• Title/Summary/Keyword: bacterial cell selectivity

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Structure-Activity Relationships of Peptide Antibiotics with Improved Bacterial Cell Selectivity of Pseudin

  • Lee, Yeongjoon;Jeon, Dasom;Kim, Jin-Kyoung;Kim, Yangmee
    • Journal of the Korean Magnetic Resonance Society
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    • v.21 no.3
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    • pp.78-84
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    • 2017
  • Pseudin is a naturally occurring 24 amino-acid-residue antimicrobial peptide derived from the skin of paradoxical frog Pseud's paradoxa. It shows potency against the bacteria and antibiotic-resistant bacteria strain, but has high cytotoxicity against mammalian cell. In our previous study, substitution of $Pro^{11}$ for Gly (Ps-P) increased bacterial cell selectivity but decreased the antibacterial activity of pseudin. In this study, we designed pseudin analogue, Ps-4K-P with increased cationicity up to +7 in Ps-P by substituting Glu14, Gln10, Gln24, and Leu18 with Lys. Ps-4K-P showed improved potent antibacterial activity with high bacterial cell selectivity. We determined the tertiary structure of Ps-4K-P in the presence of DPC micelles by NMR spectroscopy and it has a hinge structure at $Pro^{11}$ followed by three turn helices from $Pro^{11}$ to $Val^{23}$ at the C-terminus. Amphipathicity with increased cationicity as well as helix-hinge-helix structural motif provided by introduction of a Pro at position $Gly^{11}$ are the crucial factors which confer antibacterial activity with bacterial cell selectivity to Ps-4K-P.

Structure and Bacterial Cell Selectivity of a Fish-Derived Antimicrobial Peptide, Pleurocidin

  • Yang Ji-Young;Shin Song-Yub;Lim Shin-Saeng;Hahm Kyung-Soo;Kim Yang-Mee
    • Journal of Microbiology and Biotechnology
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    • v.16 no.6
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    • pp.880-888
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    • 2006
  • Pleurocidin, an $\alpha$-helical cationic antimicrobial peptide, was isolated from skin mucosa of winter flounder (Pleuronectes americamus). It had strong antimicrobial activities against Gram-positive and Gram-negative bacteria, but had very weak hemolytic activity. The Gly$^{13,17}\rightarrow$Ala analog (pleurocidin-AA) showed similar antibacterial activities, but had dramatically increased hemolytic activity. The bacterial cell selectivity of pleurocidin was confirmed through the membrane-disrupting and membrane-binding affinities using dye leakage, tryptophan fluorescence blue shift, and tryptophan quenching experiments. However, the non-cell-selective antimicrobial peptide, pleurocidin-AA, interacts strongly with both negatively charged and zwitterionic phospholipid membranes, the latter of which are the major constituents of the outer leaflet of erythrocytes. Circular dihroism spectra showed that pleurocidin-AA has much higher contents of $\alpha$-helical conformation than pleurocidin. The tertiary structure determined by NMR spectroscopy showed that pleurocidin has a flexible. structure between the long helix from $Gly^3$ to $Gly^{17}$ and the short helix from $Gly^{17}$ to $Leu^{25}$. Cell-selective antimicrobial peptide pleurocidin interacts strongly with negatively charged phospholipid membranes, which mimic bacterial membranes. Structural flexibility between the two helices may play a key role in bacterial cell selectivity of pleurocidin.

Structure-Activity Relationships of 9-mer Antimicrobial Peptide analogue of Protaetiamycine, 9Pbw2

  • Kim, Jin-Kyoung;Lee, Eun-Jung;Jung, Ki-Woong;Kim, Yang-Mee
    • Journal of the Korean Magnetic Resonance Society
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    • v.15 no.1
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    • pp.1-13
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    • 2011
  • 9Pbw2 is a 9-mer analog of protaetiamycine derived from the larvae of the beetle Protaetia brevitarsis. Previously, we designed four 9-mer peptide analogues to optimize the balance between the hydrophobicity and cationicity of the peptides and to increase bacterial cell selectivity. Among them, 9Pbw2 has high antibacterial activity without cytotoxicity. The results obtained in previous study suggest that the bactericidal action of 9Pbw2 may be attributed to the inhibition of the functions of intracellular components after penetration of the bacterial cell membrane. In order to understand structure-activity relationships, we determined the three-dimensional structure of 9Pbw2 in 200 mM DPC micelle by NMR spectroscopy. 9Pbw2 has one hydrophobic turn helix from $Trp^3$ to $Arg^8$ and positively charged residues at the N- and C-terminus. This result suggested that positively charged residues from position at the C-terminus in 9Pbw2 may be important for the primary binding to the negatively charged phospholipid head groups in bacterial cell membranes and hydrophobic residues in the middle portion face toward the acyl chains of the hydrophobic lipid in the bacterial cell membrane.

