Uncoupling Amphipathicity and Hydrophobicity: Role of Charge Clustering in Membrane Interactions of Cationic Antimicrobial Peptides.

Cationic antimicrobial peptides (CAPs) occur as important innate immunity agents in many organisms, including humans, and offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane lysis and eventually cell death. In this work, we studied the biophysical and microbiological characteristics of designed CAPs varying in hydrophobicity levels and charge distributions by a variety of biophysical and biochemical approaches, including in-tandem atomic force microscopy, attenuated total reflection-FTIR, CD spectroscopy, and SDS-PAGE. Peptide structural properties were correlated with their membrane-disruptive abilities and antimicrobial activities. In bacterial lipid model membranes, a time-dependent increase in aggregated β-strand-type structure in CAPs with relatively high hydrophobicity (such as KKKKKKALFALWLAFLA-NH(2)) was essentially absent in CAPs with lower hydrophobicity (such as KKKKKKAAFAAWAAFAA-NH(2)). Redistribution of positive charges by placing three Lys residues at both termini while maintaining identical sequences minimized self-aggregation above the dimer level. Peptides containing four Leu residues were destructive to mammalian model membranes, whereas those with corresponding Ala residues were not. This finding was mirrored in hemolysis studies in human erythrocytes, where Ala-only peptides displayed virtually no hemolysis up to 320 μM, but the four-Leu peptides induced 40-80% hemolysis at the same concentration range. All peptides studied displayed strong antimicrobial activity against Pseudomonas aeruginosa (minimum inhibitory concentrations of 4-32 μM). The overall findings suggest optimum routes to balancing peptide hydrophobicity and charge distribution that allow efficient penetration and disruption of the bacterial membranes without damage to mammalian (host) membranes.

The three-dimensional structure in dodecyl phosphocholine micelles of the 26-mer membrane-permeabilizing bacteriocin-like pheromone plantaricin A (PlnA) has been determined by use of nuclear magnetic resonance spectroscopy. The peptide was unstructured in water but became partly structured upon exposure to micelles. An amphiphilic alpha-helix stretching from residue 12 to 21 (possibly also including residues 22 and 23) was then formed in the C-terminal part of the peptide, whereas the N-terminal part remained largely unstructured. PlnA exerted its membrane-permeabilizing antimicrobial activity through a nonchiral interaction with the target cell membrane because the d-enantiomeric form had the same activity as the natural l-form. This nonchiral interaction involved the amphiphilic alpha-helical region in the C-terminal half of PlnA because a 17-mer fragment that contains the amphiphilic alpha-helical part of the peptide had antimicrobial potency that was similar to that of the l- and d-enantiomeric forms of PlnA. Also the pheromone activity of PlnA depended on this nonchiral interaction because both the l- and d-enantiomeric forms of the 17-mer fragment inhibited the pheromone activity. The pheromone activity also involved, however, a chiral interaction between the N-terminal part of PlnA and its receptor because high concentrations of the l-form (but not the d-form) of a 5-mer fragment derived from the N-terminal part of PlnA had pheromone activity. The results thus reveal a novel mechanism whereby peptide pheromones such as PlnA may function. An initial nonchiral interaction with membrane lipids induces alpha-helical structuring in a segment of the peptide pheromone. The peptide becomes thereby sufficiently structured and properly positioned in the membrane interface, thus enabling it to engage in a chiral interaction with its receptor in or near the membrane water interface. This membrane-interacting mode of action explains why some peptide pheromones/hormones such as PlnA sometimes display antimicrobial activity in addition to their pheromone activity.

