Identifying Mechanisms of Resistance to Pathogen-Specific Antibacterial Antisense Compounds and Strategies to Circumvent Them

PROJECT SUMMARY/ABSTRACT The need for new antimicrobials is increasingly urgent. The rate of multidrug resistant pathogens continues to increase, leading to significant morbidity and mortality throughout the world. Furthermore, the current pipeline for new antimicrobials remains very narrow. The CDC has identified a number of Gram-negative pathogens as Urgent or Serious threats. A new paradigm in antibiotic discovery and design has recently been shown effective against numerous bacteria. This new approach is based on a platform technology called peptide- phosphorodiamidate morpholino oligomers (PPMOs). PPMOs are synthetic DNA mimics that bind to RNA in a sequence-specific, antisense manner and inhibit translation of target bacterial genes. PPMOs have already been used successfully to kill a variety of bacterial pathogens including the Gram-negative bacteria Pseudomonas aeruginosa, Salmonella typhimurium, Burkholderia cepacia complex (Bcc), Acinetobacter baumannii, Klebsiella pneumoniae and Escherichia coli. PPMOS are bactericidal in culture, and reduce bacteremia and improve survival in animal models of infection. In addition, PPMOs retain activity in both multidrug-resistant strains and biofilm settings. The mechanisms of resistance to PPMOs remains understudied. In addition, it is not clear whether common themes of resistance will be seen across many of these medically important pathogens. The goal of this project is to determine the mechanism of resistance for lead PPMOs in the pathogens Klebsiella pneumoniae, Acinetobacter baumannii and members of the Bcc. These representative members of different Proteobacteria classes will allow us to broadly study possible shared mechanisms of resistance. Utilizing species-specific lead PPMOs that target the same essential gene, mutants will be generated and characterized. Resistance circumventing strategies will be developed depending on the mechanism of resistance. The hypothesis is that drivers of resistance will be related to peptide- mediated entry or target gene sequence mutations and strategies to overcome these will be tested through new PPMO design and testing.