title: The Rab-binding Profiles of Bacterial Virulence Factors during Infection.
abstract: Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection.
authors.affiliation: From the MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom,; Department of Chemistry, South Kensington Campus, Imperial College, London SW7 2AZ, United Kingdom,; Department of Chemistry, Institute of Chemical Biology, Imperial College, London SW7 2AZ, United Kingdom.
authors.forename: Ernest C
authors.initials: EC
authors.lastname: So
authors.affiliation: From the MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom.
authors.forename: Gunnar N
authors.initials: GN
authors.lastname: Schroeder
authors.affiliation: From the MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom.
authors.forename: Danielle
authors.initials: D
authors.lastname: Carson
authors.affiliation: From the MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom.
authors.forename: Corinna
authors.initials: C
authors.lastname: Mattheis
authors.affiliation: From the MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Mousnier
authors.affiliation: Department of Chemistry, South Kensington Campus, Imperial College, London SW7 2AZ, United Kingdom.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Broncel
authors.affiliation: Department of Chemistry, South Kensington Campus, Imperial College, London SW7 2AZ, United Kingdom,; Department of Chemistry, Institute of Chemical Biology, Imperial College, London SW7 2AZ, United Kingdom.
authors.forename: Edward W
authors.initials: EW
authors.lastname: Tate
authors.affiliation: From the MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom,; Department of Chemistry, Institute of Chemical Biology, Imperial College, London SW7 2AZ, United Kingdom. Electronic address: g.frankel@imperial.ac.uk.
authors.forename: Gad
authors.initials: G
authors.lastname: Frankel
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D016952:Legionella pneumophila / Q000502:physiology*; D007877:Legionnaires' Disease / Q000378:metabolism*; D011485:Protein Binding; D060066:Protein Interaction Maps; D037521:Virulence Factors / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; C539733:SidM protein, Legionella pneumophila; D037521:Virulence Factors; D020691:rab GTP-Binding Proteins
keywords: Legionella pneumophila; LidA; Rab GTPases; SidM; Type IV secretion system effectors; bacterial pathogenesis; mass spectrometry (MS); protein complex; protein cross-linking; protein purification