INVESTIGATION OF IRON’S ROLE IN ALGINATE PRODUCTION AND MUCOIDY BY PSEUDOMONAS AERUGINOSA by JACINTA ROSE SCHRAUFNAGEL B.S., University of Colorado Denver, 2003 M.S., University of Colorado Denver, 2006 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Molecular Biology Program 2013 This thesis for the Doctor of Philosophy degree by Jacinta Rose Schraufnagel has been approved for the Molecular Biology Program by David Barton, Chair Michael Vasil, Advisor Mair Churchill Jerry Schaack Michael Schurr Martin Voskuil Date 10/30/13 ii Schraufnagel, Jacinta R. (Ph.D., Molecular Biology) Investigation of Iron’s Role in Alginate Production and Mucoidy by Pseudomonas aeruginosa. Thesis directed by Professor Michael Vasil. ABSTRACT Iron, an essential nutrient, influences the production of major virulence factors and the development of structured non-alginate biofilms in Pseudomonas aeruginosa. Fur, pyoverdine and various concentrations of iron sources have all been shown to affect non-alginate biofilm formation. Remarkably however, the role of iron in alginate biofilm formation and mucoidy is unknown. Because of alginate’s association with biofilms in chronic cystic fibrosis (CF) lung infections, we examined the influence of iron on alginate production and mucoidy in P. aeruginosa. Herein, we identify and characterize a novel iron-regulated protein that affects the transcription of alginate biosynthesis genes. Further, contrary to what is known about non-alginate biofilms, we discovered that biologically relevant concentrations of iron (10 µM – 300 µM), in various forms (e.g. FeCl3, Ferric Citrate, Heme), led to the decreased expression of alginate biosynthesis genes and the dispersion of alginate biofilms in mucoid P. aeruginosa PAO1, PA14, PAKS-1 and PS388 non-CF isolates. Examination of mucoid CF clinical isolates revealed that late-stage isolates acquired the ability to maintain the mucoid phenotype even in the presence of iron. Traditional iron acquisition (e.g. Pyoverdine and Pyochelin) systems in these nonresponders were often dysregulated, suggesting that these isolates may be less efficient at acquiring iii iron. Deletion of pvdS did not affect the ability of mucoid strains to produce alginate and fur mutations were not present in the nonresponders. Furthermore, in the presence of iron, mucoid non-CF isolates retained their ability to form non- alginate biofilms in a microtiter plate assay. Finally, we assessed the anti- microbial activity of gallium on mucoid strains and clinical CF isolates as well as the effects of gallium on alginate production. We found that gallium did not have any effect on the production of alginate, but did exhibit anti-microbial activity regardless of the organism’s ability to regulate alginate production in response to iron. This dissertation represents the first in-depth examination of iron’s effect on alginate production. We demonstrate that, unlike non-alginate biofilms that require iron to form structured biofilms, alginate biofilms form in the absence of this important micronutrient. The form and content of this abstract are approved. I recommend its publication. Approved: Michael L. Vasil iv DEDICATION This dissertation is dedicated to my Grandmother, Mrs. Helen Rose Schraufnagel, for playing such a significant and influential role throughout my life. To my parents, Daniel and Helga Schraufnagel, for their endless love, support, encouragement and for teaching me the value of hard work and determination. To my husband, Adam Wiens, who is the love of my life. v ACKNOWLEDGMENTS First and foremost, I would like to thank my parents for their love and support throughout my life. Thank you for working hard to provide a stable foundation for my life and future, you have taught me invaluable lessons about work, marriage, parenting, perseverance and integrity. I want to thank my husband for his love, understanding, for his unlimited patience and for always believing in me. Importantly, I would like to thank my advisor, Michael L. Vasil. Dr. Vasil warmly welcomed me into his lab and gave me the independence to think for myself and develop my own project. He had confidence in my abilities, offered encouragement and advice when it was needed most and conferred enough discipline to help me develop into a strong, moral, quality scientist. His guidance and persistence have not only made me a better scientist, but also a more patient and confident person. I’d like to thank Adriana Vasil for her support and assistance over the years. Adriana’s technical expertise and dedication to making quality mutants were invaluable. Thank you to all the other members of Dr. Vasil’s lab who made the lab an entertaining place to be. Thanks to Rhea May, Martin Stonehouse, Art Pritchard and Zach Wilson for being great colleagues. I would also like to thank Amanda Oglesby for helping me construct my very first mutant (PA4704) during my rotation, and Sarah Parker who offered her protein expression technical expertise. I would also like to thank my thesis committee David Barton, Mair Churchill, Jerry Schaack, Michael Schurr and vi Martin Voskuil. Thank you all for your direction, advice, encouragement, pep talks and making me think critically about science. Lastly, I would like to thank all of my friends, old and new, who have been both understanding and supportive throughout this journey. You have all been instrumental in this body of work and in my graduate career. Thank you all. vii CONTENTS CHAPTER I. INTRODUCTION..........................................................................................1 Pseudomonas aeruginosa Biology and Pathogenesis.................................2 Cystic Fibrosis..............................................................................................4 Host and Bacterial Factors That Contribute to P. aeruginosa Lung Infection in Individuals With Cystic Fibrosis…………...................................7 Iron and the CF lung...................................................................................12 P. aeruginosa Iron Acquisition....................................................................14 Regulation of Iron Homeostasis in P. aeruginosa......................................17 P. aeruginosa biofilms................................................................................19 Psl..................................................................................................22 Pel..................................................................................................23 Alginate..........................................................................................24 Iron and P. aeruginosa Biofilms..................................................................31 Purpose of This Study................................................................................34 II. MATERIALS AND METHODS...................................................................36 Materials and Chemicals...........................................................................36 Bacterial Strains, Plasmids and Growth Conditions..................................36 Construction of Mutants……………………………………………………….37 P. aeruginosa Chromosomal DNA Isolation..............................................41 Polymerase Chain Reaction (PCR)...........................................................41 Plasmid Purification...................................................................................42 DNA Ligation.............................................................................................43 viii DNA Gel Purification.................................................................................43 Escherichia coli Chemically Competent Cells and Transformation……………………………………………….........................44 Tri-Parental Mating....................................................................................45 Confirmation of Mutants............................................................................45 RNA Isolation and qRT-PCR……….………………………………………...47 Affymetrix GeneChip Microarray……………………………………………..47 Uronic Acids Assay…………………………………………………………....48 Glycosyl Composition Analysis of Extracellular Polysaccharides........................................................................................49 Purification of PA2384………………………………………………………...50 SDS Polyacrylamide Gel Electrophoresis.................................................51 Gel Mobility Shift Assay……………………………………………………….51 Affinity Purification of Antibodies...............................................................52 Western Blot and Chemiluminescence.....................................................53 Microtiter Plate Biofilm Assay………………………………………………...54 Analysis of Siderophore Production…………………………………………54 Growth Curves…………………………………………………………………55 Minimum Inhibitory Concentration (MIC)……………………………………55 Zone of Inhibition………………………………………………………………56 III. STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF PA2384……………………………………............................................57 Structural Features of PA2384…………………………………..................58 Iron-Regulated Expression of PA2384……………………………………..61 ix Global Gene Expression Profile of ∆PA2384 in Response to Iron-Limitation………………...……………………………………………….63 Purification of PA2384………………………………………………………..67 Does PA2384 Protein Bind to the algD Promoter?..................................70 PA2384 May Function as a Dimer…………………………………………..72 Functional Analysis