Novel non-helical antimicrobial peptides insert into and fuse lipid model membranes.

This research addresses the growing menace of antibiotic resistance by exploring antimicrobial peptides (AMPs) as alternatives to conventional antibiotics. Specifically, we investigate two linear amphipathic AMPs, LE-53 (12-mer) and LE-55 (16-mer), finding that the shorter LE-53 exhibits greater bactericidal activity against both Gram-negative (G(-)) and Gram-positive (G(+)) bacteria. Remarkably, both AMPs are non-toxic to eukaryotic cells. The heightened effectiveness of LE-53 is attributed to its increased hydrophobicity (H) compared to LE-55. Circular dichroism (CD) reveals that LE-53 and LE-55 both adopt ß-sheet and random coil structures in lipid model membranes (LMMs) mimicking G(-) and G(+) bacteria, so secondary structure is not the cause of the potency difference. X-ray diffuse scattering (XDS) reveals increased lipid chain order in LE-53, a potential key distinction. Additionally, XDS study uncovers a significant link between LE-53's upper hydrocarbon location in G(-) and G(+) LMMs and its efficacy. Neutron reflectometry (NR) confirms the AMP locations determined using XDS. Solution small angle X-ray scattering (SAXS) demonstrates LE-53's ability to induce vesicle fusion in bacterial LMMs without affecting eukaryotic LMMs, offering a promising strategy to combat antibiotic-resistant strains while preserving human cell integrity, whereas LE-55 has a smaller ability to induce fusion.

SARS-CoV-2 Accessory Protein Orf7b Induces Lung Injury via c-Myc Mediated Apoptosis and Ferroptosis.

The pandemic of coronavirus disease 2019 (COVID-19) has been the foremost modern global public health challenge. The airway is the primary target in severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) infection, with substantial cell death and lung injury being signature hallmarks of exposure. The viral factors that contribute to cell death and lung injury remain incompletely understood. Thus, this study investigated the role of open reading frame 7b (Orf7b), an accessory protein of the virus, in causing lung injury. In screening viral proteins, we identified Orf7b as one of the major viral factors that mediates lung epithelial cell death. Overexpression of Orf7b leads to apoptosis and ferroptosis in lung epithelial cells, and inhibitors of apoptosis and ferroptosis ablate Orf7b-induced cell death. Orf7b upregulates the transcription regulator, c-Myc, which is integral in the activation of lung cell death pathways. Depletion of c-Myc alleviates both apoptotic and ferroptotic cell deaths and lung injury in mouse models. Our study suggests a major role of Orf7b in the cell death and lung injury attributable to COVID-19 exposure, supporting it as a potential therapeutic target.

In Vitro Investigation of the Antibacterial Activity of Salamander Skin Peptides.

Given the current and future costs of antibiotic-resistant bacteria to human health and economic productivity, there is an urgent need to develop new antimicrobial compounds. Antimicrobial peptides are a promising alternative to conventional antibiotics and other antimicrobials. Amphibian skin is a rich source of bioactive compounds, but the antibacterial properties of salamander skin peptides have been neglected. Here, we examined the in vitro ability of skin peptides from 9 species of salamander representing 6 salamander families to inhibit the growth of ESKAPE pathogens, which are bacteria that have developed resistance to conventional antibiotics. We also examined whether the skin peptides caused lysis of human red blood cells. Skin peptides from Amphiuma tridactylum had the greatest antimicrobial properties, completely inhibiting the growth of all bacterial strains except for Enterococcus faecium. Likewise, skin peptides from Cryptobranchus alleganiensis completely inhibited the growth of several of the bacterial strains. In contrast, skin peptide mixtures from Ambystoma maculatum, Desmognathus fuscus, Eurycea bislineata, E. longicauda, Necturus beyeri, N. maculosus, and Siren intermedia did not completely inhibit bacterial growth even at the highest concentrations. Finally, none of the skin peptide mixtures caused lysis of human red blood cells. Together, we demonstrate that salamander skin produces peptides with potent antibacterial properties. It remains to elucidate the peptide sequences and their antibacterial mechanisms.

