Development and validation of a high-throughput cell-based screen to identify activators of a bacterial two-component signal transduction system

Julia J. Van Rensburg, Kate R. Fortney, Lan Chen, Andrew J. Krieger, Bruno P. Lima, Alan J. Wolfe, Barry Katz, Zhong-Yin Zhang, Stanley Spinola

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)3789-3799
Number of pages11
JournalAntimicrobial Agents and Chemotherapy
Volume59
Issue number7
DOIs
StatePublished - Jul 1 2015

Fingerprint

Signal Transduction
Haemophilus ducreyi
Salmonella enterica
Gene Deletion
Virulence Factors
Gram-Negative Bacteria
Phosphoric Monoester Hydrolases
Genes
Libraries
Up-Regulation
Down-Regulation
Cell Membrane
Escherichia coli
Glucose
Membranes
Serum
Pharmaceutical Preparations
Serogroup

ASJC Scopus subject areas

  • Pharmacology (medical)
  • Pharmacology
  • Infectious Diseases

Cite this

Development and validation of a high-throughput cell-based screen to identify activators of a bacterial two-component signal transduction system. / Van Rensburg, Julia J.; Fortney, Kate R.; Chen, Lan; Krieger, Andrew J.; Lima, Bruno P.; Wolfe, Alan J.; Katz, Barry; Zhang, Zhong-Yin; Spinola, Stanley.

In: Antimicrobial Agents and Chemotherapy, Vol. 59, No. 7, 01.07.2015, p. 3789-3799.

Research output: Contribution to journalArticle

Van Rensburg, Julia J. ; Fortney, Kate R. ; Chen, Lan ; Krieger, Andrew J. ; Lima, Bruno P. ; Wolfe, Alan J. ; Katz, Barry ; Zhang, Zhong-Yin ; Spinola, Stanley. / Development and validation of a high-throughput cell-based screen to identify activators of a bacterial two-component signal transduction system. In: Antimicrobial Agents and Chemotherapy. 2015 ; Vol. 59, No. 7. pp. 3789-3799.
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