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Title:Mechanisms of signal integration: Regulation of the SPI1 T3SS
Author(s):Palmer, Alexander Disterhoft
Director of Research:Slauch, James M
Doctoral Committee Chair(s):Slauch, James M
Doctoral Committee Member(s):Imlay, James A; Vanderpool, Carin K; Kehl-Fie, Thomas E.
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Salmonella, SPI1 T3SS, regulation
Abstract:Salmonella must rapidly adapt to various niches in the host during infection. Relevant virulence factors must be appropriately induced, and systems that are detrimental in a particular environment must be turned off. Salmonella infects intestinal epithelial cells using a type 3 secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). The system is controlled by three AraC-like regulators HilD, HilC, and RtsA, which form a complex feed forward loop to activate expression of hilA, encoding the main transcriptional regulator of T3SS structural genes. This system is tightly regulated, with many of the activating signals acting at the level of hilD translation or HilD protein activity. Once inside the phagosome of epithelial cells, or in macrophages during systemic stages of disease, the SPI1 T3SS is no longer required or expressed. Here we show that the PhoPQ two-component system, critical for intracellular survival, appears to be the primary mechanism by which Salmonella shuts down the SPI1 T3SS. PhoP negatively regulates hilA through multiple distinct mechanisms: direct transcriptional repression of the hilA promoter, indirect transcriptional repression of both the hilD and rtsA promoters, and activation of the sRNA PinT. Genetic analyses and electrophoretic mobility shift assays suggest that PhoP specifically binds the hilA promoter to block binding of activators HilD, HilC, and RtsA as a mechanism of repression. Another signal that integrates into SPI1 regulation is deletion of the -barrel assembly machinery component bamB, but mechanistically how this occurs was unknown. We show that encumbered -barrel assembly activates the regulator of capsule synthesis (Rcs), a system that responds to a wide range of outer membrane insults, and represses SPI1. Though Rcs has been previously shown to repress SPI1 when sensing disulfide bond status, we show that the mechanism of sensing is distinct. Repression in a bamB background is dependent on the canonical sensor protein RcsF, whereas disulfide bond status is sensed independently of RcsF. Activation of Rcs also decreases transcription of both the hilD and hilC promoters. Motility assays demonstrated that dsbA and bamB mutants both had motility defects. Deletion of Rcs only restored motility in the bamB background, demonstrating the effect is simply regulatory. This demonstrates how Salmonella takes a global approach to regulation of the SPI1 T3SS, sensing levels of outer membrane stress before activating invasion, and emphasizes the sensitivity of SPI1 regulation. Because HilD protein activity is a key integration point for SPI1 regulation, we performed a screen for proteins that interact with HilD. By fusing HilD to a promiscuous biotin protein ligase, we were able to affinity-purify biotinylated proximal proteins and identify them via mass spectrometry. We elucidated the HilD interactome, and identified three genes that are important for SPI1 activation. MreB, a cytoskeletal protein, and SeqA, a cell division regulatory protein both affected SPI1 expression. UbiK, a ubiquinone biosynthesis factor was identified and appears to regulate SPI1 in a HilD dependent manner. UbiK could interact directly with HilD to sense and respond to ubiquinone biosynthesis to regulate SPI1. Together, we have deepened our understanding of how signals can integrate to regulate SPI1, and identified new proteins that integrate into SPI1 regulation, possibly through direct interaction with HilD.
Issue Date:2020-05-07
Type:Thesis
URI:http://hdl.handle.net/2142/108323
Rights Information:Copyright 2020 Alexander Palmer
Date Available in IDEALS:2020-08-27
Date Deposited:2020-05


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