Key virulence determinants of frank and opportunistic bacterial pathogens are secreted via the Twin Arginine Translocase (TAT) secretion system encoded by only three genes (i.e. tatABC). TAT-deficient mutants of Pseudomonas aeruginosa, Yersinia pseudotuberculosis and E. coli O157:O7 are significantly attenuated in relevant models of infection. We have now also shown (see data in this application) that the tatABC genes of Burkholderia pseudomallei functionally substitute for those of a AtatABC P. aeruginosa mutant, and that Burkholderia thailandensis TAT mutants are attenuated in their growth and survival in macrophage. These findings support the concept that TAT is an appealing target for the development of novel therapeutic agents, to diminish the pathogenic potential of 8. pseudomallei by interfering with TAT function. The increased availability of extensive compound libraries now makes it possible to conduct highthroughput screens on a more routine basis. Accordingly, we performed such a screen to identify inhibitors of TAT function. An assay, which exploits the TAT-dependent secretion of extracellular phospholipases (PLC), was designed for this purpose. High-throughput screening of -83,986 compounds identified 147 """"""""hits"""""""", which showed a significant reduction in the amount of PLC activity detected. Secondary assays were performed to distinguish between those compounds that only inhibited PLC activity and those that inhibited secretion of PLC via TAT. Additional phenotypic assays were then employed to more directly assess TAT function in the presence of the inhibitor. Follow-up screening of the initial 147 """"""""hits"""""""" with more specific assays of TAT function revealed that 39 of these induced phenotypic patterns most reflective of those seen in a TAT deletion mutant. Other approaches (e.g. over expression of TAT, cell fractionation) are currently being evaluated to determine whether any of these compounds will prove to be efficient and specific inhibitors of TAT to mitigate the pathogenic potential of B. pseudomallei. We are also evaluating the inhibitory effects of pools of 6-mer synthetic peptides that may compete with a consensus peptide in the signal sequence of TAT secreted proteins. Each amino acid is being substituted with 20 others (including ornithine) for each round of screening. We have already screened two pools of these synthetic peptides and found that the four with the highest inhibitory activity are WF, WN, WNIe, and WW in the first two positions of the consensus sequence. We will ultimately identify the most potent 6-mer peptides in terms of inhibiting TAT function. The small molecular weight compounds and the peptides from these experiments will be evaluated for their ability to inhibit the growth, trafficking and survival of B. pseudomallei in eukaryotic cells. This research project fits within the RMRCE Integrated Research Focus on Bacterial Therapeutics, and will interact directly with RP 2.1, 2.6 and will utilize the resources of the Core D (Product Development and Manufacturing Core) and Core E (Genomics, Proteomics Core).
The development new agents for the treatment of infections has significantly trailed behind the rapid expansion of resistant pathogens. This is also exacerbated by the failure to identify novel antimicrobial targets during the past two decades. Accordingly, we have identified a worthy target in the Select Agent Burkholderia pseudomallei and, we are identifying and characterizing specific inhibitors directed at this target to ultimately use as antimicrobial agents in the treatment B. pseudomallei infections (e.g. melioidosis).
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