Synergistic Biophysical Techniques Reveal Structural Mechanisms of Engineered Cationic Antimicrobial Peptides in Lipid Model Membranes.

In the quest for new antibiotics, two novel engineered cationic antimicrobial peptides (eCAPs) have been rationally designed. WLBU2 and D8 (all 8 valines are the d-enantiomer) efficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxic to eukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structure and pore evidence. We suggest that protein secondary structure is not a critical determinant of bactericidal activity, but that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydrocarbon region may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model, both locate deep into its hydrocarbon region where they are primed for self-promoted uptake into the periplasm. The partially α-helical secondary structure of WLBU2 in a red blood cell (RBC) membrane model containing 50 % cholesterol, could play a role in destabilizing this RBC membrane model causing pore formation that is not observed with the D8 random coil, which correlates with RBC hemolysis caused by WLBU2 but not by D8.

Elastic behavior of model membranes with antimicrobial peptides depends on lipid specificity and d-enantiomers.

In an effort to provide new treatments for the global crisis of bacterial resistance to current antibiotics, we have used a rational approach to design several new antimicrobial peptides (AMPs). The present study focuses on 24-mer WLBU2 and its derivative, D8, with the amino acid sequence, RRWVRRVRRWVRRVVRVVRRWVRR. In D8, all of the valines are the d-enantiomer. We use X-ray low- and wide-angle diffuse scattering data to measure elasticity and lipid chain order. We show a good correlation between in vitro bacterial killing efficiency and both bending and chain order behavior in bacterial lipid membrane mimics; our results suggest that AMP-triggered domain formation could be the mechanism of bacterial killing in both Gram-positive and Gram-negative bacteria. In red blood cell lipid mimics, D8 stiffens and orders the membrane, while WLBU2 softens and disorders it, which correlate with D8's harmless vs. WLBU2's toxic behavior in hemolysis tests. These results suggest that elasticity and chain order behavior can be used to predict mechanisms of bactericidal action and toxicity of new AMPs.

Memory T cells specific for murine cytomegalovirus re-emerge after multiple challenges and recapitulate immunity in various adoptive transfer scenarios.

Reconstitution of CMV-specific immunity after transplant remains a primary clinical objective to prevent CMV disease, and adoptive immunotherapy of CMV-specific T cells can be an effective therapeutic approach. Because of viral persistence, most CMV-specific CD8(+) T cells become terminally differentiated effector phenotype CD8(+) T cells (TEFF). A minor subset retains a memory-like phenotype (memory phenotype CD8(+) T cells [TM]), but it is unknown whether these cells retain memory function or persist over time. Interestingly, recent studies suggest that CMV-specific CD8(+) T cells with different phenotypes have different abilities to reconstitute sustained immunity after transfer. The immunology of human CMV infections is reflected in the murine CMV (MCMV) model. We found that human CMV- and MCMV-specific T cells displayed shared genetic programs, validating the MCMV model for studies of CMV-specific T cells in vivo. The MCMV-specific TM population was stable over time and retained a proliferative capacity that was vastly superior to TEFF. Strikingly, after transfer, TM established sustained and diverse T cell populations even after multiple challenges. Although both TEFF and TM could protect Rag(-/-) mice, only TM persisted after transfer into immune replete, latently infected recipients and responded if recipient immunity was lost. Interestingly, transferred TM did not expand until recipient immunity was lost, supporting that competition limits the Ag stimulation of TM. Ultimately, these data show that CMV-specific TM retain memory function during MCMV infection and can re-establish CMV immunity when necessary. Thus, TM may be a critical component for consistent, long-term adoptive immunotherapy success.

Engineered cationic antimicrobial peptide (eCAP) prevents Pseudomonas aeruginosa biofilm growth on airway epithelial cells.

