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

Susan PuckettMentor: Dr. Bruce GellerAVI BioPharmaHoward Hughes Medical Institute

Antibiotics Today

Race against antibiotic resistance

MRSAMDR & 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)

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

A U G A G C A C U A U C G A A G A

RNAPMO

http://www.stat.stanford.edu/~susan/courses/s166/node2.html

N

O

PO NMe

Me

N

O

O

O

O

P

O

O

O

O O

PMO DNA

Base

Base

Base

Base

_

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

N

N

P NMe

Me

N

O

O

NH2-RFFRFFRFFRXB

O

C

CH3

O

Base

11

QuestionsWhat 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?

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

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

AS19 mutant testing

VancomycinRevertant test antibiotic. Picked 10 mutants that were susceptible.

Rifampin, erythromycin 100 x Resistant to AcpP PMOMutants also resistant to FtsZ and GyrA

PMOs

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

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

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 phagehttp://www.steve.gb.com/science/model_organisms.html

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

KpnI

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

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

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

Acknowledgements

Howard Hughes Medical InstituteURISCAVI BioPharmaOregon State UniversityDr. Kevin AhernDr. Bruce GellerBrett MellbyeGeorgi Mitev