Tachiyama, ShoichiSkaar, RyanChang, YunjieCarroll, Brittany L.Muthuramalingam, MeenakumariWhittier, Sean K.Barta, Michael L.Picking, Wendy L.Liu, JunPicking, William D.2021-12-102021-12-102021-06-03Tachiyama S, Skaar R, Chang Y, Carroll BL, Muthuramalingam M, Whittier SK, Barta ML, Picking WL, Liu J and Picking WD (2021) Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System. Front. Cell. Infect. Microbiol. 11:682635. doi: 10.3389/fcimb.2021.682635https://hdl.handle.net/1808/32282Shigella 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.© 2021 Tachiyama, Skaar, Chang, Carroll, Muthuramalingam, Whittier, Barta, Picking, Liu and Picking. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).http://creativecommons.org/licenses/by/4.0/Type III secretion systemInjectisomeSpa33MxiNMxiKSorting platformShigella flexneriArticle10.3389/fcimb.2021.682635PMC8211105openAccess