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.
Highlights
Many bacterial pathogens use secretion systems to translocate virulence factors, termed effectors, into the host cell where they subvert cell signaling to facilitate bacterial survival and proliferation [1]
Infected cells were crosslinked with 1% formaldehyde, lysed in phosphate buffer containing 1% Triton X-100 and SidM complexes were isolated either by a Neutravidin singleaffinity purification (SAP) or a Ni2ϩ-nitrilotriacetic acid (NTA)/Neutravidin tandem-affinity purification (TAP) and analyzed by LC-MS/MS
In this work we studied the interactomes of SidM and LidA during infection
Summary
Many bacterial pathogens use secretion systems to translocate virulence factors, termed effectors, into the host cell where they subvert cell signaling to facilitate bacterial survival and proliferation [1]. Central to the Legionella virulence strategy is its ability to manipulate the function of multiple Rab GTPases, which are important regulators of vesicular trafficking. Critical to the manipulation of Rab is the effector SidM (DrrA), which contains three functional domains. Upon translocation, it anchors via a phosphatidylinositol 4-phosphate (PI4P) binding domain onto the LCV [8], where it recruits and activates Rab using its guanine nucleotide exchange factor (GEF) domain [9]. The Rab-binding Profiles of Legionella Effectors erned by spatial mobility constraints It is currently unknown which of these reported effector-Rab GTPase interactions are relevant during infection
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