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Date of Award

Spring 5-15-2013

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Bacterial pathogens have evolved multiple strategies for intracellular survival. One example is the modification of host cells to establish a replicative niche. This often requires the transfer of proteins from the bacterium into the host cell, a process dependent upon specialized secretion systems. This thesis focuses on the molecular mechanism of secretion by the type IVB secretion system (T4SS), termed Dot/Icm, of the intracellular pathogen Legionella pneumophila. Dot/Icm is a membrane-spanning macromolecular complex composed of 27 proteins, which secretes approximately 275 effectors into the host cell that are required for intracellular survival and replication. One of these proteins, DotL, is the proposed type IV coupling protein (T4CP) for the Dot/Icm T4SS. In the majority of T4SS, T4CPs are thought to play three important roles: (1) to provide energy for effector translocation through ATPase activity, (2) to act as a receptor for substrates at the inner membrane, and (3) to function as a structural link that `couples' substrates to the T4SS apparatus. We hypothesized that DotL functions as a critical component of the L. pneumophila T4SS apparatus for substrate recognition and translocation.

In order to characterize DotL's molecular function(s), a collection of dotL mutants unable to replicate within the host Acanthamoeba castellanii were isolated. These mutants have revealed information regarding DotL's role in monitoring the secretion pore, regulating the rate of substrate transfer, mediating the export of specific classes of Dot/Icm substrates and determining the folding status of effectors during export. One key dotL mutant, dotLY725Stop, was instrumental in the identification of a direct interaction between DotL and the type IV adaptor proteins IcmS/IcmW, and this interaction was shown to be essential for the export of the IcmS/IcmW-dependent proteins. These experiments also led to key insights regarding the stoichiometry of various components of the Dot/Icm T4SS, particularly the ratio of DotL to IcmS/IcmW. These data have resulted in fundamental advances regarding L. pneumophila Dot/Icm function. A new model of T4SS is presented in which DotL plays a central role in export by directly binding IcmS/IcmW to mediate the secretion of Dot/Icm substrates.

Language

English (en)

Chair and Committee

Joseph P. Vogel

Committee Members

Michael G. Caparon, Barbara N. Kunkel, Petra A. Levin, L. David Sibley, Heather L. True-Krob

Comments

Permanent URL: https://doi.org/10.7936/K7XW4H7F

Available for download on Monday, May 15, 2113

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