Antimicrobial Polypeptides Research Articles

Structural design and antimicrobial properties of polypeptides and saccharide-polypeptide conjugates.

The problems of microbial infections and the emergence of drug-resistant microbes are increasingly serious, causing countless loss of lives and economic loss. The discovery and study of host defense peptides opened a new avenue in developing antimicrobial regents, and have attracted a lot of attention in recent years. Compared with natural host defense peptides, synthetic antimicrobial polypeptides can be conveniently synthesized in large scale and with low cost. Furthermore, saccharide-polypeptide conjugates have been valued for their optimal effect on antimicrobial properties and biocompatibility. In this review article, we provide an overview of the development and progress of antimicrobial polypeptides and saccharide-polypeptide conjugates regarding their structural design, biological functions and antimicrobial mechanism. By pointing out the challenges, we also provide future prospects of this research field from our perspectives.

Antimicrobial Polypeptides: Molecular Architecture and Charging Effects Enhance the In Vitro and In Vivo Performance of Multi‐Arm Antimicrobial Agents Based on Star‐Shaped Poly(l‐lysine) (Adv. Therap. 12/2019)

Antimicrobial Polypeptides: Molecular Architecture and Charging Effects Enhance the In Vitro and In Vivo Performance of Multi‐Arm Antimicrobial Agents Based on Star‐Shaped Poly(<scp>l</scp>‐lysine) (Adv. Therap. 12/2019)

Bacterial Virus Lambda Gpd-Fusions to Cathelicidins, α- and β-Defensins, and Disease-Specific Epitopes Evaluated for Antimicrobial Toxicity and Ability to Support Phage Display.

We showed that antimicrobial polypeptides, when translated as gene fusions to the bacteriophage lambda capsid decoration protein gpD, formed highly toxic molecules within E. coli, suggesting that they can retain their antimicrobial activity conformation when fused to gpD. These include gpD-fusions to human and porcine cathelicidins LL37 and PR39, β-defensins HBD3 and DEFB126-Δ (deleted for its many COOH-terminal glycosylation sites), and α-defensin HD5. Antimicrobial toxicity was only observed when the peptides were displayed from the COOH-terminal, and not the NH2-terminal end, of gpD. This suggests that COOH-terminal displayed polypeptides of gpD-fusions can more readily form an active-state conformation than when they are displayed from the NH2-terminal end of gpD. The high toxicity of the COOH-displayed gpD-defensins suggests either that the fused defensin peptides can be oxidized, forming three correct intramolecular disulfide bonds within the cytosol of bacterial cells, or that the versions without disulfide bonds are highly toxigenic. We showed the high efficiency of displaying single epitope 17 amino-acid fusions to gpD on LDP (lambda display particles), even when the gpD-fusion protein was toxic. The efficient formation of high display density LDP, displaying a single disease specific epitope (DSE), suggests the utility of LDP-DSE constructs for use as single epitope vaccines (SEV).

Coordination-Assisted Self-Assembled Polypeptide Nanogels to Selectively Combat Bacterial Infection.

In the present scenario, the invention of bacteria-selective antimicrobial agent comprising negligible toxicity and hemolytic effect is a great challenge. To surmount this challenge, here, a series of polypeptide nanogels (PNGs) have been fabricated by a coordination-assisted self-assembly of a mannose-conjugated antimicrobial polypeptide, poly(arginine-r-valine)-mannose (poly(Arg-r-Val)-M2), with Zn2+ ions. The fabricated PNGs are spherical in shape with a unique structural appearance similar to that of Taxus baccata fruits. PNGs, with a unique structural arrangement and threshold surface charge density, selectively interact with the bacterial membrane and exhibit potent antimicrobial activity, as reflected in their lower minimum inhibitory concentration values (varies from 2 to 16 μg/mL). PNGs show a remarkably high binding constant, 6.02 × 105 M-1 (from isothermal titration calorimetry, ITC), with the bacterial membrane which manifests its potent bactericidal effect. PNGs are nontoxic against mammalian and red blood cells as reflected from their higher cell viability and insignificant hemolytic effect. PNGs are taken up by the bacterial membrane and selectively undergo structural deformation (scrutinized by ITC) followed by an exposure of free poly(Arg-r-Val)-M2 molecules. The free poly(Arg-r-Val)-M2 molecules are enforced to lyse the bacterial membrane (visualized by cryo-transmission electron microscopy) followed by the diffusion of the cytoplasmic component out of the membrane which culminates in the final death of the bacterium.

