ABSTRACT
Innate defense regulator-1002 (IDR-1002) is a synthetic peptide with promising immunomodulatory and antibiofilm properties. An appreciable body of work exists around its mechanism of action at the cellular and molecular level, along with its efficacy across several infection and inflammation models. However, little is known about its absorption, distribution, and excretion in live organisms. Here, we performed a comprehensive biodistribution assessment with a gallium-67 radiolabeled derivative of IDR-1002 using nuclear tracing techniques. Various dose levels of the radiotracer (2-40 mg/kg) were administered into the blood, peritoneal cavity, and subcutaneous tissue, or instilled into the lungs. The peptide was well tolerated at all subcutaneous and intraperitoneal doses, although higher levels were associated with delayed absorption kinetics and precipitation of the peptide within the tissues. Low intratracheal doses were rapidly absorbed systemically, and small increases in the dose level were lethal. Intravenous doses were rapidly cleared from the blood at lower levels, and upon escalation, were toxic with a high proportion of the dose accumulating within the lung tissue. To improve biocompatibility and prolong its circulation within the blood, IDR-1002 was further formulated onto high molecular weight hyperbranched polyglycerol (HPG) polymers. Constructs prepared at 5:1 and 10:1 peptide-to-polymer ratios were colloidally stable, maintained the biological profile of the peptide payload and helped reduce red blood cell lysis. The 5:1 construct circulated well in the blood, but higher peptide loading was associated with rapid clearance by the reticuloendothelial system. Many peptides face pharmacokinetic and biocompatibility challenges, but formulations such as those with HPG have the potential to overcome these limitations.
Subject(s)
Gallium Radioisotopes , Animals , Tissue Distribution , Mice , Gallium Radioisotopes/pharmacokinetics , Gallium Radioisotopes/chemistry , Gallium Radioisotopes/administration & dosage , Lung/metabolism , Lung/drug effects , Peptides/chemistry , Peptides/pharmacokinetics , Female , Nanoparticles/chemistry , Mice, Inbred C57BL , Male , Immunity, Innate/drug effects , Antimicrobial Cationic Peptides/pharmacokinetics , Antimicrobial Cationic Peptides/chemistryABSTRACT
OBJECTIVE: To investigate the effectiveness of a d-enantiomeric antibiofilm peptide (DJK-5) on the anticorrosion ability of titanium (Ti) with different surface roughness against Streptococcus mutans biofilms. METHODS: Commercially pure Ti disks with machined (MA, smooth) or sandblasted + acid-etched (SLA, rough) surfaces were prepared and characterized. All disks were divided into three groups: a positive control (PC) group with S. mutans, a DJK-5-treated group, and a negative control (NC) group without S. mutans. Biofilm formation and corrosion on Ti surfaces were determined by confocal laser scanning microscopy and scanning electron microscopy after 2 and 6 days, and the electrochemical properties were evaluated. RESULTS: Ten µg/mL of DJK-5 killed 83.3 % and 87.4 % of biofilms on SLA and MA Ti surfaces, respectively after 2 days, and 72.9 % and 77.7 % after 6 days, with more bacteria surviving on SLA surfaces with higher roughness (p < 0.05). DJK-5 treatment induced less surface defects with tiny pit corrosion than PC. DJK-5 treatment when compared to PC, led to electrochemical properties more reflecting NC surfaces, including significantly less negative corrosion potential, lower corrosion current, and higher passive film resistance (p < 0.05). SLA surfaces exhibited higher current density and lower resistance than MA surfaces (p < 0.05). CONCLUSION: DJK-5 effectively enhanced the corrosion resistance of Ti with different surface roughness while killing S. mutans biofilms, and smooth surfaces were more susceptible to peptide treatment. CLINICAL SIGNIFICANCE: The antibiofilm peptide is promising for promoting the anticorrosion ability of Ti against biofilms, thereby preventing biofilm-related infections.
