Development of novel LL-37 derived antimicrobial peptides with LPS and LTA neutralizing and antimicrobial activities for therapeutic application

New peptides for lipopolysaccharide (LPS) and lipoteichoic acid (LTA) neutralization in upper respiratory tract infections were developed and evaluated in terms of efficacy and safety for application in humans. Based on the sequence of the human antimicrobial peptide LL-37 we developed and investigated length variants, substitution analogues and modifications to stabilize the peptides to prevent enzymatic degradation and to improve efficacy. The most promising peptide appears P60.4, a 24 amino acid peptide with similar efficacy as LL-37 in terms of LPS and LTA neutralization and lower pro-inflammatory activity. In addition, the acetylated and amidated version of this peptide shows no toxicity and displays higher or equal antimicrobial activity compared to LL-37.     

Neutralization of bacterial endotoxins by frog antimicrobial peptides

The ability of skin antimicrobial peptides of the southern bell frog, Litoria raniformis, to neutralize in vitro the endotoxin, proinflammatory lipopolysaccharide (LPS) complex, from two different gram‐negative bacterial pathogens, human pathogen Escherichia coli (0111:B4) and frog pathogen Klebsiella pneumoniae, was investigated. The LPS neutralization activity of the natural mixture of skin antimicrobial peptides was measured using chromogenic Limulus amebocyte lysate assays. These skin antimicrobial peptides neutralized the LPSs from both pathogens at physiologically relevant concentrations (IC₅₀ < 100 µg/mL) showing their potential for non‐specific LPS neutralization in vivo in the skin of infected frogs and for development of anti‐endotoxin agents.     

Design of multifunctional peptides expressing both antimicrobial activity and shiga toxin neutralization activity

We have designed novel short peptides expressing both antimicrobial and Shiga-toxin (Stx) neutralization activities by combining nuclear localization signal (NLS) peptides (RIRKKLR, PKKKRKV, and PRRRK) tandemly with globotriaoside (Gb3) mimic peptide (WHWTWL). These fusion peptides exhibited excellent antimicrobial activity against both gram-positive and gram-negative bacteria. A peptide WHWTWLRIRKKLR (Trp-His-Trp-Thr-Trp-Leu-Arg-Ile-Arg-Lys-Lys-Leu-Arg), especially, exhibited about 100 times higher activity than the original NLS peptide. SPR analysis demonstrated that the binding of this peptide to both Stxs was strong: K(d) = 6.6 x 10(-6) to Stx-1 and 6.8 x 10(-6) to Stx-2. The in vitro assay against Stx-1 using HeLa cells showed that this peptide increased the survival rate of HeLa cells against the infection of Stx-1. The peptide has been found to maintain high antimicrobial activity, Stx neutralization activity, and no cytotoxicity at its concentration of 7.8-31.3 microg/mL (4.2-16.7 microM). The present peptide design has a prospect of developing potent multifunctional drugs to destroy proteinaceous toxin-producing bacteria and to simultaneously neutralize the toxins released by bacteriolysis.     

Quality of Antimicrobial Products Used in Striped Catfish (Pangasianodon hypophthalmus) Aquaculture in Vietnam

Antimicrobial usage is common in Asian aquaculture. This study aimed to determine the quality of antimicrobial products used by Vietnamese striped catfish (Pangasianodon hypophthalmus) farmers. Twenty one antimicrobial products (11 products contained a single antimicrobial and 10 products contained a mixture of two different antimicrobials) commonly used by catfish farmers were obtained from so-called chemical shops located in the Mekong Delta, Vietnam. Ultra High Performance Liquid Chromatography Mass Spectrometry was used to analyze concentration of sulfonamides, trimethoprim, amoxicillin, cefalexin and ciprofloxacin whereas concentrations of florfenicol and doxycycline were analyzed by High Performance Liquid Chromatography with UV detection. Results revealed that only 4/11 products with a single antimicrobial and 2/10 products with a mixture of antimicrobials contained active substances within ±10% of the concentration declared on the product label. Two products with antimicrobial mixtures did not contain any of the declared antimicrobials. Comparing two batches, analysis of 11 products revealed that only one product contained a concentration of active compound that varied with less than 10% in both batches. Several product labels provided inadequate information on how to calculate therapeutic dosage and further stated withdrawal time despite lack of pharmacokinetic data on the antimicrobials in catfish. There is an urgent need to strengthen approval procedures and in particular regularly to monitor the quality of antimicrobials used in Vietnamese aquaculture.     

