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Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa
Antibiotics are becoming less effective in treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa. Antimicrobial therapies based on the inhibition of specific virulence-related traits, as opposed to growth inhibitors, constitute an innovative and appealing approach to tackle th...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635566/ https://www.ncbi.nlm.nih.gov/pubmed/31355152 http://dx.doi.org/10.3389/fcimb.2019.00250 |
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author | Massai, Francesco Saleeb, Michael Doruk, Tugrul Elofsson, Mikael Forsberg, Åke |
author_facet | Massai, Francesco Saleeb, Michael Doruk, Tugrul Elofsson, Mikael Forsberg, Åke |
author_sort | Massai, Francesco |
collection | PubMed |
description | Antibiotics are becoming less effective in treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa. Antimicrobial therapies based on the inhibition of specific virulence-related traits, as opposed to growth inhibitors, constitute an innovative and appealing approach to tackle the threat of P. aeruginosa infections. The twin-arginine translocation (Tat) pathway plays an important role in the pathogenesis of P. aeruginosa, and constitutes a promising target for the development of anti-pseudomonal drugs. In this study we developed and optimized a whole-cell, one-well assay, based on native phospholipase C activity, to identify compounds active against the Tat system. Statistical robustness, sensitivity and consequently suitability for high-throughput screening (HTS) were confirmed by a dry run/pre-screening test scoring a Z′ of 0.82 and a signal-to-noise ratio of 49. Using this assay, we evaluated ca. 40,000 molecules and identified 59 initial hits as possible Tat inhibitors. Since phospholipase C is exported into the periplasm by Tat, and subsequently translocated across the outer membrane by the type II secretion system (T2SS), our assay could also identify T2SS inhibitors. To validate our hits and discriminate between compounds that inhibited either Tat or T2SS, two separate counter assays were developed and optimized. Finally, three Tat inhibitors and one T2SS inhibitor were confirmed by means of dose-response analysis and additional counter and confirming assays. Although none of the identified inhibitors was suitable as a lead compound for drug development, this study validates our assay as a simple, efficient, and HTS compatible method for the identification of Tat and T2SS inhibitors. |
format | Online Article Text |
id | pubmed-6635566 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-66355662019-07-26 Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa Massai, Francesco Saleeb, Michael Doruk, Tugrul Elofsson, Mikael Forsberg, Åke Front Cell Infect Microbiol Cellular and Infection Microbiology Antibiotics are becoming less effective in treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa. Antimicrobial therapies based on the inhibition of specific virulence-related traits, as opposed to growth inhibitors, constitute an innovative and appealing approach to tackle the threat of P. aeruginosa infections. The twin-arginine translocation (Tat) pathway plays an important role in the pathogenesis of P. aeruginosa, and constitutes a promising target for the development of anti-pseudomonal drugs. In this study we developed and optimized a whole-cell, one-well assay, based on native phospholipase C activity, to identify compounds active against the Tat system. Statistical robustness, sensitivity and consequently suitability for high-throughput screening (HTS) were confirmed by a dry run/pre-screening test scoring a Z′ of 0.82 and a signal-to-noise ratio of 49. Using this assay, we evaluated ca. 40,000 molecules and identified 59 initial hits as possible Tat inhibitors. Since phospholipase C is exported into the periplasm by Tat, and subsequently translocated across the outer membrane by the type II secretion system (T2SS), our assay could also identify T2SS inhibitors. To validate our hits and discriminate between compounds that inhibited either Tat or T2SS, two separate counter assays were developed and optimized. Finally, three Tat inhibitors and one T2SS inhibitor were confirmed by means of dose-response analysis and additional counter and confirming assays. Although none of the identified inhibitors was suitable as a lead compound for drug development, this study validates our assay as a simple, efficient, and HTS compatible method for the identification of Tat and T2SS inhibitors. Frontiers Media S.A. 2019-07-10 /pmc/articles/PMC6635566/ /pubmed/31355152 http://dx.doi.org/10.3389/fcimb.2019.00250 Text en Copyright © 2019 Massai, Saleeb, Doruk, Elofsson and Forsberg. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cellular and Infection Microbiology Massai, Francesco Saleeb, Michael Doruk, Tugrul Elofsson, Mikael Forsberg, Åke Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa |
title | Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa |
title_full | Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa |
title_fullStr | Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa |
title_full_unstemmed | Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa |
title_short | Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa |
title_sort | development, optimization, and validation of a high throughput screening assay for identification of tat and type ii secretion inhibitors of pseudomonas aeruginosa |
topic | Cellular and Infection Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635566/ https://www.ncbi.nlm.nih.gov/pubmed/31355152 http://dx.doi.org/10.3389/fcimb.2019.00250 |
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