Subject(s)
Streptococcus mutans , Titanium , Titanium/pharmacology , Titanium/chemistry , Biofilms , Peptides , Microscopy, Electron, Scanning , Surface PropertiesABSTRACT
OBJECTIVE: To evaluate the dynamics of killing of oral multispecies biofilms grown on dental restorative materials by commercially available mouthrinses and a d-enantiomeric peptide. METHODS: Four composite resins (3 M Supreme, 3 M Supreme flow, Kerr Sonicfill, and Shofu Beautifil II) and one glass ionomer (GC Fuji II) were used as restorative materials. Plaque biofilms were grown on the surfaces of restorative material discs for 1 week. The surface roughness and biofilm attachment were assessed by atomic force microscopy and scanning electron microscopy. One-week-old biofilms grown anaerobically at 37 °C were exposed to each of five solutions for one minute (twice daily for seven days): Listerine Total care and Paroex Gum mouthrinses, 0.12% chlorhexidine, 0.001% d-enantiomeric peptide DJK-5, and sterile water. The dynamic variation of the biovolume of the biofilms and the percentage of dead bacteria were monitored and analyzed using confocal laser scanning microscopy. RESULTS: All restorative materials had similar surface roughness with intact biofilm attachment. The percentage of dead bacteria and biovolume of biofilms treated by each oral rinse solution remained constant between days 1 and 7, with no statistically significant difference. DJK-5 showed the highest percentage of dead bacteria (up to 75.7%; cf. â¼20-40% for other mouthrinses) of all solutions tested within 7 days. CONCLUSIONS: DJK-5 outperformed conventional mouthrinses in killing bacteria in oral multispecies biofilms grown on dental restorative materials. CLINICAL SIGNIFICANCE: The antimicrobial peptide DJK-5 is effective against oral biofilms and serves as a promising candidate for the development of future mouthrinses to improve long-term oral hygiene.
Subject(s)
Chlorhexidine , Dental Plaque , Humans , Chlorhexidine/pharmacology , Peptides , Mouthwashes/pharmacology , Bacteria , BiofilmsABSTRACT
Antimicrobial peptides (AMPs) are promising next-generation antibiotics that can be used to combat drug-resistant pathogens. However, the high cost involved in AMP synthesis and their short plasma half-life render their clinical translation a challenge. To address these shortcomings, we report efficient production of bioactive amidated AMPs by transient expression of glycine-extended AMPs in Nicotiana benthamiana line expressing the mammalian enzyme peptidylglycine α-amidating mono-oxygenase (PAM). Cationic AMPs accumulate to substantial levels in PAM transgenic plants compare to nontransgenic N. benthamiana. Moreover, AMPs purified from plants exhibit robust killing activity against six highly virulent and antibiotic resistant ESKAPE pathogens, prevent their biofilm formation, analogous to their synthetic counterparts and synergize with antibiotics. We also perform a base case techno-economic analysis of our platform, demonstrating the potential economic advantages and scalability for industrial use. Taken together, our experimental data and techno-economic analysis demonstrate the potential use of plant chassis for large-scale production of clinical-grade AMPs.
Subject(s)
Antimicrobial Cationic Peptides , Antimicrobial Peptides , Animals , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/biosynthesis , Antimicrobial Cationic Peptides/pharmacology , Antimicrobial Peptides/biosynthesis , Mammals , Plants , Nicotiana/chemistry , Nicotiana/genetics , Drug Resistance, Bacterial/drug effectsABSTRACT
Titanium alloys have gained popularity in implant dentistry for the restoration of missing teeth and related hard tissues because of their biocompatibility and enhanced strength. However, titanium corrosion and infection caused by microbial biofilms remains a significant clinical challenge leading to implant failure. This study aimed to evaluate the effectiveness of antibiofilm peptides 1018 and DJK-5 on the corrosion resistance of titanium in the presence of Streptococcus mutans. Commercially pure titanium disks were prepared and used to form biofilms. The disks were randomly assigned to different treatment groups (exposed to S. mutans supplied with sucrose) including a positive control with untreated biofilms, peptides 1018 or DJK-5 at concentrations of 5 µg/mL or 10 µg/mL, and a negative control with no S. mutans. Dynamic biofilm growth and pH variation of all disks were measured after one or two treatment periods of 48 h. After incubation, the dead bacterial proportion, surface morphology, and electrochemical behaviors of the disks were determined. The results showed that peptides 1018 and DJK-5 exhibited significantly higher dead bacterial proportions than the positive control group in a concentration dependent manner (p < 0.01), as well as far less defects in microstructure. DJK-5 at 10 µg/mL killed 84.82% of biofilms and inhibited biofilm growth, preventing acidification due to S. mutans and maintaining a neutral pH. Potential polarization and electrochemical impedance spectroscopy data revealed that both peptides significantly reduced the corrosion and passive currents on titanium compared to titanium surfaces with untreated biofilms, and increased the resistance of the passive film (p < 0.05), with 10 µg/mL of DJK-5 achieving the greatest effect. These findings demonstrated that antibiofilm peptides are effective in promoting corrosion resistance of titanium against S. mutans, suggesting a promising strategy to enhance the stability of dental implants by endowing them with antibiofilm and anticorrosion properties.