融合有酸性片段的抗菌肽串连重复基因的构建

为提高抗菌肽的表达,设计在抗菌肽基因的N端融合一段编码酸性肽的片段以及减轻表达产物对宿主的毒性,通过含有酶切位点的接头将该融合肽基因以同向串连的方式连接成多拷贝基因,克隆至pUC19载体。为此,分段设计合成了编码天蚕素A-蜂毒素杂合肽和酸性肽的DNA片段。首先将其连接成融合肽全基因,然后分别与含相同粘性末端的前后接头连接。通过控制基因和接头加入的量及次序,可得到两侧有EcoRI和SalI酶切位点的同向串连的多拷贝基因。选取合适拷贝数的基因,将其克隆至pUC19载体,PCR扩增和DNA测序证明多拷贝基因构建成功且基因方向相同。结果表明,该方法能简捷高效地获得所需的多拷贝基因,为提高表达产物的量打下基因。     

Studies on the potency test of antiserum for Weils disease by the intracutaneous method.

A method for estimating the leptospiricidal activity of therapeutic antiserum for Weil's disease was improved by using the intracutaneous method in guinea pigs. The neutralization curves between the leptospiral suspensions for challenge and the antisera were shown to be linear over a wide range of the dosis of the pathogen. Although the reproducibility of the neutralization experiments was high, a significant divergence was observed among the slopes in various test samples. However, the trouble due to such a discrepancy in the slope may practically be reduced if such an antiserum preparation that has a regression coefficient close to the mean of those of various neutralization lines constructed by many different products is adopted as the reference. A practical method for the potency test was suggested.     

The antimicrobial activity and acute toxicity of the polymer salts of gentamycin]

By neutralization of copolymers of N-(2-hydroxypropyl) methacrylamide with acrylic acid and copolymers of N-vinylpyrrolidone with crotonic and p-crotonoylaminophenoxyacetic acids in the presence of gentamycin, water-soluble polymer salts containing from 10 to 25 mass% of gentamycin were obtained. These salts regardless of gentamycin content completely retain high level of antimicrobial activity of gentamycin against Staphylococcus spp. and Escherichia coli and are characterized by less (by more than one order of magnitude) acute toxicity.     

Factors, determining the activity of a new antimicrobial substance from fish oils from various species

The induction of antimicrobial activity of a new preparation, an aqueous fraction of water-oil emulsion oxidized by air oxygen, was studied. The effect of various factors (the degree of unsaturation of the initial oil and the content of oil oxidation products in obtained preparation) on the antimicrobial activity was determined. The antimicrobial activity of the preparation was induced by oil oxidation. The preparation produced from sardine Sardinops melanostica oil (33.95% of polyunsaturated fatty acids) displayed the highest antimicrobial activity. The antimicrobial activity was shown in water-soluble oil oxidation products.     

Plasmid analysis of bacteria of Bacillus genus used in the development of probiotics

Presence of plasmid-contained strains in probiotics prepared on the basis of Bacillus bacteria was assessed. Plasmid analysis was performed by the method based on neutralization of bacterial suspension after alkaline treatment with lithium chloride. Presence of 4-6 plasmids with molecular weight from 1 to 75 MDa was revealed in 4 commercially available probiotics. Since plasmids can facilitate transfer of genes of the antimicrobial resistance to pathogenic microorganisms it was recommended to perform thorough search of probiotic microorganisms without plasmids or ensure the elimination of R-plasmids in start cultures for the most promising biopreparations.     

Activity of a new antimicrobial preparation from fish oils from various sources: Effects of different factors

The induction of antimicrobial activity of a new preparation, an aqueous fraction of water-oil emulsion oxidized by atmospheric oxygen, was studied. The effect of varions factors (the degree of unsaturation of the initial oil and the content of oil oxidation products in the preparation) on antimicrobial activity was determined. The antimicrobial activity of the preparation was induced by oil oxidation. The preparation produced from sardineSardinops melanostica oil (33.95% polyunsaturated fatty acids) displayed the highest antimicrobial activity. Antimicrobial activity was shown in water-soluble oil oxidation products.     

