct cytosolic complexes containing the type III secretion system ATPase resolved by 3D single-molecule tracking in liveYersinia enterocolitica

“…The D ~ 1.3 µm 2 /s diffusive state is consistent with our previous result obtained with PAmCherry-YeSctL, based on which we 10 concluded that both YeSctQ and YeSctL diffuse at the same slow rate as part of the same complex (18). The SctLQ interaction is also supported by a number of previous studies (17,19,20). On the other hand, the D ~ 3.1 µm 2 /s YeSctL diffusive state has not been previously observed.…”

“…Subsequent work has focused on reconstituting such pod complexes in vitro . Se SctNLQ(19) and Sf SctLQK complexes(20) were successfully isolated, and the estimated size and shapes of these complexes were qualitatively consistent with cryo-ET pod densities. Based on these results, we speculate that the D ~ 1.3 μm 2 /s diffusive state we observe in wild-type cells and in Δ sctD mutants is likely not just a SctNLQ complex, but in fact a SctNLQK heterooligomer.…”

“…12 ). In vitro light-scattering measurements on Sf SctQ-containing complexes by Tachiyama et al 21 suggested the ability of Sf SctQ to adopt different conformational states depending on its interacting partner. A small, globular complex (hydrodynamic diameter = 10.2 nm) was observed upon co-expression and co-purification of Sf SctQ and K, while a larger complex (hydrodynamic diameter = 13.4 nm) was observed for co-purified Sf SctQ and Q C .…”

“…Importantly however, a Se SctL2: Se SctN heterotrimer was consistently observed in any complex that included these two proteins (19). Dynamic light scattering was used to determine the relative size of cytosolic injectisome proteins and their respective complexes (20). Key differences were observed for reconstituted complexes containing Sf SctK and S’f SctQ and complexes containing Sf SctQ and Sf SctL ( Sf prefix indicates Shigella flexneri ).…”

Assembly and architecture of the type III secretion sorting platform

“…6C). Overall, this result indicates that one molecule of SpaO per pod is able to simultaneously link the OrgA docking platform with the OrgB cradle suggesting a topological arrangement for the sorting platform pods (Fig 6D ), which differs from a previously proposed model 47 .…”