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Nasrallah, Gheyath K.
Potential roles of the chaperonin (HtpB), polyamines, and the polyamine binding protein (PotD) in Legionella pneumophila pathogenesis /by Gheyath K. Nasrallah.
Ph. D. -- Dalhousie University, 2011
Halifax, NS :Dalhousie University
1 online resource (xxi, 283 leaves) :illustrations
Title from PDF title page (viewed Dec. 3, 2013).
Thesis supervisors: Dr. Rafael Garduno and Dr. Lois Murray.
Includes bibliographical references (leaves 243-282).
ABSTRACT: The intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV) where it abundantly releases its chaperonin HtpB, suggesting that HtpB may have virulence-related functions. To assess these functions, I attempted to construct an L. pneumophila?htpBmutant but was unable to do so, likely because htpB is essential. In the absence of genetic deletion, functional tests were used to study the released HtpB. A small portion of the HtpB in L. pneumophila-infected cells was found in the cytoplasm of the infected cells, as judged by the CyaA reporter assay. To identify potential functions of the HtpB present in the eukaryotic cytoplasm, htpB was ectopically expressed in Saccharomyces cerevisiae. HtpB induced pseudohyphal growth (PHG) in yeast, suggesting it interacts with eukaryotic targets. A yeast two-hybrid screen showed that HtpB interacted with SAMDC, an essential yeast enzyme encoded by SPE2 that is required for polyamine biosynthesis. Overexpression of SPE2 induced PHG in S. cerevisiae, suggesting that HtpB induces PHG by activating polyamine synthesis, and that L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC reduced L. pneumophila replication in host cells, whereas exogenous polyamines enhanced intracellular growth. Bioinformatics revealed that most known enzymes required for polyamine biosynthesis in bacteria are absent in L. pneumophila, suggesting that L. pneumophila depends on exogenous polyamines transported from host cells. L. pneumophila possesses only one putative operon, potABCD, which encodes a polyamine transporter. Using GFP as a reporter of potABCD promoter (PpotA), we found that PpotA activity was turned on during exponential phase of growth in vitro. To test the potential function of this transporter in pathogenesis, potD was deleted.
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