RESUMO
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.
Assuntos
Peptídeos Antimicrobianos , Humanos , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Interações Hidrofóbicas e Hidrofílicas , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacosRESUMO
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.
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COVID-19 , Ferroptose , Lesão Pulmonar , Proteínas Virais , Animais , Camundongos , Apoptose , Lesão Pulmonar/virologia , Fases de Leitura Aberta , SARS-CoV-2 , Proteínas Virais/genéticaRESUMO
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.
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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.
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Anti-Infecciosos , Urodelos , Animais , Humanos , Antibacterianos/farmacologia , Peptídeos/farmacologia , Pele/microbiologia , Bactérias , Testes de Sensibilidade MicrobianaRESUMO
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 â¼0.5 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 â¼1 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.
Assuntos
Peptídeos Antimicrobianos , Simulação de Dinâmica Molecular , Peptídeos Catiônicos Antimicrobianos/química , Bactérias/metabolismo , Bicamadas Lipídicas/química , Lipídeos , Fosfatos , ÁguaRESUMO
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.
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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.
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Peptídeos Catiônicos Antimicrobianos , Infecções Bacterianas , Animais , Peptídeos Antimicrobianos , Radioisótopos de Carbono , CamundongosRESUMO
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.
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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.
Assuntos
Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Biofilmes/efeitos dos fármacos , Infecções Relacionadas à Prótese/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Concentração de Íons de Hidrogênio , Camundongos , Concentração Osmolar , Solução SalinaRESUMO
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.
Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Lipopolissacarídeos/química , Lipídeos de Membrana/química , Pseudomonas aeruginosa/química , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Hemólise , Lipopolissacarídeos/metabolismo , Lipídeos de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Secundária de ProteínaRESUMO
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.
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Anti-Infecciosos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Produtos Biológicos/uso terapêutico , Glicoproteínas/metabolismo , Fosfoproteínas/metabolismo , Infecções por Pseudomonas/tratamento farmacológico , Infecções Respiratórias/tratamento farmacológico , Animais , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Modelos Animais de Doenças , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Resultado do TratamentoRESUMO
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.
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Peptídeos Catiônicos Antimicrobianos/química , Elasticidade , Lipídeos/química , Membranas Artificiais , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Estereoisomerismo , Valina/químicaRESUMO
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.
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Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Glicoproteínas/química , Peptídeos/química , Peptídeos/farmacologia , Fosfoproteínas/química , Staphylococcus aureus/efeitos dos fármacos , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
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.
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Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Biofilmes/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Adulto , Infecções Bacterianas/microbiologia , Biofilmes/crescimento & desenvolvimento , Criança , Pré-Escolar , Perfilação da Expressão Gênica , Violeta Genciana/análise , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/crescimento & desenvolvimento , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Coloração e Rotulagem , Adulto Jovem , CatelicidinasRESUMO
Antibiotics are unable to remove biofilms from surgical implants. This high antibiotic tolerance is related to bacterial persisters, a sub-population of bacteria phenotypically tolerant to antibiotics secondary to a reduced metabolic state. WLBU2 is an engineered cationic amphipathic peptide designed to maximize antimicrobial activity with minimal mammalian cell toxicity. The objective of this study was to test the ability of WLBU2 to remove Staphylococcus aureus surgical implant biofilms. WLBU2 effectively treated S. aureus biofilms formed by a variety of clinical MSSA and MRSA strains and created culture-negative implants in the in vitro biofilm model. Blocking bacterial metabolism by inhibiting oxidative phosphorylation did not affect WLBU2 killing compared to decreased killing by cefazolin. In the surgical implant infection animal model, WLBU2 decreased biofilm mass as compared to control, untreated samples. WLBU2 could rapidly eliminate implants in vitro and had sufficient efficacy in vivo with minimal systemic toxicity.
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Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Infecções Relacionadas à Prótese/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Modelos Animais de Doenças , Camundongos , Fosforilação Oxidativa/efeitos dos fármacosRESUMO
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.
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Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Relação Estrutura-AtividadeRESUMO
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.
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Antimicrobial-resistant infections are an urgent public health threat, and development of novel antimicrobial therapies has been painstakingly slow. Polymicrobial infections are increasingly recognized as a significant source of severe disease and also contribute to reduced susceptibility to antimicrobials. Chronic infections also are characterized by their ability to resist clearance, which is commonly linked to the development of biofilms that are notorious for antimicrobial resistance. The use of engineered cationic antimicrobial peptides (eCAPs) is attractive due to the slow development of resistance to these fast-acting antimicrobials and their ability to kill multidrug-resistant clinical isolates, key elements for the success of novel antimicrobial agents. Here, we tested the ability of an eCAP, WLBU2, to disrupt recalcitrant Pseudomonas aeruginosa biofilms. WLBU2 was capable of significantly reducing biomass and viability of P. aeruginosa biofilms formed on airway epithelium and maintained activity during viral coinfection, a condition that confers extraordinary levels of antibiotic resistance. Biofilm disruption was achieved in short treatment times by permeabilization of bacterial membranes. Additionally, we observed simultaneous reduction of infectivity of the viral pathogen respiratory syncytial virus (RSV). WLBU2 is notable for its ability to maintain activity across a broad range of physiological conditions and showed negligible toxicity toward the airway epithelium, expanding its potential applications as an antimicrobial therapeutic. IMPORTANCE Antimicrobial-resistant infections are an urgent public health threat, making development of novel antimicrobials able to effectively treat these infections extremely important. Chronic and polymicrobial infections further complicate antimicrobial therapy, often through the development of microbial biofilms. Here, we describe the ability of an engineered antimicrobial peptide to disrupt biofilms formed by the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen Pseudomonas aeruginosa during coinfection with respiratory syncytial virus. We also observed antiviral activity, indicating the ability of engineered antimicrobial peptides to act as cross-kingdom single-molecule combination therapies.
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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.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Células Epiteliais/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Técnicas de Cocultura , Humanos , Engenharia de Proteínas , Pseudomonas aeruginosa/fisiologia , Proteínas Recombinantes/genéticaRESUMO
We previously reported a series of de novo engineered cationic antibiotic peptides (eCAPs) consisting exclusively of arginine and tryptophan (WR) that display potent activity against diverse multidrug-resistant (MDR) bacterial strains. In this study, we sought to examine the influence of arginine compared to lysine on antibacterial properties by direct comparison of the WR peptides (8-18 residues) with a parallel series of engineered peptides containing only lysine and tryptophan. WR and WK series were compared for antibacterial activity by bacterial killing and growth inhibition assays and for mechanism of peptide-bacteria interactions by surface plasmon resonance and flow cytometry. Mammalian cytotoxicity was also assessed by flow cytometry, haemolytic and tetrazolium-based assays. The shortest arginine-containing peptides (8 and 10 mers) displayed a statistically significant increase in activity compared to the analogous lysine-containing peptides. The WR and WK peptides achieved maximum antibacterial activity at the 12-mer peptide (WK12 or WR12). Further examination of antibacterial mechanisms of the optimally active 12-mer peptides using surface plasmon resonance and flow cytometry demonstrates stronger interactions with Pseudomonasaeruginosa, greater membrane permeabilizing activity, and lower inhibitory effects of divalent cations on activity and membrane permeabilization properties of WR12 compared to WK12 (P < 0.05). Importantly, WK12 and WR12 displayed similar negligible haemolytic and cytotoxic effects at peptide concentrations up to ten times the MIC or 20 times the minimum bactericidal concentration. Thus, arginine, compared to lysine, can indeed yield enhanced antibacterial activity to minimize the required length to achieve functional antimicrobial peptides.