Shigella species cause shigellosis (bacillary dysentery) with high global morbidity and childhood mortality. Shigella uses a type III secretion system (T3SS) to deliver virulence proteins into host cells to promote pathogen entry as the first step in establishing infection. The T3S apparatus (T3SA) consists of: 1) an external needle and its tip complex for delivering translocator and effector proteins; 2) a basal body that spans the bacterial envelope; and 3) a poorly defined cytoplasmic sorting platform that energizes secretion and controls the temporal delivery of secretion substrates. We recently provided the most detailed structural analysis of the Shigella T3SA sorting platform to date, which has allowed us to generate a model for the placement of the Spa47 ATPase, its interaction with the basal body via Spa13, and its association with the other key soring platform components MxiN and Spa33. From the available data, we now hypothesize that the interactions occurring between these components are required for stabilizing the T3SA and that the dynamic nature of these interactions is pivotal for controlling secretion status. Such interactions should be identifiable through ultrastructural analysis of the T3SA in Shigella minicells and by biochemical/molecular analyses. To test our hypothesis, the specific aims of this investigation are 1) to identify the specific protein-protein interactions and biochemical properties of the sorting platform components to determine their roles in controlling type III secretion, 2) to determine the importance of structural changes within the Shigella T3SA sorting platform during different stages of secretion induction and in mutants, and 3) to test proposed models of Shigella type III secretion that implicate sorting platform interactions in activation of the T3SA.
These aims will allow us to demonstrate the physical requirements for Spa33 interaction with the cytoplasmic domain of MxiG and the sorting platform component MxiN. Such interactions are necessary for the assembly and stabilization (and thus function) of the T3SA sorting platform and the way these interactions influence the platforms structure as determined by cryo-electron tomography. In parallel, we will test two potentially complementary models by which the platform contributes mechanistically to type III secretion activation. T3SSs are essential virulence determinants for many important human pathogens, but our understanding of the mechanics of these nanomachines remains poor. We will use the Shigella T3SS as a model to uncover the link between secretion status and the role of the T3SA cytoplasmic complex in driving secretion. The interdisciplinary team assembled to complete this work will reveal the fundamental mechanisms by which type III secretion occurs and identify steps that might serve as future targets for anti-infective drugs.

Public Health Relevance

Shigellosis or bacillary dysentery remains a serious global public health problem, especially for children under the age of five. The ability for Shigella species to cause dysentery requires a functional type III secretion apparatus (T3SA) which delivers host-altering effector proteins into the cells of the large intestine. This research will explore the assembly of the cytoplasmic portion of the T3SA nanomachine by identifying key protein-protein interactions that contribute to T3SA assembly, stability and function. These findings will lay the foundation for future development of small molecule inhibitors that will help in the treatment of bacillary dysentery.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Bacterial Pathogenesis Study Section (BACP)
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Mills, Melody
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University of Kansas Lawrence
Schools of Pharmacy
United States
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Barta, Michael L; Tachiyama, Shoichi; Muthuramalingam, Meenakumari et al. (2018) Using disruptive insertional mutagenesis to identify the in situ structure-function landscape of the Shigella translocator protein IpaB. Protein Sci 27:1392-1406