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Identifying Genes in E. coli Required for Susceptibility to Antisense Antibiotics

Identifying Genes in E. coli Required for Susceptibility to Antisense Antibiotics. Susan Puckett Mentor: Dr. Bruce Geller AVI BioPharma Howard Hughes Medical Institute. Antibiotics Today. Race against antibiotic resistance MRSA MDR & XDR Tuberculosis

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Identifying Genes in E. coli Required for Susceptibility to Antisense Antibiotics

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

  4. Getting PMOs into the cell peptide • 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

  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 Gram-Negative Envelope http://www.conceptdraw.com/en/sampletour/medical/

  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. KpnI KpnI 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

  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

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