Role of <i>bax</i>, <i>ibpA</i>, <i>ibpB</i> and <i>cspH</i> Genes in Protecting CFT073 (Uropathogenic <i>Escherichia coli</I>) Against Salt and Urea Stress

Uropathogenic <i>Escherichia coli</i> (UPEC) are the primary cause of 80 to 90% of uncomplicated urinary tract infections. After entering the urinary tract, uropathogenic <i>E.coli</i> 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 <i>bax</i>, <i>ibpAB</i> and <i>cspH</i> gene sequences were shown to be well conserved among commensal <i>E.coli</i> MG1655 and 3 different UPEC strains CFT073, UTI89 and <i>E.coli</i> 536. Then GFP transcriptional fusion plasmids were constructed for <i>bax</i>, <i>ibpAB</i> and <i>cspH</i> genes and promoter activity assays were performed in K medium. Results showed that in CFT073, expression of <i>bax</i> was induced significantly in the presence of 0.2 M and 0.3 M NaCl, and the expression of <i>ibpAB</i> and <i>cspH</i> genes were induced in the presence of 0.3 M and 0.6 M urea in K medium, though the induction in <i>cspH</i> 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 <i>bax</i>, <i>ibpAB</i> and <i>cspH</i> knockouts of each strain. Results showed that <i>bax</i> deletion reduced the salt tolerance of BW25113 but had no effect on the salt tolerance of CFT073. Deletion of <i>ibpAB</i> operon did not affect the urea tolerance of CFT073 and BW25113. However <i>cspH</i> 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 <i>cspH</i> knockout would produce a similar effect in CFT073. Based on the current study, it can be concluded that even though the expression of <i>bax</i> and <i>ibpAB</i> 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.

Name thesis:degree_name

Master of Science (MS)

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Pharmacology and Toxicology

Grantor dc:publisher

Wright State University

Year dc:date

2013

Author dc:creator

• Beesetty, Pavani

Contributors dc:contributor

• Paliy, Oleg

• Microbiology
• Bax
• ibpAB
• cspH
• CFT073
• salt tolerance
• urea tolerance

Statement dc:rights

• unrestricted
• This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Language dc:language

English

Repository record dc:identifier

http://rave.ohiolink.edu/etdc/view?acc_num=wright1364042868

OAI identifier oai:identifier

oai:etd.ohiolink.edu:wright1364042868