Date on Master's Thesis/Doctoral Dissertation

12-2019

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Abu Kwaik, Yousef

Committee Co-Chair (if applicable)

Miller, Richard

Committee Member

Miller, Richard

Committee Member

Lawrenz, Matthew

Committee Member

Lamont, Richard

Committee Member

Mitchell, Thomas

Author's Keywords

Legionnaires disease; legionella; AnkH; LARP7; transcription; LCV; T4SS; 7SK snRNP

Abstract

Legionella pneumophila is a Gram-negative facultative intracellular bacterium that can be found dispersed throughout freshwater environments, where it primarily parasitizes amoebae and other protozoan species. Humans are an accidental host for L. pneumophila, and infection occurs upon inhalation of aerosolized water droplets that contain the bacteria. L. pneumophila is the causative agent of Legionnaires’ Disease, which is the result of intracellular proliferation within alveolar macrophages. Pathogenesis of L. pneumophila is dependent on the Dot/Icm type 4 secretion system (T4SS) apparatus, which is comprised of 27 proteins and is responsible for translocating over 330 effector proteins into the host cell. Many of these effector proteins contain eukaryotic-like domains and motifs, which have been acquired through interkingdom horizontal gene transfer from various aquatic eukaryotic hosts. While L. pneumophila contains such a large repertoire of effector proteins, most of them are not required for survival and proliferation in mammalian macrophages, since single deletion of most effectors does not result in a defect in intracellular replication. Although this could be explained by effector redundancy, it is more likely that these effector proteins constitute a tool box utilized by L. pneumophila to survive and replicate within numerous species of protozoa. One effector identified, that when deleted results in a defect in intracellular replication, is the AnkH effector. It has been shown that AnkH is required for robust intracellular replication of L. pneumophila within amoebae, human macrophages and the A/J mouse model of infection. It has previously been shown that AnkH is an effector that contains ankyrin repeats, which are eukaryotic-like domains, and function as a scaffold for protein-protein interactions. Other than requirement of AnkH during intracellular replication, its function and host targets remain unknown and are the focus of this work. We further characterized AnkH to elucidate its host target and function during infection of macrophages. Using a yeast 2 hybrid system, seven potential host interacting partners have been identified and one interacting partner, human La related protein 7 (LARP7), has been confirmed via co-immunoprecipitation. LARP7 is a component of a transcriptional regulatory complex, 7SK snRNP complex that negatively regulates transcriptional elongation. The AnkH -LARP7 interaction blocks LARP7 binding to components of the 7SK snRNP complex, resulting in the disruption of the complex. Knockdown of LARP7 using LARP7 specific RNAi results in a significant growth defect of the WT strain during infection of macrophages, and the growth defect of the ∆ankH null mutant becomes more severe. RNAseq has been performed on macrophages infected with either WT or ∆ankH strains of L. pneumophila to determine modulation of transcription during infection. The data show that there are a total of 405 genes that are differentially regulated in cells infected with WT versus the ∆ankH mutant. The crystal structure of AnkH has been resolved, and it revealed that AnkH contains 4 ankyrin repeats, 2 asparagine hydroxylation motifs, a cysteine-like protease domain and a cap domain. When residues are substituted within the ankyrin repeats, asparagine hydroxylation sites and cysteine-like protease domain, a decrease in intracellular replication is observed, indicating these domains are critical for the function of AnkH. A substitution within the β-hairpin loop of the third ankyrin repeat results in diminished LARP7-AnkH interactions, and phenocopies the ΔankH null mutant defect in intracellular growth. Taken together, these data suggest that the β-hairpin loop of the third ankyrin repeat of AnkH interacts with the host LARP7, which disrupts host cell transcription elongation by inhibiting assembly of the 7SK snRNP complex resulting in global modulation of transcription. This interaction is important for the intracellular replication of L. pneumophila in human macrophages. The ARDs, asparagine hydroxylation motifs and cysteine-like protease pocket are all required for the function of AnkH in intracellular replication of WT L. pneumophila. AnkH is an important effector protein that aids in the survival and replication of L. pneumophila in all hosts, the study of which would result in a better understanding of how L. pneumophila creates an environment within host cells that supports robust intracellular replication.

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