1. Peter Kirby Dept. of Microbiology Resistance to Peptide - Phosphorodiamidate Morpholino Oligomers in E. Coli 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

5. 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

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

7. PPMO Action

8. 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)

9. 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

10. Methods  Electroporate kanamycin-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

11. Results  12 resistant cells discovered  3 strongest colonies selected MIC in Luria-Bertani Broth Antibiotic RXR4-AcpP 06-0076 ColonyMIC (μM) 76.140 76.280 76.380 76.480 76.580 76.680 76.780 76.840 76.940 76.1080 76.1140 76.1240 MIC in Luria-Bertani Broth Antibiotic RXR4-AcpP 06-0076 ParentMIC (μM) XL1-Blue20

12. Mutated Gene Isolation

13. Mutated Genes  sbmA gene mutated in both 76.2 and 76.3 Function: Predicted Transporter

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

15. Mutated Genes (cont)  ADCY (Adenylate Cyclase) gene mutated in 76.10 Function: cAMP dependent pathway

16. Adenylate Cyclase  Adenylate Cylcase (ADCY) is essential to the cAMP dependent pathway Catalyzes the conversion of ATP to cyclic adenosine monophosphate (cAMP) ○ Activates CAP binding sites  CAP binding site found 200bp from sbmA gene

17. MIC’s in Mueller Hinton II and Brain Heart Infusion Broths MHII Broth AntibioticRXR4-AcpP 06-0076 ColonyMIC (μM) 76.210 76.310 76.10-- XL1-Blue1.25 BHI Broth AntibioticRXR4-AcpP 06-0076 ColonyMIC (μM) 76.220 76.320 76.1020 XL1-Blue5

18. Conclusion  We found two genes that influence E. coli’s resistance to PPMOs sbmA – transporter Adenylate Cyclase – cAMP

19. Acknowledgements  Bruce Geller  HHMI  URISC  Kevin Ahern