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Doruk, Tugrul
Publications (2 of 2) Show all publications
Massai, F., Saleeb, M., Doruk, T., Elofsson, M. & Forsberg, Å. (2019). Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa. Frontiers in Cellular and Infection Microbiology, 9, Article ID 250.
Open this publication in new window or tab >>Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa
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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
Keywords
Pseudomonas aeruginosa, high-throughput screening, twin arginine translocase, type II secretion, virulence inhibitors, phospholipase C
National Category
Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-161822 (URN)10.3389/fcimb.2019.00250 (DOI)000474778200001 ()31355152 (PubMedID)
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-13Bibliographically approved
Avican, U., Doruk, T., Östberg, Y., Fahlgren, A. & Forsberg, Å. (2017). The Tat substrate SufI is critical for the ability of Yersinia pseudotuberculosis to cause systemic infection. Infection and Immunity, 85(4), Article ID e00867-16.
Open this publication in new window or tab >>The Tat substrate SufI is critical for the ability of Yersinia pseudotuberculosis to cause systemic infection
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2017 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 85, no 4, article id e00867-16Article in journal (Refereed) Published
Abstract [en]

The twin arginine translocation (Tat) system targets folded proteins across the inner membrane and is crucial for virulence in many important humanpathogenic bacteria. Tat has been shown to be required for the virulence of Yersinia pseudotuberculosis, and we recently showed that the system is critical for different virulence-related stress responses as well as for iron uptake. In this study, we wanted to address the role of the Tat substrates in in vivo virulence. Therefore, 22 genes encoding potential Tat substrates were mutated, and each mutant was evaluated in a competitive oral infection of mice. Interestingly, a.sufI mutant was essentially as attenuated for virulence as the Tat-deficient strain. We also verified that SufI was Tat dependent for membrane/periplasmic localization in Y. pseudotuberculosis. In vivo bioluminescent imaging of orally infected mice revealed that both the.sufI and Delta tatC mutants were able to colonize the cecum and Peyer's patches (PPs) and could spread to the mesenteric lymph nodes (MLNs). Importantly, at this point, neither the Delta tatC mutant nor the Delta sufI mutant was able to spread systemically, and they were gradually cleared. Immunostaining of MLNs revealed that both the Delta tatC and Delta sufI mutants were unable to spread from the initial infection foci and appeared to be contained by neutrophils, while wild-type bacteria readily spread to establish multiple foci from day 3 postinfection. Our results show that SufI alone is required for the establishment of systemic infection and is the major cause of the attenuation of the Delta tatC mutant.

Keywords
Yersinia pseudotuberculosis, Tat pathway, virulence, SufI, mesenteric lymph nodes, neutrophils
National Category
Microbiology Immunology
Identifiers
urn:nbn:se:umu:diva-128087 (URN)10.1128/IAI.00867-16 (DOI)000397581800003 ()28115509 (PubMedID)
Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2018-06-09Bibliographically approved
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