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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
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).ORCID iD: 0000-0002-3219-4669
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2019 (English)In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 9, article id 250Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019. Vol. 9, article id 250
Keywords [en]
Pseudomonas aeruginosa, high-throughput screening, twin arginine translocase, type II secretion, virulence inhibitors, phospholipase C
National Category
Infectious Medicine
Identifiers
URN: urn:nbn:se:umu:diva-161822DOI: 10.3389/fcimb.2019.00250ISI: 000474778200001PubMedID: 31355152OAI: oai:DiVA.org:umu-161822DiVA, id: diva2:1342404
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-13Bibliographically approved

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Massai, FrancescoSaleeb, MichaelDoruk, TugrulElofsson, MikaelForsberg, Åke

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Department of Molecular Biology (Faculty of Medicine)Molecular Infection Medicine Sweden (MIMS)Umeå Centre for Microbial Research (UCMR)Department of ChemistryDepartment of Molecular Biology (Faculty of Science and Technology)
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