ABSTRACT
Bacterial biofilm infections associated with wounded skin are prevalent, recalcitrant, and in urgent need of treatments. Additionally, host responses in the skin to biofilm infections are not well understood. Here we employed a human organoid skin model to explore the transcriptomic changes of thermally-injured epidermis to methicillin-resistant Staphylococcus aureus (MRSA) biofilm colonization. MRSA biofilm impaired skin barrier function, enhanced extracellular matrix remodelling, elicited inflammatory responses including IL-17, IL-12 family and IL-6 family interleukin signalling, and modulated skin metabolism. Synthetic antibiofilm peptide DJK-5 effectively diminished MRSA biofilm and associated skin inflammation in wounded human ex vivo skin. In the epidermis, DJK-5 shifted the overall skin transcriptome towards homeostasis including modulating the biofilm induced inflammatory response, promoting the skin DNA repair function, and downregulating MRSA invasion of thermally damaged skin. These data clarified the underlying immunopathogenesis of biofilm infections and revealed the intrinsic promise of synthetic peptides in reducing inflammation and biofilm infections.
Subject(s)
Methicillin-Resistant Staphylococcus aureus , Humans , Methicillin-Resistant Staphylococcus aureus/physiology , Anti-Bacterial Agents/pharmacology , Biofilms , Epidermis/metabolism , Peptides/metabolism , Inflammation/metabolismABSTRACT
Host defense peptides (HDPs) represent an alternative way to address the emergence of antibiotic resistance. Crocodylians are interesting species for the study of these molecules because of their potent immune system, which confers high resistance to infection. Profile hidden Markov models were used to screen the genomes of four crocodylian species for encoded cathelicidins and eighteen novel sequences were identified. Synthetic cathelicidins showed broad spectrum antimicrobial and antibiofilm activity against several clinically important antibiotic-resistant bacteria. In particular, the As-CATH8 cathelicidin showed potent in vitro activity profiles similar to the last-resort antibiotics vancomycin and polymyxin B. In addition, As-CATH8 demonstrated rapid killing of planktonic and biofilm cells, which correlated with its ability to cause cytoplasmic membrane depolarization and permeabilization as well as binding to DNA. As-CATH8 displayed greater antibiofilm activity than the human cathelicidin LL-37 against methicillin-resistant Staphylococcus aureus in a human organoid model of biofilm skin infection. Furthermore, As-CATH8 demonstrated strong antibacterial effects in a murine abscess model of high-density bacterial infections against clinical isolates of S. aureus and Acinetobacter baumannii, two of the most common bacterial species causing skin infections globally. Overall, this work expands the repertoire of cathelicidin peptides known in crocodylians, including one with considerable therapeutic promise for treating common skin infections.
