Redefining Peptide 14D: Substitutional Analysis for Accelerated TB Diagnosis and Enhanced Activity against Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a predominant cause of mortality, especially in low- and middle-income nations. Recently, antimicrobial peptides have been discovered that at low concentrations could stimulate the growth of M. tuberculosis (hormetic response). In this study, such a peptide was used to investigate the effects on the time to positivity (TTP). A systematic substitution analysis of peptide 14D was synthesized using Spot synthesis technology, resulting in 171 novel peptides. Our findings revealed a spectrum of interactions, with some peptides accelerating M. tuberculosis growth, potentially aiding in faster diagnostics, while others exhibited inhibitory effects. Notably, peptide NH2-wkivfiwrr-CONH2 significantly reduced the TTP by 25 h compared to the wild-type peptide 14D, highlighting its potential in improving TB diagnostics by culture. Several peptides demonstrated potent antimycobacterial activity, with a minimum inhibitory concentration (MIC) of 20 µg/mL against H37Rv and a multidrug-resistant M. tuberculosis strain. Additionally, for two peptides, a strongly diminished formation of cord-like structures was observed, which is indicative of reduced virulence and transmission potential. This study underscores the multifaceted roles of antimicrobial peptides in TB management, from enhancing diagnostic efficiency to offering therapeutic avenues against M. tuberculosis.

Specific Cationic Antimicrobial Peptides Enhance the Recovery of Low-Load Quiescent Mycobacterium tuberculosis in Routine Diagnostics.

The culture confirmation of Mycobacterium tuberculosis (MTB) remains the gold standard for the diagnosis of Tuberculosis (TB) with culture conversion representing proof of cure. However, over 40% of TB samples fail to isolate MTB even though many patients remain infectious due to the presence of viable non-culturable forms. Previously, we have shown that two short cationic peptides, T14D and TB08L, induce a hormetic response at low concentrations, leading to a stimulation of growth in MTB and the related animal pathogen Mycobacterium bovis (bTB). Here, we examine these peptides showing they can influence the mycobacterial membrane integrity and function through membrane potential reduction. We also show this disruption is associated with an abnormal reduction in transcriptomic signalling from specific mycobacterial membrane sensors that normally monitor the immediate cellular environment and maintain the non-growing phenotype. We observe that exposing MTB or bTB to these peptides at optimal concentrations rapidly represses signalling mechanisms maintaining dormancy phenotypes, which leads to the promotion of aerobic metabolism and conversion into a replicative phenotype. We further show a practical application of these peptides as reagents able to enhance conventional routine culture methods by stimulating mycobacterial growth. We evaluated the ability of a peptide-supplemented sample preparation and culture protocol to isolate the MTB against a gold standard routine method tested in parallel on 255 samples from 155 patients with suspected TB. The peptide enhancement increased the sample positivity rate by 46% and decreased the average time to sample positivity of respiratory/faecal sampling by seven days. The most significant improvements in isolation rates were from sputum smear-negative low-load samples and faeces. The peptide enhancement increased sampling test sensitivity by 19%, recovery in samples from patients with a previously culture-confirmed TB by 20%, and those empirically treated for TB by 21%. We conclude that sample decontamination and culture enhancement with D-enantiomer peptides offer good potential for the much-needed improvement of the culture confirmation of TB.

Discovery of Antimicrobial Peptides That Can Accelerate Culture Diagnostics of Slow-Growing Mycobacteria Including Mycobacterium tuberculosis.

Antimicrobial peptides (AMPs) can directly kill Gram-positive bacteria, Gram-negative bacteria, mycobacteria, fungi, enveloped viruses, and parasites. At sublethal concentrations, some AMPs and also conventional antibiotics can stimulate bacterial response increasing their resilience, also called the hormetic response. This includes stimulation of growth, mobility, and biofilm production. Here, we describe the discovery of AMPs that stimulate the growth of certain mycobacteria. Peptide 14 showed a growth stimulating effect on Mycobacteria tuberculosis (MTB), M. bovis, M. avium subsp. paratuberculosis (MAP), M. marinum, M. avium-intracellulare, M. celatum, and M. abscessus. The effect was more pronounced at low bacterial inocula. The peptides induce a faster transition from the lag phase to the log phase and keep the bacteria longer in the log phase before entering stationary phase when compared to nontreated controls. In some cases, an increase in the division rate was observed. An initial screen using MAP and a collection of 75 peptides revealed 13 peptides with a hormetic effect. For MTB, a collection of 25 artificial peptides were screened and 13 were found to reduce the time to positivity (TTP) by at least 5%, improving growth. A screen of 43 naturally occurring peptides, 11 fragments of naturally occurring peptides and 5 designed peptides, all taken from the database APD3, identified a further 44 peptides that also lowered TTP by at least 5%. Lasioglossin LL-III (Bee) and Ranacyclin E (Frog) were the most active natural peptides, and the human cathelicidin LL37 fragment GF-17 and a porcine cathelicidin protegrin-1 fragment were the most active fragments of naturally occurring peptides. Peptide 14 showed growth-stimulating activity between 10 ng/mL and 10 µg/mL, whereas the stability-optimised Peptide 14D had a narrow activity range of 0.1-1 µg/mL. Peptides identified in this study are currently in commercial use to improve recovery and culture for the diagnostics of mycobacteria in humans and animals.