We compared the properties of two peptides of identical size and amino acid composition, Ac-(LKKL)(5)-NHEt and Ac-(KL)(10)-NHEt. Both are amphipathic, but only Ac-(LKKL)(5)-NHEt is a potent promoter of negative curvature. CD studies performed in the presence of lipids confirmed that under these conditions Ac-(LKKL)(5)-NHEt forms an alpha-helix, and Ac-(KL)(10)-NHEt adopts a beta structure. We studied their binding affinity by centrifugation and isothermal titration calorimetry techniques. The Ac-(LKKL)(5)-NHEt bound to zwitterionic and anionic liposomes, while Ac-(KL)(10)-NHEt interacted mainly with anionic liposomes. Ac-(LKKL)(5)-NHEt was more lytic than Ac-(KL)(10)-NHEt for zwitterionic palmitoyloleoylphosphatidylcholine (POPC) liposomes, and for liposomes composed of lipids extracted from either sheep or human erythrocytes (RBC). Both peptides had similar lytic and lipid mixing activities for liposomes containing anionic lipids. Both peptides were highly hemolytic, with Ac-(LKKL)(5)-NHEt active against sheep RBC and Ac-(KL)(10)-NHEt more active against human RBC. From their respective minimal effective concentrations (MECs) as antimicrobial agents, we judged Ac-(KL)(10)-NHEt to be 2 to 5-fold more potent than Ac-(LKKL)(5)-NHEt in media that contained physiological concentrations of NaCl. Notwithstanding, both peptides had MECs <1 microg/mL for Escherichia coli and Pseudomonas aeruginosa and <4 microg/mL for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. Although selectivity of antimicrobial peptides for bacterial membranes may result, in part, from the preferential display of anionic residues in these membranes, inability to interact with or bind to zwitterionic phospholipids offers no guarantee that the peptide will lack appreciable cytotoxicity for host cells.

We propose a (3, 2)D CT-HCCH-COSY experiment to rapidly collect the data and provide significant dispersion in the spectral region containing (13)C-(1)H cross peaks of CH(3) groups belonging to Ala, Ile, Leu, Met, Thr and Val residues. This enables one to carry out chemical shift based editing and grouping of all the (13)C-(1)H cross peaks of CH(3) groups belonging to Ala, Ile, Leu, Met, Thr and Val residues in fractionally (10%) (13)C-labelled proteins, which in turn aids in the sequence-specific resonance assignments in general and side-chain resonance assignments in particular, in any given protein. Further, we demonstrate the utility of this experiment for stereospecific assignments of the pro-R and pro-S methyl groups belonging to the Leu and Val residues in fractionally (10%) (13)C-labelled proteins. The proposed experiment opens up a wide range of applications in resonance assignment strategies and structure determination of proteins.

The complement system is a central component of host defense but can also contribute to the inflammation seen in pathological conditions. The C1s protease of the first complement component, the C1 complex, initiates the pathway. In this study we have elucidated the full specificity of the enzyme for the first time using a randomized phage display library. It was found that, aside from the crucial P(1) position, the S(3) and S(2) subsites (in that order) played the greatest role in determining specificity. C1s prefers Leu or Val at P(3) and Gly or Ala residues at P(2). Apart from the S(2)' position, which showed specificity for Leu, prime subsites did not greatly affect specificity. It was evident, however, that together they significantly contributed to the efficiency of cleavage of a peptide. A peptide substrate based on the top sequence obtained in the phage display validated these results and produced the best kinetics of any C1s substrate to date. The results allow an understanding of the active site specificity of the C1s protease for the first time and provide a basis for the development of specific inhibitors aimed at controlling inflammation associated with complement activation in adverse pathological situations.

Temperature-Dependent Antimicrobial Activity of Menhaden Fish Oil In Vitro and on Pet Food Kibbles against Salmonella