Structure-Activity Relationship of the N-terminal Helix Analog of Papiliocin, PapN

  • Jeon, Dasom;Jeong, Min-Cheol;Kim, Jin-Kyoung;Jeong, Ki-Woong;Ko, Yoon-Joo;Kim, Yangmee
    • Journal of the Korean Magnetic Resonance Society
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    • v.19 no.2
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    • pp.54-60
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    • 2015
  • Papiliocin, from the swallowtail butterfly, Papilio xuthus, shows high bacterial cell selectivity against Gram-negative bacteria. Recently, we designed a 22mer analog with N-terminal helix from $Lys^3$ to $Ala^{22}$, PapN. It shows outstanding antimicrobial activity against Gram-negative bacteria with low toxicity against mammalian cells. In this study, we determined the 3-D structure of PapN in 300 mM DPC micelle using NMR spectroscopy and investigated the interactions between PapN and DPC micelles. The results showed that PapN has an amphipathic ${\alpha}$-helical structure from $Lys^3$ to $Lys^{21}$. STD-NMR and DOSY experiment showed that this helix is important in binding to the bacterial cell membrane. Furthermore, we tested antibacterial activities of PapN in the presence of salt for therapeutic application. PapN was calcium- and magnesium-resistant in a physiological condition, especially against Gram-negative bacteria, implying that it can be a potent candidate as peptide antibiotics.

Molecular Motions of [N(C2H5)4]+ and [N(CH3)4]+ ions by 1H Nuclear Magnetic Resonance Relaxation in [N(C2H5)4]2CoCl4 and [N(CH3)4]2CoCl4 Single Crystals

  • Yoon, Su-A;Lim, Ae-Ran
    • Journal of the Korean Magnetic Resonance Society
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    • v.15 no.2
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    • pp.146-156
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    • 2011
  • The line widths and spin-lattice relaxation times of protons in $[N(C_2H_5)_4]_2CoCl_4$ and $[N(CH_3)_4]_2CoCl_4$ single crystals were investigated in the temperature range 160-400 K. The temperature dependences of the spin-lattice relaxation times are attributed to the molecular motions of the ethyl and methyl groups in the $[N(C_2H_5)_4]^+$ and $[N(CH_3)_4]^+$ ions respectively. The NMR line widths indicate that the ethyl groups in $[N(C_2H_5)_4]_2CoCl_4$ have one more degree of freedom than the methyl groups in $[N(CH_3)_4]_2CoCl_4$. The experimental results are interpreted in terms of the reorientations of the methyl and ethyl groups.

Structure-activity relationships of cecropin-like peptides and their interactions with phospholipid membrane

  • Lee, Eunjung;Jeong, Ki-Woong;Lee, Juho;Shin, Areum;Kim, Jin-Kyoung;Lee, Juneyoung;Lee, Dong Gun;Kim, Yangmee
    • BMB Reports
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    • v.46 no.5
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    • pp.282-287
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    • 2013
  • Cecropin A and papiliocin are novel 37-residue cecropin-like antimicrobial peptides isolated from insect. We have confirmed that papiliocin possess high bacterial cell selectivity and has an ${\alpha}$-helical structure from $Lys^3$ to $Lys^{21}$ and from $Ala^{25}$ to $Val^{35}$, linked by a hinge region. In this study, we demonstrated that both peptides showed high antimicrobial activities against multi-drug resistant Gram negative bacteria as well as fungi. Interactions between these cecropin-like peptides and phospholipid membrane were studied using CD, dye leakage experiments, and NMR experiments, showing that both peptides have strong permeabilizing activities against bacterial cell membranes and fungal membranes as well as $Trp^2$ and $Phe^5$ at the N-terminal helix play an important role in attracting cecropin-like peptides to the negatively charged bacterial cell membrane. Cecropin-like peptides can be potent peptide antibiotics against multi-drug resistant Gram negative bacteria and fungi.

