Pseudomonas aeruginosa bronchitis and pneumonia represent a major cause of morbidity and mortality in patients with cystic fibrosis (CF). The incidence of drug resistant strains is on the rise making effective treatment of P. aeruginosa infection increasingly difficult and has created a need for new therapeutic approaches. This application proposes to investigate the use of novel synthetic oligonucleotides as antimicrobial agents for P. aeruginosa and assess its effect in a model of airway epithelial attachment. The challenge is to develop a strategy to target P. aeruginosa without any impairment to the host airway epithelial cells. The oligonucleotide target is the 3'-end of the 16S ribosomal RNA (rRNA) of P. aeruginosa which contains the anti-Shine-Dalgarno sequence. This sequence is not present in the analogous human 18S rRNA suggesting a pathogen specific therapeutic strategy. The 3'-end of the 16S rRNA containing the anti-Shine-Dalgarno sequence serves as a messenger RNA (mRNA) ribosomal binding site for subsequent translation. The oligonucleotide is designed to bind to the 3'-end of the rRNA and act as a competitive inhibitor for protein synthesis in P. aeruginosa. Preliminary data demonstrate that an oligonucleotide coupled with a covalently linked 5'-alanine is readily transported into P. aeruginosa resulting in a significant reduction in bacterial growth. Other amino acids may improve transport as well. Further, phosphorothioate oligonucleotides are used to greatly increase the nuclease resistance. Other backbone modifications such as phosphorodithioate or methylphophonate oligonucleotides may be more nuclease resistant. Finally, preliminary data demonstrate that the competitive oligonucleotide dramatically reduces attachment to host epithelial cells. Therefore, the following hypothesis will be tested: Synthetic oligonucleotides directed against the anti-Shine-Dalgarno sequence slows growth and decreases attachment of P. aeruginosa to lung epithelial cells. This hypothesis will be addressed by the following Specific Aims: 1) To determine if synthetic oligonucleotides are biostatic for P. aeruginosa; 2) To determine the stability and transport kinetics of synthetic oligonucleotides in P. aeruginosa; 3) To determine the effect of synthetic oligonucleotides on P. aeruginosa cell-free translation systems; 4) To determine if synthetic oligonucleotides can inhibit P. aeruginosa attachment to lung epithelial cells; and 5) to investigate possible resistant mechanisms of P. aeruginosa to synthetic oligonucleotides. If successful, completion of these Specific Aims will result in the development of an oligonucleotide-based therapy for P. aeruginosa, which may become a basically new therapeutic approach to this debilitating infection.
