• Characterization of Heme Transport in Pseudomonas aeruginosa and the Preferential Pathway for Heme Uptake

      Smith, Aaron Dennison; Wilks, Angela (2015)
      Bacterial 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.
    • Mechanisms of Resistance to the Fms-like Tyrosine Kinase 3 Inhibitor Crenolanib

      Mathias, Trevor James; Baer, Maria R. (2014)
      Mechanisms of Resistance to the Fms-like Tyrosine Kinase 3 Inhibitor Crenolanib: Trevor J. Mathias, Master of Science, 2014 Dissertation Directed by: Dr. Maria R. Baer, Professor, Department of Medicine and Molecular Medicine Acute myeloid leukemia (AML) is a bone marrow cancer in which myeloblasts fail to differentiate into downstream functional cell types. A number of genetic abnormalities can contribute to the development of AML. One of the most common and important is a mutation in the gene encoding Fms-like tyrosine kinase 3 (FLT3), a type 3 receptor tyrosine kinase, which is expressed on AML cells. The most common FLT3 mutation is an internal tandem duplication in the juxtamembrane domain, known as FLT3-ITD, resulting in constitutive and aberrant signaling. FLT3-ITD is present in ~30% of AML cases and is associated with poor treatment outcomes. Several FLT3 inhibitors are currently in clinical trials. One of these FLT3 inhibitors, crenolanib, is a potent and specific type I inhibitor, targeting the activated form of the kinase. My work is focused on the mechanisms of resistance and sensitization to crenolanib. Expression of ABC proteins associated with drug resistance on AML cells generally correlates with poor treatment response. I found that crenolanib is not an inhibitor of any of the three commonly upregulated ABC transporters, ABCB1, ABCG2, and ABCC1, but is a substrate of the ABCB1 transporter, indicating the potential for crenolanib to be effluxed from cells expressing ABCB1. Other FLT3 inhibitors have been shown to induce point mutations in FLT3-ITD, reducing the effectiveness of the treatment. Random mutagenesis in the presence of crenolanib produced point mutations in FLT3-ITD that only conferred mild resistance to crenolanib, with preserved sensitivity at effective and well tolerated concentrations in Phase I clinical trials. In cells containing FLT3-ITD there is increased expression of Pim-1, a proto-oncogene that is activated by a downstream target of FLT3, STAT5. Additionally, our laboratory has shown that Pim-1 promotes aberrant signaling of FLT3-ITD, and it is therefore an attractive therapeutic target. A Pim kinase inhibitor was used for in vitro combination treatments with crenolanib. This inhibitor synergized with crenolanib to produce increased levels of apoptosis. The mechanism behind this interaction is under further study. These studies helped to further elucidate the role of crenolanib in the treatment of FLT3-ITD positive AML.