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dc.contributor.advisor Stathopoulos, Christos en
dc.contributor.author Halsey, Taylor en
dc.date.accessioned 2019-02-08T23:29:10Z en
dc.date.available 2019-02-08T23:29:10Z en
dc.date.issued 2019-02-08 en
dc.identifier.uri http://hdl.handle.net/10211.3/207964 en
dc.description.abstract Autotransporter proteins, typically associated with virulence in pathogenic bacteria, are thought to serve a different function in probiotic bacteria. One such protein, secreted autotransporter toxin (Sat), has been shown in numerous pathogenic bacteria to cause vacuolization and cytotoxic effects on cells. Sat is also secreted in high amounts by the non-pathogenic, probiotic Escherichia coli Nissle 1917 (EcN). The use of EcN is under review as a candidate probiotic supplement in the United States and approval has been delayed in part because of genomic similarities to uropathogenic E. coli (UPEC) CFT073, which is responsible for approximately 70% of non-hospital acquired urinary tract infections nationwide. Previous generalized studies have indicated that when Sat functions collaboratively with other native proteins in EcN, its cytotoxic effects are negated, but the mechanisms behind these actions are unknown. To independently study the effects of Sat, we used a recombinant sat overexpression plasmid derived from UPEC CFT073 (pSat) transformed into the non-pathogenic E. coli strain HB101 (HB101 pSat). Concentrated cell-free supernatant was obtained from wildtype EcN and wildtype UPEC CFT073, as well as EcNDSat for assays and an SDS-PAGE and Bradford protein concentration assay were performed to confirm the expression, concentration, and secretion profiles. In vitro infection challenges using the HeLa and Vero cell lines were performed and analyzed using the MTT cytotoxicity assay. Our preliminary results showed that Sat of UPEC CFT073 decreased viability of undifferentiated epithelial cells when subjected to challenges with concentrated cell-free supernatant. When the same challenges were given to Vero cells, a greater level of toxicity was conferred on the differentiated epithelial cell line. Cytotoxic effects in Vero cells were amplified following crude purification of cell- iv free supernatant samples using ammonium sulfate precipitation. These results suggest that Sat of UPEC CFT073 confers damage to host epithelial cells and may have greater effects on relevant differentiated cell lines. It is also suggested that purified Sat protein from both EcN and UPEC CFT073 confer similar levels of cytotoxicity to Vero cells and reduce viability compared to the concentrated supernatant samples alone. Further experiments are needed to determine the mechanisms of action behind these results and target the specific factors that prevent Sat of EcN from displaying cytotoxic features in host cells. en
dc.description.statementofresponsibility by Taylor M. Halsey en
dc.format.extent 109 pgs. en
dc.language.iso en en
dc.publisher California State Polytechnic University, Pomona en
dc.rights.uri http://www.cpp.edu/~broncoscholar/rightsreserved.html en
dc.subject Microbiology, bacterial toxins, hela cells, vero cells, E. coli nissle 1917, probiotic en
dc.subject microbiology en
dc.subject bacterial toxins en
dc.subject hela cells en
dc.subject vero cells en
dc.subject E. coli nissle 1917 en
dc.subject probiotic en
dc.title Comparative Functional Analysis of Secreted Autotransporter Toxin (Sat) in Uropathogenic E. coli CFT073 and probiotic E. coli Nissle 1917 in mammalian cells en
dc.type Thesis en
dc.contributor.department Department of Biological Sciences en
dc.description.degree M.S. en
dc.contributor.committeeMember Buckley, Nancy en
dc.contributor.committeeMember Liu, Junjun en
dc.rights.license All rights reserved en

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