The ability of pathogenic bacteria to sense the changes in their environment and respond by expressing essential virulence is one of the key components of the infectious process. The long term objectives of the proposal are to examine the patterns of gene expression of two important human uropathogens-Escherichia coli and Pseudomonas aeruginosa-during various growth conditions, including growth in the urinary tract of infected patients. Specifically, a library will be prepared of cloned DNA from a uropathogenic E. coli strain containing sequences which are absent in the genomes of laboratory E. coli strains. Similarly, a genomic library of uropathogenic P. aeruginosa strain will be constructed. These libraries will be used to prepare filters containing high-density DNA arrays of clones, and they will be probed with cDNA probes derived from mRNAs isolated under various conditions that mimic the human urinary tract. Two such conditions will include growth in human urine, and bacterial attachment to human bladder epithelial cells in culture. In order to identify genes that are differentially expressed, the hybridization patterns of two different probes with the cloned DNA in arrays will be compared. RNA will be isolated from the pathogens propagated in urine or in the presence of primary bladder epithelial cells, and the same organisms grown in standard laboratory media. Probes from E. coli and P. aeruginosa, directly isolated in urine samples from patients with urinary tract infections will also be prepared, and these will be used for the analysis of gene expression during natural infections of humans. Genes of E. coli and P. aeruginosa, which are expressed in isolates from patients with urinary tract infections-or under conditions mimicking such infections-will be cloned and sequenced. The transcriptional start sites of these genes will be determined, in order to identify any urine-tract specific regulatory genes, which may be targets of transcriptional regulators. It is believed that the proposed research will lead to the identification of genes and regulatory circuits which are responsible for the expression of virulence factors in the human urinary tract infected by uropathogenic E. coli and P. aeruginosa.
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