Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification
The Dot/Icm type IV secretion system (T4SS) is essential for the pathogenesis of Legionella species and translocates a multitude of effector proteins into host cells. The identification of host cell targets of these effectors is often critical to unravel their roles in controlling the host. Here we describe a method to characterize the protein complexes associated with effectors in infected host cells. To achieve this, Legionella expressing an effector of interest fused to a Bio-tag, a combination of hexahistidine tags and a specific recognition sequence for the biotin ligase BirA, are used to infect host cells expressing BirA, which leads to biotinylation of the translocated effector. Following chemical cross-linking, effector interactomes are isolated by tandem affinity purification employing metal affinity and NeutrAvidin resins and identified by western blotting or mass spectrometry.
Key wordsLegionella Dot/Icm type IV secretion system Effectors Interactome Host cell targets Tandem affinity purification Bio-tag
This research and manuscript were enabled by Wellcome Trust and Medical Research Council UK grants (MR/L018225/1) for GF, AM, ECS, GNS, as well as additional institutional funding for GNS from Queen’s University Belfast and MRF/Asthma UK Research Grant (MRFAUK-2015-311) funding for AM.
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