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dc.contributor.advisorSmith, Jason G
dc.contributor.authorHolly, Mayumi Kate
dc.date.accessioned2019-08-14T22:36:50Z
dc.date.submitted2019
dc.identifier.otherHolly_washington_0250E_20308.pdf
dc.identifier.urihttp://hdl.handle.net/1773/44392
dc.descriptionThesis (Ph.D.)--University of Washington, 2019
dc.description.abstractThe human gastrointestinal (GI) tract is a major site of host-pathogen interactions. As such, many human pathogens have evolved to either use the GI tract as a portal of entry to gain access to distal sites in the body or cause acute disease within the GI tract. For many years we lacked a suitable cell culture system for modeling the epithelial celluarlity of the human GI tract, thus our understanding of how the human intestinal epithelium responds to infection has been limited by previous models. Recently, a new system for culturing untransformed intestinal epithelial cells was developed, termed enteroids. Human enteroids are a powerful tool for understanding the complex interactions between human intestinal epithelial cells and enteric pathogens. We used human enteroids to investigate infection of intestinal epithelial cells by two important human pathogens, human adenovirus (HAdV) and Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium). Enteric HAdV are notoriously difficult to culture in vitro despite being excreted in high numbers from infected individuals. We found that both prototype strains and clinical isolates of enteric and nonenteric HAdVs productively replicate in human enteroids. Additionally, we show that HAdV-5p, a respiratory pathogen, and HAdV-41p, an enteric pathogen, are both sensitive to type I and III interferons in human enteroid monolayers but not A549 cells, a transformed cell line commonly employed in adenovirology. And, HAdV-5p but not HAdV-41p was potently neutralized by the enteric human alpha-defensin HD5. Unique to enteroids is the diversity of intestinal epithelial cell types found in the gut, thus enteroids can reveal novel aspects of HAdV tropism. Intriguingly, HAdV-5p, but not HAdV-41p, preferentially infected goblet cells. These studies highlight new facets of HAdV biology that are uniquely revealed by primary intestinal epithelial cell culture. To extend our studies on innate immune sensing in untransformed intestinal epithelial cells, we chose to study S. Typhimurium, another enteric pathogen. S. Typhimurium has been studied extensively in transformed cell lines, immune cells, and mice, but there is a dearth of knowledge on how S.Typhimurium infects and is sensed by human intestinal epithelial cells. We showed that S. Typhimurium elicits rapid and robust IL-18 secretion from untransformed intestinal epithelial cells compared to C2Bbe1 cells, a common transformed cell line derived from colorectal adenocarcinoma. Interestingly, IL-18 secretion is dependent on caspase-4, but not caspase-1 or caspase-5. Additionally, lack of caspase-4 in human enteroid monolayers resulted in increased intracellular colony forming units and increased numbers of intracellular bacteria per cell. These data indicate that caspase-4 is critical caspase for sensing and responding to S. Typhimurium infection, and plays a vital role in restricting intracellular bacterial numbers. Taken together, human enteroids uncovered novel and interesting aspects of enteric pathogen biology and intestinal epithelial innate immune responses.
dc.format.mimetypeapplication/pdf
dc.language.isoen_US
dc.rightsCC BY-NC-ND
dc.subjectAdenovirus
dc.subjectEnteroids
dc.subjectGut
dc.subjectInflammasome
dc.subjectSalmonella
dc.subjectMicrobiology
dc.subject.otherMicrobiology
dc.titleHuman enteroids as a model for infectious disease
dc.typeThesis
dc.embargo.termsDelay release for 1 year -- then make Open Access
dc.embargo.lift2020-08-13T22:36:50Z


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