ABSTRACT
Innate defense regulators (IDRs) are synthetic host-defense peptides (HDPs) with broad-spectrum anti-infective properties, including immunomodulatory, anti-biofilm and direct antimicrobial activities. A lack of pharmacokinetic data about these peptides hinders their development and makes it challenging to fully understand how they work in vivo since their mechanism of action is dependent on tissue concentrations of the peptide. Here, we set out to define in detail the pharmacokinetics of a well-characterized IDR molecule, IDR-1018. To make the peptide traceable, it was radiolabeled with the long-lived gamma-emitting isotope gallium-67. After a series of bench-top characterizations, the radiotracer was administered to healthy mice intravenously (IV) or subcutaneously (SQ) at various dose levels (2.5-13 mg/kg). Nuclear imaging and ex-vivo biodistributions were used to quantify organ and tissue uptake of the radiotracer over time. When administered as an IV bolus, the distribution profile of the radiotracer changed as the dose was escalated. At 2.5 mg/kg, the peptide was well-tolerated, poorly circulated in the blood and was cleared predominantly by the reticuloendothelial system. Higher doses (7 and 13 mg/kg) as an IV bolus were almost immediately lethal due to respiratory arrest; significant lung uptake of the radiotracer was observed from nuclear scans of these animals, and histological examination found extensive damage to the pulmonary vasculature and alveoli. When administered SQ at a dose of 3 mg/kg, radiolabeled IDR-1018 was rapidly absorbed from the site of injection and predominately cleared renally. Apart from the SQ injection site, no other tissue had a concentration above the minimum inhibitory concentration that would enable this peptide to exert direct antimicrobial effects against most pathogenic bacteria. Tissue concentrations were sufficient, however, to disrupt microbial biofilms and alter the host immune response. Overall, this study demonstrated that the administration of synthetic IDR peptide in vivo is best suited to local administration which avoids some of the issues associated with peptide toxicity that are observed when administered systemically by IV injection, an issue that will have to be addressed through formulation.
Subject(s)
Anti-Infective Agents , Antimicrobial Cationic Peptides , Animals , Antimicrobial Cationic Peptides/toxicity , Immunity, Innate , Mice , Microbial Sensitivity Tests , Tissue DistributionABSTRACT
Rotifers are used as the first feed for marine fish larvae and are grown in large cultures that have high loads of organic matter and heterotrophic bacteria; these bacteria are passed on to the developing fish larvae and can potentially lead to bacterial infections. A modified minimum inhibitory concentration (MIC) protocol for antimicrobial peptides was used to determine the potency of ten antimicrobial peptides (AMPs) in artificial seawater relevant to a rotifer culture (salinity of 25) against common marine pathogens. All of the AMPs had antimicrobial activity against the bacterial isolates when the salt concentration was approximately zero. However, in high salt concentrations, the majority of the AMPs had an MIC value greater than 65 µg mL-1 in artificial seawater (25). The only exceptions were 2009 (32.5 µg mL-1) and 3002 (32.5 µg mL-1) against Vibrio rotiferianus and Tenacibaculum discolor, respectively. The selected synthetic AMPs were not effective at reducing the bacterial load in brackish salt concentrations of a typical commercial rotifer culture (25).
Subject(s)
Antimicrobial Peptides , Rotifera , Animals , Larva/microbiology , Rotifera/microbiology , SeawaterABSTRACT
Multifunctional scaffolds with host defense peptides designed for regenerative endodontics are desirable nanobiotechnological tools for dentistry. Here, different scaffolds were tested for use during the pulp revascularization process, including poly(vinyl alcohol)-PVA hydrogels or resins, collagen hydrogels and poly(vinyl alcohol) PVA/Chitosan (PVA/CS) nanofibers. Based on time to degradation (21 days), nanofibers were chosen to be incorporated with ciprofloxacin and IDR-1002 (each at 50 mg/g). Nanofibers containing ciprofloxacin and IDR-1002 had anti-biofilm activity against Enterococcus faecalis, Staphylococcus aureus and a multispecies oral biofilm, besides anti-inflammatory activities. The in vivo subcutaneous tissue response to tooth fragments filled with nanofibers demonstrated a pulp-like tissue formation, when compared to empty teeth fragments. Thus, we designed a strong antimicrobial, immunomodulatory and regenerative candidate for pulp revascularization and regeneration procedures.
