Dissertation
FUNCTIONAL CHARACTERIZATION OF CAMPYLOBACTER JEJUNI VIRULENCE RESPONSES TO HOST FACTORS USING IN VITRO AND IN VIVO MODELS
Doctor of Philosophy (PhD), Washington State University
01/2020
Handle:
https://hdl.handle.net/2376/111798
Abstract
Campylobacter jejuni is the most common bacterial cause of diarrhea worldwide. Human infection often results from consumption of raw poultry products, as C. jejuni is a frequent component of the avian intestinal microbiota. Despite its worldwide prevalence, there is not a deep mechanistic understanding of how C. jejuni survives antimicrobial host factors such as intestinal bile, promotes invasion into host epithelial cells, and causes inflammation in the intestine. Here, we found that growth of C. jejuni in deoxycholate, an intestinal bile component, results in a buildup of reactive oxygen species which causes oxidative DNA damage in the form of 8-Oxo-2’-deoxyguanosine and double-strand breaks. We also found that the AddAB DNA repair proteins were responsible for the repair of double-strand DNA breaks and facilitates colonization of the chicken cecum by C. jejuni.
An important aspect of C. jejuni pathogenesis in a human is the ability to invade intestinal epithelial cells. To uncover potential virulence factors, the C. jejuni proteome and transcriptome was determined during co-culture with two immortalized human epithelial cell lines, INT 407 and Caco-2. It was found that genes involved in chemotaxis (Cj0448c, cheBR, and cetAB) were dispensable for invasion, while proteins that regulate flagellar assembly (flhF, flaAB and flgL) were required for invasion. Studies were also conducted with the C. jejuni flagellar secreted effector protein CiaD, which is essential for bacterial invasion. It was found that CiaD promotes invasion by binding to the host cell protein IQGAP1 and displacing RacGAP1 from the IQGAP1 complex. This interaction results in increased Rac1 activity, leading to bacterial uptake.
Furthermore, the initial immune response to C. jejuni was studied in a novel porcine ligated intestinal loop model. We found that inflammatory markers including IL-8 and TNF-α appear in the intestinal lumen as early as 12 hours after infection and is accompanied by a marked influx of neutrophils. It was also found that a C. jejuni ∆ciaD mutant was attenuated in its ability to promote intestinal inflammation. In summary, these studies have defined important factors that C. jejuni utilizes to survive in the intestinal environment, promote bacterial uptake, and mediate inflammation during disease.
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Details
- Title
- FUNCTIONAL CHARACTERIZATION OF CAMPYLOBACTER JEJUNI VIRULENCE RESPONSES TO HOST FACTORS USING IN VITRO AND IN VIVO MODELS
- Creators
- Nicholas Negretti
- Contributors
- Michael E Konkel (Advisor)Lisa M Gloss (Committee Member)Jean Celli (Committee Member)Steven A Roberts (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- School of Molecular Biosciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 421
- Identifiers
- 99900581810501842
- Language
- English
- Resource Type
- Dissertation