Ocins database: a database of bug-busters from Bifidobacterium, Lactobacillus, and Enterococcus.

The ocins are antimicrobial polypeptides produced by probiotic microbes, such as Lactobacillus , Enterococcus , Streptococcus , Leuconostoc and Bifidobacterium . They are produced in response to stress and for the self-defense of the bacterium. It is indispensable to understand their mechanistic characteristics, structures, and functions, if the food industry is to reduce contamination levels and produce germfree foods. Databases of the ocins that are readily accessible to the food industry are scarce, but urgently required. Therefore, we established a very useful, unique, and a simple ocin database, which not merely provides information about ocins, but also directs their utilisation in the food industry. The database includes information about each ocin, its amino acid sequence, molecular weight, and isoelectric point. The database also possesses all the currently known ocin (probiotic origin only) sequences and structures, target organisms, and relevant to food industries (aqua culture, dairy and meat industries), which is hard to obtain in other databases. The database is free for public and accessed at http://ocins.cftri.com/ocins/.

Design and Synthesis of Biocompatible, Hemocompatible, and Highly Selective Antimicrobial Cationic Peptidopolysaccharides via Click Chemistry.

Despite the excellent antimicrobial activity, the high toxicity and low selectivity of cationic antimicrobial peptides (AMPs) and their synthetic analogues impede their biomedical applications. In this study, we report a series of cationic peptidopolysaccharides synthesized by thiol-ene click chemistry of grafting antimicrobial polypeptides, methacrylate-ended poly(lysine- random-phenylalanine) (Me-K nF m), onto a thiolated polysaccharide (dextran, Dex) backbone. Their copolymers (Dex- g-K nF m) exhibit potent broad-spectrum antibacterial and antifungal activity against Gram-negative bacteria ( Pseudomonas aeruginosa and Escherichia coli), Gram-positive bacteria [methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis], and fungi ( Candida albicans) with minimal inhibitory concentrations in the range of 31.25-500 μg·mL-1. More importantly, Dex- g-K nF m copolymers did not induce drug resistance of MRSA up to 17 passages. In addition, these copolymers have an improved hemocompatibility and exhibit good in vitro biocompatibility with murine myoblast (C2C12) cells. Among the synthesized peptidopolysaccharides, DexL- g-K12.5F12.5-50%, as the optimal agent, displayed a selectivity more than 200 times the maximum value of polypeptide molecules. Furthermore, a strong in vivo antimicrobial efficacy with a log reduction above 3 in a mouse bacterial sepsis model has been obtained. These excellent biological properties present a promising prospect for Dex- g-K nF m in biomedical applications.

Computational protein design of bacteriocins based on structural scaffold of aureocin A53

Bacteriocins are highly potent anti-microbial polypeptides and proteins produced by bacteria. They are rapidly degraded in the environment after their use, due to their proteinaceous nature. Some bacteriocins are used as preservatives in foods. Native and engineered bacteriocins are of potential interest as replacements for conventional antibiotics that are losing their efficacy due to the development of antibiotic-resistant strains. Aureocin A53 is a class II bacteriocin. It is a broad spectrum microbicide, with demonstrated ability to inhibit the growth of methicillin-resistant Staphylococcus aureus. Validated computational protein design tools have been used for reengineering of the aureocin A53 sequence to produce novel sequence variants of the bacteriocins aureocin A53 and lacticin Q. The novel proteins are expected to possess an altered spectrum of bactericidal specificity and potency.

