Yoonkyung Park

The Role of Antimicrobial Peptides in Preventing Multidrug-Resistant Bacterial Infections and Biofilm Formation

Over the last decade, decreasing effectiveness of conventional antimicrobial-drugs has caused serious problems due to the rapid emergence of multidrug-resistant pathogens. Furthermore, biofilms, which are microbial communities that cause serious chronic infections and dental plaque, form environments that enhance antimicrobial resistance. As a result, there is a continuous search to overcome or control such problems, which has resulted in antimicrobial peptides being considered as an alternative to conventional drugs. Antimicrobial peptides are ancient host defense effector molecules in living organisms. These peptides have been identified in diverse organisms and synthetically developed by using peptidomimic techniques. This review was conducted to demonstrate the mode of action by which antimicrobial peptides combat multidrug-resistant bacteria and prevent biofilm formation and to introduce clinical uses of these compounds for chronic disease, medical devices, and oral health. In addition, combinations of antimicrobial peptides and conventional drugs were considered due to their synergetic effects and low cost for therapeutic treatment.

Protease Inhibitors from Plants with Antimicrobial Activity

Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents.

A Leu-Lys-rich antimicrobial peptide: activity and mechanism.

To develop novel antibiotic peptides useful as therapeutic drugs, the analogues were designed to increase not only net positive charge by Lys substitution but also hydrophobic helix region by Leu substitution from cecropin A (1-8)-magainin 2 (1-12) hybrid peptide (CA-MA). In particular, CA-MA analogue P5 (P5), designed by flexible region (GIG-->P) substitution, Lys (positions 4, 8, 14, 15) and Leu (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. Confocal microscopy showed that P5 was located in the plasma membrane. The antibacterial effects of analogues were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene as a plasma membrane probe. Flow cytometric analysis revealed that P5 acted in an energy-independent manner. This interaction is also independent of the ionic environment. Furthermore, P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy and showed strong membrane disrupting activity when examined using liposomes (phosphatidyl choline/cholesterol; 10:1, w/w). Its potent antibiotic activity suggests that P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents.

Fungicidal effect of indolicidin and its interaction with phospholipid membranes.

The fungicidal effect and mechanism of a tryptophan-rich 13-mer peptide, indolicidin derived from granules of bovine neutrophils, were investigated. Indolicidin displayed a strong fungicidal activity against various fungi. In order to understand the fungicidal mechanism(s) of indolicidin, we examined the interaction of indolicidin with the pathogenic fungus Trichosporon beigelii. Fluorescence confocal microscopy and flow cytometry analysis revealed that indolicidin acted rapidly on the plasma membrane of the fungal cells in an energy-independent manner. This interaction is also dependent on the ionic environment. Furthermore, indolicidin caused significant morphological changes when tested for the membrane disrupting activity using liposomes (phosphatidylcholine/cholesterol; 10:1, w/w). The results suggest that indolicidin may exert its fungicidal activity by disrupting the structure of cell membranes, via direct interaction with the lipid bilayers, in a salt-dependent and energy-independent manner.

Higher biofilm formation in multidrug-resistant clinical isolates of Staphylococcus aureus.

The biofilm-forming capacity of Staphylococcus aureus contributes to antibiotic resistance, but whether antibiotic-resistant strains have the capacity to form biofilms has not yet been determined. Therefore, we recovered 101 clinical isolates of S. aureus and performed antibiotic susceptibility testing for 30 antibiotics using a VITEK II automatic system. We then carried out a biofilm assay on 96-well polystyrene plates. In addition, the presence of IS256 involved in the variation of biofilm phases of S. aureus was determined by polymerase chain reaction. The prevalence of IS256 was significantly related to multidrug resistance as well as biofilm expression, with biofilm positivity in 27 (39.7%) of the 68 IS256-positive strains and 3 (9.1%) of the 33 IS256-negative strains. In our analysis of the relationship between meticillin resistance and biofilm formation, we found that the rate of biofilm positivity was 37.9% (25/66) for meticillin-resistant strains and 14.3% (5/35) for meticillin-susceptible strains (P<0.05). Staphylococcal cassette chromosome mec (SCCmec) typing found that SCCmec type IV was most prevalent, comprising 14 (56.0%) of the 25 biofilm-positive, meticillin-resistant strains. A statistical analysis testing the relationship between multidrug resistance and biofilm formation revealed a significantly higher rate of biofilm development in strains with greater multiresistance compared with strains with less multiresistance. Our results suggest that the multidrug-resistant clinical isolates of S. aureus have a greater likelihood of developing biofilms on medical devices.

