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

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

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

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

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

PPMO Action

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)

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

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

Results  12 resistant cells discovered  3 strongest colonies selected MIC in Luria-Bertani Broth Antibiotic RXR4-AcpP ColonyMIC (μM) MIC in Luria-Bertani Broth Antibiotic RXR4-AcpP ParentMIC (μM) XL1-Blue20

Mutated Gene Isolation

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

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

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

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

MIC’s in Mueller Hinton II and Brain Heart Infusion Broths MHII Broth AntibioticRXR4-AcpP ColonyMIC (μM) XL1-Blue1.25 BHI Broth AntibioticRXR4-AcpP ColonyMIC (μM) XL1-Blue5

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

Acknowledgements  Bruce Geller  HHMI  URISC  Kevin Ahern