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Characterization of the Pseudomonas aeruginosa NQR Complex, a Novel Form of Bacterial Proton Pump, and the Ubiquinone Binding Site
Title
Characterization of the Pseudomonas aeruginosa NQR Complex, a Novel Form of Bacterial Proton Pump, and the Ubiquinone Binding Site
Creator
Raba, Daniel Alexander
Date
2019
Description
The proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of...
Show moreThe proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of bacteria, including pathogenic species such as Vibrio cholera, Pseudomonas aeruginosa, Chlamydia trachomatis, as well as others. Residing in the cytoplasmic membrane of these bacteria, the enzyme couples the transfer of electrons to the pumping of cations across the cell membrane. In all previously studied homologues, the enzyme generates a sodium gradient through its pumping activity that can be utilized by the cell to power essential homeostatic processes. Furthermore, the electrochemical gradient generated by this enzyme has been shown to regulate the production of virulent factors and the efficacy of antibiotic extrusion and elimination. Although certain homologues have been investigated, particularly that of V. cholerae (Vc-NQR), the NQR homologues belonging to important pathogenic species have not been well studied. In the research detailed in this thesis, the first characterization of the NQR of P. aeruginosa (Pa-NQR) is described which identified this homologue as a new form of bacterial proton pump, differentiating it from all other studied homologues of NQR. Additionally, as part of this study our research group characterized the mechanism of inhibition of Pa-NQR by the molecule HQNO which is produced by P. aeruginosa and is known to be a strong inhibitor of Vc-NQR. Our results show that Pa-NQR possesses resistance to inhibition by this molecule compared to Vc-NQR, pinpointing residue F155 of subunit D as being important to resistance and the type of inhibition to be partial-mixed. Moreover, in further developing the understanding of the NQR of V. cholerae, we investigated the binding site of ubiquinone, the final electron acceptor of NQR’s electron transfer process, determining residues P185, L190, and F193 to be important for maintaining the structural composition of the ubiquinone pocket, ensuring efficient substrate binding and catalysis.
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MITOCHONDRIA RELOCALIZATION IN CHLAMYDIA TRACHOMATIS INFECTED HFF-1 CELLS
Title
MITOCHONDRIA RELOCALIZATION IN CHLAMYDIA TRACHOMATIS INFECTED HFF-1 CELLS
Creator
Shuppara, Alexander Mitchell
Date
2021
Description
Chlamydia trachomatis is an infectious, gram-negative, obligate intracellular human bacterial pathogen. With over eight hundred million cases...
Show moreChlamydia trachomatis is an infectious, gram-negative, obligate intracellular human bacterial pathogen. With over eight hundred million cases worldwide, C. trachomatis is the most prevalent sexually transmitted infection. It manifests as either trachoma, lymphogranuloma venereum, or other urogenital tract sequelae. As an intracellular pathogen, Chlamydia must scavenge for essential metabolites from establishing networks with its host’s organelles including Golgi apparatus, endoplasmic reticulum, endocytic vesicles, mitochondria, and the cytoskeleton. C. trachomatis was considered an “energy parasite” that is entirely dependent on their host’s ATP production. Yet, recent mitochondrial inhibitor-based evidence suggests that C. trachomatis possess a sodium-based energy gradient for ATP production. Despite this finding, literature on specific interactions between host cell mitochondria and C. trachomatis requires further definition. This project evaluates mitochondrial dynamics changes from C. trachomatis infection in the human foreskin fibroblast cell line, HFF-1. We first defined C. trachomatis growth characteristics in HFF-1 over 36 hours-post infection. Next, we determined changes in mitochondrial dynamics and content throughout infection using immunofluorescent and immunoblotting techniques. observations on infected cells show mitochondrial morphology changes from an elongated appearance at the early stages of infection to fragmented in the late infection stages. Unlike in HeLa cells, HFF-1 remains in a normal distribution throughout the cell and we do not observe mitochondria relocalizing toward the inclusion. By studying mitochondrial relocalization dynamics, new insights into the dynamic and parasitic relationship of Chlamydia and its host can be discovered.
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