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  • Cell-penetrating antimicrobial peptides - prospectives for targeting intracellular infections.

Cell-penetrating antimicrobial peptides - prospectives for targeting intracellular infections.

Pharmaceutical research (2015-03-18)
Jesper S Bahnsen, Henrik Franzyk, Edward J Sayers, Arwyn T Jones, Hanne M Nielsen
ABSTRACT

To investigate the suitability of three antimicrobial peptides (AMPs) as cell-penetrating antimicrobial peptides. Cellular uptake of three AMPs (PK-12-KKP, SA-3 and TPk) and a cell-penetrating peptide (penetratin), all 5(6)-carboxytetramethylrhodamine-labeled, were tested in HeLa WT cells and analyzed by flow cytometry and confocal microscopy. Furthermore, the effects of the peptides on eukaryotic cell viability as well as their antimicrobial effect were tested. In addition, the disrupting ability of the peptides in the presence of bilayer membranes of different composition were analyzed. AMP uptake relative to penetratin was ~13% (PK-12-KKP), ~66% (SA-3) and ~50% (TPk). All four peptides displayed a punctate uptake pattern in HeLa WT cells with co-localization to lysosomes and no indication that clathrin-mediated endocytosis was the predominant uptake mechanism. TPk showed the highest antibacterial activity. SA-3 exhibited selective disruption of liposomes mimicking Gram-positive and Gram-negative membranes. PK-12-KKP is an unlikely candidate for targeting intracellular bacteria, as the eukaryotic cell-penetrating ability is poor. SA-3, affected the cellular viability to an unacceptable degree. TPk showed acceptable uptake efficiency, high antimicrobial activity and relatively low toxicity, and it is the best potential lead peptide for further development.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
HEPES, BioUltra, Molecular Biology, ≥99.5% (T)
Sigma-Aldrich
5(6)-Carboxytetramethylrhodamine, BioReagent, suitable for fluorescence, ≥85% (sum of isomers, HPCE)
Sigma-Aldrich
Sodium dodecyl sulfate, BioUltra, Molecular Biology, ≥99.0% (GC)
Supelco
N,N-Dimethylformamide, analytical standard
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Sodium dodecyl sulfate, tested according to NF, mixture of sodium alkyl sulfates consisting mainly of sodium dodecyl sulfate
Sigma-Aldrich
Sodium dodecyl sulfate, ≥98.0% (GC)
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Sodium dodecyl sulfate, suitable for ion pair chromatography, LiChropur, ≥99.0%
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Sodium dodecyl sulfate, ≥90% ((Assay))
Supelco
2,2,2-Trifluoroethanol, analytical standard, suitable for NMR (spectroscopy)
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N,N-Dimethylformamide, anhydrous, 99.8%
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N-Chlorosuccinimide, 98%
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Cholesterol, tested according to Ph. Eur.
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Glycerol solution, 83.5-89.5% (T)
Supelco
Cholesterol solution, certified reference material, 10 mg/mL in chloroform
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SyntheChol® NS0 Supplement, 500 ×, synthetic cholesterol, animal component-free, aqueous solution, sterile-filtered, suitable for cell culture
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Sodium dodecyl sulfate, ACS reagent, ≥99.0%
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Sodium dodecyl sulfate, ≥99.0% (GC), dust-free pellets
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2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine, ≥99.0% (TLC)
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Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, Molecular Biology, ≥98.5% (GC)
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Sodium dodecyl sulfate, ReagentPlus®, ≥98.5% (GC)
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2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine, ≥95.5% (GC), ≥98% (TLC)
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Sodium pyruvate, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99%
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L-α-Phosphatidylethanolamine, dioleoyl, ≥99% (GC), ≥98% (TLC), lyophilized powder
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Cholesterol, powder, BioReagent, suitable for cell culture, ≥99%
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Cholesterol, Sigma Grade, ≥99%
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HEPES, BioXtra, pH 5.0-6.5 (1 M in H2O), ≥99.5% (titration)
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HEPES, ≥99.5% (titration)
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HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
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Sodium pyruvate, powder, BioXtra, suitable for mouse embryo cell culture
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Sodium pyruvate, BioXtra, ≥99%