Steps for Shigella Gatekeeper Protein MxiC Function in Hierarchical Type III Secretion Regulation

• Published in: The Journal of biological chemistry Vol. 292; no. 5; pp. 1705 - 1723
• Main Authors: Roehrich, A. Dorothea, Bordignon, Enrica, Mode, Selma, Shen, Da-Kang, Liu, Xia, Pain, Maria, Murillo, Isabel, Martinez-Argudo, Isabel, Sessions, Richard B., Blocker, Ariel J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.02.2017; American Society for Biochemistry and Molecular Biology
• Subjects: Antigens, Bacterial - genetics; Antigens, Bacterial - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Secretion Systems - genetics; Bacterial Secretion Systems - metabolism; biophysics; cellular regulation; electron paramagnetic resonance (EPR); Gram-negative bacteria; Microbiology; molecular genetics; Mutation; MxiC; protein secretion; Shigella; Shigella flexneri - genetics; Shigella flexneri - metabolism; type III secretion system (T3SS); molecular genetics; MxiC; type III secretion system (T3SS); cellular regulation; Gram-negative bacteria; electron paramagnetic resonance (EPR); biophysics; protein secretion; Shigella
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.746826

Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.