The objective of this research is to discover drugs that prevent infection caused by a variety of clinically and economically important disease causing bacteria. Recent work by Dr. David Low~s group has shown that the expression of fimbraie involved in the pathogenesis of urinary tract infections (UTIs) and diarrheal disease caused by a number of organisms is controlled by a methylation- dependent phase variation mechanism. This methylation-dependent regulatory mechanism is controlled by a number of proteins including deoxyadenosine methylase (Dam). Small perturbations in the levels of Dam abrogate the expression of Pap fimbraie (involved in UTI), and presumably, due to conservation in the regulation mechanism, the other methylation-regulated fimbraie. Dr. Low has developed an assay to measure Dam activity. We propose to modify this assay to utilize a Scintillation Proximity Assay (SPA) format in order to perform high- throughput screening of combinatorial compounds. We propose to screen 10,000 compounds to identify a compound that perturbs Dam activity. These compounds will be investigated for their ability to abrogate fimbraie expression. Compounds that affect Dam levels, and hence abrogate fimbraie expression by these pathogenic bacteria, will potentially cause these organisms to lose their ability to adhere to cells of the gut, ureter, and bladder. These non-adherent cells could then be flushed from these areas by normal clearance mechanisms.
Compounds identified by this work would be effective against UTI- causing E.coli. About 35% of the nosocomial infections are UTI~s and 25% of those are caused by E.coli. E.coli is the causative agent of about 75% of the community derived UTI~s and UTI causing E.coli are responsible for about 20% of community-acquired bacteremias. It is likely that an inhibitor of Dam would have broad spectrum activity. Dam is involved in normal bacterial replication and the Dam controlled phase variation mechanism used by uropathogenic E.coli is also used by other fimbrial expression systems associated with clinically and economically important pathogens.
