High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors

PloS One
Umesh K BageshwarSiegfried M Musser

Abstract

The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric fi...Continue Reading

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Citations

Jun 26, 2018·Angewandte Chemie·Markus LakemeyerStephan A Sieber
Aug 6, 2017·Current Microbiology·Deepanjan GhoshSureshkumar Ramasamy
Apr 21, 2021·Antimicrobial Agents and Chemotherapy·Hanh N LamVictoria Auerbuch
Sep 10, 2021·PloS One·Umesh K BageshwarSiegfried M Musser
Oct 14, 2021·Chembiochem : a European Journal of Chemical Biology·Upasana S PottethIshu Saraogi
Dec 4, 2021·ACS Infectious Diseases·Victoria E ColesLori L Burrows

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Methods Mentioned

BETA
X-ray
Fluorescence
fluorescence microscopy
fluorescence imaging
Assay
confocal microscopy

Software Mentioned

Genedata Assay Analyzer

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