Analiza vrednosti vitamina D u serumu u akutnom virusnom bronhiolitisu

Introduction: Bronchiolitis is an acute viral disease of lower respitatory pathways in children under 2 years of age. It is manifested by coughing, whistling and refusing food intake. The most common cause of bronchiolitis is a respiratory syncytial virus. Vitamin D is a steroid hormone which is synthesized in the skin after sun exposure or food intake. With discovery of Vitamin D receptors and hydroxylated enzymes in immune cells, a potential role for Vitamin D in maintaining immune homeostasis was seen. It has been established that Vitamin D induces a change of the antimicrobial polypeptide, cathelicidin, which has both antibacterial and antiviral roles. Objective: Determine whether the serum Vitamin D levels in acute viral bronchiolitis patients from Central Kosovo is lowered. Methods: This prospective study included 34 children aged up to 2 years, that have been diagnosed with the heavy form of acute viral bronchiolitis. Blood was drawn from all subjects to analyze serum Vitamin D levels. Results:Average levels of Vitamin D, measured in all subjects, was 35.5 ng/ml. After Vitamin D levels stratification, 62% of the subjects had normal levels of serum Vitamin D, while 38% of the subjects had lower levels of serum Vitamin D. 15% of children had lower levels than 20 ng/ml. With children of ages up to 12 months there was a statistical significance of negative direction between Vitamin D serum levels and body height (r=-0.683, p=0.010) and between Vitamin D serum levels and body weight (r=-0.725, p=0.005). With children of ages up to 24 months there was only a statistical significance of negative direction between Vitamin D levels and body height (r=-0.540, p=0.011). Conclusion: Our research confirmed that children with acute viral bronchiolitis have a significant deficiency of Vitamin D, although it's a required addition to nutrition for children under the age of 2 years.

Biocompatible antimicrobial electrospun nanofibers functionalized with ε-poly-l-lysine

The antimicrobial polypeptide ε-poly(l-lysine) (ε-PL) was electrostatically incorporated to poly(acrylic acid) (PAA)/poly(vinyl alcohol) (PVA) electrospun nanofibers. ε-PL loading and distribution was assessed by infrared spectra, ζ-potential measurements and the primary amino reactive dye fluorescamine. Functionalized fibers with 485 ± 140 nm diameter, could be loaded with 0.57–0.74 g ε-PL (g dressing)−1 that released at a constant rate of 5.4 ± 2.8 mg ε-PL (g dressing day)−1. Such a dressings resulted in two orders of magnitude lower bacterial colonization than non-functionalized PAA-PVA after 14 days of incubation. Bacterial impairment was attributed to the damage of cell membranes and the formation of intracellular reactive oxygen species. ε-PL functionalized nanofibers did not display cytotoxicity to human corneal epithelial cells, HCEpC, in 24 h MTT assays. However, the viability of rapidly growing tumoral HeLa cells decreased >50% under the same conditions. The prepared biocompatible nanofibrous dressings with durable antibacterial activity show potential application as wound dressings and other biomedical uses.

Theoretical Insights into the Interactions between Star-Shaped Antimicrobial Polypeptides and Bacterial Membranes.

A structurally nanoengineered antimicrobial polypeptide consisting of lysine and valine residues is a new class of antimicrobial agent with superior antibacterial activity against multidrug-resistant bacteria and low toxicity toward mammalian cells. Utilizing coarse-grained models, we studied the interactions of microbial cytoplasmic membranes with polypeptides of either (K2V1)5 (star-KV) or CM15 (star-CM15). Our computational results verify the low toxicity of polypeptides of (K2V1)5 toward the dipalmitoyl phosphatidylcholine bilayer. This low toxicity is demonstrated to originate from weakened hydrophobicity combined with its random coil conformation for (K2V1)5 because of the highly abundant valine residues, compared with the typical antimicrobial peptides, such as CM15. In the interactions with a palmitoyl-oleoyl-phosphatidylethanolamine/palmitoyl-oleoyl-phosphatidylglycerol bilayer, star-KV has greater ability in phase separation and generation of phase boundary defects not only in lipid redistribution but also in lateral dynamic movements, although both star-KV and star-CM15 can extract the phosphatidylglycerol lipids and purify the phosphatidylethanolamine lipids into continuum domains. We suggest that the polypeptide of (K2V1)5 can nondisruptively kill bacteria by hampering bacterial metabolism through reorganizing lipid domain distribution and simultaneously "freezing" lipid movement.