Can BioSAXS detect ultrastructural changes of antifungal compounds in Candida albicans?-an exploratory study.

The opportunistic yeast Candida albicans is the most common cause of candidiasis. With only four classes of antifungal drugs on the market, resistance is becoming a problem in the treatment of fungal infections, especially in immunocompromised patients. The development of novel antifungal drugs with different modes of action is urgent. In 2016, we developed a groundbreaking new medium-throughput method to distinguish the effects of antibacterial agents. Using small-angle X-ray scattering for biological samples (BioSAXS), it is now possible to screen hundreds of new antibacterial compounds and select those with the highest probability for a novel mode of action. However, yeast (eukaryotic) cells are highly structured compared to bacteria. The fundamental question to answer was if the ultrastructural changes induced by the action of an antifungal drug can be detected even when most structures in the cell stay unchanged. In this exploratory work, BioSAXS was used to measure the ultrastructural changes of C. albicans that were directly or indirectly induced by antifungal compounds. For this, the well-characterized antifungal drug Flucytosine was used. BioSAXS measurements were performed on the synchrotron P12 BioSAXS beamline, EMBL (DESY, Hamburg) on treated and untreated yeast C. albicans. BioSAXS curves were analysed using principal component analysis (PCA). The PCA showed that Flucytosine-treated and untreated yeast were separated. Based on that success further measurements were performed on five antifungal peptides {1. Cecropin A-melittin hybrid [CA (1-7) M (2-9)], KWKLFKKIGAVLKVL; 2. Lasioglossin LL-III, VNWKKILGKIIKVVK; 3. Mastoparan M, INLKAIAALAKKLL; 4. Bmkn2, FIGAIARLLSKIFGKR; and 5. optP7, KRRVRWIIW}. The ultrastructural changes of C. albicans indicate that the peptides may have different modes of action compared to Flucytosine as well as to each other, except for the Cecropin A-melittin hybrid [CA (1-7) M (2-9)] and optP7, showing very similar effects on C. albicans. This very first study demonstrates that BioSAXS shows promise to be used for antifungal drug development. However, this first study has limitations and further experiments are necessary to establish this application.

BioSAXS-an emerging method to accelerate, enrich and de-risk antimicrobial drug development.

Antimicrobial resistance is a worldwide threat to modern health care. Low-profit margin and high risk of cross-resistance resulted in a loss of interest in big pharma, contributing to the increasing threat. Strategies to address the problem are starting to emerge. Novel antimicrobial compounds with novel modes of action are especially valued because they have a lower risk of cross-resistance. Up to now determining the mode of action has been very time and resource consuming and will be performed once drug candidates were already progressed in preclinical development. BioSAXS is emerging as a new method to test up to thousands of compounds to classify them into groups based on ultra-structural changes that correlate to their modes of action. First experiments in E. coli (gram-negative) have demonstrated that using conventional and experimental antimicrobials a classification of compounds according to their mode of action was possible. Results were backed up by transmission electron microscopy. Further work showed that also gram-positive bacteria (Staphylococcus aureus) can be used and the effects of novel antimicrobial peptides on both types of bacteria were studied. Preliminary experiments also show that BioSAXS can be used to classify antifungal drugs, demonstrated on Candida albicans. In summary, BioSAXS can accelerate and enrich the discovery of antimicrobial compounds from screening projects with a novel mode of action and hence de-risk the development of urgently needed antimicrobial drugs.

In silico identification of two peptides with antibacterial activity against multidrug-resistant Staphylococcus aureus.