OBJECTIVES: Chronic infections with the opportunistic pathogen Pseudomonas aeruginosa are responsible for the majority of the morbidity and mortality in patients with cystic fibrosis (CF). While P. aeruginosa infections may initially be treated successfully with standard antibiotics, chronic infections typically arise as bacteria transition to a biofilm mode of growth and acquire remarkable antimicrobial resistance. To address the critical need for novel antimicrobial therapeutics that can effectively suppress chronic bacterial infections in challenging physiological environments, such as the CF lung, we have rationally designed a de novo engineered cationic antimicrobial peptide, the 24-residue WLBU2, with broad-spectrum antibacterial activity for pan-drug-resistant P. aeruginosa in liquid culture. In the current study, we tested the hypothesis that WLBU2 also prevents P. aeruginosa biofilm growth. METHODS: Using abiotic and biotic biofilm assays, co-culturing P. aeruginosa with polarized human airway epithelial cells, we examined the ability of WLBU2 to prevent biofilm biogenesis alone and in combination with currently used antibiotics. RESULTS: We observed a dose-dependent reduction in biofilm growth on an abiotic surface and in association with CF airway epithelial cells. WLBU2 prevented P. aeruginosa biofilm formation when co-cultured with mucus-producing primary human CF airway epithelial cells and using CF clinical isolates of P. aeruginosa, even at low pH and high salt conditions that mimic the CF airway. When used in combination, WLBU2 significantly increases killing by the commonly used antibiotics tobramycin, ciprofloxacin, ceftazidime and meropenem. CONCLUSIONS: While other studies have demonstrated the ability of natural and synthetic antimicrobial peptides to prevent abiotic bacterial biofilm formation, the current studies for the first time demonstrate the effective peptide treatment of a biotic bacterial biofilm in a setting similar to the CF airway, and without negative effects on human airway epithelial cells, thus highlighting the unique potential of this engineered cationic antimicrobial peptide for treatment of human respiratory infections.

Engineered cationic antimicrobial peptides to overcome multidrug resistance by ESKAPE pathogens.

Multidrug resistance constitutes a threat to the medical achievements of the last 50 years. In this study, we demonstrated the abilities of two de novo engineered cationic antibiotic peptides (eCAPs), WLBU2 and WR12, to overcome resistance from 142 clinical isolates representing the most common multidrug-resistant (MDR) pathogens and to display a lower propensity to select for resistant bacteria in vitro compared to that with colistin and LL37. The results warrant an exploration of eCAPs for use in clinical settings.

Rational design of engineered cationic antimicrobial peptides consisting exclusively of arginine and tryptophan, and their activity against multidrug-resistant pathogens.

The emergence of multidrug-resistant (MDR) pathogens underscores the need for new antimicrobial agents to overcome the resistance mechanisms of these organisms. Cationic antimicrobial peptides (CAPs) provide a potential source of new antimicrobial therapeutics. We previously characterized a lytic base unit (LBU) series of engineered CAPs (eCAPs) of 12 to 48 residues demonstrating maximum antibacterial selectivity at 24 residues. Further, Trp substitution in LBU sequences increased activity against both P. aeruginosa and S. aureus under challenging conditions (e.g., saline, divalent cations, and serum). Based on these findings, we hypothesized that the optimal length and, therefore, the cost for maximum eCAP activity under physiologically relevant conditions could be significantly reduced using only Arg and Trp arranged to form idealized amphipathic helices. Hence, we developed a novel peptide series, composed only of Arg and Trp, in a sequence predicted and verified by circular dichroism to fold into optimized amphipathic helices. The most effective antimicrobial activity was achieved at 12 residues in length (WR12) against a panel of both Gram-negative and Gram-positive clinical isolates, including extensively drug-resistant strains, in saline and broth culture and at various pH values. The results demonstrate that the rational design of CAPs can lead to a significant reduction in the length and the number of amino acids used in peptide design to achieve optimal potency and selectivity against specific pathogens.

Novel Helical Trp- and Arg-Rich Antimicrobial Peptides Locate Near Membrane Surfaces and Rigidify Lipid Model Membranes.