Studies on the Production of Broad Spectrum Antimicrobial Compound Polypeptide (Actinomycins) and Lipopeptide (Fengycin) from Streptomyces sp. K-R1 Associated with Root of Abutilon indicum against Multidrug Resistant Human Pathogens

Endophytic actinomycetes associated with medicinal plants of Chhattisgarh are rich source of novel antimicrobial compounds. The aim of this study was to evaluate the production of antibiotics by endophytic Streptomyces sp. K-R1 associated with root of Abutilon indicum against multidrug resistant human pathogens. The 16S rRNA sequence of the isolate K-R1 has been identified as Streptomyces parvulus NBRC 13193T (AB184326) with the GenBank database (accession number KY777591). Streptomyces parvulus K-R1 grown under submerged fermentation condition optimized by central composite design (glucose—11.25 g/l, soybean meal 12.17 g/l, calcium carbonate—1.47 g/l, sodium chloride—11.25 g/l; pH 7.18 at 30.51 °C with 4% seed inoculum for 10 days of incubation) exhibited highest activity against multidrug resistant Staphylococcus aureus JNMC-3, Staphylococcus epidermidis JNMC-4, Klebsiella pneumoniae MTCC-3384, Klebsiella pneumoniae JNMC-6, Pseudomonas aeruginosa MTCC-741, Proteus vulgaris JNMC-7, Candida albicans MTCC-183 and Aspergillus niger MTCC-872. The structures of the active compounds were elucidated by UV–Vis spectroscopy, 1H and 13C NMR, FTIR and ESIMS. Actinomycin D, actinomycin X0β and fengycin were detected in crude extracts and major components were eluted by HPLC at 10.63, 10.88 and 6.3 min respectively. In this case, a high yield of antibiotics was achieved with Streptomyces parvulus K-R1 fermented in glucose soybean meal broth media with a production of 240 mg/l after 5 days, 290 mg/l after 8 days and 340 mg/l after 11 days, which can be used in industrial fermentation process to obtain high yields. To the author’s best knowledge, it is the first report of fengycin production by Streptomyces parvulus.

Co-occurrence of mcr-1, mcr-4 and mcr-5 genes in multidrug-resistant ST10 Enterotoxigenic and Shiga toxin-producing Escherichia coli in Spain (2006-2017)

Colistin is an antimicrobial polypeptide commonly employed for controlling and treating neonatal and post-weaning diarrhoea (PWD) diseases caused by Enterotoxigenic and Shiga toxin-producing Escherichia coli (ETEC and STEC). The plasmid-mediated colistin resistance gene, mcr-1 was first described in late 2015 and, since then, multiple studies have reported its global distribution. In addition, five different mcr genes have been identified. The aim of this study was to characterise the colistin-resistant E. coli clonal groups implicated in PWD in farms of intensive pig production. Of 186 ETEC and STEC isolated in Spain from 2006 to 2017, 76.9% showed resistance to colistin. Of those, 102 were mcr-4 carriers, 37 mcr-1 and 5 mcr-5, with co-occurrence of mcr-1/mcr-4, mcr-1/mcr-5 and mcr-4/mcr-5 in five isolates. Three different mcr-4 variants were detected, including the new mcr-4.4 and mcr-4.5 described here. Interestingly, the clonal group ST10-A (CH11-24) appears to be primarily responsible for the spread of mcr-4. In summary, our results show that the pig industry is an important reservoir of colistin-resistant E. coli, carriers of other additional risk genes, such as blaESBL. These food-producing animals might be spreading a cocktail of multiple resistances, posing a worrisome threat to human health.

Antimicrobial and anticancer potential of low molecular weight polypeptides extracted and characterized from leaves of Azadirachta indica

