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Published on: June 30, 2016
Development and validation of a high-throughput cell-based screen to identify activators of a bacterial two-component
Julia J van Rensburg1, Kate R Fortney1, Lan Chen2
1Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Abstract:
CpxRA is a two-component signal transduction system (2CSTS) found in many drug-resistant Gram-negative bacteria. In response to periplasmic stress, CpxA autophosphorylates 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, the 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 the 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 that of the initial hit. This HTS is amenable for the screening of larger libraries to find compounds that activate CpxRA by other mechanisms, and it could be adapted to find activators of other two-component systems.
Insights
Researchers developed a novel high-throughput screen to find activators of the CpxRA two-component system, crucial for combating drug-resistant bacteria. A unique compound was identified that activates CpxRA, offering a potential new antivirulence strategy.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Drug Discovery
Background:
- The CpxRA two-component signal transduction system (2CSTS) regulates bacterial responses to periplasmic stress.
- Activation of CpxRA leads to avirulence in Gram-negative bacteria, suggesting its potential as an antivirulence target.
- Targeting CpxRA offers a novel antimicrobial and antivirulence strategy against drug-resistant pathogens.
Purpose of the Study:
- To develop and validate a cell-based high-throughput screen (HTS) for identifying activators of the CpxRA system.
- To discover novel compounds that activate CpxRA, specifically targeting its phosphatase activity.
- To explore the potential of CpxRA activators as a new class of antimicrobial or antivirulence agents.
Main Methods:
- Development of a CpxR-P-responsive lacZ reporter assay in Escherichia coli strains with deletions in CpxRA genes.
- Screening of 36,000 compounds using the developed HTS assay to identify CpxRA activators.
- Characterization of hit compounds, including activity profiling in mutant strains and assessment of cytotoxicity and serum stability.
Main Results:
- A novel cell-based HTS for CpxRA activators was successfully developed and validated.
- One hit compound was identified that activates CpxRA by inhibiting CpxA phosphatase activity, leading to CpxR-P accumulation.
- A derivative of the hit compound showed retained activity with reduced cytotoxicity and improved serum stability.
Conclusions:
- The developed HTS is effective for discovering CpxRA activators and can be adapted for other 2CSTS.
- The identified compound represents the first reported activator of a 2CSTS, demonstrating a new mechanism for modulating bacterial virulence.
- CpxRA activation holds significant promise for developing novel antivirulence therapies against drug-resistant Gram-negative bacteria.
