Biochemistry and Pharmacology - Theses
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ItemTargeting the mitochondrion in Coxiella burnetii infection( 2020)Mitochondria are essential organelles, fundamental to eukaryotic cell function and survival. Perhaps best known for their role in energy production, mitochondria are also central to many cellular processes, including calcium homeostasis, immune and cell death signalling. With such diverse cellular roles, it is no surprise that microbial virulence factors target the mitochondrion during infection. Coxiella burnetii is a unique intracellular bacterial pathogen and the causative agent of Q fever. The bacterium infects alveolar macrophages and replicates within a highly acidic, lysosome-like vacuole, termed the Coxiella-containing vacuole (CCV). C. burnetii translocates over 150 bacterial effector proteins into the host cytosol via a Type 4 Secretion System (T4SS). Effector proteins translocated into the cell modulate cellular functions to facilitate CCV development and bacterial replication. This study has aimed to deepen our understanding of the host-pathogen interactions occurring between C. burnetii and the host cell mitochondrion during infection. In doing so, we have developed new methods and adapted current technologies to the study of this fascinating bacterial pathogen. We uncovered the effector protein CBU0077 (later renamed Mitochondrial Coxiella effector A (MceA)) that localised to the mitochondrial network during infection. This established the mitochondrion is a bona fide target during C. burnetii infection. To investigate whether additional C. burnetii proteins associate with the organelle during infection, we analysed mitochondria purified from infected cells by high-sensitivity mass spectrometry. This unbiased, proteomic approach identified an additional 7 effector proteins associated with mitochondria in the context of infection and we further went on to biochemically characterise CBU1425. We were able to demonstrate that CBU1425 is imported into the mitochondria and interacts with key components of the organelle quality control pathway, revealing a new aspect of C. burnetii biology. A complex interplay exists between the host cell and the pathogenic agent during infection. To disentangle the impact of C. burnetii infection on the host cell and the mitochondrion, we utilised SILAC quantitative proteomics to investigate changes to the cellular and organellar proteome. This revealed a global effect on cellular and mitochondrial pathways. Our findings compare and contrast these organelle-wide changes between cell types and another distinct pathogen, providing a deeper understanding of the role of the mitochondrion during C. burnetii infection and, more broadly in the host cell response to this assault. This research has contributed comprehensive new insights into our understanding of the interaction between the host cell mitochondria and C. burnetii during infection. With a foundational knowledge of the host-pathogen interactions occurring during infection, we can begin to further probe the molecular requirements and outcomes of our own cellular pathways in both health and disease.