Publication Date

2013

Document Type

Thesis

Committee Members

David Cool (Committee Member), James Lucot (Committee Member), Oleg Paliy (Advisor)

Degree Name

Master of Science (MS)

Abstract

Uropathogenic Escherichia coli (UPEC) are the primary cause of 80 to 90% of uncomplicated urinary tract infections. After entering the urinary tract, uropathogenic E.coli has to tolerate high levels of salt and urea in urine to cause a successful infection. The osmotic stress imposed by urea is different from that by NaCl, as urea can freely move across the cell membrane and is a protein denaturant. Hence, microarray experiments were performed to observe the differential expression of genes in CFT073 (UPEC) due to the presence of 0.3 M NaCl and 0.6 M urea in K medium individually. Based on the results, Bax (a hypothetical protein), IbpAB (small heat shock chaperone proteins) and CspH (speculated to be DNA/RNA chaperone protein) were chosen for the current study in which their role in protecting CFT073 against salt or urea stress was investigated. First bax, ibpAB and cspH gene sequences were shown to be well conserved among commensal E.coli MG1655 and 3 different UPEC strains CFT073, UTI89 and E.coli 536. Then GFP transcriptional fusion plasmids were constructed for bax, ibpAB and cspH genes and promoter activity assays were performed in K medium. Results showed that in CFT073, expression of bax was induced significantly in the presence of 0.2 M and 0.3 M NaCl, and the expression of ibpAB and cspH genes were induced in the presence of 0.3 M and 0.6 M urea in K medium, though the induction in cspH expression needs further confirmation. It was also shown that RpoS sigma factor does not regulate the expression of these genes during high levels of salt or urea in K medium. To study the importance of each gene in protecting CFT073 and BW25113 (K-12 strain) against salt or urea stress, growth curve experiments were performed using bax, ibpAB and cspH knockouts of each strain. Results showed that bax deletion reduced the salt tolerance of BW25113 but had no effect on the salt tolerance of CFT073. Deletion of ibpAB operon did not affect the urea tolerance of CFT073 and BW25113. However cspH null mutant of BW25113 showed reduced growth rate in the presence of 0.9 M urea in M9 medium compared to its wild type strain, and further experiments are needed to determine if cspH knockout would produce a similar effect in CFT073. Based on the current study, it can be concluded that even though the expression of bax and ibpAB genes are induced during salt and urea stress respectively, these genes are dispensable for CFT073 to tolerate high levels of salt or urea in K medium and M9 medium.

Page Count

93

Department or Program

Department of Pharmacology and Toxicology

Year Degree Awarded

2013

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