Isolation Of Antimicrobial Peptides Research Articles

Diversity of Antimicrobial Peptides in Silkworm.

Antimicrobial resistance is a phenomenon that the present-day world is witnessing that poses a serious threat to global health. The decline in the development of novel therapeutics over the last couple of decades has exacerbated the situation further. In this scenario, the pursuit of new alternative therapeutics to commonly used antibiotics has gained predominance amongst researchers across the world. Antimicrobial peptides (AMPs) from natural sources have drawn significant interest in the recent years as promising pharmacological substitutes over the conventional antibiotics. The most notable advantage of AMPs is that microorganisms cannot develop resistance to them. Insects represent one of the potential sources of AMPs, which are synthesized as part of an innate immune defence against invading pathogens. AMPs from different insects have been extensively studied, and silkworm is one of them. Diverse classes of AMPs (including attacins, cecropins, defensins, enbocins, gloverins, lebocins and moricins) were identified from silkworm that exhibit antimicrobial property against bacteria, fungi and viruses, indicating their potential therapeutic benefits. This review briefs about the immune responses of silkworm to invading pathogens, the isolation of AMPs from silkworms, AMPs reported in silkworms and their activity against various microorganisms.

Isolation of Antimicrobial Peptides (AMPs) From Different Sources: A Review

Antimicrobial peptides (AMPs) are small peptide molecules produced by a wide range of species, from bacteria to higher vertebrates, as a part of the innate immune system. These naturally produced peptides fight against intrusive microbes like viruses, bacteria, and fungi. AMPs are produced in various body parts by different organisms, such as skin secretion of amphibians; leaf, stem, fruits, or whole parts of plants; glandular secretion of mammals; the venom of reptiles and arthropods, etc. Due to this source variation, there is no universal method for the isolation, i.e., extraction and purification of the AMPs. Therefore, several techniques have been established to extract and purify these natural peptides from various sources. Generally, solid-phase extraction and chemical extraction techniques are the most common methods used for the extraction of AMPs. For purification and identification purposes, chromatography, gel electrophoresis, filtration, Edman degradation, circular dichroism, mass spectrometry, etc. are mainly adopted. In this review, we provide a brief discussion on the isolation process of AMPs based on their sources. This study would be helpful in isolating newer AMPs that could be used as potential antimicrobial agents. Bangladesh Pharmaceutical Journal 26(1): 99-111, 2023 (January)

Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag.

Antimicrobial resistance is an increasing global threat, demanding new therapeutic biomolecules against multidrug-resistant bacteria. Antimicrobial peptides (AMPs) are promising candidates for a new generation of antibiotics, but their potential application is still in its infancy, mostly due to limitations associated with large-scale production. The use of recombinant DNA technology for the production of AMPs fused with polymer tags presents the advantage of high-yield production and cost-efficient purification processes at high recovery rates. Owing to their unique properties, we explored the use of an elastin-like recombinamer (ELR) as a fusion partner for the production and isolation of two different AMPs (ABP-CM4 and Synoeca-MP), with an interspacing formic acid cleavage site. Recombinant AMP-ELR proteins were overproduced in Escherichia coli and efficiently purified by temperature cycles. The introduction of a formic acid cleavage site allowed the isolation of AMPs, resorting to a two-step methodology involving temperature cycles and a simple size-exclusion purification step. This simple and easy-to-implement purification method was demonstrated to result in high recovery rates of bioactive AMPs. The minimum inhibitory concentration (MIC) of the free AMPs was determined against seven different bacteria of clinical relevance (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two Burkholderia cenocepacia strains), in accordance with the EUCAST/CLSI antimicrobial susceptibility testing standards. All the bacterial strains (except for Pseudomonas aeruginosa) were demonstrated to be susceptible to ABP-CM4, including a resistant Burkholderia cenocepacia clinical strain. As for Synoeca-MP, although it did not inhibit the growth of Pseudomonas aeruginosa or Klebsiella pneumoniae, it was demonstrated to be highly active against the remaining bacteria. The present work provides the basis for the development of an efficient and up-scalable biotechnological platform for the production and purification of active AMPs against clinically relevant bacteria.