Here we report two antimicrobial peptides (AMPs), HG2 and HG4 identified from a rumen microbiome metagenomic dataset, with activity against multidrug-resistant (MDR) bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) strains, a major hospital and community-acquired pathogen. We employed the classifier model design to analyse, visualise, and interpret AMP activities. This approach allowed in silico discrimination of promising lead AMP candidates for experimental evaluation. The lead AMPs, HG2 and HG4, are fast-acting and show anti-biofilm and anti-inflammatory activities in vitro and demonstrated little toxicity to human primary cell lines. The peptides were effective in vivo within a Galleria mellonella model of MRSA USA300 infection. In terms of mechanism of action, HG2 and HG4 appear to interact with the cytoplasmic membrane of target cells and may inhibit other cellular processes, whilst preferentially binding to bacterial lipids over human cell lipids. Therefore, these AMPs may offer additional therapeutic templates for MDR bacterial infections.

Rational Designed Hybrid Peptides Show up to a 6-Fold Increase in Antimicrobial Activity and Demonstrate Different Ultrastructural Changes as the Parental Peptides Measured by BioSAXS.

Antimicrobial peptides (AMPs) are a promising class of compounds being developed against multi-drug resistant bacteria. Hybridization has been reported to increase antimicrobial activity. Here, two proline-rich peptides (consP1: VRKPPYLPRPRPRPL-CONH2 and Bac5-v291: RWRRPIRRRPIRPPFWR-CONH2) were combined with two arginine-isoleucine-rich peptides (optP1: KIILRIRWR-CONH2 and optP7: KRRVRWIIW-CONH2). Proline-rich antimicrobial peptides (PrAMPs) are known to inhibit the bacterial ribosome, shown also for Bac5-v291, whereas it is hypothesized a "dirty drug" model for the arginine-isoleucine-rich peptides. That hypothesis was underpinned by transmission electron microscopy and biological small-angle X-ray scattering (BioSAXS). The strength of BioSAXS is the power to detect ultrastructural changes in millions of cells in a short time (seconds) in a high-throughput manner. This information can be used to classify antimicrobial compounds into groups according to the ultrastructural changes they inflict on bacteria and how the bacteria react towards that assault. Based on previous studies, this correlates very well with different modes of action. Due to the novelty of this approach direct identification of the target of the antimicrobial compound is not yet fully established, more research is needed. More research is needed to address this limitation. The hybrid peptides showed a stronger antimicrobial activity compared to the proline-rich peptides, except when compared to Bac5-v291 against E. coli. The increase in activity compared to the arginine-isoleucine-rich peptides was up to 6-fold, however, it was not a general increase but was dependent on the combination of peptides and bacteria. BioSAXS experiments revealed that proline-rich peptides and arginine-isoleucine-rich peptides induce very different ultrastructural changes in E. coli, whereas a hybrid peptide (hyP7B5GK) shows changes, different to both parental peptides and the untreated control. These different ultrastructural changes indicated that the mode of action of the parental peptides might be different from each other as well as from the hybrid peptide hyP7B5GK. All peptides showed very low haemolytic activity, some of them showed a 100-fold or larger therapeutic window, demonstrating the potential for further drug development.

Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens.

According to the World Health Organization (WHO) the development of resistance against antibiotics by microbes is one of the most pressing health concerns. The situation will intensify since only a few pharmacological companies are currently developing novel antimicrobial compounds. Discovery and development of novel antimicrobial compounds with new modes of action are urgently needed. Antimicrobial peptides (AMPs) are known to be able to kill multidrug-resistant bacteria and, therefore, of interest to be developed into antimicrobial drugs. Proteolytic stability and toxicities of these peptides are challenges to overcome, and one strategy frequently used to address stability is cyclization. Here we introduced a disulfide-bond to cyclize a potent and nontoxic 9mer peptide and, in addition, as a proof-of-concept study, grafted this peptide into loop 6 of the cyclotide MCoTI-II. This is the first time an antimicrobial peptide has been successfully grafted onto the cyclotide scaffold. The disulfide-cyclized and grafted cyclotide showed moderate activity in broth and strong activity in 1/5 broth against clinically relevant resistant pathogens. The linear peptide showed superior activity in both conditions. The half-life time in 100% human serum was determined, for the linear peptide, to be 13 min, for the simple disulfide-cyclized peptide, 9 min, and, for the grafted cyclotide 7 h 15 min. The addition of 10% human serum led to a loss of antimicrobial activity for the different organisms, ranging from 1 to >8-fold for the cyclotide. For the disulfide-cyclized version and the linear version, activity also dropped to different degrees, 2 to 18-fold, and 1 to 30-fold respectively. Despite the massive difference in stability, the linear peptide still showed superior antimicrobial activity. The cyclotide and the disulfide-cyclized version demonstrated a slower bactericidal effect than the linear version. All three peptides were stable at high and low pH, and had very low hemolytic and cytotoxic activity.