Different mechanisms of action of antimicrobial peptides: insights from fluorescence spectroscopy experiments and molecular dynamics simulations†

Most antimicrobial peptides exert their activity by interacting with bacterial membranes, thus perturbing their permeability. They are investigated as a possible solution to the insurgence of bacteria resistant to the presently available antibiotic drugs. However, several different models have been proposed for their mechanism of membrane perturbation, and the molecular details of this process are still debated. Here, we compare fluorescence spectroscopy experiments and molecular dynamics (MD) simulations regarding the association with lipid bilayers and lipid perturbation for two different amphiphilic helical antimicrobial peptides, PMAP‐23 and trichogin GA IV. PMAP‐23, a cationic peptide member of the cathelicidin family, is considered to induce membrane permeability according to the Shai‐Matsuzaki‐Huang “carpet” model, while trichogin GA IV is a neutral peptide, member of the peptaibol family. Although several lines of evidence suggest a “barrel‐stave” mechanism of pore formation for the latter peptide, its length is only half the normal thickness of a lipid bilayer. Both fluorescence spectroscopy experiments and MD simulations indicated that PMAP‐23 associates with membranes close to their surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. By contrast, trichogin GA IV can undergo a transition from a surface‐bound state to a transmembrane orientation. In the first arrangement, it does not cause any strong membrane perturbation, while in the second orientation it might be able to span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane. Copyright

Design of novel analogue peptides with potent antibiotic activity based on the antimicrobial peptide, HP (2–20), derived from N-terminus of Helicobacter pylori ribosomal protein L1

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is the antimicrobial sequence derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RPL1). In order to develop novel antibiotic peptides useful as therapeutic agents, potent antibiotic activities against bacteria, fungi and cancer cells without a cytotoxic effect are essential. To this end, several analogues with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, the substitution of Trp for the hydrophobic amino acids, Gln and Asp at positions 17 and 19 of HP (2-20) (Anal 3), caused a dramatic increase in antibiotic activity without a hemolytic effect. In contrast, the decrease of hydrophobicity brought about by substituting Ser for Leu and Phe at positions 12 and 19 of HP (2-20), respectively (Anal 4, Anal 5), did not have a significant effect on the antibiotic activity. The antibiotic effects of these synthetic peptides were further investigated by treating prepared protoplasts of Candida albicans and conducting an artificial liposomal vesicle (PC/PS; 3:1, w/w) disrupting activity test. The results demonstrated that the Anal 3 prevented the regeneration of fungal cell walls and induced an enhanced release of fluorescent dye (carboxyfluorescein) trapped in the artificial membrane vesicles to a greater degree than HP (2-20). The potassium-release test conducted on C. albicans indicated that Anal 3 induced greater amounts of potassium ion to be released than the parent peptide, HP (2-20) did. These results indicated that the hydrophobic region of peptides is prerequisite for its effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.

