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Title:Survival of Salmonella in the macrophage phagosome
Author(s):Fenlon, Luke Andrew
Director of Research:Slauch, James M
Doctoral Committee Chair(s):Slauch, James M
Doctoral Committee Member(s):Imlay, James; Cronan, John; Whitaker, Rachel
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Salmonella
Superoxide
SodC
Macrophage
Phagosome
TnSeq
Abstract:Salmonella Typhimurium is a gram-negative facultative anaerobe bacterium capable of causing life threatening disease in humans. Transmitted via the fecal-oral route, S. Typhimurium infection typically results in self-limiting gastroenteritis. However, in immunocompromised patients, Salmonella can invade the gut epithelial barrier and cause systemic infection resulting in septicemia and lethal infection. Key to establishment of systemic infection is the ability of Salmonella to survive intracellularly within host phagocytes, particularly macrophages. Salmonella requires an arsenal of virulence factors and defense mechanisms to survive the onslaught of host antimicrobial defenses that typically kill bacteria. One important antimicrobial defense is the oxidative burst, in which high levels of superoxide are generated within the Salmonella containing vacuole. SodCI, a periplasmic superoxide dismutase, is required to defend Salmonella against phagocytic superoxide. Work from our lab and others has established that SodCI functions to defend an extracytoplasmic target from the oxidative burst. The work presented herein aimed to provide additional insight to the target of phagocytic superoxide in S. Typhimurium. To this end I utilized differential TnSeq, a novel genome wide approach, to identify genetic interactions with sodCI. This involved generating over 40,000 random transposon mutants in a wild type and sodC background, passaging those mutants through an intraperitoneal mouse infection model, then sequencing the input and output populations. By comparing individual gene fitness between wild type and sodC, novel genetic interactions can be discovered. Thus far we have identified 3 genetic interactions. Of particular interest is YtfL. An inner membrane protein of unknown function, ytfL is epistatic to sodC and presumably a target of phagocytic superoxide. This experiment was complimented with RNA-Seq data from a sodC mutant and wild type during tissue culture macrophage infection. Surprisingly, results suggested that a sodC mutant has decreased expression of key virulence factors relative to wild type, implying a global alteration in the Salmonella-phagocyte environment. Additionally, I characterized the contribution of cytoplasmic copper export and the periplasmic copper chaperone, CueP, in activation of SodCI in vitro. While thisseemingly circuitous route of copper trafficking can contribute to activation of SodCI, it is not absolutely required. In addition to serving as a cofactor in enzymatic reactions, copper has been shown to be utilized by the host as an antimicrobial defense mechanism. I show that S. Typhimurium is fully virulent in the absence of cytoplasmic copper export in vivo, implying Salmonella surviving systemic infection do not encounter significant cytoplasmic copper stress.
Issue Date:2020-07-16
Type:Thesis
URI:http://hdl.handle.net/2142/108613
Rights Information:Copyright 2020 Luke Fenlon
Date Available in IDEALS:2020-10-07
Date Deposited:2020-08


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