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Susceptibility

Salt-Resistant Alpha-Helical Cationic Antimicrobial Peptides

Carol Friedrich, Monisha G. Scott, Nedra Karunaratne, Hong Yan, Robert E. W. Hancock
Carol Friedrich
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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Monisha G. Scott
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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Nedra Karunaratne
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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Hong Yan
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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Robert E. W. Hancock
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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DOI: 10.1128/AAC.43.7.1542
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  • Fig. 1.
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    Fig. 1.

    CD spectra of cationic peptides in the presence of 90-nm liposomes (POPC-POPG, 7:3) and CP26 in phosphate buffer (random coil). The peptides are represented as follows: CP201, round dots; CP208, solid lines; CEMA, dash-dots; CEME, long dashes; CP26, dashes; and CP29, square dots.

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    Fig. 2.

    Survival of logarithmic-phase (A) and stationary-phase (B) E. coli UB1005 in MH broth after addition of fourfold the MIC of peptide, gentamicin, or ceftazidime. This corresponds to 2 μg of CP26, CP29, and ceftazadime per ml, 4 μg of CEME and CEMA per ml, and 0.5 μg of gentamicin per ml. Actual initial concentrations of bacteria ranged from 0.5 × 108 to 2.5 × 108/ml but were corrected to an initial concentration of 1 × 108/ml for clarity. A typical experiment out of three trials is shown. Symbols: ▵, no peptide; ▴, CP26; ▾, CP29; ●, CEME; ○, CEMA; ■, ceftazidime; □, gentamicin. Results for CP26 in panel A were almost superimposable with the ceftazidime results and were thus omitted for clarity. No evidence of bacterial aggregation was observed when viewed under a light microscope or in light-scattering experiments.

  • Fig. 3.
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    Fig. 3.

    Permeabilization of cytoplasmic membrane of E. coli ML35 by cationic peptides as determined by their ability to unmask cytoplasmic β-galactosidase. The hydrolysis of ONPG was measured spectrophotometrically at 420 nm. Symbols: ▴, CP26 at 12.8 μg/ml; ●, CEME at 6.4 μg/ml; ○, CEMA at 6.4 μg/ml; ▾, CP29 at 6.4 μg/ml; ■, polymyxin B at 12.8 μg/ml; □, CP201 at 12.8 μg/ml; ▵, CP208 at 12.8 μg/ml.

  • Fig. 4.
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    Fig. 4.

    Effects of NaCl, MgCl2, and CCCP on the cytoplasmic membrane permeabilization activity of cationic peptides CP29 (solid), CEME (stippled), and CP26 (striped).

Tables

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  • Table 1.

    Amino acid sequences of antimicrobial cationic peptides used in this study

    PeptideAmino acid sequenceLength (no. of amino acids)Charge% Hydrophobic amino acids
    CP26KWKSFIKKLTSAAKKVVTTAKPLISS26+746
    CP29KWKSFIKKLTTAVKKVLTTGLPALIS26+650
    CP201KWKSFIKNLTKGGSKILTTGLPALIS26+542
    CP208KKKSFIKLLTSAKVSVLTTAKPLISS26+642
    CEMEKWKLFKKIGIGAVLKVLTTGLPALIS26+569
    CEMAKWKLFKKIGIGAVLKVLTTGLPALKLTK28+664
  • Table 2.

    α-Helicity in various environments as assessed by CD spectroscopy interpreted according to the K2D algorithm (2)

    Condition% α-Helix
    CP26CP29CP201CP208CEMACEME
    Phosphate buffer688747
    Liposomesa26, 3550, 5720817, 3329, 30
    SDS (40:1)422313265035
    50% TFE3020NDND3225
    • ↵a The first number is for 90-nm liposomes, and the second is for 60-nm liposomes. ND, not determined.

  • Table 3.

    Broth dilution MICs of cationic peptides against various gram-negative bacteria

    SpeciesStrainRelevant phenotype or genotypeMIC (μg/ml)
    CP26CP29CP201CP208CEMECEMA
    P. aeruginosaPAO1Wild type2286422
    K385nfxB4486422
    1008OCR01nalB221612822
    PAO963nalA22812822
    PA-83-48β-Lactamase derepressed4232>12824
    Z61Antibiotic sensitive223212824
    E. coliUB1005Wild type0.50.543211
    S. typhimurium14028sParent of MS7953s2232>6424
    MS7953sDefensin sensitive0.50.252811
  • Table 4.

    Influence of NaCl, MgCl2, and polyanionic alginate on MICs of cationic antimicrobial peptides forP. aeruginosa PAO1

    AdditiveConcnMIC (μg/ml)a
    CP26CP29CEMECEMA
    None2222
    NaCl100 mM4222
    200 mM16244
    300 mM32444
    MgCl21 mM8444
    3 mM1616164
    5 mM>32323232
    Sodium alginate0.01%2244
    0.05%881632
    0.1%16326464
    • ↵a These are the average values of three experiments.

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Salt-Resistant Alpha-Helical Cationic Antimicrobial Peptides
Carol Friedrich, Monisha G. Scott, Nedra Karunaratne, Hong Yan, Robert E. W. Hancock
Antimicrobial Agents and Chemotherapy Jul 1999, 43 (7) 1542-1548; DOI: 10.1128/AAC.43.7.1542

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Salt-Resistant Alpha-Helical Cationic Antimicrobial Peptides
Carol Friedrich, Monisha G. Scott, Nedra Karunaratne, Hong Yan, Robert E. W. Hancock
Antimicrobial Agents and Chemotherapy Jul 1999, 43 (7) 1542-1548; DOI: 10.1128/AAC.43.7.1542
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KEYWORDS

Anti-Bacterial Agents
Antimicrobial Cationic Peptides
Carrier Proteins
Protein Structure, Secondary

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