Tachiyama Shoichi, Skaar Ryan, Chang Yunjie, Carroll Brittany L, Muthuramalingam Meenakumari, Whittier Sean K, Barta Michael L, Picking Wendy L, Liu Jun, Picking William D
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, United States.
Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, United States.
Front Cell Infect Microbiol. 2021 Jun 3;11:682635. doi: 10.3389/fcimb.2021.682635. eCollection 2021.
, 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 MxiK and connect to the Spa47 ATPase MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33). The structure of Spa33 is known, but the precise makeup and structure of the pods 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 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 and 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.
痢疾杆菌（志贺氏菌病的病原体）将III型分泌系统（T3SS）用作其主要毒力因子。T3SS注射体将效应蛋白输送到宿主细胞中以促进进入并创造一个重要的细胞内生态位。注射体的细胞质分选平台（SP）是一个关键组件，有助于底物选择和分泌能量供应。SP由寡聚的Spa33“荚”组成，这些“荚”与基体MxiK相关联并连接到Spa47 ATP酶MxiN。这些“荚”包含Spa33的异源三聚体，其中一个全长拷贝与两个C端结构域（Spa33）拷贝相关联。Spa33的结构是已知的，但“荚”的确切组成和结构仍然难以捉摸。我们在此表明，重组野生型Spa33可以制备成与MxiK和MxiN形成不同稳定复合物的异源三聚体。在双杂交分析中，Spa33复合物与这些蛋白质的结合发生在全长Spa33组件上。此外，这些复合物各自具有不同的生物物理特性。基于这些特性、新的高分辨率冷冻电子断层扫描数据以及Spa33与鞭毛FliM-FliN复合物之间的结构相似性，我们提供了SP“荚”内Spa33异源三聚体的初步模型。根据这些发现以及SP界面和T3SS中动力学的不断发展的模型，我们提出了一个SP功能模型，其中两种不同的复合物在SP的背景下聚集在一起，有助于在T3SS分泌底物募集过程中形成完整的“荚”结构。