Haloganan: A novel antimicrobial peptide for treatment of wound infections

Multidrug-resistant bacteria are a global health problem. One of the last-resort antibiotics against Gram-negative bacteria is the cyclic lipopeptide colistin, displaying a flexible linker with a fatty acid moiety. The aim of the present project was to investigate the effect on antimicrobial activity of introducing fatty acid moieties of different lengths and in different positions in a cyclic peptide, S3(B), containing a flexible linker. The lipidated analogues of S3(B) were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis. Following assembly of the linear peptide by Fmoc solid-phase peptide synthesis, on-resin head-to-tail cyclization and fatty acid acylation were performed. The antimicrobial activity was determined against the ESKAPE pathogens, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. Furthermore, hemolytic activity was determined against human erythrocytes. A total of 18 cyclic lipopeptides were synthesized and characterized. It was found that introduction of fatty acids in positions next to the flexible linker was more strongly linked to antimicrobial activity. The fatty acid length altered the overall hydrophobicity, which was the driving force for both high antimicrobial and hemolytic activity. Peptides became highly hemolytic when carbon-chain length exceeded 10 (i.e., C10), overlapping with the optimum for antimicrobial activity (i.e., C8–C12). The most promising candidate (C8)5 showed antimicrobial activity corresponding to that of S3(B), but with an improved hemolytic profile. Finally, (C8)5 was further investigated in a time-kill experiment.

Mutations were introduced by oligonucleotide-directed mutagenesis into the cDNA of poliovirus type 1 (Mahoney) in the region coding for the first five amino acids (myristoylation signal) of the viral capsid protein precursor P1. The cDNAs were then transcribed in vitro and the properties of the transcripts carrying the mutations studied in vitro by translation in a reticulocyte lysate or in vivo upon transfection of primate cells. Mutation of amino acid residue number 5 (Ser5----Thr) did not affect the viral phenotype, whereas mutations of residues number 1 (Gly1----Arg), 2 (Ala2----Pro) or 5 (Ser5----Pro) prevented myristoylation of P1 and were lethal. However, delayed production of virus was occasionally observed as the result of reverse mutations, which were found to restore a functional myristoylation signal as well as a wild-type virus phenotype. Thus, the myristoylation signal of the poliovirus polyprotein can accommodate Ala, Ser, Thr or Leu residues at position 2 and Ser, Thr or Ala residues at position 5. Mutations that altered myristoylation of P1 and affected virus viability did not prevent replication of the viral RNA but severely impeded in vitro processing of P1. This suggests that myristoylation plays a role in poliovirus capsid protein assembly.

Amino acid compositions of carp kidney metallothionein

Background The leaves of Leea rubra contain an abundance of phenolic constituents and have medicinal uses as antipyretic and diaphoretic agents and are also used in the treatment of stomach ache, rheumatism, arthritis etc. In spite of the traditional uses, data on the scientific evaluation of the plant are not sufficient. So, the present study was designed to evaluate the protective role of the extract against oxidative damage to DNA and human erythrocytes as well as antitumor and antibacterial activities against some resistant bacteria. Methods The protective activity of the ethyl acetate fraction (EAF) of the extract was investigated by evaluating the inhibition of oxidative damage of pUC19 plasmid DNA as well as hemolysis and lipid peroxidation damage to human erythrocytes induced by 2,2′-azobis-2-amidinopropane (AAPH). Antitumor activity was assessed by evaluating the percentage inhibition of cell growth, morphological changes of Ehrlich's ascites carcinoma (EAC) cells, and hematological parameters. Antimicrobial activity was determined by the disc diffusion method against different resistant microorganisms. Results EAF effectively inhibited AAPH-induced oxidative damage to DNA because it can inhibit the transformation of the supercoiled form of plasmid DNA to open circular and further linear form. The oxidative hemolysis caused by AAPH in human erythrocytes was inhibited by EAF extract in a time-dependent manner, and the production of malondialdehyde (MDA) was significantly reduced, which indicates the prevention of lipid peroxidation. In antitumor assay, 76% growth of inhibition of EAC was observed compared with the control mice (p < 0.05) at a dose of 100 mg/kg body weight. Antimicrobial activity was evaluated against two pathogenic resistant microorganisms (Escherichia coli and Pseudomonas aeruginosa), and the highest antimicrobial activity was observed against Pseudomonas spp. Conclusion EAF may have great importance in preventing oxidative damage to DNA, erythrocytes, and other cellular components as well as can be a good candidate in cancer chemotherapy and treating infectious diseases caused by antibiotic-resistant bacteria.

