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Novel antibiotic drugs are urgently needed because of the increase in drug-resistant bacteria. The use of antimicrobial peptides has been suggested to replace antibiotics as they have strong antimicrobial activity and can be extracted from living organisms such as insects, marine organisms, and mammals. HPA3NT3-A2 ([Ala 1,8 ] HPA3NT3) is an antimicrobial peptide that is an analogue of the HP (2-20) peptide derived from Helicobacter pylori ribosomal protein L1. Although this peptide was shown to have strong antimicrobial activity against drug-resistant bacteria, it also showed lower toxicity against sheep red blood cells (RBCs) and HaCaT cells compared to HPA3NT3. The l-Lys residues of HPA3NT3-A2 was substituted with d-Lys residues (HPA3NT3-A2D; [d-Lys 2,5,6,9,10,15 ] HPA3NT3-A2) to prevent the cleavage of peptide bonds by proteolytic enzymes under physiological conditions. This peptide showed an increased half-life and maintained its antimicrobial activity in the serum against Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ) (pathogen). Furthermore, the antimicrobial activity of HPA3NT3-A2D was not significantly affected in the presence of mono- or divalent ions (Na + , Mg 2+ , Ca 2+ ). Finally, l- or d-HPA3NT3-A2 peptides exhibited the strongest antimicrobial activity against antibiotic-resistant bacteria and failed to induce resistance in Staphylococcus aureus after 12 passages.

Bacterial biofilms formed through secretion of extracellular polymeric substances (EPS) have been implicated in many serious infections and can increase antibiotic resistance by a factor of more than 1000. Here, we examined the abilities of the antimicrobial peptide HPA3NT3-A2 to inhibit and reduce biofilm formation, eliminate EPS, and suppress inflammation in mice infected with clinical isolates of drug-resistant Pseudomonas aeruginosa strains. HPA3NT3-A2 was developed from a desirable analogue peptide, HPA3NT3, derived from residues 2-20 of the Helicobacter pylori ribosomal protein L1. HPA3NT3-A2 showed stronger activity against planktonic cells (MIC: 8 μM) compared to ciprofloxacin or tobramycin (>512 μM), and a favorable minimum biofilm inhibition and elimination concentration. This peptide also neutralized LPS; decreased levels of EPS; inhibited the production of pro-inflammatory cytokines in the lung, kidney, and spleen; decreased white blood cell counts; and increased survival among infected mice.

Drug-resistant microorganism infections cause serious disease and can lead to mortality and morbidity. In particular, Staphylococcus aureus induces pyrogenic and toxigenic infections, and drug-resistance occurs rapidly. Multidrug-resistant S. aureus, such as methicillin-resistant S. aureus and methicillin-sensitive S. aureus, can also cause immunodeficiency and immune deficiency syndrome from lipoteichoic acid. However, antimicrobial peptides, such as KW4, have strong antimicrobial activity, low cytotoxicity, and high neutralization activity against endotoxin substances from Gram-negative bacteria. The objective of this study was to use a synthetic KW4 antimicrobial peptide to evaluate the inhibition of drug-resistance development, antimicrobial activity, and neutralizing activity in S. aureus Gram-positive bacteria. The KW4 peptide showed strong antimicrobial activity against drug-resistant S. aureus strains and significantly increased the anti-neutralizing activity of lipoteichoic acid in S. aureus 1630 drug-resistant bacteria. In addition, S. aureus ATCC 29213 did not develop resistance to KW4 as with other antibiotic drugs. These results suggest that the KW4 peptide is an effective antibiotic and anti-neutralizing agent against multidrug-resistant S. aureus strains.

Antimicrobial peptides (AMPs) are a promising new class of antibacterial compounds. However, their applications in the treatment of intracellular pathogenic bacteria have been limited by their in vivo instability and low penetrating ability into mammalian cells. Here, we report that gold nanoparticles conjugated with DNA aptamer (AuNP-Apt) efficiently delivered AMPs into mammalian living systems with enhanced stability of the AMPs. C-terminally hexahistidine-tagged A3-APO (A3-APO(His)) AMPs were loaded onto AuNPs conjugated with His-tag DNA aptamer (AuNP-Apt(His)) by simple mixing and were delivered into Salmonella enterica serovar Typhimurium (S. Typhimurium)-infected HeLa cells, resulting in the increased viability of host cells due to the elimination of intracellular S. Typhimurium cells. Furthermore, the intravenous injection of AuNP-Apt(His) loaded with A3-APO(His) into S. Typhimurium-infected mice resulted in a complete inhibition of S. Typhimurium colonization in the mice organs, leading to 100% survival of the mice. Therefore, AuNP-Apt(His) can serve as an innovative platform for AMP therapeutics to treat intracellular bacterial infections in mammals., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