Low molecular weight antimicrobial polypeptides were extracted and purified from the young fresh leaves of Azadirachta indica (neem). The total protein extracted was precipitated with 15% TCA-Acetone. The total purified proteins yielded from the two extraction methods were 122.33±2.21 and 115.09±1.88mg/g of the total fresh weight. The SDS-PAGE analysis identified the presence of eight low molecular weight polypeptide bands. The antimicrobial activity of the resolved bands was detected by Polyacrylamide gel-Agar overlay diffusion assay (PAG-ADA). Their broad-spectrum bactericidal activity was confirmed using the same technique and found three low molecular weight bands from 11 to 14kDa collectively exhibiting superior bactericidal activities against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermis, Enterococcus faecalis, Pseudomonas aeruginosa and fungicidal activity against Candida tropicalis. The FTIR spectrum of the protein bands depicted the presence of hydroxyl and carbonyl groups in the protein bands. These polypeptides were characterized by MALDI-TOF/TOF analysis. Further, the purified protein extract was found to be active against HELA, BT-549 and Neuro-2a cell lines with IC50 value of 74.03±2.31, 64.82±1.64, 238.32±2.12 and 109.94±2.96, 59.61±0.75 for 24h and 48h, respectively. The results of present study indicate that these polypeptides exhibit broad spectrum antimicrobial and anticancer activity and can therefore be explored for their therapeutic potential.

The Saposin-Like Protein AplD Displays Pore-Forming Activity and Participates in Defense Against Bacterial Infection During a Multicellular Stage of Dictyostelium discoideum.

Due to their archaic life style and microbivor behavior, amoebae may represent a source of antimicrobial peptides and proteins. The amoebic protozoon Dictyostelium discoideum has been a model organism in cell biology for decades and has recently also been used for research on host-pathogen interactions and the evolution of innate immunity. In the genome of D. discoideum, genes can be identified that potentially allow the synthesis of a variety of antimicrobial proteins. However, at the protein level only very few antimicrobial proteins have been characterized that may interact directly with bacteria and help in fighting infection of D. discoideum with potential pathogens. Here, we focus on a large group of gene products that structurally belong to the saposin-like protein (SAPLIP) family and which members we named provisionally Apls (amoebapore-like peptides) according to their similarity to a comprehensively studied antimicrobial and cytotoxic pore-forming protein of the protozoan parasite Entamoeba histolytica. We focused on AplD because it is the only Apl gene that is reported to be primarily transcribed further during the multicellular stages such as the mobile slug stage. Upon knock-out (KO) of the gene, aplD− slugs became highly vulnerable to virulent Klebsiella pneumoniae. AplD− slugs harbored bacterial clumps in their interior and were unable to slough off the pathogen in their slime sheath. Re-expression of AplD in aplD− slugs rescued the susceptibility toward K. pneumoniae. The purified recombinant protein rAplD formed pores in liposomes and was also capable of permeabilizing the membrane of live Bacillus megaterium. We propose that the multifarious Apl family of D. discoideum comprises antimicrobial effector polypeptides that are instrumental to interact with bacteria and their phospholipid membranes. The variety of its members would allow a complementary and synergistic action against a variety of microbes, which the amoeba encounters in its environment.

Isolation, Characterization, and Antimicrobial Activity of Extracellular Polypeptides from Diazotrophic Strain Rhizobium radiobacter

Antimicrobial polypeptide fractions were isolated from Rhizobium radiobacter culture medium and purified. Mass-spectrometric analysis showed that the most active biocidal peptide fraction contained 24 components with molecular masses in the range 1171.14–3626.78 Da. The isolated peptide fractions contained 60 and 56% protein, respectively, and inhibited the growth of test pathogens Candida albicans (CA) and Escherichia coli (EC). These polypeptides demonstrated protective properties.

Transferability of MCR-1/2 Polymyxin Resistance: Complex Dissemination and Genetic Mechanism.

Polymyxins, a group of cationic antimicrobial polypeptides, act as a last-resort defense against lethal infections by carbapenem-resistant Gram-negative pathogens. Recent emergence and fast spread of mobilized colistin resistance determinant mcr-1 argue the renewed interest of colistin in clinical therapies, threatening global public health and agriculture production. This mini-review aims to present an updated overview of mcr-1, covering its global dissemination, the diversity of its hosts/plasmid reservoirs, the complexity in the genetic environment adjacent to mcr-1, the appearance of new mcr-like genes, and the molecular mechanisms for mobilized colistin resistance determinant 1/2 (MCR-1/2).