ABSTRACT
Host defense peptides (HDPs) have been the subject of great interest for the treatment of multidrug-resistant bacterial infections due to their multimodal activity and low induction of resistance. However, aggregation, toxicity, and short biological half-life have limited their applicability for clinical treatment. Many methods have been explored to alleviate these issues, such as polymer (e.g., polyethylene glycol (PEG)) conjugation, but these are often accompanied by reductions in the activity of the HDP. Here, we detail the design of a novel PEG-HDP conjugate incorporating an enzymatic cleavage sequence targeting matrix metalloproteinases (MMPs) that accumulate at sites of inflammation and infection. Addition of the cleavage sequence onto either the N- or the C-terminal region of the parent peptide (peptide 73, a derivative of the HDP aurein 2.2) was explored to determine the location for optimal antimicrobial activity following MMP cleavage; furthermore, the susceptibility of the peptide to MMP cleavage after conjugation to 2 kDa or 5 kDa PEG was examined. The top candidate, L73, utilized an N-terminal cleavage site that was subsequently conjugated to a 2 kDa PEG polymer. Both L73 and the conjugate exhibited no antimicrobial activity in vitro until cleaved by purified MMP, which liberated a peptide fragment with 16- or 63-fold improved activity, respectively, corresponding to a minimum inhibitory concentration (MIC) of 8 µg/mL, comparable to that of peptide 73 (4 µg/mL). Furthermore, PEG conjugation improved the blood compatibility and reduced the aggregation tendency of the HDP in vitro, indicating enhanced biocompatibility. When administered as a single subcutaneous dose (~3.6 mg, or a peptide concentration of 142 mg/kg) in a mouse abscess model of high-density methicillin-resistant Staphylococcus aureus (MRSA) infection, the conjugate displayed strong activity, reducing abscess size and bacterial load by 73.3% and 58-fold, respectively. This activity was completely lost when the cleavage site was rendered resistant to MMPs by the substitution of two d-amino acids, supporting the hypothesis that antimicrobial activity was dependent on cleavage by MMPs, which were shown here to increasingly accumulate at the abscess site up to 18 h post infection. Finally, the conjugate displayed biocompatibility in vivo, with no identifiable toxicity or aggregation.
Subject(s)
Antimicrobial Cationic Peptides , Methicillin-Resistant Staphylococcus aureus , Animals , Drug Resistance, Multiple, Bacterial , Mice , Microbial Sensitivity Tests , Polyethylene GlycolsABSTRACT
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).
Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Biofouling/prevention & control , Coated Materials, Biocompatible/pharmacology , Immobilized Proteins/pharmacology , Acrylamides/chemistry , Animals , Anti-Bacterial Agents/chemical synthesis , Antimicrobial Cationic Peptides/chemical synthesis , Catheters/microbiology , Coated Materials, Biocompatible/chemical synthesis , Humans , Immobilized Proteins/chemical synthesis , Indoles/chemistry , Male , Mice, Inbred BALB C , Polymers/chemistry , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/physiology , Staphylococcus saprophyticus/drug effects , Staphylococcus saprophyticus/physiology , Urinary Tract Infections/prevention & controlABSTRACT
Host defence peptides (HDPs) are integral components of innate immunity across all living organisms. These peptides can exert direct antibacterial effects, targeting planktonic cells (referred to as antimicrobial peptides), and exhibit antibiofilm (referred to as antibiofilm peptides), antiviral, antifungal and host-directed immunomodulatory activities. In this Review, we discuss how the complex functional attributes of HDPs provide many opportunities for the development of antimicrobial therapeutics, focusing particularly on their emerging antibiofilm properties. The mechanisms of action of antibiofilm peptides are compared and contrasted with those of antimicrobial peptides. Furthermore, obstacles for the practical translation of candidate peptides into therapeutics and the potential solutions are discussed. Critically, HDPs have the value-added assets of complex functional attributes, particularly antibiofilm and anti-inflammatory activities and their synergy with conventional antibiotics.
