1. Identifying Genes in E. coli Required for Susceptibility to Antisense Antibiotics Susan Puckett Mentor: Dr. Bruce Geller AVI BioPharma Howard Hughes Medical Institute

2. Antibiotics Today  Race against antibiotic  resistance  MRSA  MDR & XDR Tuberculosis  According to the CDC, “more than 70% of the bacteria that cause hospital-acquired infections are resistant to at least one of the drugs most commonly used to treat them.” (http://www.cdc.gov/ncidod/dhqp/ar.html)

3. DNA mimics as antibiotics  Phosphorodiamidate morpholino oligomers (PMOs)  Antisense mechanism  Disrupts protein synthesis (translation) by blocking ribosome  Man-made, can be targeted GTGATAGCTTC AUGAGCACUAUCGAAGA RNA PMO http://www.stat.stanford.edu/~susan/courses /s166/node2.html

4. Getting PMOs into the cell  Naked PMO ineffective  Cationic peptides  Mechanism of PMO entry unknown  Last year work:  Found mutants resistant to peptide-PMOs  Resistance was linked to peptide and not to PMO PMO peptide

5. Questions  What happens to the PMO once it gets into the cell?  broken down?  How does the PMO get in?  transporter?  Mutants: what is mutating?  Are there genes that code for proteins necessary for the PMOs to be effective? What are these genes?

6. Escherichia coli strain AS19  E. coli lab strains good for experiments  AS19: permeable outer membrane  Naked PMOs (without peptides) can get in http://www.conceptdraw.com/en/sampletour/medical/ Gram-Negative Envelope

7. AS19 mutations  AS19 was plated on agar plate containing enough naked PMO to prevent growth  However, after 24 hour incubation there were several colonies growing

8. AS19 mutant testing  Vancomycin  Revertant test antibiotic.  Picked 10 mutants that were susceptible.  Rifampin, erythromycin  100 x Resistant to AcpP PMO  Mutants also resistant to FtsZ and GyrA PMOs

9. Library Experiment  Making competent cells of mutants and introducing an E. coli library  Library: plasmids containing different pieces of the genome  One plasmid per competent cell  Hypothesis: one plasmid will contain gene that has mutated and that this gene will cause the PMO to once again become effective  After 40 plate sets, no susceptible strains found

10. Conclusions  AS19 mutants resistant to naked AcpP PMO have been found  Mutants picked are not revertants back to the non-leaky E. coli strain  Mutations have not been in the target region of the PMO  Library experiment did not result in finding any susceptible strains

11. Gene Knockout Experiment with W3110  Use phage to insert transposon to knock out gene  Hypothesis: A gene or genes is (are) necessary for PMO efficacy & can be knocked out to produce a PMO- resistant phenotype Lambda phage http://www.steve.gb.com/science/model_organisms.html

12. Gene Knockout Steps  Insert Tn5 transposon randomly in W3110 genome through phage infection  Plate on kanamycin/(RFF)3R-AcpP PMO  Purify DNA from colony of interest  Digest genomic DNA and pUC19 DNA with KpnI, ligate  Use competent cells, plate on kanamycin KpnI

13. Results  2 colonies, named pUCTn5-1 and pUCTn5-2  Purify plasmid DNA, select primers that would surround insert  Sequence into insert about 500 nucleotides  BLAST search comparing to W3110 genome http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=Retrieve&dopt=Overview&list_uids=16351 Tn5 insert was found to be here

14. Conclusions  AS19 was more difficult to experiment with despite the PMOs not needing peptides and gene for PMO susceptibility was not identified  Gene knockout experiment: one gene necessary for the (RFF)3R-AcpP PMO to be effective is or is near the yehL gene in the E. coli W3110 genome

15. What does this mean?  Gene function in region might suggest mechanism of susceptibility  This could show how the PMO is getting into the cell, how the PMO is degraded, or other mechanisms to inactivate the PMO  Peptide-PMOs could then be designed with this new knowledge in mind

16. Acknowledgements  Howard Hughes Medical Institute  URISC  AVI BioPharma  Oregon State University  Dr. Kevin Ahern  Dr. Bruce Geller  Brett Mellbye  Georgi Mitev