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Experimental Therapeutics

Bacterial Resistance to Antisense Peptide Phosphorodiamidate Morpholino Oligomers

Susan E. Puckett, Kaleb A. Reese, Georgi M. Mitev, Valerie Mullen, Rudd C. Johnson, Kyle R. Pomraning, Brett L. Mellbye, Lucas D. Tilley, Patrick L. Iversen, Michael Freitag, Bruce L. Geller
Susan E. Puckett
aDepartment of Microbiology, Oregon State University, Corvallis, Oregon, USA
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Kaleb A. Reese
aDepartment of Microbiology, Oregon State University, Corvallis, Oregon, USA
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Georgi M. Mitev
aDepartment of Microbiology, Oregon State University, Corvallis, Oregon, USA
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Valerie Mullen
aDepartment of Microbiology, Oregon State University, Corvallis, Oregon, USA
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Rudd C. Johnson
aDepartment of Microbiology, Oregon State University, Corvallis, Oregon, USA
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Kyle R. Pomraning
bDepartment of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon, USA
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Brett L. Mellbye
cAVI BioPharma, Inc., Corvallis, Oregon, USA
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Lucas D. Tilley
cAVI BioPharma, Inc., Corvallis, Oregon, USA
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Patrick L. Iversen
cAVI BioPharma, Inc., Corvallis, Oregon, USA
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Michael Freitag
bDepartment of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon, USA
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Bruce L. Geller
aDepartment of Microbiology, Oregon State University, Corvallis, Oregon, USA
cAVI BioPharma, Inc., Corvallis, Oregon, USA
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DOI: 10.1128/AAC.00850-12
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    Fig 1

    Viable cell count of 18-h cultures. Stationary cultures of W3110 or PR200.1 were diluted to 5 × 105 CFU/ml in Mueller-Hinton broth and divided in three. (RFF)3RXB-FtsZ (FtsZ) or scrambled-base-sequence (Scr) control PPMO (160 μM) or no PPMO was added. Cultures were grown aerobically at 37°C for 18 h, and then samples of each were diluted and plated to determine viable cells. Error bars indicate standard deviations. *, highly significant (P < 0.01) difference compared to either the no-PPMO or scrambled-base-sequence (Scr) control-treated culture.

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    Fig 2

    Luciferase activity of cultures treated with PPMOs. Growing cultures were treated for 7 h without PPMO (no PPMO) or with 3 concentrations of a PPMO [(RFF)3RXB-Luc] targeted to a luciferase reporter gene or a scrambled-base-sequence control [(RFF)3RXB-Scr]. After 7 h, samples of each culture were measured for luciferase activity by luminometry. The experiment was repeated 3 times, and the error bars indicate standard deviations. *, a highly significant (P < 0.01) difference compared to cultures of PR200.1 with the same concentrations of (RFF)3RXB-Luc, the cultures of W3110 with the same concentrations of (RFF)3RXB-Scr, or the culture without PPMO.

Tables

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  • Table 1

    MIC of AcpP PPMOs in pure cultures of E. coli

    Motif, PPMO no.aConjugated peptidebMIC (μM [μg/ml])
    W3110PR200.1
    Motif 1 (C-N-N)
        NG-05-0200RFFRFFRFFRXB2.5 (14)40 (222)
        NG-05-0653DRDFDFDRDFDFDRDFDFDRXB2.5 (14)40 (222)
        NG-23-248RXXRXXRXXRXB20 (102)80 (204)
        NG-06-0199KFFKFFKFFKXB10 (54)80 (435)
    Motif 2 (C-N), NG-06-0073RXRXRXRXRXRXB1.25 (7)1.25 (7)
    Motif 3 (C-N-C)
        NG-06-0076RXRRXRRXRRXRXB1.25 (7)1.25 (7)
        NG-07-0795RFRRFRRFRRFRXB1 (6)16 (94)
    No motif, NG-05-0246RTRTRFLRRTXB20 (111)40 (111)
    • ↵a Motif 1 is cationic-nonpolar-nonpolar (C-N-N). Motif 2 is cationic-nonpolar (C-N). Motif 3 is cationic-nonpolar-cationic (C-N-C).

    • ↵b X is 6-aminohexanoic acid, B is β-alanine, O is ornithine, and D indicates the isomeric form or the residue that follows.

  • Table 2

    MIC of (RFF)3R-AcpP (NG-05-0200) using oligopeptide transport mutants

    E. coli strainMutation/phenotypeMIC
    μMμg/ml
    Morse 2034Wild-type oligopeptide transport528
    PA0183opp knockout, oligopeptide permease deletion528
    PA0333dpp and opp knockout, PA0183 plus dipeptide permease deletion528
    PA0643tpp, dpp, and opp knockout, PA0333 plus tripeptide permease mutant528
  • Table 3

    MICs of standard antibiotics and PPMOs using transposome mutant RR3, isogenic parent strain XL1-Blue MRF′, and their sbmA complemented strains

    PPMO no.Antibiotic or PPMOMIC (μM [μg/ml])
    XL1-Blue MRF′RR3XL1-Blue MRF′ (pSbmA)RR3(pSbmA)
    Polymyxin B0.8 (1)0.8 (1)0.8 (1)0.8 (1)
    Colistin0.9 (1)0.9 (1)0.9 (1)0.9 (1)
    Erythromycin34 (25)34 (25)34 (25)34 (25)
    Rifampin6 (5)6 (5)6 (5)6 (5)
    Bleomycin0.2 (0.25)0.7 (1)0.01 (0.016)0.01 (0.016)
    Phleomycin0.3 (0.5)1.3 (2)0.02 (0.03)0.02 (0.03)
    NG-05-0200(RFF)3RXB-AcpP2 (11)64 (355)2 (11)2 (11)
    NG-05-0653[d-(RFF)3R]XB-AcpP2 (11)64 (355)2 (11)2 (11)
    NG-06-0076(RXR)4XB-AcpP1 (6)8 (48)0.1 (0.6)0.1 (0.6)
    NG-05-0655(RFF)3RXB-Scr>128 (>714)>128 (>714)>128 (>714)>128 (>714)
    NG-06-0078(RXR)4XB-Scr>128 (>714)>128 (>714)>128 (>714)>128 (>714)
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Bacterial Resistance to Antisense Peptide Phosphorodiamidate Morpholino Oligomers
Susan E. Puckett, Kaleb A. Reese, Georgi M. Mitev, Valerie Mullen, Rudd C. Johnson, Kyle R. Pomraning, Brett L. Mellbye, Lucas D. Tilley, Patrick L. Iversen, Michael Freitag, Bruce L. Geller
Antimicrobial Agents and Chemotherapy Nov 2012, 56 (12) 6147-6153; DOI: 10.1128/AAC.00850-12

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Bacterial Resistance to Antisense Peptide Phosphorodiamidate Morpholino Oligomers
Susan E. Puckett, Kaleb A. Reese, Georgi M. Mitev, Valerie Mullen, Rudd C. Johnson, Kyle R. Pomraning, Brett L. Mellbye, Lucas D. Tilley, Patrick L. Iversen, Michael Freitag, Bruce L. Geller
Antimicrobial Agents and Chemotherapy Nov 2012, 56 (12) 6147-6153; DOI: 10.1128/AAC.00850-12
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