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High throughput screening methods for assessing antibiofilm and immunomodulatory activities of synthetic peptides.

ABSTRACT: The recent observation that certain cationic peptides possess potent antibiofilm activity demonstrated that small peptides could be used to treat biofilm-associated infections. Other so-called innate defense regulator peptides possess potent immunomodulatory properties such as leukocyte recruitment and suppression of harmful inflammation. A peptide that directly targets biofilm cells while favorably modulating the immune response would be particularly advantageous for treating serious skin infections caused by Staphylococcus aureus. In the present work, using SPOT-synthesized peptide arrays on cellulose membranes, we outline a strategy for systematically assessing the antibiofilm activity of hundreds of IDR-1002 (VQRWLIVWRIRK-NH2) and IDR-HH2 (VQLRIRVAVIRA-NH2) peptide variants against MRSA biofilms. In addition, the ability of these peptides to stimulate production of a monocyte chemoattractant protein (MCP-1) and suppress LPS-induced interleukin (IL)-1? production in human peripheral blood mononuclear cells (PBMCs) was evaluated. These results informed the synthesis of second-generation peptides resulting in a new peptide, IDR-2009 (KWRLLIRWRIQK-NH2), with enhanced MCP-1 stimulatory activity, favorable IL-1? suppression characteristics and strong antibiofilm activity against MRSA and Pseudomonas aeruginosa biofilms. This work provides a proof-of-concept that multiple peptide activities can be optimized simultaneously to generate novel sequences that possess a variety of biological properties.

SUBMITTER: Haney EF

PROVIDER: S-EPMC4581888 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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High throughput screening methods for assessing antibiofilm and immunomodulatory activities of synthetic peptides.

Haney Evan F EF Mansour Sarah C SC Hilchie Ashley L AL de la Fuente-Núñez César C Hancock Robert E W RE

Peptides 20150331

The recent observation that certain cationic peptides possess potent antibiofilm activity demonstrated that small peptides could be used to treat biofilm-associated infections. Other so-called innate defense regulator peptides possess potent immunomodulatory properties such as leukocyte recruitment and suppression of harmful inflammation. A peptide that directly targets biofilm cells while favorably modulating the immune response would be particularly advantageous for treating serious skin infec ...[more]

PMID: 25836992

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Project description:Pseudomonas aeruginosa is the most common pathogen infecting cystic fibrosis (CF) lungs, causing acute and chronic infections. Intrinsic and acquired antibiotic resistance allow P. aeruginosa to colonize and persist despite antibiotic treatment, making new therapeutic approaches necessary. Combining high-throughput screening and drug repurposing is an effective way to develop new therapeutic uses for drugs. This study screened a drug library of 3,386 drugs, mostly FDA approved, to identify antimicrobials against P. aeruginosa under physicochemical conditions relevant to CF-infected lungs. Based on the antibacterial activity, assessed spectrophotometrically against the prototype RP73 strain and 10 other CF virulent strains, and the toxic potential evaluated toward CF IB3-1 bronchial epithelial cells, five potential hits were selected for further analysis: the anti-inflammatory and antioxidant ebselen, the anticancer drugs tirapazamine, carmofur, and 5-fluorouracil, and the antifungal tavaborole. A time-kill assay showed that ebselen has the potential to cause rapid and dose-dependent bactericidal activity. The antibiofilm activity was evaluated by viable cell count and crystal violet assays, revealing carmofur and 5-fluorouracil as the most active drugs in preventing biofilm formation regardless of the concentration. In contrast, tirapazamine and tavaborole were the only drugs actively dispersing preformed biofilms. Tavaborole was the most active drug against CF pathogens other than P. aeruginosa, especially against Burkholderia cepacia and Acinetobacter baumannii, while carmofur, ebselen, and tirapazamine were particularly active against Staphylococcus aureus and B. cepacia. Electron microscopy and propidium iodide uptake assay revealed that ebselen, carmofur, and tirapazamine significantly damage cell membranes, with leakage and cytoplasm loss, by increasing membrane permeability. IMPORTANCE Antibiotic resistance makes it urgent to design new strategies for treating pulmonary infections in CF patients. The repurposing approach accelerates drug discovery and development, as the drugs' general pharmacological, pharmacokinetic, and toxicological properties are already well known. In the present study, for the first time, a high-throughput compound library screening was performed under experimental conditions relevant to CF-infected lungs. Among 3,386 drugs screened, the clinically used drugs from outside infection treatment ebselen, tirapazamine, carmofur, 5-fluorouracil, and tavaborole showed, although to different extents, anti-P. aeruginosa activity against planktonic and biofilm cells and broad-spectrum activity against other CF pathogens at concentrations not toxic to bronchial epithelial cells. The mode-of-action studies revealed ebselen, carmofur, and tirapazamine targeted the cell membrane, increasing its permeability with subsequent cell lysis. These drugs are strong candidates for repurposing for treating CF lung P. aeruginosa infections.

Project description:BackgroundVisceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis.Methodology/principal findingsImmunodominant proteins from L. infantum were mapped in silico to predict B cell epitopes, and the 360 predicted peptides were synthesized on cellulose membranes. Immunoassays were used to select the most reactive peptides, which were then investigated with canine sera. Next, the 10 most reactive peptides were synthesized using solid phase peptide synthesis protocol and tested using ELISA. The sensitivity and specificity of these peptides were also compared to the EIE-LVC Bio-Manguinhos kit, which is recommended by the Brazilian Ministry of Health for use in leishmaniasis control programs. The sensitivity and specificity of the selected synthesized peptides was as high as 88.70% and 95.00%, respectively, whereas the EIE-LVC kit had a sensitivity of 13.08% and 100.00% of specificity. Although the tests based on synthetic peptides were able to diagnose up to 94.80% of asymptomatic dogs with leishmaniasis, the EIE-LVC kit failed to detect the disease in any of the infected asymptomatic dogs.Conclusions/significanceOur study shows that ELISA using synthetic peptides is a technique with great potential for diagnosing CVL; furthermore, the use of these peptides in other diagnostic methodologies, such as immunochromatographic tests, could be beneficial to CVL control programs.

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High throughput screening methods for assessing antibiofilm and immunomodulatory activities of synthetic peptides.

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High throughput screening methods for assessing antibiofilm and immunomodulatory activities of synthetic peptides.

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