Subject(s)
Anti-Infective Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Antimicrobial Peptides/pharmacology , Biofilms/drug effects , Immunomodulating Agents/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/chemistry , Anti-Inflammatory Agents/chemistry , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Peptides/chemistry , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Immunity, Innate , Immunomodulating Agents/chemistryABSTRACT
Bacterial biofilms demonstrate high broad-spectrum adaptive antibiotic resistance and cause two thirds of all infections, but there is a lack of approved antibiofilm agents. Unlike the standard minimal inhibitory concentration assay to assess antibacterial activity against planktonic cells, there is no standardized method to evaluate biofilm inhibition and/or eradication capacity of novel antibiofilm compounds. The protocol described here outlines simple and reproducible methods for assessing the biofilm inhibition and eradication capacities of novel antibiofilm agents against adherent bacterial biofilms grown in 96-well microtiter plates. It employs two inexpensive dyes: crystal violet to stain adhered biofilm biomass and 2,3,5-triphenyl tetrazolium chloride to quantify metabolism of the biofilm cells. The procedure is accessible to any laboratory with a plate reader, requires minimal technical expertise or training and takes 4 or 5 d to complete. Recommendations for how biofilm inhibition and eradication results should be interpreted and presented are also described.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Physiological Phenomena/drug effects , Biofilms/drug effects , Biofilms/growth & development , Biological Assay/methods , Microtechnology/methods , Bacterial Adhesion/drug effects , Time FactorsABSTRACT
Peptides are promising drug candidates because of their diversity, biocompatibility and spectrum of activities. Here, we describe a protocol for high-throughput screening of SPOT-peptide arrays to assess the antibiofilm, antimicrobial and immunomodulatory activities of synthetic peptides. It is a Protocol Extension of our previous Nature Protocols article, which describes the synthesis of SPOT-peptide arrays and assays for screening antimicrobial activity. This latest protocol allows the simultaneous assessment of hundreds of synthetic host defense peptides to define their overall activity profiles and identify candidate sequences that are suitable for further characterization and development as anti-infectives. When coupled with the SPOT-array technology for peptide synthesis, the described procedures are rapid, inexpensive and straightforward for peptide library screening. The protocols can be implemented in most microbiology or immunology research laboratories without the need for specialists. The time to complete each step ranges between 1 and 4 h with overnight pauses, and datasets related to the antibiofilm and immunomodulatory activities of a large set of peptide sequences can be generated in a few days.
Subject(s)
Antimicrobial Cationic Peptides/chemical synthesis , Biofilms/drug effects , Immunologic Factors/pharmacology , Protein Array Analysis/methods , Antimicrobial Cationic Peptides/pharmacology , Cell Death/drug effects , HumansABSTRACT
ß-defensin host defense peptides are important components of the innate immune system of vertebrates. Although evidence of their broad antimicrobial, antibiofilm and immunomodulatory activities in mammals have been presented, ß-defensins from other vertebrate species, like crocodylians, remain largely unexplored. In this study, five new crocodylian ß-defensin variants from Alligator mississippiensis and Crocodylus porosus were selected for synthesis and characterization based on their charge and hydrophobicity values. Linear peptides were synthesized, folded, purified and then evaluated for their antimicrobial and antibiofilm activities against the bacterial pathogens, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Enterobacter cloacae and Acinetobacter baumannii. The Am23SK variant (SCRFSGGYCIWNWERCRSGHFLVALCPFRKRCCK) from A. mississippiensis displayed promising activity against both planktonic cells and bacterial biofilms, outperforming the human ß-defensin 3 under the experimental conditions. Moreover, Am23SK exhibited no cytotoxicity towards mammalian cells and exerted immunomodulatory effects in vitro, moderately suppressing the production of proinflammatory mediators from stimulated human bronchial epithelial cells. Overall, our results have expanded the activity landscape of crocodylian and reptilian ß-defensin in general.