Cationic antimicrobial peptides (AMPs) have attracted a great interest as novel class of antibiotics that might help in the treatment of infectious diseases caused by pathogenic bacteria. However, some AMPs with high antimicrobial activities are also highly hemolytic and subject to proteolytic degradation from human and bacterial proteases that limit their pharmaceutical uses. In this work a D-diastereomer of Pandinin 2, D-Pin2, was constructed to observe if it maintained antimicrobial activity in the same range as the parental one, but with the purpose of reducing its hemolytic activity to human erythrocytes and improving its ability to resist proteolytic cleavage. Although, the hydrophobic and secondary structure characteristics of L- and D-Pin2 were to some extent similar, an important reduction in D-Pin2 hemolytic activity (30-40 %) was achieved compared to that of L-Pin2 over human erythrocytes. Furthermore, D-Pin2 had an antimicrobial activity with a MIC value of 12.5 μM towards Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and two strains of Pseudomonas aeruginosa in agar diffusion assays, but it was half less potent than that of L-Pin2. Nevertheless, the antimicrobial activity of D-Pin2 was equally effective as that of L-Pin2 in microdilution assays. Yet, when D- and L-Pin2 were incubated with trypsin, elastase and whole human serum, only D-Pin2 kept its antimicrobial activity towards all bacteria, but in diluted human serum, L- and D-Pin2 maintained similar peptide stability. Finally, when L- and D-Pin2 were incubated with proteases from P. aeruginosa DFU3 culture, a clinical isolated strain, D-Pin2 kept its antibiotic activity while L-Pin2 was not effective.

To find candidates with high antimicrobial and low hemolytic activities, many gratisin (GR) analogues have been designed and synthesized. In the present account, we synthesized novel derivatives of GR having both the polycationic and fatty acyl groups, cyclo{-Val(1)-Orn(2)-Leu(3)-D-Phe(4)-Pro(5)-D-Lys(6)(X)-Val(7)-Orn(8)-Leu(9)-D-Phe(10)-Pro(11)-D-Lys(12)-} {X=-CO(CH(2))(6)CH(3) (1), -Lys-CO(CH(2))(6)CH(3) (2), -(Lys)(2)-CO(CH(2))(6)CH(3) (3), and -(Lys)(3)-CO(CH(2))(6)CH(3) (4)}, and examined the biological activities. Among them, we found that 2-4 have differential ionic interaction against the prokaryotic membrane and eukaryotic membrane. In other words, the dissociation with high antimicrobial activity and low hemolytic activity is caused by the addition of D-Lys(6)-{(Lys)(n)-CO(CH(2))(6)CH(3)} residues at position 6 of [D-Lys(6,12)]-GR. Our findings should be helpful in finding drug candidates with high antimicrobial activity and low hemolytic activity that are capable of combating microbial resistance.