Antibiotics are losing effectiveness as bacteria become resistant to conventional drugs. To find new alternatives, antimicrobial peptides (AMPs) are rationally designed with different lengths, charges, hydrophobicities (H), and hydrophobic moments (µH), containing only three types of amino acids: arginine, tryptophan, and valine. Six AMPs with low minimum inhibitory concentrations (MICs) and <25% toxicity to mammalian cells are selected for biophysical studies. Their secondary structures are determined using circular dichroism (CD), which finds that the % α-helicity of AMPs depends on composition of the lipid model membranes (LMMs): gram-negative (G(-)) inner membrane (IM) >gram-positive (G(+)) > Euk33 (eukaryotic with 33 mol% cholesterol). The two most effective peptides, E2-35 (16 amino acid [AA] residues) and E2-05 (22 AAs), are predominantly helical in G(-) IM and G(+) LMMs. AMP/membrane interactions such as membrane elasticity, chain order parameter, and location of the peptides in the membrane are investigated by low-angle and wide-angle X-ray diffuse scattering (XDS). It is found that headgroup location correlates with efficacy and toxicity. The membrane bending modulus KC displays nonmonotonic changes due to increasing concentrations of E2-35 and E2-05 in G(-) and G(+) LMMs, suggesting a bacterial killing mechanism where domain formation causes ion and water leakage.

Rational Framework for the Design of Trp- and Arg-Rich Peptide Antibiotics Against Multidrug-Resistant Bacteria.

The threat of antibiotic resistance warrants the discovery of agents with novel antimicrobial mechanisms. Antimicrobial peptides (AMPs) directly disrupting bacterial membranes may overcome resistance to traditional antibiotics. AMP development for clinical use has been mostly limited to topical application to date. We developed a rational framework for systematically addressing this challenge using libraries composed of 86 novel Trp- and Arg-rich engineered peptides tested against clinical strains of the most common multidrug-resistant bacteria known as ESKAPE pathogens. Structure-function correlations revealed minimum lengths (as low as 16 residues) and Trp positioning for maximum antibacterial potency with mean minimum inhibitory concentration (MIC) of 2-4 µM and corresponding negligible toxicity to mammalian cells. Twelve peptides were selected based on broad-spectrum activity against both gram-negative and -positive bacteria and <25% toxicity to mammalian cells at maximum test concentrations. Most of the selected PAX remained active against the colistin-resistant clinical strains. Of the selected peptides, the shortest (the 16-residue E35) was further investigated for antibacterial mechanism and proof-of-concept in vivo efficacy. E35 killed an extensively-resistant isolate of Pseudomonas aeruginosa (PA239 from the CDC, also resistant to colistin) by irreversibly disrupting the cell membranes as shown by propidium iodide incorporation, using flow cytometry and live cell imaging. As proof of concept, in vivo toxicity studies showed that mice tolerated a systemic dose of up to 30 mg/kg peptide and were protected with a single 5 mg/kg intravenous (IV) dose against an otherwise lethal intraperitoneal injection of PA239. Efficacy was also demonstrated in an immune-compromised Klebsiella pneumoniae infection model using a daily dose of 4mg/kg E35 systemically for 2 days. This framework defines the determinants of efficacy of helical AMPs composed of only cationic and hydrophobic amino acids and provides a path for a potential departure from the restriction to topical use of AMPs toward systemic application.

Antimicrobial Peptide Mechanism Studied by Scattering-Guided Molecular Dynamics Simulation.