Subject(s)
Alligators and Crocodiles , Anti-Bacterial Agents/pharmacology , beta-Defensins/chemistry , beta-Defensins/pharmacology , Animals , Anti-Bacterial Agents/chemistry , Biofilms/drug effects , Cell Line , Epithelial Cells , Humans , Immunomodulating Agents/chemistry , Immunomodulating Agents/pharmacology , Microbial Sensitivity Tests , Protein Folding , beta-Defensins/chemical synthesisABSTRACT
Bacterial biofilms cause 65% of all human infections and are highly resistant to antibiotic therapy but lack specific treatments. To provide a human organoid model for studying host-microbe interplay and enabling screening for novel antibiofilm agents, a human epidermis organoid model with robust methicillin-resistant Staphylococcus aureus (MRSA) USA300 and Pseudomonas aeruginosa PAO1 biofilm was developed. Treatment of 1-day and 3-day MRSA and PAO1 biofilms with antibiofilm peptide DJK-5 significantly and substantially reduced the bacterial burden. This model enabled the screening of synthetic host defense peptides, revealing their superior antibiofilm activity against MRSA compared to the antibiotic mupirocin. The model was extended to evaluate thermally wounded skin infected with MRSA biofilms resulting in increased bacterial load, cytotoxicity, and pro-inflammatory cytokine levels that were all reduced upon treatment with DJK-5. Combination treatment of DJK-5 with an anti-inflammatory peptide, 1002, further reduced cytotoxicity and skin inflammation.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Models, Biological , Organoids/microbiology , Anti-Bacterial Agents/therapeutic use , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Bacterial Load/drug effects , Biofilms/growth & development , Burns/drug therapy , Burns/immunology , Burns/microbiology , Drug Evaluation, Preclinical , Drug Therapy, Combination , Humans , Methicillin-Resistant Staphylococcus aureus/drug effects , Oligopeptides/pharmacology , Oligopeptides/therapeutic use , Organoids/drug effects , Organoids/immunology , Organoids/injuries , Pseudomonas aeruginosa/drug effects , Skin/drug effects , Skin/immunology , Skin/injuries , Skin/microbiologyABSTRACT
Host-defense peptides (HDPs) are vital components of innate immunity in all vertebrates. While their antibacterial activity toward bacterial cells was the original focus for research, their ability to modulate immune and inflammatory processes has emerged as one of their major functions in the host and as a promising approach from which to develop novel therapeutics targeting inflammation and innate immunity. In this review, with particular emphasis on the cathelicidin family of peptides, the roles of natural HDPs are examined in managing immune activation, cellular recruitment, cytokine responses, and inflammation in response to infection, as well as their contribution(s) to various inflammatory disorders and autoimmune diseases. Furthermore, we discuss current efforts to develop synthetic HDPs as therapeutics aimed at restoring balance to immune responses that are dysregulated and contribute to disease pathologies.
ABSTRACT
BACKGROUND: Bacterial infections represent a major worldwide health problem the antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the antimicrobial activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes through the Joker algorithm. METHODS: Antimicrobial activity was measured by broth microdilution method. Membrane integrity was measured using fluorescent probes and through scanning electron microscopy imaging. A sliding window of truncated peptides was used to determine the EcDBS1R6 active core. Molecular dynamics in TFE/water environment was used to assess the EcDBS1R6 structure. RESULTS: Signal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker transformed this peptide into a membrane-active agent capable of killing bacteria. The C-terminus was unable to fold into an α-helix whereas its fragments showed poor or no antimicrobial activity, suggesting that the EcDBS1R6 antibacterial core was located at the helical N-terminus, corresponding to the signal peptide portion of the parent peptide. CONCLUSION: The strategy of transforming signal peptides into AMPs appears to be promising and could be used to produce novel antimicrobial agents. GENERAL SIGNIFICANCE: The process of transforming an inactive signal peptide into an antimicrobial peptide could open a new venue for creating new AMPs derived from signal peptides.
Subject(s)
Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Escherichia coli/chemistry , Protein Sorting Signals , Amino Acid Sequence , Gram-Negative Bacteria/drug effects , Microbial Sensitivity Tests , Molecular Dynamics Simulation , Protein ConformationABSTRACT
Antimicrobial peptides (AMPs) constitute a diverse family of peptides with the ability to protect their host against microbial infections. In addition to their ability to kill microorganisms, several AMPs also exhibit selective cytotoxicity towards cancer cells and are collectively referred to as anticancer peptides (ACPs). Here a large library of AMPs, mainly derived from the porcine cathelicidin peptide, tritrpticin (VRRFPWWWPFLRR), were assessed for their anticancer activity against the Jurkat T cell leukemia line. These anticancer potencies were compared to the cytotoxicity of the peptides towards normal cells isolated from healthy donors, namely peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs; where hemolytic activity was assessed). Among the active tritrpticin derivatives, substitution of Arg by Lys enhanced the selectivity of the peptides towards Jurkat cells when compared to PBMCs. Additionally, the side chain length of the Lys residues was also optimized to further enhance the tritrpticin ACP selectivity at low concentrations. The mechanism of action of the peptides with high selectivity involved the permeabilization of the cytoplasmic membrane of Jurkat cells, without formation of apoptotic bodies. The incorporation of non-natural Lys-based cationic amino acids could provide a new strategy to improve the selectivity of other synthetic ACPs to enhance their potential for therapeutic use against leukemia cells.