Simple SummaryThe global crisis of increased mortality rates due to the emergence of antimicrobial resistance and cancers has increased researchers’ efforts to find new, potent solutions through implementing natural products in the pharmaceutical industry. The present investigation produced echinenone (yellowish-orange pigment) from Micrococcus lylae MW407006 with potent pharmacological activities. A response surface methodology statistical design was used to optimize the biomass production, pigment concentration, and antimicrobial activity. The Spearman correlation coefficient was assessed, which indicated a strong linear relationship between biomass production, pigment concentration, and antimicrobial activity. Nano-echinenone was physically synthesized through the ball-milling technique. The synthesized nano-echinenone showed higher pharmacological activities (antimicrobial, antioxidant, and antitumor activities) in comparison with the crude pigment. The significantly high selectivity index of the synthesized nano-echinenone proved its safety and paved the way for its possible use in the pharmaceutical industry.Bacterial pigments (e.g., melanin and carotenoids) are considered to be among the most important secondary metabolites due to their various pharmacological activities against cancer and microbial resistance. Different pigmented bacterial strains were isolated from soil samples from El Mahmoudiyah governance and screened for their antimicrobial activity. The most promising pigment producer was identified as Micrococcus lylae MW407006; furthermore, the produced pigment was identified as echinenone (β-carotene pigment). The pigment production was optimized through a central composite statistical design to maximize the biomass production, pigment concentration, and the antimicrobial activity. It was revealed that the most significant fermentation parameters were the glucose (as a carbon source) and asparagine (as a nitrogen source) concentrations. Nano-echinenone was synthesized using the ball milling technique, characterized, and finally assessed for potential antimicrobial, antioxidant, and antitumor activities. The data revealed that the synthesized nano-echinenone had higher antimicrobial activity than the crude pigment. The cytotoxic potency of echinenone and nano-echinenone was investigated in different cell lines (normal and cancer cells). The inhibition of cell proliferation and induction of cell death was observed in Caco-2 and Hep-G2 cells. The data proved that nano-echinenone is a suitable candidate for use as a safe antimicrobial and anti-hepatocellular-carcinoma agent.