Rapid Assembly of Infection-Resistant Coatings: Screening and Identification of Antimicrobial Peptides Works in Cooperation with an Antifouling Background.

Bacterial adhesion and the succeeding biofilm formation onto surfaces are responsible for implant- and device-associated infections. Bifunctional coatings integrating both nonfouling components and antimicrobial peptides (AMPs) are a promising approach to develop potent antibiofilm coatings. However, the current approaches and chemistry for such coatings are time-consuming and dependent on substrates and involve a multistep process. Also, the information is limited on the influence of the coating structure or its components on the antibiofilm activity of such AMP-based coatings. Here, we report a new strategy to rapidly assemble a stable, potent, and substrate-independent AMP-based antibiofilm coating in a nonfouling background. The coating structure allowed for the screening of AMPs in a relevant nonfouling background to identify optimal peptide combinations that work in cooperation to generate potent antibiofilm activity. The structure of the coating was changed by altering the organization of the hydrophilic polymer chains within the coatings. The coatings were thoroughly characterized using various surface analytical techniques and correlated with the efficiency to prevent biofilm formation against diverse bacteria. The coating method that allowed the conjugation of AMPs without altering the steric protection ability of hydrophilic polymer structure results in a bifunctional surface coating with excellent antibiofilm activity. In contrast, the conjugation of AMPs directly to the hydrophilic polymer chains resulted in a surface with poor antibiofilm activity and increased adhesion of bacteria. Using this coating approach, we further established a new screening method and identified a set of potent surface-tethered AMPs with high activity. The success of this new peptide screening and coating method is demonstrated using a clinically relevant mouse infection model to prevent catheter-associated urinary tract infection (CAUTI).

Peptide Inhibitors of Bacterial Protein Synthesis with Broad Spectrum and SbmA-Independent Bactericidal Activity against Clinical Pathogens.

Proline-rich antimicrobial peptides (PrAMPs) are promising lead compounds for developing new antimicrobials; however, their narrow spectrum of action is limiting. PrAMPs kill bacteria binding to their ribosomes and inhibiting protein synthesis. In this study, 133 derivatives of the PrAMP Bac7(1-16) were synthesized to identify the crucial residues for ribosome inactivation and antimicrobial activity. Then, five new Bac7(1-16) derivatives were conceived and characterized by antibacterial and membrane permeabilization assays, X-ray crystallography, and molecular dynamics simulations. Some derivatives displayed broad spectrum activity, encompassing Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Staphylococcus aureus. Two peptides out of five acquired a weak membrane-perturbing activity while maintaining the ability to inhibit protein synthesis. These derivatives became independent of the SbmA transporter, commonly used by native PrAMPs, suggesting that they obtained a novel route to enter bacterial cells. PrAMP-derived compounds could become new-generation antimicrobials to combat antibiotic-resistant pathogens.

The effect of lipidation and glycosylation on short cationic antimicrobial peptides.