In an effort to combat rising antimicrobial resistance, our labs have rationally designed cationic, helical, amphipathic antimicrobial peptides (AMPs) as alternatives to traditional antibiotics since AMPs incur bacterial resistance in weeks, rather than days. One highly positively charged AMP, WLBU2 (+13e), (RRWV RRVR RWVR RVVR VVRR WVRR), has been shown to be effective in killing both Gram-negative (G(-)) and Gram-positive (G(+)) bacteria by directly perturbing the bacterial membrane nonspecifically. Previously, we used two equilibrium experimental methods: synchrotron X-ray diffuse scattering (XDS) providing lipid membrane thickness and neutron reflectometry (NR) providing WLBU2 depth of penetration into three lipid model membranes (LMMs). The purpose of the present study is to use the results from the scattering experiments to guide molecular dynamics (MD) simulations to investigate the detailed biophysics of the interactions of WLBU2 with LMMs of Gram-negative outer and inner membranes, and Gram-positive cell membranes, to elucidate the mechanisms of bacterial killing. Instead of coarse-graining, backmapping, or simulating without bias for several microseconds, all-atom (AA) simulations were guided by the experimental results and then equilibrated for ∼0.5 µs. Multiple replicas of the inserted peptide were run to probe stability and reach a combined time of at least 1.2 µs for G(-) and also 2.0 µs for G(+). The simulations with experimental comparisons help rule out certain structures and orientations and propose the most likely set of structures, orientations, and effects on the membrane. The simulations revealed that water, phosphates, and ions enter the hydrocarbon core when WLBU2 is positioned there. For an inserted peptide, the three types of amino acids, arginine, tryptophan, and valine (R, W, V), are arranged with the 13 Rs extending from the hydrocarbon core to the phosphate group, Ws are located at the interface, and Vs are more centrally located. For a surface state, R, W, and V are positioned relative to the bilayer interface as expected from their hydrophobicities, with Rs closest to the phosphate group, Ws close to the interface, and Vs in between. G(-) and G(+) LMMs are thinned ∼1 Å by the addition of WLBU2. Our results suggest a dual anchoring mechanism for WLBU2 both in the headgroup and in the hydrocarbon region that promotes a defect region where water and ions can flow across the slightly thinned bacterial cell membrane.

Mass Balance Study of the Engineered Cationic Antimicrobial Peptide, WLBU2, Following a Single Intravenous Dose of 14C-WLBU2 in Mice.

BACKGROUND: To address multidrug resistance, we developed engineered Cationic Antimicrobial Peptides (eCAPs). Lead eCAP WLBU2 displays potent activity against drug-resistant bacteria and effectively treats lethal bacterial infections in mice, reducing bacterial loads to undetectable levels in diverse organs. OBJECTIVE: To support the development of WLBU2, we conducted a mass balance study. METHODS: CD1 mice were administered 10, 15, 20 and 30 mg/kg of QDx5 WLBU2 or a single dose of [14C]-WLBU2 at 15 mg/kg IV. Tolerability, tissue distribution and excretion were evaluated with liquid scintillation and HPLC-radiochromatography. RESULTS: The maximum tolerated dose of WLBU2 is 20 mg/kg IV. We could account for greater than >96% of the radioactivity distributed within mouse tissues at 5 and 15 min. By 24h, only ~40-50% of radioactivity remained in the mice. The greatest % of the dose was present in liver, accounting for ~35% of radioactivity at 5 and 15 min, and ~ 8% of radioactivity remained at 24h. High radioactivity was also present in kidneys, plasma, red blood cells and lungs, while less than 0.2% of radioactivity was present in brain, fat, or skeletal muscle. Urinary and fecal excretion accounted for 12.5 and 2.2% of radioactivity at 24h. CONCLUSION: WLBU2 distributes widely to mouse tissues and is rapidly cleared with a terminal radioactivity half-life of 22 h, a clearance of 27.4 mL/h/kg, and a distribution volume of 0.94 L/kg. At 2-100 µg-eq/g, the concentrations of 14C-WLBU2 appear high enough in the tissues to account for the inhibition of microbial growth.

Direct antimicrobial activity of cationic amphipathic peptide WLBU2 against Staphylococcus aureus biofilms is enhanced in physiologic buffered saline.

Periprosthetic joint infection of total knee arthroplasties represents a major challenge to the field of orthopedic surgery. These infections are commonly associated with antibiotic-tolerant Staphylococcus aureus biofilms. Engineered cationic amphipathic peptide WLBU2 has shown the ability to kill antibiotic-resistant pathogens and drug-tolerant bacterial biofilms. The novelty of using WLBU2 during the direct irrigation and debridement of periprosthetic joint infections led our group to investigate the optimal washout conditions for treatment of S. aureus biofilms. S. aureus mature biofilms were grown on metal implant material and treated with WLBU2 dissolved in differing irrigation solvents. Mature biofilms were treated both in vitro as well as in a periprosthetic joint infection murine model. WLBU2 activity against S. aureus biofilms was increased when dissolved in diphosphate-buffered saline (dPBS) with pH of 7.0 compared with normal saline with pH of 5.5. WLBU2 activity was decreased in acidic dPBS and increased in alkaline dPBS. WLBU2 activity could be decreased in hypertonic dPBS and increased in hypotonic dPBS. WLBU2 dissolved in less acidic dPBS displayed increased efficacy in treating periprosthetic joint infection (PJI) implants ex vivo. WLBU2 demonstrated the ability to eliminate PJI associated S. aureus biofilms on arthroplasty material. The efficacy of engineered cationic amphipathic peptide WLBU2 for intraoperative elimination of S. aureus biofilms can be further optimized when kept in a less acidic and more physiologic pH adjusted saline. Understanding optimal physical washout conditions are vital for the success of WLBU2 in treating S. aureus biofilms in PJI clinical trials going forward.

