Browsing Theses and Dissertations School of Pharmacy by Subject "ABC transporter"
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Characterization of Heme Transport in Pseudomonas aeruginosa and the Preferential Pathway for Heme UptakeBacterial pathogens require iron for their survival and virulence and have evolved multiple mechanisms to acquire this scarce micro-nutrient. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa acquires heme as an iron source through the Phu (Pseudomonas heme utilization) and Has (Heme assimilation system) systems. The studies herein detail the initial purification and characterization of the outer membrane (OM) HasR and PhuR receptors. A series of site-directed mutagenesis and spectroscopic studies confirmed HasR, in keeping with previously characterized OM receptors, coordinates heme through the conserved N-terminal plug His-221 and His-624 of the surface exposed FRAP-loop. In contrast PhuR coordinates heme through His-124 and Tyr-519 ligands not previously reported in OM receptors but associated with high affinity heme binding proteins. In vivo studies utilizing a combination of bacterial genetics, isotopic labeling (13C-heme), and qRT-PCR further revealed that both receptors are required for optimal heme uptake. However, whereas deletion of hasR leads to an inability to regulate heme uptake, loss of PhuR results in decreased efficiency in heme uptake, despite a significant up regulation in HasR protein levels. The results are consistent with PhuR being the major heme uptake receptor, while HasR senses and regulates extracellular heme uptake. Thus PhuR and HasR represent non-redundant receptors required for accessing and regulating heme uptake across a wide range of physiological conditions found upon infection. The research presented herein also involved optimization of the ABC-transporter ShuUV along with the soluble periplasmic heme binding ShuT proteins from Shigella dysenteriae, which are involved in the transport of heme across the cytoplasmic membrane and into the cell. By generating and screening a series of expression constructs we were able to obtain a construct that resulted in increased expression levels of ShuUV homodimer. Reconstitution of ShuUV in lipososmes with heme loaded ShuT trapped in the interior of the liposome gave a functional system that could transport heme on activation with ATP. Taken together, the current research lays the foundation for future spectroscopic and structural studies aimed at understanding the molecular mechanisms of membrane bound heme transport proteins.