Host factors in Shigella flexneri infection

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Project summary. Host interactions play critical roles during infection with bacterial pathogens. For many bacterial pathogens, type 3 secretion systems (T3SSs) mediate numerous interactions with the host. Host interactions, in turn, regulate the activation of the T3SSs. Once activated, T3SS effectors are delivered into host cells via pores in mammalian plasma membranes (translocons) that consist of two T3SS translocases. T3SS effectors modulate host cells in ways that promote infection. We found that engagement of host factors leads to specific conformational changes in the translocon that are required for activation of effector secretion. We found that residues in the host cytosolic domains of the bacterial translocases reside at least transiently within the pore channel. We found that Shigella OspB manipulates mTORC1, the mammalian central regulator of cell growth and proliferation, leading to OspB alteration of cell proliferation during Shigella infection. We propose to investigate the mechanisms by which cellular interactions with natively delivered plasma membrane-embedded Shigella flexneri translocons contribute to translocon activation prior to cell invasion, with the goal of uncovering mechanisms of pre-invasion roles of plasma-membrane embedded translocons, including the mechanisms of pore opening, activation of effector secretion, effector translocation, and membrane ruffling. Additionally, we propose to plan to leverage our recent findings to determine the mechanisms of OspB manipulation of mTORC1, specifically identifying mammalian substrate(s), characterizing the mechanism of mammalian substrate cleavage, and interrogating the mechanism by which OspB mammalian substrate(s) alter mTORC1 activity. Our overall goal is to determine how mammalian cell proteins and interactions modulate bacterial infection and pathogenesis, specifically the activation of the T3SS, and how T3SS effectors interact with and alter mammalian cells. Given the conservation of T3SSs and OspB among medically important bacterial pathogens, it is highly likely that our insights will have broad implications for host-pathogen interactions more generally, therefore be of considerable interest to the pathogenesis community.