Transcript Resistance to Peptide - Phosphorodiamidate E. Coli Peter Kirby Dept. of Microbiology

Resistance to Peptide - Phosphorodiamidate
Morpholino Oligomers in E. Coli
Peter Kirby
Dept. of Microbiology
Mentor: Bruce Geller
Dept. of Microbiology
Oregon State University
Modern Antibiotics and Bacteria

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

Antibiotics target proteins or
macromolecular complexes
Bacteria can develop resistance
to antibiotics
70 percent of the bacteria that
cause infections in hospitals
are resistant to at least one of
the drugs most commonly
used for treatment
New antibiotics are needed to
combat bacteria
Phosphorodiamidate Morpholino Oligomers

New form of antibiotic
PMO’s are synthetic DNA
compounds

Molecules contains:

 Phosphorodiamidate backbone
 Morpholine rings
 4 nucleotide bases
PMO-Peptide Conjugates

PMO’s have a high molecular weight (MW)
 Have trouble entering Gram-negative bacteria
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Solution: PMO’s were attached to membranepenetrating peptides
PPMO Function
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PPMO’s are sequence
specific
PPMO’s hybridize to mRNA
near AUG start codons
PPMO’s inhibit translation
PPMO Action
Our Research Project
Aim 1: Identify and characterize the mechanisms of
resistance to PPMO’s by E. coli
 Hypothesis: One mechanism of resistance to PPMO’s
is caused by mutations in a transporter
 Strategy: Make random mutation in E. coli, select for
resistance to PPMO’s, and identify mutated gene(s)

Framework
Electroporation:
 Place bacteria between two electrodes and charging the
electrodes with strong potential to open cell wall
 Transposome:
 DNA with ability to insert itself into a chromosomal DNA
sequence
 Contains a gene to code for kanamycin resistance
 Result:
 Inserting a transposome causes random mutations
 Produces mutations in all genes in chromosomal DNA

Methods
Electroporate kanamycin-marked transposomes into
E. coli and grow in the presence of PPMO and
kanamycin
 Isolate chromosomal DNA from PPMO-resistant
mutants
 Clone and sequence DNA flanking transposome
 BLAST E. coli genome to identify gene responsible for
mutation and PPMO resistance
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Results
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12 resistant cells discovered
3 strongest colonies selected
MIC in Luria-Bertani Broth
Antibiotic
MIC in Luria-Bertani Broth
RXR4-AcpP
Antibiotic
06-0076
Parent
MIC (μM)
XL1-Blue
20
Colony
76.1
76.2
76.3
76.4
76.5
76.6
76.7
76.8
76.9
76.10
76.11
76.12
RXR4-AcpP
06-0076
MIC (μM)
40
80
80
80
80
80
80
40
40
80
40
40
Mutated Gene Isolation
Mutated Genes
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sbmA gene mutated in both 76.2 and 76.3
 Function: Predicted Transporter
sbmA

sbmA: predicted ABC transporter located in inner
membrane
 ABC transporters: Transmembrane protein that uses
Adenosine Triphosphate hydrolysis to translocate substances
across the membrane
Mutated Genes (cont)

ADCY (Adenylate Cyclase) gene mutated in 76.10
 Function: cAMP dependent pathway
Adenylate Cyclase
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Adenylate Cylcase (ADCY) is essential to the cAMP
dependent pathway
 Catalyzes the conversion of ATP to cyclic adenosine
monophosphate (cAMP)
○ Activates CAP binding sites

CAP binding site found 200bp from sbmA gene
MIC’s in Mueller Hinton II and Brain
Heart Infusion Broths
MHII Broth
Antibiotic
RXR4-AcpP 06-0076
Colony
MIC (μM)
76.2
10
76.3
10
76.10
--
XL1-Blue
1.25
BHI Broth
Antibiotic
RXR4-AcpP 06-0076
Colony
MIC (μM)
76.2
20
76.3
20
76.10
20
XL1-Blue
5
Conclusion

We found two genes that influence E. coli’s resistance to
PPMOs
 sbmA – transporter
 Adenylate Cyclase – cAMP
Acknowledgements
Bruce Geller
 HHMI
 URISC
 Kevin Ahern
