Shigella species cause diarrhea and dysentery in humans by infecting the colon. Pathogenesis depends on regulated interactions between the host and pathogen. Whereas identification and characterization of Shigella virulence proteins has advanced considerably, it has been more challenging to identify host proteins that participate in infection. During the current funding period, we defined the roles of several host proteins in S. flexneri pathogenesis and identified dozens of other host proteins in broad-based screens designed to discover host genes that promote S. flexneri infection. Our goal is to continue to build on these discoveries by conducting in depth investigations into how a two of these host proteins function in Shigella pathogenesis. 1. Analyze the topology, organization, and function of the translocon pore relevant to S. flexneri docking and effector translocation. We discovered that docking of the T3SS needle apparatus onto the membrane-embedded translocon pore depends on interaction of the pore protein IpaC with cellular intermediate filaments, yet pore formation per se is independent of this interaction. Based on these findings, we are now able to allow pore formation while preventing docking of bacteria, thereby enabling us to (1) map the topology, symmetry, and stoichioletry of the translocon pore proteins upon delivery by the T3SS, (2) define the relative positions of and interactions between the two translocon proteins in membrane-embedded helices, (3) test our postulate that C-terminal sequences in IpaC enter the interior of the pore, and (4) investigate how interaction of intermediate filaments with IpaC enables docking of the needle on the pore. 2. Determine the function of NLRP11 in responses of human macrophages to S. flexneri infection. Whereas some NLRP proteins are constituents of inflammasomes, NLRP11 has no known function. Our data show that NLRP11 is required for efficient cell death induced by S. flexneri infection and by cytosolic LPS (cLPS). cLPS triggers the activation of an inflammasome that contains caspases-4 and 5, for which no NLRP or other sensor molecule has been identified. Because LPS binds caspases-4 and 5 directly in vitro, it has been postulated that the cLPS inflammasome may not require an NLRP or sensor molecule. Based on our data, we hypothesize that NLRP11 functions as a sensor molecule for the cLPS inflammasome pathway. We will test this hypothesis using a combination of genetic and biochemical approaches to (1) determine the function of NLRP11 in macrophage responses to S. flexneri and cLPS, (2) determine whether acylation state of LPS, which modulates innate immune responses to S. flexneri, modulates NLRP11 recognition and/or signaling, and (3) define the breadth of human pathogens that trigger NLRP11-dependent responses.

Public Health Relevance

Bacteria that cause infections in humans exploit human cellular factors to promote disease. We have identified human factors that are used by Shigella, a bacterium that causes severe diarrhea, and propose to investigate how these factors contribute to disease. Understanding these processes will potentially reveal new avenues for treatments.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Mills, Melody
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Massachusetts General Hospital
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Miller, Kelly A; Garza-Mayers, Anna Cristina; Leung, Yiuka et al. (2018) Identification of interactions among host and bacterial proteins and evaluation of their role early during Shigella flexneri infection. Microbiology 164:540-550
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