Slowing down single-molecule trafficking through a protein nanopore reveals intermediates for peptide translocation

The microscopic details of how peptides translocate one at a time through nanopores are crucial determinants for transport through membrane pores and important in developing nano-technologies. To date, the translocation process has been too fast relative to the resolution of the single molecule techniques that sought to detect its milestones. Using pH-tuned single-molecule electrophysiology and molecular dynamics simulations, we demonstrate how peptide passage through the α-hemolysin protein can be sufficiently slowed down to observe intermediate single-peptide sub-states associated to distinct structural milestones along the pore, and how to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide. Molecular dynamics simulations of peptide translocation reveal the time- dependent ordering of intermediate structures of the translocating peptide inside the pore at atomic resolution. Calculations of the expected current ratios of the different pore-blocking microstates and their time sequencing are in accord with the recorded current traces.

Marine peptides and their anti-infective activities.

Marine bioresources are a valuable source of bioactive compounds with industrial and nutraceutical potential. Numerous clinical trials evaluating novel chemotherapeutic agents derived from marine sources have revealed novel mechanisms of action. Recently, marine-derived bioactive peptides have attracted attention owing to their numerous beneficial effects. Moreover, several studies have reported that marine peptides exhibit various anti-infective activities, such as antimicrobial, antifungal, antimalarial, antiprotozoal, anti-tuberculosis, and antiviral activities. In the last several decades, studies of marine plants, animals, and microbes have revealed tremendous number of structurally diverse and bioactive secondary metabolites. However, the treatments available for many infectious diseases caused by bacteria, fungi, and viruses are limited. Thus, the identification of novel antimicrobial peptides should be continued, and all possible strategies should be explored. In this review, we will present the structures and anti-infective activity of peptides isolated from marine sources (sponges, algae, bacteria, fungi and fish) from 2006 to the present.

Cell specificity, anti-inflammatory activity, and plausible bactericidal mechanism of designed Trp-rich model antimicrobial peptides.

To develop novel short Trp-rich antimicrobial peptides (AMPs) with potent cell specificity (targeting bacteria but not eukaryotic cells) and anti-inflammatory activity, a series of 11-meric Trp-rich model peptides with different ratios of Leu and Lys/Arg residues, XXWXXWXXWXX-NH(2) (X indicates Leu or Lys/Arg), was synthesized. K(6)L(2)W(3) displayed an approximately 40-fold increase in cell specificity, compared with the natural Trp-rich AMP indolicidin (IN). Lys-containing peptides (K(8)W(3), K(7)LW(3) and K(6)L(2)W(3)) showed approximately 2- to 4-fold higher cell specificities than did their counterparts, the Arg-containing peptides (R(8)W(3), R(7)LW(3) and R(6)L(2)W(3)), indicating that multiple Lys residues are more important than multiple Arg residues in the design of AMPs with good cell specificity. The excellent resistance of d-enantiomers (K(6)L(2)W(3)-D and R(6)L(2)W(3)-D) and Orn/Nle-containing peptides (O(6)L(2)W(3) and O(6)L(2)W(3)) to trypsin digestion compared with the rapid breakdown of the l-enantiomers (K(6)L(2)W(3) and R(6)L(2)W(3)), highlights the clinical potential of such peptides. K(6)L(2)W(3), R(6)L(2)W(3), K(6)L(2)W(3)-D and R(6)L(2)W(3)-D caused weak dye leakage from bacterial membrane-mimicking negatively charged EYPG/EYPE (7:3, v/v) liposomes. Confocal microscopy showed that these peptides penetrated the cell membrane of Escherichia coli and accumulated in the cytoplasm, as observed for buforin-2. Gel retardation studies revealed that the peptides bound more strongly to DNA than did IN. These results suggested that one possible peptide bactericidal mechanism may relate to the inhibition of intracellular functions via interference with DNA/RNA synthesis. Furthermore, some model peptides, containing K(6)L(2)W(3), K(5)L(3)W(3), R(6)L(2)W(3), O(6)L(2)W(3), O(6)L(2)W(3), and K(6)L(2)W(3)-D inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA expression, the release of nitric oxide (NO) following LPS stimulation in RAW264.7 cells and had powerful LPS binding activities at bactericidal concentrations. Collectively, our results indicated that these peptides have potential for future development as novel antimicrobial and anti-inflammatory agents.