CA-MA is a hybrid antimicrobial peptide (AMP) derived from two naturally occurring AMPs, cecropin A and magainin 2. CA-MA shows strong antimicrobial activity against Gram-negative and Gram-positive bacteria but also exhibits cytotoxicity toward mammalian cells. Our objective was to identify CA-MA analogues with reduced cytotoxicity by systematic replacement of amino acids with positively charged R groups (His and Lys), aliphatic R groups (Leu), or polar R groups (Glu). Among the CA-MA analogues studied (CMA1 to -6), CMA3 showed the strongest antimicrobial activity, including against drug-resistant Escherichia coli and Pseudomonas aeruginosa strains isolated from hospital patients. CMA3 appeared to act by inducing pore formation (toroidal model) in the bacterial membrane. In cytotoxicity assays, CMA3 showed little cytotoxicity toward human red blood cells (hRBCs) or HaCaT cells. Additionally, no fluorescence was released from small or giant unilamellar vesicles exposed to 60 μM CMA3 for 80 s, whereas fluorescence was released within 35 s upon exposure to CA-MA. CMA3 also exerted strong lipopolysaccharide (LPS)-neutralizing activity in RAW 264.7 cells, and BALB/c mice exposed to LPS after infection by Escherichia coli showed improved survival after administration of one 0.5-mg/kg of body weight or 1-mg/kg dose of CMA3. Finally, in a mouse model of septic shock, CMA3 reduced the levels of proinflammatory factors, including both nitric oxide and white blood cells, and correspondingly reduced lung tissue damage. This study suggests that CMA3 is an antimicrobial/antiendotoxin peptide that could serve as the basis for the development of anti-inflammatory and/or antimicrobial agents with low cytotoxicity., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In response to the growing problem of multidrug-resistant pathogenic microbes, much attention is being paid to naturally occurring and synthetic antimicrobial peptides (AMPs) and the effects of their structural modification. Among these modifications, amino acid substitution is a simple approach to enhancing biological activity and reducing cytotoxicity. An earlier study indicated that HPA3, an analog of HP (2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1, forms large pores and shows considerable cytotoxicity. However, HPA3P, in which a proline (Pro) is substituted for glutamic acid (Glu) at position 9 of HPA3, shows markedly less cytotoxicity. This may be attributable to the presence of a Pro-kink into middle of the HPA3P structure within the membrane environment. Unfortunately, HPA3P is not an effective antibacterial agent in vivo. We therefore designed a helix-PXXP-helix structure (HPA3P2), in which Pro was substituted for the Glu and phenylalanine (Phe) at positions 9 and 12 of HPA3, yielding a molecule with a flexible central hinge. As compared to HPA3P, HPA3P3 exhibited dramatically increased antibacterial activity in vivo. ICR mice infected with clinically isolated multidrug-resistant Pseudomonas aeruginosa showed 100% survival when administered one 0.5-mg/kg dose of HPA3P2 or three 0.1-mg/kg doses of HPA3P2. Moreover, in a mouse model of septic shock induced by P. aeruginosa LPS, HPA3P2 reduced production of pro-inflammatory mediators and correspondingly reduced lung (alveolar) and liver tissue damage. The changes in HPA3 behavior with the introduction of Pro likely reflects alterations of the mechanism of action: i) HPA3 forms pores in the bacterial cell membranes, ii) HPA3P permeates the cell membranes and binds to intracellular RNA and DNA, and iii) HPA3P2 acts on the outer cellular membrane component LPS. Collectively, these results suggest HPA3P2 has the potential to be an effective antibiotic for use against multidrug-resistant bacterial strains., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

HP (2-20) is a 19-aa, amphipathic, α-helical peptide with antimicrobial properties that was derived from the N-terminus of Helicobacter pylori ribosomal protein L1. We previously showed that increasing the net hydrophobicity of HP (2-20) by substituting Trp for Gln(17) and Asp(19) (Anal 3) increased the peptide's antimicrobial activity. In hydrophobic medium, Anal 3 forms an amphipathic structure consisting of an N-terminal random coil region (residues 2-5) and an extended helical region (residues 6-20). To investigate the structure-activity relationship of Anal 3, we substituted Pro for Glu(9) (Anal 3-Pro) and then examined the new peptide's three-dimensional structure, antimicrobial activity and mechanism of action. Anal 3-Pro had an α-helical structure in the presence of trifluoroethanol (TFE) and sodium dodecyl sulfate (SDS). NMR spectroscopic analysis of Anal 3-Pro's tertiary structure in SDS micelles confirmed that the kink potential introduced by Pro(10) was responsible for the helix distortion. We also found that Anal 3-Pro exhibited about 4 times greater antimicrobial activity than Anal 3. Fluorescence activated flow cytometry and confocal fluorescence microscopy showed that incorporating a Pro-hinge into Anal 3 markedly reduced its membrane permeability so that it accumulated in the cytoplasm without remaining in the cell membrane. To investigate the translocation mechanism, we assessed its ability to release of FITC-dextran. The result showed Anal 3-Pro created a pore <1.8 nm in diameter, which is similar to buforin II. Notably, scanning electron microscopic observation of Candida albicans revealed that Anal 3-Pro and buforin II exert similar effects on cell membranes, whereas magainin 2 exerts a different, more damaging, effect. In addition, Anal 3-Pro assumed a helix-hinge-helix structure in the presence of biological membranes and formed micropores in both bacterial and fungal membranes, through which it entered the cytoplasm and tightly bound to DNA. These results indicate that the bending region of Anal 3- Pro peptide is prerequisite for effective antibiotic activity and may facilitate easy penetration of the lipid bilayers of the cell membrane.