Selective killing of Helicobacter pylori with pH-responsive helix–coil conformation transitionable antimicrobial polypeptides

Current clinical treatment of Helicobacter pylori infection, the main etiological factor in the development of gastritis, gastric ulcers, and gastric carcinoma, requires a combination of at least two antibiotics and one proton pump inhibitor. However, such triple therapy suffers from progressively decreased therapeutic efficacy due to the drug resistance and undesired killing of the commensal bacteria due to poor selectivity. Here, we report the development of antimicrobial polypeptide-based monotherapy, which can specifically kill H. pylori under acidic pH in the stomach while inducing minimal toxicity to commensal bacteria under physiological pH. Specifically, we designed a class of pH-sensitive, helix-coil conformation transitionable antimicrobial polypeptides (HCT-AMPs) (PGA)m-r-(PHLG-MHH)n, bearing randomly distributed negatively charged glutamic acid and positively charged poly(γ-6-N-(methyldihexylammonium)hexyl-l-glutamate) (PHLG-MHH) residues. The HCT-AMPs showed unappreciable toxicity at physiological pH when they adopted random coiled conformation. Under acidic condition in the stomach, they transformed to the helical structure and exhibited potent antibacterial activity against H. pylori, including clinically isolated drug-resistant strains. After oral gavage, the HCT-AMPs afforded comparable H. pylori killing efficacy to the triple-therapy approach while inducing minimal toxicity against normal tissues and commensal bacteria, in comparison with the remarkable killing of commensal bacteria by 65% and 86% in the ileal contents and feces, respectively, following triple therapy. This strategy renders an effective approach to specifically target and kill H. pylori in the stomach while not harming the commensal bacteria/normal tissues.

Macromol. Biosci. 9/2017

Back Cover: Acne caused by Propionibacterium acnes is treated with antibiotics but effectiveness is often compromised by resistance and development of “superbugs”. An alternative antimicrobial polycation without resistance buildup is described. The results point to a promising pathway for polycation antimicrobial polypeptide (AMP) mimics against P. acnes. This is reported by Sithara S. Nair, Olga Y. Zolotarskaya, Matthew J. Beckwith, Dennis E. Ohman and Kenneth J. Wynne, article number 1700090.

Effect of guar gum conjugation on functional, antioxidant and antimicrobial activity of egg white lysozyme

This study was undertaken to analyze the effect of conjugation of egg-white lysozyme with guar gum. Lysozyme is an antimicrobial polypeptide that can be used for food preservation. Its antibacterial activity is limited to gram positive bacteria. Conjugation with polysaccharides like guar gum may broaden its activity against gram negatives. Conjugate was developed through Maillard reaction. Assays carried out included sugar estimation, SDS-PAGE, GPC, color, FT-IR, DSC, thermal stability, solubility, emulsifying, foaming and antioxidant activity. In addition, antimicrobial activity of the conjugate was determined against two gram positive (Staphyllococcus aureus and Enterococcus) and two gram negative pathogens (E. coli and Salmonella). Results showed higher functional properties of lysozyme-guar gum conjugate. The antioxidant properties increased from 2.02–35.80% (Inhibition of DPPH) and 1.65–4.93AAE/g (reducing power) upon guar gum conjugation. Conjugate significantly inhibited gram negative bacteria and the antibacterial activity also increased significantly against gram positive pathogens.

Bacteria‐Assisted Activation of Antimicrobial Polypeptides by a Random‐Coil to Helix Transition

AbstractThe application of antimicrobial peptides (AMPs) is largely hindered by their non‐specific toxicity against mammalian cells, which is usually associated with helical structure, hydrophobicity, and charge density. A random coil‐to‐helix transition mechanism has now been introduced into the design of AMPs, minimizing the toxicity against mammalian cells while maintaining high antimicrobial activity. By incorporating anionic phosphorylated tyrosine into the cationic polypeptide, the helical structure of AMPs was distorted owing to the side‐chain charge interaction. Together with the decreased charge density, the AMPs exhibited inhibited toxicity against mammalian cells. At the infectious site, the AMPs can be activated by bacterial phosphatase to restore the helical structure, thus contributing to strong membrane disruptive capability and potent antimicrobial activity. This bacteria‐activated system is an effective strategy to enhance the therapeutic selectivity of AMPs.