Download Identifying Genes in E. coli

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
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
O
O
Base
Base
O
N
Me
O
P
O
N
_
P
O
Me
O
O
O
O
Base
Base
O
N
PMO
DNA
A U G A G C A C U A U C G A A G A
RNA
PMO
G T G A T A G C T T C
http://www.stat.stanford.edu/~susan/courses
/s166/node2.html
Getting PMOs into the cell
Naked PMO ineffective NH2-RFFRFFRFFRXB
N
 Cationic peptides
 Mechanism of PMO entry
N
unknown
P
O
 Last year work:
peptide

O
Me
N
Me
Base
O
 Found
mutants resistant to
peptide-PMOs
 Resistance was linked to peptide
and not to PMO
N
C
CH3
PMO
11
O
Questions
 What
happens to the PMO once it gets
into the cell?
 broken
 How
down?
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

Gram-Negative Envelope
http://www.conceptdraw.com/en/sampletour/medical/
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
 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
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 PMOresistant phenotype

Lambda phage
http://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

KpnI
KpnI

Use competent cells, plate on kanamycin
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 Institute
 URISC
 AVI
BioPharma
 Oregon State University
 Dr. Kevin Ahern
 Dr. Bruce Geller
 Brett Mellbye
 Georgi Mitev