Performance profiles of topical antimicrobials in vitro

The in-vitro efficacy of commercially available topical antimicrobial products against control strains and those from clinical material are compared with an agar diffusion model. The MICs of the constituent antimicrobial compounds have been determined for the same organisms. Plotting the inhibition zone diameters produced by the topical products against the log₁₀ MICs of their constituent antimicrobial compound(s) gives overall product performance profiles for a range of organisms. These profiles confirm that the formulation of a topical product clearly modifies the response obtained with a specific antimicrobial compound.     

Performance profiles of topical antimicrobials in vitro

The in-vitro efficacy of commercially available topical antimicrobial products against control strains and those from clinical material are compared with an agar diffusion model. The MICs of the constituent antimicrobial compounds have been determined for the same organisms. Plotting the inhibition zone diameters produced by the topical products against the log10 MICs of their constituent antimicrobial compound(s) gives overall product performance profiles for a range of organisms. These profiles confirm that the formulation of a topical product clearly modifies the response obtained with a specific antimicrobial compound.     

Antimicrobial activity of human α-defensin 5 and its linear analogs: N-terminal fatty acylation results in enhanced antimicrobial activity of the linear analogs

Human α-defensin 5 (HD5) exhibits broad spectrum antimicrobial activity and plays an important role in mucosal immunity of the small intestine. Although there have been several studies, the structural requirements for activity and mechanism of bacterial killing is yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD5 and linear analogs. Cysteine deletions attenuated the antibacterial activity considerably. Candidacidal activity was affected to a lesser extent. Fatty acid conjugated linear analogs showed antimicrobial activity comparable activity to HD5. Effective surface charge neutralization of bacteria was observed for HD5 as compared to the non-fatty acylated linear analogs. Our results show that HD5 and non-fatty acylated linear analogs enter the bacterial cytoplasm without causing damage to the bacterial inner membrane. Although fatty acylated peptides exhibited antimicrobial activity comparable to HD5, their mechanism of action involved permeabilization of the Escherichia coli inner membrane. HD5 and analogs had the ability to bind plasmid DNA. HD5 had greater binding affinity to plasmid DNA as compared to the analogs. The three dimensional structure of HD5 favors greater interaction with the bacterial cell surface and also with DNA. Antibacterial activity of HD5 involves entry into bacterial cytoplasm and binding to DNA which would result in shut down of the bacterial metabolism leading to cell death. We show how a moderately active linear peptide derived from the α-defensin HD5 can be engineered to enhance antimicrobial activity almost comparable to the native peptide.     

Pilot-scale extraction and antimicrobial activity of crude extract from ovine neutrophils

Antimicrobial peptides have potential to be a high-value product purified from waste ovine blood. Previous work characterised antimicrobial peptides isolated from ovine neutrophils and determined the mechanisms of action. Here it is shown that the crude antimicrobial extract can be produced on a pilot-scale while retaining the antimicrobial activity. This crude extract could be used as a biopreservative for chilled lamb meat products or in a topical cream for treating cuts and grazes.     

Anticancer Peptide SVS-1: Efficacy Precedes Membrane Neutralization

Anticancer peptides are polycationic amphiphiles capable of preferentially killing a wide spectrum of cancer cells relative to noncancerous cells. Their primary mode of action is an interaction with the cell membrane and subsequent activation of lytic effects; however, the exact mechanism responsible for this mode of action remains controversial. Using zeta potential analyses we demonstrate the interaction of a small anticancer peptide with membrane model systems and cancer cells. Electrostatic interactions have a pivotal role in the cell killing process, and in contrast to the antimicrobial peptides action cell death occurs without achieving full neutralization of the membrane charge.     

Antibacterial metabolites of lactic acid bacteria: their diversity and properties

The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.     