Isolation of Antimicrobial Peptide from Food Protein Hydrolysates: An Overview

Antimicrobial resistance (AMR) is one of the most serious public health threats that results mostly from the selective pressure exerted by antibiotic use and abuse. AMR has become a major problem with global human deaths due to antibiotic resistant infections predicted to reach 10 million by 2050. Antimicrobial peptides (AMPs) has been discovered to have capabilities to kill microorganisms and can take other roles as an alternative to antibiotics which is favorable to the need of minimizing the usage of antibiotics as they lead to the increasing of AMR. AMP can be found naturally in almost all domains of life as part of the innate immune system to combat virus, bacteria, fungi and even cancer cells. It can also be extracted from food proteins using enzymatic hydrolysis. High antimicrobial properties/activities are depend on the degree of hydrolysis (DH) of peptides, peptide source/origin, and type of enzyme used for the hydrolysis process. There are several other variables that can be manipulated to optimum condition to obtain high DH. Variables such as temperature, pH, enzyme concentration and hydrolysis time have proven to bring impact to the DH of peptides. Other bioactive peptides that have been discovered during the process have great potential to bring benefit in medicinal and nutraceutical areas.

Recent Advances in the Development of Antimicrobial Peptides (AMPs): Attempts for Sustainable Medicine?

Since the first isolation of antimicrobial peptides (AMPs) they have attracted extensive interest in medicinal chemistry. However, only a few AMP-based drugs are currently available on the market. Despite their effectiveness, biodegradability, and versatile mode of action that is less likely to induce resistance compared to conventional antibiotics, AMPs suffer from major issues that need to be addressed to broaden their use. Notably, AMPs can lack selectivity leading to side effects and cytotoxicity, and also exhibit in vivo instability. Several strategies are being actively considered to overcome the limitations that restrain the success of AMPs. In the current work, recent strategies reported for improving AMPs in the context of drug design and delivery were surveyed, and also their possible impact on patients and the environment was assessed. As a major advantage AMPs possess an easily tunable skeleton offering opportunities to improve their properties. Strategic structural modifications and the beneficial properties of cyclic or branched AMPs in term of stability have been reported. The conjugation of AMPs with nanoparticles has also been explored to increase their in vivo stability. Other techniques such as the coupling of AMPs with specific antibodies aim to increase the selectivity of the potential drug towards the target. These strategies were evaluated for their effect on the environment highlighting green technologies. Although further research is needed taking into account both environmental and human health consequences of novel AMPs, several of these compounds are promising drug candidates for use in sustainable medicine.

Proteomics assisted profiling of antimicrobial peptide signatures from black pepper (Piper nigrum L.).

Plant antimicrobial peptides are the interesting source of studies in defense response as they are essential components ofinnate immunity which exert rapid defense response. In spite of abundant reports on the isolation of antimicrobial peptides (AMPs) from many sources, the profile of AMPs expressed/identified from single crop species under certain stress/physiological condition is still unknown. This work describes the AMP signature profile of black pepper and their expression upon Phytophthora infection using label-free quantitative proteomics strategy. The differential expression of 24 AMPs suggests that a combinatorial strategy is working in the defense network. The 24 AMP signatures belonged to the cationic, anionic, cysteine-rich and cysteine-free group. As the first report on the possible involvement of AMP signature in Phytophthora infection, our results offer a platform for further study on regulation, evolutionary importance and exploitation of theses AMPs as next generation molecules against pathogens.

Purification, characterization and application of a novel antimicrobial peptide from Andrias davidianus blood.

This is the pioneer study on screening and isolation of antimicrobial peptide from the blood of Andrias davidianus. Here, we have developed a novel method by combining magnetic liposomes adsorption with reversed-phase high-performance liquid chromatography to purify and screen the antimicrobial peptides. From this screen, we identified a novel antimicrobial peptide which we name as andricin B. Andricin B is unique as it checks the growth of both Gram-positive and Gram-negative bacteria as well as few fungal species.

