Identifying Genes in E. coli Required for Susceptibility to Antisense Antibiotics Susan Puckett...
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Transcript of Identifying Genes in E. coli Required for Susceptibility to Antisense Antibiotics Susan Puckett...
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