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 tandemaffinity
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 infectiondependent
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