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 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.
authors: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors: Julia J
authors: JJ
authors: van Rensburg
authors: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors: Kate R
authors: KR
authors: Fortney
authors: Chemical Genomics Core, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors: Lan
authors: L
authors: Chen
authors: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors: Andrew J
authors: AJ
authors: Krieger
authors: Department of Microbiology and Immunology, Loyola University Chicago, Chicago, Illinois, USA.
authors: Bruno P
authors: BP
authors: Lima
authors: Department of Microbiology and Immunology, Loyola University Chicago, Chicago, Illinois, USA.
authors: Alan J
authors: AJ
authors: Wolfe
authors: Department of Biostatistics, Indiana University School of Medicine, Indianapolis, Indiana, USA Department of Biostatistics, Richard M. Fairbanks School of Public Health, Indiana University, Indianapolis, Indiana, USA.
authors: Barry P
authors: BP
authors: Katz
authors: Department of Biochemistry, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors: Zhong-Yin
authors: ZY
authors: Zhang
authors: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA Center for Immunobiology, Indiana University School of Medicine, Indianapolis, Indiana, USA sspinola@iu.edu.
authors: Stanley M
authors: SM
authors: Spinola
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000819:agonists* / Q000235:genetics; D002227:Carbazoles / Q000494:pharmacology*; D045744:Cell Line, Tumor; D055808:Drug Discovery; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter / Q000235:genetics; D006191:Haemophilus ducreyi / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007763:Lac Operon / Q000235:genetics; D008826:Microbial Sensitivity Tests; D011494:Protein Kinases / Q000235:genetics; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D001617:beta-Glucosidase / Q000378:metabolism
chemical_list: C000606253:2,3,4,9-tetrahydro-1H-carbazol-1-amine; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002227:Carbazoles; C085504:CpxR protein, Bacteria; D011494:Protein Kinases; C092472:CpxA protein, bacteria; D001617:beta-Glucosidase