The first identified cathelicidin from tree frogs possesses anti-inflammatory and partial LPS neutralization activities

As of February 2017, approximately 7639 amphibian species have been described in the AmphibiaWeb database. However, only 20 cathelicidin-like antimicrobial peptides have been identified to date from 10 amphibian species. Half of these peptides were identified from genome sequences and have not yet been functionally characterized. In this study, a novel cathelicidin-like peptide designated cathelicidin-PP was purified from the skin of tree frog Polypedates puerensis. Cathelicidin-PP is a 32 residue peptide of sequence ASENGKCNLLCLVKKKLRAVGNVIKTVVGKIA. Circular dichroism spectroscopy indicated that cathelicidin-PP mainly adopts a β-sheet structure in membrane-mimetic solutions. Cathelicidin-PP exhibits potent antimicrobial activity against bacteria and fungi, especially Gram-negative bacteria. Meanwhile, it shows low cytotoxicity toward mammalian cells. Scanning electron microscopy analysis indicated that cathelicidin-PP kills bacteria through the disruption of the bacterial cell membrane integrity. Furthermore, cathelicidin-PP exerts significant anti-inflammatory functions by inhibiting the lipopolysaccharide (LPS)-mediated generation of nitric oxide and pro-inflammatory cytokines, tumor necrosis factor-α, interleukin-1β, and interleukin-6. The MAPKs (ERK, JNK, and p38) and NF-κB signaling pathways are involved in the anti-inflammatory effect. Cathelicidin-PP caused partial neutralization of LPS in a dose-dependent manner. Quantitative PCR indicated that infection of tree frogs with bacteria causes increased expression of cathelicidin-PP in immune-related tissues. Taken together, cathelicidin-PP is the first identified cathelicidin-like peptide from tree frogs. Our findings demonstrate that in addition to direct bactericidal capacity, cathelicidin-PP also possesses immunomodulatory properties, including partial neutralization of LPS, and inhibiting the production of inflammatory cytokines.

     

Antibacterial metabolites of lactic acid bacteria: Their diversity and properties

The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.     

Effect of simulated gastrointestinal conditions and epithelial transport on extracts of green tea and sage

Few in vitro screening studies on the biological activities of plant extracts that are intended for oral administration consider the effect of the gastrointestinal system. This study investigated this aspect on extracts of Camellia sinensis (green tea) and Salvia officinalis (sage) using antimicrobial activity as a model for demonstration. Both the crude extracts and their products after exposure to simulated gastric fluid (SGF) as well as simulated intestinal fluid (SIF) were screened for antimicrobial activity. The chromatographic profiles of the crude plant extracts and their SGF as well as SIF products were recorded and compared qualitatively by means of high performance liquid chromatography coupled to mass spectrometry. The effect of epithelial transport on the crude plant extracts was determined by applying them to an in vitro intestinal epithelial model (Caco-2). The crude extracts for both plants exhibited reduced antimicrobial activity after exposure to SGF, while no antimicrobial activity was detected after exposure to SIF. These results suggested chemical modification or degradation of the antimicrobial compounds when exposed to gastrointestinal conditions. This was confirmed by a reduction of the peak areas on the LC–UV–MS chromatograms. From the chromatographic profiles obtained during the transport study, it is evident that some compounds in the crude plant extracts were either not transported across the cell monolayer or they were metabolised during passage through the cells. It can be deduced that the gastrointestinal environment and epithelial transport process can dramatically affect the chromatographic profiles and biological activity of orally ingested natural products.     

Lipid saturation and head group composition have a pronounced influence on the membrane insertion equilibrium of amphipathic helical polypeptides

The histidine-rich peptides of the LAH4 family were designed using cationic antimicrobial peptides such as magainin and PGLa as templates. The LAH4 amphipathic helical sequences exhibit a multitude of interesting biological properties such as antimicrobial activity, cell penetration of a large variety of cargo and lentiviral transduction enhancement. The parent peptide associates with lipid bilayers where it changes from an orientation along the membrane interface into a transmembrane configuration in a pH-dependent manner. Here we show that LAH4 adopts a transmembrane configuration in fully saturated DMPC membranes already at pH 3.5, i.e. much below the pK_a of the histidines whereas the transition pH in POPC correlates closely with histidine neutralization. In contrast in POPG membranes the in-planar configuration is stabilized by about one pH unit. The differences in pH can be converted into energetic contributions for the in-plane to transmembrane transition equilibrium, where the shift in the transition pH due to lipid saturation corresponds to energies which are otherwise obtained by the exchange of several cationic with hydrophobic residues. A similar dependence on lipid saturation has also been observed when the PGLa and magainin antimicrobial peptides interact within lipid bilayers suggesting that the quantitative evaluation presented in this paper also applies to other membrane polypeptides.