Engineered Cationic Antimicrobial Peptides (eCAPs) to Combat Multidrug-Resistant Bacteria.

The increasing rate of antibiotic resistance constitutes a global health crisis. Antimicrobial peptides (AMPs) have the property to selectively kill bacteria regardless of resistance to traditional antibiotics. However, several challenges (e.g., reduced activity in the presence of serum and lack of efficacy in vivo) to clinical development need to be overcome. In the last two decades, we have addressed many of those challenges by engineering cationic AMPs de novo for optimization under test conditions that typically inhibit the activities of natural AMPs, including systemic efficacy. We reviewed some of the most promising data of the last two decades in the context of the advancement of the field of helical AMPs toward clinical development.

Antibacterial Properties and Efficacy of a Novel SPLUNC1-Derived Antimicrobial Peptide, α4-Short, in a Murine Model of Respiratory Infection.

Multidrug resistance (MDR) by bacterial pathogens constitutes a global health crisis, and resistance to treatment displayed by biofilm-associated infections (e.g., cystic fibrosis, surgical sites, and medical implants) only exacerbates a problem that is already difficult to overcome. Antimicrobial peptides (AMPs) are a promising class of therapeutics that may be useful in the battle against antibiotic resistance, although certain limitations have hindered their clinical development. The goal of this study was to examine the therapeutic potential of novel AMPs derived from the multifunctional respiratory host defense protein SPLUNC1. Using standard growth inhibition and antibiofilm assays, we demonstrated that a novel structurally optimized AMP, α4-short, was highly effective against the most common group of MDR bacteria while showing broad-spectrum bactericidal and antibiofilm activities. With negligible hemolysis and toxicity to white blood cells, the new peptide also demonstrated in vivo efficacy when delivered directly into the airway in a murine model of Pseudomonas aeruginosa-induced respiratory infection. The data warrant further exploration of SPLUNC1-derived AMPs with optimized structures to assess the potential application to difficult-to-cure biofilm-associated infections.IMPORTANCE The rise of superbugs underscores the urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs) have the ability to kill superbugs regardless of resistance to traditional antibiotics. However, AMPs often display a lack of efficacy in vivo. Sequence optimization and engineering are promising but may result in increased host toxicity. We report here the optimization of a novel AMP (α4-short) derived from the multifunctional respiratory protein SPLUNC1. The AMP α4-short demonstrated broad-spectrum activity against superbugs as well as in vivo efficacy in the P. aeruginosa pneumonia model. Further exploration for clinical development is warranted.

Enhanced biofilm prevention activity of a SPLUNC1-derived antimicrobial peptide against Staphylococcus aureus.

SPLUNC1 is a multifunctional protein of the airway with antimicrobial properties. We previously reported that it displayed antibiofilm activities against P. aeruginosa. The goal of this study was to determine whether (1) the antibiofilm property is broad (including S. aureus, another prevalent organism in cystic fibrosis); (2) the α4 region is responsible for such activity; and (3), if so, this motif could be structurally optimized as an antimicrobial peptide with enhanced activities. We used S. aureus biofilm-prevention assays to determine bacterial biomass in the presence of SPLUNC1 and SPLUNC1Δα4 recombinant proteins, or SPLUNC1-derived peptides (α4 and α4M1), using the well-established crystal-violet biofilm detection assay. The SPLUNC1Δα4 showed markedly reduced biofilm prevention compared to the parent protein. Surprisingly, the 30-residue long α4 motif alone demonstrated minimal biofilm prevention activities. However, structural optimization of the α4 motif resulted in a modified peptide (α4M1) with significantly enhanced antibiofilm properties against methicillin-sensitive (MSSA) and-resistant (MRSA) S. aureus, including six different clinical strains of MRSA and the well-known USA300. Hemolytic activity was undetectable at up to 100µM for the peptides. The data warrant further investigation of α4-derived AMPs to explore the potential application of antimicrobial peptides to combat bacterial biofilm-related infections.

