Characterization of Heme Transport in Pseudomonas aeruginosa and the Preferential Pathway for Heme Uptake Item Type dissertation Authors Smith, Aaron Dennison Publication Date 2015 Abstract 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 ... Keywords ABC transporter; iron acquisition; outer membrane receptor; transporter; ATP-Binding Cassette Transporters; Heme; Pseudomonas aeruginosa Download date 24/09/2021 16:37:31 Link to Item http://hdl.handle.net/10713/4622 Curriculum Vitae Aaron Dennison Smith Education: University of Maryland, Baltimore (2008-present) Department of Pharmaceutical Sciences School of Pharmacy GPA: 3.92 Degree: Ph.D. Expected Date of Degree: May 2015 Dissertation: Characterization of Heme Transport in Pseudomonas aeruginosa and the Preferential Pathway for Heme Uptake University of Maryland Baltimore County (2002-2007) Department of Biochemistry and Molecular Biology GPA: 3.1 Degree: B.S. Date of Degree: July 2007 Employment: Graduate Research Assistant (2008-present) Department of Pharmaceutical Sciences School of Pharmacy, University of Maryland, Baltimore Technical Director (2006-2008) Chemspec, Inc. Baltimore, MD Teaching Experience: Teaching Assistant (2008-2009) School of Pharmacy, University of Maryland, Baltimore PharmD courses: Medicinal Chemistry, Microbiology, Pharmacokinetics Resource Teacher (2006) Vertically Integrated Partnerships K-16 Internship University of Maryland Baltimore County Leadership Opportunities: Graduate Recruitment Strategies Taskforce Committee (2012-2013) President: Pharmacy Graduate Student Association (2009-2010) Team Captain: PSC Race for the Cure (2008-2010) Presentations: Characterization of the outer membrane heme receptors and the preferential pathway for heme uptake in Pseudomonas aeruginosa. Tetrapyrroles Gordon Research Conference, Newport, Rhode Island. July 2014. Poster presentation. Characterization of the outer membrane receptor PhuR and the ABC-transporter ShuUV and their contributions to heme utilization. UMB School of Pharmacy Alumni Reunion Day, University of Maryland Baltimore. Fall 2012. Poster presentation. The DNA Binding Properties of the Cytoplasmic Heme Binding Protein PhuS of Pseudomonas aeruginosa. Graduate Research Day, University of Maryland Baltimore. April 2009. Poster presentation. The DNA Binding Properties of the Cytoplasmic Heme Binding Protein PhuS of Pseudomonas aeruginosa. Bioinorganic Chemistry Gordon Research Seminar, Ventura, California. January 2009. Poster presentation. Publications: Book Chapters: Smith AD, Wilks A. (2012) Extracellular Heme Uptake and the Challenges of Bacterial Cell Membranes. In S. Lutsenko & J.M. Arguello (Eds.), Metal Transporters (pp. 359-392). Elsevier Inc.: Academic Press. Submitted Publications: Smith AD, Modi AR, Sun S, Dawson JH, and Wilks A. (2014) Spectroscopic Determination of Distinct Heme Ligands in the Outer Membrane Receptors PhuR and HasR of Pseudomonas aeruginosa. Biochem. Submitted. Smith AD and Wilks A (2014) Differential Contributions of the Outer Membrane Receptors PhuR and HasR to Heme Acquisition in Pseudomonas aeruginosa. J Biol Chem. Submitted. Awards and Honors: PSC Departmental Fellowship Research Award Recipient (2012) PSC Departmental Merit Research Award Recipient (2011) Rho Chi Induction, Pharmacy Honor Society (2009) All-America East Conference Academic Honor Roll (4 semesters) Maryland Saltwater Sportfishermen’s Association (MSSA) Scholarship Award (2002-2006) NCAA Div. I Athletic Scholarship (2002-2006) Affiliations: American Chemical Society (ACS) Member Rho Chi, Pharmacy Honors Society Abstract Title of Dissertation: Characterization of Heme Transport in Pseudomonas aeruginosa and the Preferential Pathway for Heme Uptake Aaron Dennison Smith, Doctor of Philosophy, 2015 Dissertation Directed by: Dr. Angela Wilks, Professor, Department of Pharmaceutical Sciences, School of Pharmacy 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. Characterization of Heme Transport in Pseudomonas aeruginosa and the Preferential Pathway for Heme Uptake by Aaron Dennison Smith Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2015 Dedicated to My Family And everyone who has shared in this journey iii Acknowledgements It has been an extremely difficult and challenging journey, full of sacrifices and hardships, to finally have arrived at this point in my graduate career. However, there have also been many rewards and accomplishments that greatly overshadow the many trials and hardships faced. I cannot look back on my graduate career without remembering all of the positive influences that helped me succeed and shape me into the independent scientist I am today. The current work presented within was not the sole accomplishments of a single individual, but a compilation of thoughts and ideas, discussions and guidance, and collaborations and friendships from so many exceptional individuals that made this manuscript possible and greatly influenced my goal of pursuing a PhD. I must first thank my advisor and mentor Angela Wilks. She has been a constant driving force towards the accomplishment of this goal and could not have hoped for a better mentor to train and learn from. I sincerely appreciate all of her guidance and support, from scientific discussions and bouncing ideas off her, to attending scientific conferences and being able to present my work to the scientific community. She has always been supportive in allowing me to pursue my intellectual curiosities, even so far as allowing me to learn and conduct new research and techniques with another lab in California. Thank you also for believing in me and not giving up on me when times were rough and seemed quite impossible to overcome, both professionally and personally. I will forever be grateful for everything you have done for me and consider it a privilege and honor to have worked with and learned from your expertise. iv I must also thank my committee members Sarah Michel, C.S. Raman, and Peter Swaan for their guidance and insightful discussions throughout my graduate career, as well as my external member Herschel Wade for accepting to sit on my committee. Their support has been vital to the success of finishing my PhD. There have been many others as well who have greatly helped with the research presented herein. Maureen Kane, Bob Ernst, and Jace Jones were extremely helpful with the mass spectrometry training and analysis and were always present for discussion and troubleshooting. Our collaborators John Dawson and his lab members Anuja Modi and Shengfang Sun for all of their work they did for the MCD, as well as Amanda Oglesby-Sherrouse for generously providing bacterial strains utilized in the studies presents in this manuscript. I also would like to thank Tom Poulos and his lab members for bringing me into their lab and making me feel welcome for the time I spent at University of California, Irvine. Sarah Michel, Amanda Oglesby-Sherrouse,