Application of intact cell-based NFAT-β-lactamase reporter assay for Pasteurella multocida toxin-mediated activation of calcium signaling pathway

Research output: Contribution to journalArticlepeer-review

Abstract

Pasteurella multocida toxin (PMT) stimulates and subsequently uncouples phospholipase C β1 (PLCβ1) signal transduction through its selective action on the α subunit of the Gq-protein. Here, we describe the application of an NFAT-β-lactamase reporter assay as a functional readout for PMT-induced activation of the Gq-protein-coupled PLCβ1-IP3-Ca2+ signaling pathway. Use of the NFAT-β-lactamase reporter assay with a cell-permeable fluorogenic substrate provides high sensitivity due to the absence of endogenous β-lactamase activity in mammalian cells. This assay system was optimized for cell density, dose and time exposure of PMT stimulation. It is suited for quantitative characterization of PMT activity in mammalian cells and for use as a high-throughput screening method for PMT deletion and point mutants suitable for vaccine development. This method has application's for diagnostic screening of clinical isolates of toxinogenic P. multocida.

Original languageEnglish (US)
Pages (from-to)597-605
Number of pages9
JournalToxicon
Volume51
Issue number4
DOIs
StatePublished - Mar 15 2008

Keywords

  • Atrophic rhinitis
  • Dermonecrotic toxin
  • FRET
  • Gq-protein
  • NFAT
  • PLCβ1
  • PMT
  • Reporter
  • β-Lactamase

ASJC Scopus subject areas

  • Toxicology

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