Effects of C-Terminal Residues of 12-Mer Peptides on Antibacterial Efficacy and Mechanism

  • Son, Kkabi;Kim, Jieun;Jang, Mihee;Chauhan, Anil Kumar;Kim, Yangmee
    • Journal of Microbiology and Biotechnology
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    • v.29 no.11
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    • pp.1707-1716
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    • 2019
  • The development of new antimicrobial agents is essential for the effective treatment of diseases such as sepsis. We previously developed a new short peptide, Pap12-6, using the 12 N-terminal residues of papiliocin, which showed potent and effective antimicrobial activity against multidrug-resistant Gram-negative bacteria. Here, we investigated the antimicrobial mechanism of Pap12-6 and a newly designed peptide, Pap12-7, in which the 12th Trp residue of Pap12-6 was replaced with Val to develop a potent peptide with high bacterial selectivity and a different antibacterial mechanism. Both peptides showed high antimicrobial activity against Gram-negative bacteria, including multidrug-resistant Gram-negative bacteria. In addition, the two peptides showed similar anti-inflammatory activity against lipopolysaccharide-stimulated RAW 264.7 cells, but Pap12-7 showed very low toxicities against sheep red blood cells and mammalian cells compared to that showed by Pap12-6. A calcein dye leakage assay, membrane depolarization, and confocal microscopy observations revealed that the two peptides with one single amino acid change have different mechanisms of antibacterial action: Pap12-6 directly targets the bacterial cell membrane, whereas Pap12-7 appears to penetrate the bacterial cell membrane and exert its activities in the cell. The therapeutic efficacy of Pap12-7 was further examined in a mouse model of sepsis, which increased the survival rate of septic mice. For the first time, we showed that both peptides showed anti-septic activity by reducing the infiltration of neutrophils and the production of inflammatory factors. Overall, these results indicate Pap12-7 as a novel non-toxic peptide with potent antibacterial and anti-septic activities via penetrating the cell membrane.

Analogues of Hybrid Antimicrobial Peptide, CAMA-P2, Designed with Improved Antimicrobial and Synergistic Activities

  • Jeong, Ki-Woong;Shin, So-Young;Kim, Jin-Kyoung;Kim, Yang-Mee
    • Bulletin of the Korean Chemical Society
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    • v.32 no.8
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    • pp.2577-2583
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    • 2011
  • We have designed a 20-residue hybrid peptide CA(1-8)-MA(1-12) (CAMA) incorporating residues 1-8 of cecropin A (CA) and residues 1-12 of magainin 2 (MA) with high bacterial cell selectivity. CAMA-P2 is an ${\alpha}$-helical antimicrobial peptide designed from a CAMA hybrid peptide and substitution of Gly-Ile-Gly hinge sequence of CAMA to Pro influences the flexibility at central part of CAMA. Based on structure-activity relationships of CAMA peptides, to investigate the effects of the total positive charges on antimicrobial activity of CAMA-P2, the $Ser^{14}{\rightarrow}$Lys analogue (CAMA-syn1) was synthesized. The role of tryptophan at C-terminal ${\alpha}$-helix on its antimicrobial activity as well as synergistic activity was also investigated using $Ser^{14}{\rightarrow}$Lys/$Phe^{18}{\rightarrow}$Trp analogue (CAMA-syn2). Also, we designed CAMA-syn3 by substitution of $Lys^{16}$ located opposite side of substituted $Lys^{14}$ of CAMA-syn1 with Leu residue, resulting in increase of hydrophobicity and amphipathicity of the peptide. All of CAMA-syn analogues showed good antimicrobial activities similar to those of CAMA and CAMA-P2. The CAMA-syn1 and CAMA-syn2 showed low hemolytic activity and cytotoxicity against human keratinocyte Haca-T cells while CAMA-syn3 showed hemolytic activity and cytotoxicity at its MIC value. We then investigated their abilities to act synergistically in combination with the antimicrobial flavonoids and synthetic compounds screened in our laboratory. The results showed that all peptides exhibited synergistic effects with dihydrobinetin, while only CAMA-syn2 exhibited synergistic effects with YKAs3001 against both S. aureus and MRSA, suggesting that Trp residue at C-terminus of CAMA-syn2 may facilitate the polar antibiotic flavonoids and synthetic compounds to permeabilize the membrane. This study will be useful for the development of new antibiotic peptides with potent antimicrobial and synergistic activity but without cytotoxicity.