&lt;p&gt;&lt;b&gt;The wettability of a surface is determined by two factors; the chemistry of the surface, which determines the inherent chemical hydrophilicity or hydrophobicity, and the physicality of the surface, whereby the surface roughness enhances and exaggerates the inherent hydrophilicity or hydrophobicity of the surface. Hence, controlling the wettability of a surface requires manipulation of both the chemical nature of the surface and the surface roughness. This research predominantly investigated controlling the wettability of surfaces through creation and manipulation of surface roughness, but also studied alterations of the chemical hydrophilicity and hydrophobicity of surfaces. These studies were performed using two different substrates, for different potential commercial applications. Wool fabrics were studied, in order to increase their hydrophobicity and produce a stain and water repellent wool fabric. 3D printed polymers were also investigated in order to create surfaces with useful wetting behaviours, including superhydrophobicity and anisotropic wetting.&lt;/b&gt;&lt;/p&gt; &lt;p&gt;First, wool fabrics were manipulated through physical processing alone, in order to increase the surface roughness. This relied on altering the arrangement of the micrometre–sized wool fibres in the fabrics and creating a more disordered array. This was achieved through hand–felting of the wool fabrics and was found to successfully increase the surface roughness of the fabrics, and hence increase the hydrophobicity. However, it was found that the increased felting led to detectable increases in the macro–scale roughness of the surface, reducing the soft hand feel of the wool fabrics. This effect is an undesirable property in the textiles industry and another physical process of shearing protruding wool fibres was investigated in order to reduce this macro–scale roughness and increase the softness of the fabric. However, this processes was found to also reduce the micro-scale roughness of the surface and hence, the hydrophobicity of the fabrics was also decreased. This decrease in hydrophobicity was exacerbated due to the increased chemical hydrophilicity of the surface due to the exposure of more fibre ends, which are hydrophilic. Hence, the hydrophobicity of the sheared fabrics was reduced to a level lower than the untreated fabrics. Therefore, the use of physical processes to increase the roughness of wool fabrics was found to be successful in increasing the hydrophobicity of the surfaces. However, as the soft texture of the wool fabrics was compromised, the fields of application of these fabrics was determined to be somewhat limited.&lt;/p&gt; &lt;p&gt;Subsequently, the hydrophobicity of wool fabrics was increased using chemical modifications to increase the surface roughness, while not compromising the soft texture of the fabrics themselves. In these investigations, the surface roughness was again increased, but on a smaller scale, through the functionalisation of wool fibres with inorganic particles on the micro- or nano-scale. Three types of particles were investigated, gold nanoparticles, Ag/AgCl particles and CaCO3 particles. The gold nanoparticle functionalisation successfully increased the surface roughness on a very small scale, but the concurrent increase in chemical hydrophilicity was found to outweigh this increased surface roughness and result in a net decrease in hydrophobicity of the functionalised fabrics. The Ag/AgCl and CaCO3 functionalisations both successfully increased the hydrophobicity of the wool fabrics. Of these treatments, the Ag/AgCl functionalisation was found to have the greatest impact on increasing the hydrophobicity and Ag/AgCl functionalised fabrics showed increased water repellency in practical tests. The Ag/AgCl functionalisation also renders the fabrics antimicrobial, due to the known antimicrobial activity of silver nanoparticles (present as silver nanodomains on AgCl microparticles in Ag/AgCl composites). Hence, these investigations led to the development of water and stain repellent, and antimicrobial wool fabrics with great potential for commercial applications.&lt;/p&gt; &lt;p&gt;Polymer substrates and 3D printing were used in order to create surfaces with specific surface roughness patterns and changeable surface chemistry for the purpose of creating surfaces with controlled wetting behaviours. For these investigations, a fused deposition modelling (FDM) 3D printing process was used to facilitate rapid prototyping of surface roughness pattern designs and allow for optimisation of these designs. These surfaces were printed using acrylonitrile butadiene styrene (ABS). Firstly, a highly hydrophobic surface was designed. An array of droplet-shaped structures was printed onto a flat substrate. These droplet structures were on the size scale of a few hundred micrometres, but the process of melting and extruding the polymer in the printing process also gave the surface some degree of smaller scale roughness. This roughness was then increased further, by functionalising the surface with silver nanoparticles. This functionalisation was achieved in-situ and also rendered the surface antimicrobial. Due to the inherent chemical hydrophilicity of silver, the surfaces also required a subsequent treatment with a fluoroalkyl silane (FAS) to render them chemically hydrophobic. While these surfaces did not achieve the typically defined conditions of superhydrophobicity, they were found to be highly hydrophobic and were also antimicrobial. Hence, these surfaces were deemed to be potentially useful for commercial applications in healthcare industries.&lt;/p&gt; &lt;p&gt;FDM 3D printing was also used to create a surface with anisotropic wetting behaviour. This was achieved through printing a surface with 1-dimensional roughness in the form of protruding lines extending in one direction across the surface plane. These surfaces were printed using ABS or polylactic acid (PLA). Different shapes, sizes and spacings of these lines were investigated and these parameters were optimised. The resulting surface displayed significant anisotropy in its wetting behaviour with highly hydrophobic behaviour observed in the direction parallel to the protruding lines, but significantly more hydrophilic behaviour in the perpendicular direction. Hence, this surface allowed the movement of water in one direction across the surface but hindered movement in the perpendicular direction. This surface was then treated with a FAS, and subsequently treated with ion implantation in selected areas, using varied fluence of Ar+ or C+ ions. The FAS treatment served to change the surface chemistry from that of the printed polymer to a generic hydrophobic chemistry, using a surface treatment that is applicable to many different substrate materials. The ion implantation treatment significantly decreased the hydrophobicity of the treated areas of the FAS functionalised surface; in fact the ion implantation treatment was found to render the selected areas formally hydrophilic. This allowed for the design of a surface with both hydrophilic and hydrophobic areas, that allowed for movement of water in only one direction across the surface. Such a surface has potential applications in water harvesting, utilising the hydrophilic areas for condensation of water from the air, and the hydrophobic areas for movement of this water across the surface toward a collection tank.&lt;/p&gt; &lt;p&gt;The research carried out herein led to the successful increase in hydrophobicity of wool fabrics using both physical and chemical processes. 3D printing was also successfully utilised to create two different surfaces with controlled wetting behaviours. Firstly, a surface with high hydrophobicity and antimicrobial activity was produced. And secondly, a surface with anisotropic wetting behaviour and areas of hydrophilicity and areas of hydrophobicity was achieved. Possible applications of these surfaces were considered.&lt;/p&gt;