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

Toxicon : Official Journal of the International Society on Toxinology
Shuhong LuoB A Wilson

Abstract

Pasteurella multocida toxin (PMT) stimulates and subsequently uncouples phospholipase C beta1 (PLCbeta1) signal transduction through its selective action on the alpha subunit of the Gq-protein. Here, we describe the application of an NFAT-beta-lactamase reporter assay as a functional readout for PMT-induced activation of the Gq-protein-coupled PLCbeta1-IP(3)-Ca(2+) signaling pathway. Use of the NFAT-beta-lactamase reporter assay with a cell-permeable fluorogenic substrate provides high sensitivity due to the absence of endogenous beta-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.

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Citations

Mar 31, 2009·ChemMedChem·Zachary K SweeneySilvia Patrick
Mar 29, 2008·Protein Science : a Publication of the Protein Society·Leila R AminovaBrenda A Wilson

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