B-290. Identification of Novel Inhibitors of the Pseudomonas aeruginosa Twin-Arginine- Translocase (TAT) Secretion System Using a High-Throughput Approach

A. P. Tomaras, A. E. Pritchard, M. L. Vasil;
Univ. of Colorado, Aurora, CO.

Some of the major virulence determinants of Pseudomonas aeruginosa are secreted via the TAT secretion system, which moves folded proteins across the inner membrane. A TAT-deficient mutant of P. aeruginosa is severely attenuated in a chronic pulmonary model of infection. These and other findings support the concept that TAT function is an appealing target for the development of novel therapeutic agents, which could mitigate its pathogenic potential by attenuating or abrogating TAT function. The increasing availability of extensive compound libraries now makes it more possible to conduct high-throughput screening on a routine basis. Accordingly, we initiated a high-throughput screen to identify inhibitors of TAT function. An assay, which exploits the TAT-dependent secretion of the hemolytic phospholipase C (PlcH), was designed for this purpose. Using a synthetic substrate to detect the PLC activity associated with PlcH, we were able to assess the functionality of the TAT system in the presence of various compounds by measuring a yellow nitrophenyl product that was released by the PLC secreted via TAT. High-throughput screening of ~84,000 compounds identified approximately 140 “hits”, which showed a significant reduction in the amount of PlcH activity detected. Secondary assays were performed to distinguish between those compounds that only inhibited PlcH enzyme activity and those that were more likely to affect TAT function. Additional assays were then employed to more directly assess TAT function. Other, PlcH independent, but TAT-dependent phenotypes were based on those displayed by a ΔTAT mutant of P. aeruginosa. Specifically, choline utilization and copper resistance are two distinctive phenotypes altered in a ΔTAT mutant. Follow-up screening of the initial 140 hits with these assays revealed that 40 displayed choline- and copper-specific phenotypic patterns similar to those exhibited by the mutant. Other approaches are currently being designed to determine whether any of these compounds, which have answered our secondary screens, will prove promising as efficient and specific inhibitors of TAT function.