Date on Master's Thesis/Doctoral Dissertation

12-2014

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Committee Chair

Abu-Kwaik, Yousef

Committee Co-Chair (if applicable)

Demuth, Don

Committee Member

Lawrenz, Matthew

Committee Member

Miller, Richard

Committee Member

Warawa, Jonathan

Subject

Legionella pneumophila; Ubiquitin; Proteins

Abstract

Legionella pneumophila is a facultative intracellular pathogen that infects a wide array of protozoan hosts and human alveolar macrophages. L. pneumophila is dependent on a functional Dot/Icm type IVB secretion system that translocates bacterial effector proteins into the host cell cytosol. L. pneumophila genomes encode more than 250 effector proteins, many of which inhibit host cellular processes to form a favorable niche termed the Legionella-containing vacuole (LCV). The eukaryotic-like Dot/Icm translocated effector AnkB contains two eukaryotic-like ankyrin protein-protein interacting domains, one eukaryotic-like F- box domain and an eukaryotic C-terminal CaaX motif. Immediately following attachment of extracellular bacteria, AnkB is translocated into the host cell where it is rapidly farnesylated and anchored to the plasma membrane beneath the attached extracellular bacteria. AnkB recruits the host cell SCF1 E3 ubiquitin ligase machinery to the point of attachment and promotes the lysine48-linked polyubiquitination of the AnkB substrates. Interestingly, the proteasomal degradation of the lysine48-liked polyubiquitinated proteins increases the levels of intracellular free amino acids within 15 minutes of attachment of extracellular bacteria. This early increase in free cellular amino acids is needed to prevent a starvation response and inhibits differentiation into the non-replicative phase which facilitates intracellular replication. The polyubiquitinated proteins surrounding the LCV have a wide range of cellular functions, and include the amino acid transporters SLC1A4 and SLC3A2 and the sodium bicarbonate transporter SLC4A7. In addition the ubiquitinated proteome of the WT strain, the LCV contains proteins involved in the immune response, including interferon regulatory factor 7 and Interleukin-1 receptor-associated kinase 1a. The complete LCV proteome of the WT strain as well as the ankB mutant strain contained E2 ubiquitin-conjugation enzymes, E3 ubiquitin ligases and ubiquitin peptidases. Bioinformatic analysis determined the major metabolic networks within the LCV proteome, including the phosphatidylinositol 4,5 diphosphate pathway and multiple amino acid synthesis pathways. These data showed that AnkB is polyubiquitinated on lysine 67 through lysine11-linked polyubiquitination. While lysine11-linked polyubiquitination has been shown to target the modified protein for proteasomal degradation, stability of AnkB is not affected following ubiquitination. This highlights a novel example of an F-box effector protein that is modified though lysine11-linked polyubiquitination. Taken together, AnkB manipulates multiple eukaryotic cellular pathways to enable intra-vacuolar proliferation of L. pneumophila.

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