An antibacterial protein (about 12 kDa) was isolated from human amniotic fluid through dialysis, ultrafiltration and C18 reversed-phase HPLC steps. Automated Edman degradation showed that the N-terminal sequence of the antibacterial protein was NH(2)-Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-Asn-Gly-. The N-terminal sequence of the antibacterial protein was found to be identical to that of β(2)-microglobulin, a component of MHC class I molecules, which are present on all nucleated cells. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) revealed that the molecular mass of the antibacterial protein was 11,631 Da. This antibacterial protein, β(2)M, possessed potent antibacterial activity against pathogenic bacteria. Specially, antibacterial activity was observed in potassium buffer, and potassium ion was found to be critical for the antibacterial activity. Interestingly, the antibacterial action of β(2)M was associated with dissipation of the transmembrane potential, but the protein did not cause damage to the membrane that would result in SYTOX green uptake. In addition, stimulation of WISH amniotic epithelial cells with the bacterial endotoxin lipopolysaccharide (LPS) induced dose-dependent upregulation of β(2)M mRNA expression. These results suggest that β(2)M contributes to a self-defense response when amniotic cells are exposed to pathogens.

Recently, harmful algal bloom (HAB), also termed "red tide", has been recognized as a serious problem in marine environments according to climate changes worldwide. Many novel materials or methods to prevent HAB have not yet been employed except for clay dispersion, in which can the resulting sedimentation on the seafloor can also cause alteration in marine ecology or secondary environmental pollution. In the current study, we investigated that antimicrobial peptide have a potential in controlling HAB without cytotoxicity to harmless marine organisms. Here, antimicrobial peptides are proposed as new algicidal compounds in combating HAB cells. HPA3 and HPA3NT3 peptides which exert potent antimicrobial activity via pore forming action in plasma membrane showed that HPA3NT3 reduced the motility of algal cells, disrupted their plasma membrane, and induced the efflux of intracellular components. Against raphidoflagellate such as Heterosigma akashiwo, Chattonella sp., and C. marina, it displayed a rapid lysing action in cell membranes at 1~4 µM within 2 min. Comparatively, its lysing effects occurred at 8 µM within 1 h in dinoflagellate such as Cochlodium polykrikoides, Prorocentrum micans, and P. minimum. Moreover, its lysing action induced the lysis of chloroplasts and loss of chlorophyll a. In the contrary, this peptide was not effective against Skeletonema costatum, harmless algal cell, even at 256 µM, moreover, it killed only H. akashiwo or C. marina in co-cultivation with S. costatum, indicating to its selective algicidal activity between harmful and harmless algal cells. The peptide was non-hemolytic against red blood cells of Sebastes schlegeli, the black rockfish, at 120 µM. HAB cells were quickly and selectively lysed following treatment of antimicrobial peptides without cytotoxicity to harmless marine organisms. Thus, the antibiotic peptides examined in our study appear to have much potential in effectively controlling HAB with minimal impact on marine ecology.

Pseudomonas aeruginosa has eventually developed resistance against flomoxef sodium, isepamicin and cefpiramide. Therefore, in this study, the antibacterial activity and synergistic effects of the amphipathic-derived P5-18mer antimicrobial peptide were tested against pathogens associated with cholelithiasis that have developed resistance against commonly used antibiotics. The results were then compared with the activities of the amphipathic-derived peptide, P5-18mer, melittin and common antibiotics. Growth inhibition of planktonic bacteria was tested using the National Committee for Clinical Laboratory Standards (NCCLS). The bactericidal activity of the antimicrobial peptides was measured using time-kill curves. Synergistic effects were evaluated by testing the effects of P5-18mer alone and in combination with flomoxef sodium, isepamicin or cefpiramide at 0.5xMIC. P5-18mer peptide displayed strong activity against pathogens and flomoxef sodium, isepamicin and cefpiramide-resistant bacteria cell lines obtained from a patient with gallstones; however, it did not exert cytotoxicity against the human keratinocyte HaCat cell line. In addition, the results of time-kill curves indicated that P5-18mer peptide exerted bactericidal activity against four strains of P. aeruginosa. Finally, the use of P5-18mer and antibiotics exerted synergistic effects against cell lines that were resistant to commonly used antibiotics. These results indicate that this class of peptides has a rapid microbicidal effect on flomoxef sodium, isepamicin and cefpiramide-resistant strains of P. aeruginosa. Therefore, these peptides may be used as a lead drug for the treatment of acquired pathogens from patients with cholelithiasis who are affected with antibiotic-resistant bacteria., (Copyright 2010 Elsevier Inc. All rights reserved.)