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Bacterial killing mechanism of sheep myeloid antimicrobial peptide-18 (SMAP-18) and its Trp-substituted analog with improved cell selectivity and reduced mammalian cell toxicity

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Abstract

To develop short antimicrobial peptide with improved cell selectivity and reduced mammalian cell toxicity compared to sheep myeloid antimicrobial peptide-29 (SMAP-29) and elucidate the possible mechanisms responsible for their antimicrobial action, we synthesized a N-terminal 18-residue peptide amide (SMAP-18) from SMAP-29 and its Trp-substituted analog (SMAP-18-W). Due to their reduced hemolytic activity and retained antimicrobial activity, SMAP-18 and SMAP-18-W showed higher cell selectivity than SMAP-29. In addition, SMAP-18 and SMAP-18-W had no cytotoxicity against three different mammalian cells such as RAW 264.7, NIH-3T3 and HeLa cells even at 100 μM. These results suggest that SMAP-18 and SMAP-18-W have potential for future development as novel therapeutic antimicrobial agent. Unlike SMAP-29, SMAP-18 and SMAP-18-W showed relatively weak ability to induce dye leakage from bacterial membrane-mimicking liposomes, N-phenyl-1-napthylamine (NPN) uptake and o-nitrophenyl-β-galactoside (ONPG) hydrolysis. Similar to SMAP-29, SMAP-18-W led to a significant membrane depolarization (>80 %) against Staphylococcus aureus at 2 × MIC. In contrast, SMAP-18 did not cause any membrane depolarization even at 4 × MIC. In confocal laser scanning microscopy, we observed translocation of SMAP-18 across the membrane in a non-membrane disruptive manner. SMAP-29 and SMAP-18-W were unable to translocate the bacterial membrane. Collectively, we propose here that SMAP-29 and SMAP-18-W kill microorganisms by disrupting/perturbing the lipid bilayer and forming pore/ion channels on bacterial cell membranes, respectively. In contrast, SMAP-18 may kill bacteria via intracellular-targeting mechanism.

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Abbreviations

AMP:

Antimicrobial peptide

Fmoc:

9-Fluorenylmethoxycarbonyl

TFA:

Tifluoroacetic acid

DCC:

Dicyclohexylcarbodiimide

HOBt:

1-Hydroxy-benzotriazole

DiSC3-5:

3,3′-Dipropylthiadicarbocyanine iodide

EYPE:

Egg yolk l-α-phosphatidylethanolamine

EYPG:

Egg yolk l-α-phosphatidylglycerol

EYPC:

Egg yolk l-α-phosphatidylcholine

NPN:

N-phenyl-1-napthylamine

ONPG:

o-Nitrophenyl-β-galactoside

FBS:

Fetal bovine serum

MALDI-TOF MS:

Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry

RP-HPLC:

Reverse-phase high-performance liquid chromatography

CFU:

Colony forming unit

MIC:

Minimal inhibitory concentration

PBS:

Phosphate-buffered saline

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium

LUVs:

Large unilamellar vesicles

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The authors have declared that there is no conflict of interest.

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Authors and Affiliations

  1. Department of Bio-Materials, Graduate School and Department of Cellular and Molecular Medicine, School of Medicine, Chosun University, Gwangju, 501-759, Republic of Korea

    Binu Jacob, Il-Seon Park & Song Yub Shin

  2. Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul, 143-701, Republic of Korea

    Yangmee Kim

  3. Division of Electron Microscopic Research, Korea Basic Science Institute, 113 Gwahanno, Daejeon, 305-333, Republic of Korea

    Jae-Kyung Hyun

  4. Division of Magnetic Resonance, Korea Basic Science Institute, 804-1 Yangchung-ri, Ochang, Chungbuk, 363-883, Republic of Korea

    Jeong-Kyu Bang

Authors
  1. Binu Jacob
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  2. Yangmee Kim
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  3. Jae-Kyung Hyun
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  4. Il-Seon Park
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  5. Jeong-Kyu Bang
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  6. Song Yub Shin
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Correspondence to Jeong-Kyu Bang or Song Yub Shin.

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Jacob, B., Kim, Y., Hyun, JK. et al. Bacterial killing mechanism of sheep myeloid antimicrobial peptide-18 (SMAP-18) and its Trp-substituted analog with improved cell selectivity and reduced mammalian cell toxicity. Amino Acids 46, 187–198 (2014). https://doi.org/10.1007/s00726-013-1616-8

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  • DOI: https://doi.org/10.1007/s00726-013-1616-8

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