Gram-negative enteric pathogens cause significant disease burden. To establish disease, many of these organisms translocate bacterial effector proteins into the host cell cytosol. Shigella flexneri, the focus of this proposal, is a major caus of diarrheal illness, resulting in 165 million cases and 1.1 million deaths globally each year. S. flexneri translocates effectors using an apparatus known as the type III secretion system (T3SS), which is highly conserved among bacterial pathogens. Loss of the T3SS prevents virulence of Shigella, Salmonella, Yersinia, Vibrio, and E. coli species, which demonstrates its importance for bacterial pathogenesis. Bacterial contact with the host cell surface activates the T3SS to deliver two different bacterial proteins to form the translocon pore in the host cell membrane. The translocon pore is required for subsequent T3SS-mediated translocation of effectors into the host cell cytosol. Bacterial contact with the host cell surface activates the T3SS, but the molecular mechanisms responsible for T3SS activation are unknown. To identify whether host factors contribute to T3SS activity, a genome-wide selection was performed. This selection identified a role for vimentin, an intermediate filament. Intermediate filaments are the least studied of the three major types of eukaryotic cytoskeletal filaments, and their role during bacterial pathogenesis is poorly understood. My preliminary data demonstrate that intermediate filaments interact with the C-terminus of the S. flexneri translocon pore protein IpaC. Intermediate filaments are not necessary for translocon pore formation, but are required for both efficient S. flexneri docking to host cells and for efficient bacterial effector translocation by T3SSs. The observation that translocon pore formation occurs without subsequent bacterial effector translocation in cells lacking intermediate filaments shows these processes can be dissociated, which allows a unique opportunity to study how bacterial docking impacts T3SS-mediated effector delivery. My overall hypothesis is that the interaction between intermediate filaments and IpaC alters the association of S. flexneri with the host cell so as to promote translocation of effectors. I propose to test aspects of this hypothesis with the following aims: I) To assess the impact of IpaC interaction with intermediate filaments on downstream processes in T3SS effector translocation. (II) To characterize the effect of intermediate filament interactions with IpaC on processes associated with S. flexneri docking. This proposal will provide mechanistic insights into how intermediate filaments contribute both to docking by S. flexneri to host cells and translocation of effectors by its T3SS. Due to the highly conserved nature of the T3SS and its near ubiquitous requirement for the virulence of gram-negative enteric pathogens, the work proposed here is highly likely to have a broad impact on our understanding of bacterial pathogenesis.

Public Health Relevance

Many bacterial pathogens deliver proteins into human cells using specialized bacterial machinery. These bacterial proteins coopt host signaling pathways allowing the bacteria to subvert host responses and cause disease. This proposal aims to define the role of host factors in bacterial protein delivery and to use these insights to gain an understanding of how bacterial protein delivery occurs. Due to the near ubiquitous requirement of bacterial protein delivery to establish disease, this proposal is highly likely to elucidate general mechanisms in bacterial pathogenesis.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F13-C (20)L)
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Mills, Melody
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Massachusetts General Hospital
Independent Hospitals
United States
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Russo, Brian C; Stamm, Luisa M; Raaben, Matthijs et al. (2016) Intermediate filaments enable pathogen docking to trigger type 3 effector translocation. Nat Microbiol 1:16025