Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

摘要: The technique presented here allows one to analyze at which step a target protein, or alternatively small molecule, interacts with the components of signaling pathway. method is based, on hand, inducible expression specific protein initiate event defined and predetermined in selected cascade. Concomitant expression, other gene interest then investigator evaluate if activity expressed located upstream downstream initiated event, depending readout pathway that obtained. Here, apoptotic cascade was as demonstrate protocol functionality. Pathogenic bacteria, such Coxiella burnetii, translocate effector proteins interfere host cell death induction ensure bacterial survival promote their dissemination organism. C. burnetii CaeB effectively inhibits after apoptosis UV-light staurosporine. To narrow down interferes propagation signal, well-characterized pro-apoptotic were transiently doxycycline-inducible manner. If acts these proteins, will proceed unhindered. downstream, be inhibited. test Bax, level mitochondria, caspase 3, major executioner protease. induced by Bax but not 3 expression. CaeB, thus, between two proteins.