Download Mechanisms of Translocation of Legionella pneumophila Effectors

Poster No. 32
Title:
Mechanisms of Translocation of Legionella pneumophila Effectors via the Dot/Icm Type IVB Secretion System
Author:
Whitney Amyot
Presented by:
Whitney Amyot
Department:
Department of Molecular Biology and Microbiology, Tufts University School of Medicine
Abstract:
Legionella pneumophila is an intracellular pathogen that avoids fusion with lysosomal compartments
and forms a replication vacuole derived from rough endoplasmic reticulum. Biogenesis and growth within the
Legionella-containing vacuole requires a functional type IVB secretion system (T4SS) known as the Dot/Icm
complex. Considerable research is underway to identify and characterize upwards of 250 bacterial effector
proteins that are translocated through the Dot/Icm complex. However, the mechanism by which effector proteins
are transported through the Dot/Icm system to the phagosomal surface or the host cytoplasm has yet to be
elucidated.
A primary question of the mechanisms of translocation is whether this process requires unfolding of the
translocated protein. To monitor the folding state of proteins during translocation, we constructed protein
fusions of various L. pneumophila effectors to the rapidly and tightly folding dihydrofolate reductase (DHFR)
protein. Fusions to DHFR prevented the translocation of nearly all the effectors studied, including a 50 amino
acid carboxy-terminal tag of an effector protein, suggesting that tightly folded proteins may not be able to pass
through the Dot/Icm complex. Surprisingly, a DHFR fusion to the effector protein Lpg1798 is still translocated
at significant levels. Current studies are underway to characterize the folding or other inherent properties of the
protein that either allow or inhibit translocation of a DHFR fusion.
Interactions of L. pneumophila proteins IcmS and IcmW are believed to serve as adaptor proteins for the
translocation of a large subset of Dot/Icm effector proteins. We have identified varying levels of dependence on
IcmS for translocation within the effectors studied and have some evidence that this phenotype is unrelated to
direct binding of IcmS to the effector protein. Of particular interest, it was found that the translocation defect in
a DHFR-effector fusion is exacerbated in an icmS deletion mutant. These data taken together suggest a role for
IcmS in facilitating a translocation competent Dot/Icm complex.
Since the LCV is surrounded by rough ER vesicles within 5 minutes after uptake, a process that requires
Dot/Icm effectors, it is likely that translocation in Legionella occurs at a high rate. In an infection time course, a
kinetic curve demonstrated a steady increase in translocation until it reached steady state levels approximately
1 hour after infection. Additional studies using FRET to visually monitor translocation in real time are underway
to further characterize the kinetics of translocation immediately upon contact between L. pneumophila and the
host cell, as well as throughout the infection process.
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