The global health threat surrounding bacterial resistance has resulted in antibiotic researchers shifting their focus away from 'traditional' antibiotics and concentrating on other antimicrobial agents, including antimicrobial peptides. These low molecular weight "mini-proteins" exhibit broad-spectrum activity against bacteria, including multi-drug resistant strains, viruses, fungi and protozoa and constitute a major element of the innate-immune system of many multicellular organisms. Some naturally occurring antimicrobial peptides are lipidated and/or glycosylated and almost all antimicrobial peptides in clinical use are either lipopeptides (Daptomycin and Polymyxin E and B) or glycopeptides (Vancomycin). Lipidation, glycosylation and PEGylation are an option for improving stability and activity in serum and for reducing the rapid clearing via the kidneys and liver. Two broad-spectrum antimicrobial peptides NH2-RIRIRWIIR-CONH2 (A1) and NH2-KRRVRWIIW-CONH2 (B1) were conjugated via a linker, producing A2 and B2, to individual fatty acids of C8, C10, C12 and C14 and in addition, A2 was conjugated to either glucose, N-acetyl glucosamine, galactose, mannose, lactose or polyethylene glycol (PEG). Antimicrobial activity against two Gram-positive strains (methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE)) and three Gram-negative strains (Salmonella typhimurium, E. coli and Pseudomonas aeruginosa) were determined. Activity patterns for the lipidated versions are very complex, dependent on sequence, bacteria and fatty acid. Two reciprocal effects were measured; compared to the parental peptides, some combinations led to a 16-fold improvement whereas other combinations let to a 32-fold reduction in antimicrobial activity. Glycosylation decreased antimicrobial activity by 2 to 16-fold in comparison to A1, respectively on the sugar-peptide combination. PEGylation rendered the peptide inactive. Antimicrobial activity in the presence of 25% human serum of A1 and B1 was reduced 32-fold and 8-fold, respectively. The longer chain fatty acids almost completely restored this activity; however, these fatty acids increased hemolytic activity. B1 modified with C8 increased the therapeutic index by 2-fold for four bacterial strains. Our results suggest that finding the right lipid-peptide combination can lead to improved activity in the presence of serum and potentially more effective drug candidates for animal studies. Glycosylation with the optimal sugar and numbers of sugars at the right peptide position could be an alternative route or could be used in addition to lipidation to counteract solubility and toxicity issues.

Synergy Pattern of Short Cationic Antimicrobial Peptides Against Multidrug-Resistant Pseudomonas aeruginosa.

With the rise of various multidrug-resistant (MDR) pathogenic bacteria, worldwide health care is under pressure to respond. Conventional antibiotics are failing and the development of novel classes and alternative strategies is a major priority. Antimicrobial peptides (AMPs) cannot only kill MDR bacteria, but also can be used synergistically with conventional antibiotics. We selected 30 short AMPs from different origins and measured their synergy in combination with polymyxin B, piperacillin, ceftazidime, cefepime, meropenem, imipenem, tetracycline, erythromycin, kanamycin, tobramycin, amikacin, gentamycin, and ciprofloxacin. In total, 403 unique combinations were tested against an MDR Pseudomonas aeruginosa isolate (PA910). As a measure of the synergistic effects, fractional inhibitory concentrations (FICs) were determined using microdilution assays with FICs ranges between 0.25 and 2. A high number of combinations between peptides and polymyxin B, erythromycin, and tetracycline were found to be synergistic. Novel variants of indolicidin also showed a high frequency in synergist interaction. Single amino acid substitutions within the peptides can have a very strong effect on the ability to synergize, making it possible to optimize future drugs toward synergistic interaction.

Proline-Rich Peptides with Improved Antimicrobial Activity against E. coli, K. pneumoniae, and A. baumannii.

Proline-rich antimicrobial peptides (PrAMPs) are promising agents to combat multi-drug resistant pathogens due to a high antimicrobial activity, yet low cytotoxicity. A library of derivatives of the PrAMP Bac5(1-17) was synthesized and screened to identify which residues are relevant for its activity. In this way, we discovered that two central motifs -PIRXP- cannot be modified, while residues at N- and C- termini tolerated some variations. We found five Bac5(1-17) derivatives bearing 1-5 substitutions, with an increased number of arginine and/or tryptophan residues, exhibiting improved antimicrobial activity and broader spectrum of activity while retaining low cytotoxicity toward eukaryotic cells. Transcription/translation and bacterial membrane permeabilization assays showed that these new derivatives still retained the ability to strongly inhibit bacterial protein synthesis, but also acquired permeabilizing activity to different degrees. These new Bac5(1-17) derivatives therefore show a dual mode of action which could hinder the selection of bacterial resistance against these molecules.

BioSAXS Measurements Reveal That Two Antimicrobial Peptides Induce Similar Molecular Changes in Gram-Negative and Gram-Positive Bacteria.

