Development and Validation of a High-throughput Cell-based Screen to Identify Activators of a Bacterial Two-component Signal Transduction System Journal Article

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Authors: van Rensburg, J. J.; Fortney, K. R.; Chen, L; Krieger, A. J.; Lima, B. P.; Wolfe, A. J.; Katz, B. P.; Zhang, Z. Y.; Spinola, S. M.
Article Title: Development and Validation of a High-throughput Cell-based Screen to Identify Activators of a Bacterial Two-component Signal Transduction System
Abstract: CpxRA is a two-component signal transduction system (2CSTS) found in many drug-resistant Gram-negative bacteria. In response to periplasmic stress, CpxA auto-phosphorylates and donates a phosphoryl group to its cognate response regulator, CpxR. Phosphorylated CpxR (CpxR-P) upregulates genes involved in membrane repair and downregulates multiple genes that encode virulence factors, which are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and Haemophilus ducreyi are avirulent in mice and humans, respectively. Thus, activation of CpxRA has high potential as a novel antimicrobial/antivirulence strategy. Using a series of Escherichia coli strains containing a CpxR-P-responsive lacZ reporter and deletions in genes encoding CpxRA system components, we developed and validated a novel cell-based high-throughput screen (HTS) for CpxRA activators. A screen of 36,000 compounds yielded one hit compound that increased reporter activity in wild-type cells. This is the first report of a compound that activates, rather than inhibits, a 2CSTS. The activity profile of the compound against CpxRA pathway mutants in the presence of glucose suggested that the compound inhibits CpxA phosphatase activity. We confirmed that the compound induced accumulation of CpxR-P in treated cells. Although the hit compound contained a nitro group, a derivative lacking this group retained activity in serum and had lower cytotoxicity than the initial hit. This HTS is amenable for screening of larger libraries to find compounds that activate CpxRA by other mechanisms and could be adapted to find activators of other two-component systems.
Journal Title: Antimicrobial Agents and Chemotherapy
ISSN: 1098-6596; 0066-4804
Publisher: American Society for Microbiology. All Rights Reserved  
Date Published: 2015
Language: ENG
DOI/URL:

AAC.00236-15
Notes: LR: 20150415; CI: Copyright (c) 2015; JID: 0315061; aheadofprint