Prevention of ESKAPE pathogen biofilm formation by antimicrobial peptides WLBU2 and LL37.

OBJECTIVES: Bacterial biofilm-dependent infections (e.g. cystic fibrosis, surgical sites, and medical implants) are associated with enhanced drug-resistance and are thus difficult to eradicate. The goal of this study was to systematically compare three distinct classes of antimicrobial peptides (AMPs) that include the clinically used antibiotic colistin, the natural AMP LL37, the engineered cationic-AMP WLBU2, and four commonly used antibiotics with different bactericidal mechanisms (tobramycin, ciprofloxacin, ceftazidime, and vancomycin) for biofilm prevention properties. METHODS: Using biofilm-prevention assays, we detected bacterial biomass post-attachment in subinhibitory concentrations (1/3 of the minimum inhibitory concentration [MIC]) for each AMP by the crystal violet method, to distinguish the commonly known bactericidal activity from potentially distinct mechanisms of biofilm prevention. Biofilm regulatory gene expression was assessed using RT-qPCR for correlation with biofilm growth inhibition. RESULTS: Commonly used antibiotics at 1x MIC showed modest ESKAPE biofilm prevention while 1/3 MIC of AMPs demonstrated up to 90% biofilm prevention. WLBU2 was generally more effective in preventing bacterial attachment than colistin and LL37. Changes in bacterial biofilm regulatory gene expression were consistent with biofilm prevention. CONCLUSION: The data warrant further exploration of AMPs with optimized structures to fill a knowledge gap on the potential application of AMPs for difficult-to-cure bacterial biofilm-related infections.

Antimicrobial Peptides: A Potential Therapeutic Option for Surgical Site Infections.

Surgical Site Infections (SSI) represents one of the most common hospital-associated infections worldwide, and many cases of SSI are due to multidrug-resistant bacteria with the propensity to attach to tissues and form biofilm on post-surgical sites. While systemic antibiotic treatment (prophylactically and therapeutically) is usually effective, SSI can be difficult to treat when associated with drug resistance. Antimicrobial Peptides (AMPs) are an untapped resource that could serve as an effective therapeutic option, as they display broad-spectrum antimicrobial activity regardless of antibiotic resistance. In the last decade, it has become increasingly clear that AMPs also display antibiofilm properties. We reviewed herein the potential of AMPs as promising therapeutics for SSI and the need for structural optimization to develop AMPs for clinical applications.

Antimicrobial peptides with selective antitumor mechanisms: prospect for anticancer applications.

In the last several decades, there have been significant advances in anticancer therapy. However, the development of resistance to cancer drugs and the lack of specificity related to actively dividing cells leading to toxic side effects have undermined these achievements. As a result, there is considerable interest in alternative drugs with novel antitumor mechanisms. In addition to the recent approach using immunotherapy, an effective but much cheaper therapeutic option of pharmaceutical drugs would still provide the best choice for cancer patients as the first line treatment. Ribosomally synthesized cationic antimicrobial peptides (AMPs) or host defense peptides (HDP) display broad-spectrum activity against bacteria based on electrostatic interactions with negatively charged lipids on the bacterial surface. Because of increased proportions of phosphatidylserine (negatively charged) on the surface of cancer cells compared to normal cells, cationic amphipathic peptides could be an effective source of anticancer agents that are both selective and refractory to current resistance mechanisms. We reviewed herein the prospect for AMP application to cancer treatment, with a focus on modes of action of cationic AMPs.