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Peter Kirby Dept. of Microbiology

Resistance to Peptide - Phosphorodiamidate Morpholino Oligomers in E. Coli. Peter Kirby Dept. of Microbiology. Mentor: Bruce Geller Dept. of Microbiology Oregon State University. Modern Antibiotics and Bacteria. Antibiotics target proteins or macromolecular complexes

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Peter Kirby Dept. of Microbiology

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  1. Resistance to Peptide - PhosphorodiamidateMorpholinoOligomers in E. Coli Peter Kirby Dept. of Microbiology Mentor: Bruce Geller Dept. of Microbiology Oregon State University

  2. Modern Antibiotics and Bacteria • Antibiotics target proteins or macromolecular complexes • Bacteria can develop resistance to antibiotics • 70 percent of the bacteria that cause infections in hospitals are resistant to at least one of the drugs most commonly used for treatment • New antibiotics are needed to combat bacteria

  3. Phosphorodiamidate Morpholino Oligomers • New form of antibiotic • PMO’s are synthetic DNA compounds • Molecules contains: • Phosphorodiamidate backbone • Morpholine rings • 4 nucleotide bases

  4. PMO-Peptide Conjugates • PMO’s have a high molecular weight (MW) • Have trouble entering Gram-negative bacteria • Solution: PMO’s were attached to membrane-penetrating peptides

  5. PPMO Function • PPMO’s are sequence specific • PPMO’s hybridize to mRNA near AUG start codons • PPMO’s inhibit translation

  6. PPMO Action

  7. Our Research Project • Aim 1: Identify and characterize the mechanisms of resistance to PPMO’s by E. coli • Hypothesis: One mechanism of resistance to PPMO’s is caused by mutations in a transporter • Strategy: Make random mutation in E. coli, select for resistance to PPMO’s, and identify mutated gene(s)

  8. Framework • Electroporation: • Place bacteria between two electrodes and charging the electrodes with strong potential to open cell wall • Transposome: • DNA with ability to insert itself into a chromosomal DNA sequence • Contains a gene to code for kanamycin resistance • Result: • Inserting a transposome causes random mutations • Produces mutations in all genes in chromosomal DNA

  9. Methods • Electroporatekanamycin-marked transposomes into E. coli and grow in the presence of PPMO and kanamycin • Isolate chromosomal DNA from PPMO-resistant mutants • Clone and sequence DNA flanking transposome • BLAST E. coli genome to identify gene responsible for mutation and PPMO resistance

  10. Results • 12 resistant cells discovered • 3 strongest colonies selected

  11. Mutated Gene Isolation

  12. Mutated Genes • sbmA gene mutated in both 76.2 and 76.3 • Function: Predicted Transporter

  13. sbmA • sbmA: predicted ABC transporter located in inner membrane • ABC transporters: Transmembrane protein that uses Adenosine Triphosphate hydrolysis to translocate substances across the membrane

  14. Mutated Genes (cont) • ADCY (AdenylateCyclase) gene mutated in 76.10 • Function: cAMP dependent pathway

  15. AdenylateCyclase • AdenylateCylcase (ADCY) is essential to the cAMPdependent pathway • Catalyzes the conversion of ATP to cyclic adenosine monophosphate (cAMP) • Activates CAP binding sites • CAP binding site found 200bp from sbmA gene

  16. MIC’s in Mueller Hinton II and Brain Heart Infusion Broths

  17. Conclusion • We found two genes that influence E. coli’sresistance to PPMOs • sbmA – transporter • AdenylateCyclase – cAMP

  18. Acknowledgements • Bruce Geller • HHMI • URISC • Kevin Ahern

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