Isolation of antimicrobial peptides from Apis florae and Apis carnica in Saudi Arabia and investigation of the antimicrobial properties of natural honey samples

As part of the ongoing search for novel antimicrobial agents and their use in singular or combined drug therapy, peptide fractions of molecular weights about 14.500 and 15.00kDa were isolated from the hemolymph of wild (Apis florae) and carniolan (Apis carnica) bees of Saudi Arabia obtained from different regions in Riyadh (variable plant sources) during the honey seasons (spring and summer) 2008–2009. Following experimental infection with 1.1×l06 viable Escherichia coli cells (ATCC 25922), the antimicrobial peptides were purified to homogeneity by reversed-phase high performance liquid chromatography. Antibacterial activity of the isolated peptide was evaluated in vitro by an agar well diffusion method for E. coli strain (ATCC 25922) and Klebsiella pneumoniae strain (ATCC 11678), the major Gram negative pathogens causing urinary tract infections, and Staphylococcus aureus (ATCC 6538) as Gram positive bacteria. A total of 10 honey samples collected from bee hives selected arbitrary at different floral areas of south Riyadh were also investigated for their antimicrobial activities against the yeast, Candida albicans (ATCC 10231) and four standard bacteria strains, E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), S. aureus (ATCC 6538) and Bacillus subtilis (ATCC 6633) using standard antimicrobial assays. The isolated antibacterial peptides and the different honey samples revealed comparable marked variations in antimicrobial activities and their sensitivity might be depending on their variable floral sources.

Differential expression of genes encoding preprobrevinin-2, prepropalustrin-2, and preproranatuerin-2 in developing larvae and adult tissues of the mountain brown frog Rana ornativentris

Previous studies led to the isolation of antimicrobial peptides (AMPs) of the brevinin-2, palustrin-2, and ranatuerin-2 families from skin extracts and/or skin secretions of the Japanese mountain brown frog, Rana ornativentris. In the present study, we cloned cDNAs encoding the precursors of brevinin-2Oc, palustrin-2Oa, and ranatuerin-2Ob and -2Oe from skin total RNA preparations from adult R. ornativentris and established a semi-quantitative RT-PCR system to measure the concentrations of these mRNAs. The levels of preprobrevinin-2 and preproranatuerin-2 mRNAs in the skin specimens of developing R. ornativentris larva were detectable only at stages later than the onset of metamorphosis and reached peaks at the stage of metamorphic climax. In contrast, prepropalustrin-2 mRNA was detected prior to the onset of metamorphosis and levels peaked at stages earlier than those of the other two mRNAs. In adult animals, preprobrevinin-2 and preproranatuerin-2 gene transcripts were detected at low levels in the small intestine and skeletal muscle but not in the stomach, liver, or kidney, whereas prepropalustrin-2 gene transcripts were detected at relatively high concentrations in all tissues examined. These results indicate that the expression of amphibian AMP genes is correlated with metamorphosis but is subjected to differential regulation.

Isolation of antimicrobial peptides from avian heterophils

Five bactericidal peptides (chicken heterophil peptides CHP1 and CHP2; turkey heterophil peptides THP1, THP2, and THP3) were purified from avian heterophil granules. All peptides were cationic and rich in cysteine, arginine, and lysine. The complete amino acid sequence, consisting of 39 amino acids, was determined for CHP1. This peptide had a molecular weight of 4481 as determined by mass spectrometry. Partial NH2-terminal amino acid sequences were obtained for the remaining peptides. Both chicken peptides and THP1 shared sequence homology at 22 residues and a cysteine motif which was similar to that of bovine beta-defensins. THP2 and THP3 were homologous to each other but were not homologous to the other three and had a unique cysteine motif. Peptides CHP1, CHP2, and THP1 killed Staphylococcus aureus and Escherichia coli in vitro, whereas THP2 and THP3 killed only S. aureus in vitro.