Application of intact cell-based NFAT-beta-lactamase reporter assay for Pasteurella multocida toxin-mediated activation of calcium signaling pathway.
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.
Luo
Shuhong
S
Department of Microbiology, University of Illinois at Urbana-Champaign, 601 South Goodwin Avenue, B128 CLSL, Urbana, IL 61801, USA.
Ho
Mengfei
M
Wilson
Brenda A
BA
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects*; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D050778:NFATC Transcription Factors; D016979:Pasteurella multocida / Q000378:metabolism*; D017354:Point Mutation; D001618:beta-Lactamases
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D050778:NFATC Transcription Factors; C062258:Pasteurella multocida toxin; D001618:beta-Lactamases