Two highly active short broad-spectrum AMPs (14D and 69D) with unknown mode of action have been investigated in regards to their effect against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria methicillin-resistant Staphylococcus aureus (MRSA). Minimal inhibitory concentration (MIC) measurements using a cell density of 108 cfu/ml resulted in values between 16 and 32 µg/ml. Time-kill experiments using 108 cfu/ml revealed complete killing, except for 69D in combination with MRSA, where bacterial load was reduced a million times. Small-angle X-ray scattering of biological samples (BioSAXS) at 108 cfu/ml was applied to investigate the ultrastructural changes in E. coli and MRSA in response to these two broad-spectrum AMPs. In addition, electron microscopy (EM) was performed to visualize the treated and non-treated bacteria. As expected, the scattering curves generated using BioSAXS show the ultrastructure of the Gram-positive and Gram-negative bacteria to be very different (BioSAXS is not susceptible to the outer shape). After treatment with either peptide, the scattering curves of E. coli and MRSA cells are much more alike. Whereas in EM, it is notoriously difficult to observe changes for spherical Gram-positives; the BioSAXS results are superior and reveal strongly similar effects for both peptides induced in Gram-positive as well as Gram-negative bacteria. Given the high-throughput possibility and robust statistics, BioSAXS can support and speed up mode of action research in AMPs and other antimicrobial compounds, making a contribution toward the development of urgently needed drugs against resistant bacteria.

The Dolphin Proline-Rich Antimicrobial Peptide Tur1A Inhibits Protein Synthesis by Targeting the Bacterial Ribosome.

Proline-rich antimicrobial peptides (PrAMPs) internalize into susceptible bacteria using specific transporters and interfere with protein synthesis and folding. To date, mammalian PrAMPs have so far been identified only in artiodactyls. Since cetaceans are co-phyletic with artiodactyls, we mined the genome of the bottlenose dolphin Tursiops truncatus, leading to the identification of two PrAMPs, Tur1A and Tur1B. Tur1A, which is orthologous to the bovine PrAMP Bac7, is internalized into Escherichia coli, without damaging the membranes, using the inner membrane transporters SbmA and YjiL/MdM. Furthermore, like Bac7, Tur1A also inhibits bacterial protein synthesis by binding to the ribosome and blocking the transition from the initiation to the elongation phase. By contrast, Tur1B is a poor inhibitor of protein synthesis and may utilize another mechanism of action. An X-ray structure of Tur1A bound within the ribosomal exit tunnel provides a basis to develop these peptides as novel antimicrobial agents.

The rumen microbiome: an underexplored resource for novel antimicrobial discovery.

Antimicrobial peptides (AMPs) are promising drug candidates to target multi-drug resistant bacteria. The rumen microbiome presents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including AMPs. Using functional screening and computational approaches, we identified 181 potentially novel AMPs from a rumen bacterial metagenome. Here, we show that three of the selected AMPs (Lynronne-1, Lynronne-2 and Lynronne-3) were effective against numerous bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). No decrease in MRSA susceptibility was observed after 25 days of sub-lethal exposure to these AMPs. The AMPs bound preferentially to bacterial membrane lipids and induced membrane permeability leading to cytoplasmic leakage. Topical administration of Lynronne-1 (10% w/v) to a mouse model of MRSA wound infection elicited a significant reduction in bacterial counts, which was comparable to treatment with 2% mupirocin ointment. Our findings indicate that the rumen microbiome may provide viable alternative antimicrobials for future therapeutic application.

Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis.

Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.

Screening and Optimizing Antimicrobial Peptides by Using SPOT-Synthesis.

Peptide arrays on cellulose are a powerful tool to investigate peptide interactions with a number of different molecules, for examples antibodies, receptors or enzymes. Such peptide arrays can also be used to study interactions with whole cells. In this review, we focus on the interaction of small antimicrobial peptides with bacteria. Antimicrobial peptides (AMPs) can kill multidrug-resistant (MDR) human pathogenic bacteria and therefore could be next generation antibiotics targeting MDR bacteria. We describe the screen and the result of different optimization strategies of peptides cleaved from the membrane. In addition, screening of antibacterial activity of peptides that are tethered to the surface is discussed. Surface-active peptides can be used to protect surfaces from bacterial infections, for example implants.

Use of Peptide Libraries for Identification and Optimization of Novel Antimicrobial Peptides.

The increasing rates of resistance among bacteria and to a lesser extent fungi have resulted in an urgent need to find new molecules that hold therapeutic promise against multidrug-resistant strains. Antimicrobial peptides have proven very effective against a variety of multidrug-resistant bacteria. Additionally, the low levels of resistance reported towards these molecules are an attractive feature for antimicrobial drug development. Here we summarise information on diverse peptide libraries used to discover or to optimize antimicrobial peptides. Chemical synthesized peptide libraries, for example split and mix method, tea bag method, multi-pin method and cellulose spot method are discussed. In addition biological peptide library screening methods are summarized, like phage display, bacterial display, mRNA-display and ribosomal display. A few examples are given for small peptide libraries, which almost exclusively follow a rational design of peptides of interest rather than a combinatorial approach.