The Effect of Calcium Binding on Adhesion and Pilus Biogenesis in the PilC Family of Proteins Michael D. L. Johnson A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics. Chapel Hill 2011 Approved by: Professor Matthew Redinbo, Ph.D. Professor Edward Collins, Ph.D. Professor Matthew Wolfgang, Ph.D. Professor Jack Griffith, Ph.D. Professor Joseph St. Geme III, M.D. ©2011 Michael D. L. Johnson ALL RIGHTS RESERVED ii ABSTRACT MICHAEL D. L. JOHNSON: The Effect of Calcium Binding on Adhesion and Pilus Biogenesis in the PilC Family of Proteins (Under the direction of Matthew Redinbo) Pseudomonas aeruginosa is an opportunistic pathogen prevalent in people on immunosuppressants, recent open wounds, or cystic fibrosis patients. P. aeruginosa attaches to the host cell via polar type IV pili (tfp). These tfp are composed of many proteins including the major pilus subunit PilA, retraction protein PilT, and one focus of this dissertation, 1163 residue protein PilY1. PilY1 shares homology to other bacterial adhesion and pilus biogenesis proteins such as the PilC family of proteins in Neisseria gonorrhoeae, Neisseria meningitidis, and Kingella kingae thus, it is often characterized as a pilus biogenesis protein and the P. aeruginosa adhesin. However, no known mechanism for either situation had been elucidated. In this study, we have identified two calcium binding sites which affect both functional pilus biogenesis and adhesion to host cells. In addition, we have also located an RGD (integrin binding motif) that mediates PilY1 binding to integrin. The C-terminal most PilY1 calcium binding site (CBS) was found from residues 851- 859 in PAK and is conserved in other strains of P. aeruginosa. This CBS is also conserved in the PilC proteins over 100 different bacteria. We found, through mutating the bidentate aspartic acid in vitro and in vivo, that this CBS controlled functional pilus biogenesis with the calcium bound and unbound states corresponding to pilus extension and retraction respectfully. We also characterized the homologous CBSs in N. gonorrhoeae and K. kingae finding similar results. iii Upon further examination, we also located separate CBS from residues 600-608. This site was in close proximity to an RGD. This CBS was conserved in other strains of P. aeruginosa, but not in other bacteria. We found that purified PilY1 bound integrin in an RGD dependent manner. Furthermore, we found that this interaction was mediated by the calcium bound states of both CBSs. Here, we demonstrate that P. aeruginosa PilY1 uses calcium to mediate both functional pilus biogenesis factor and adhesion to host cells. Future studies will hopefully include exploiting both calcium binding domains to prevent pilus formation and binding to host cells and characterizing the in vivo effects of these three sites. iv Dedication To all the wonderful women in my life, my wife Elisha, my daughters Michaela and the one still cooking, and my mother Barbara v ACKNOWLEDGEMENTS First and foremost, I must thank God. On December 14th, 2003, in my then girlfriend, now wife’s church, I was told to pursue science. Since that moment, God has put a great number of people who have been positively essential to in my life as a researcher, a father, and a husband. Thank you to Dr. Gary Johnson for your wonderful advice, which guided me to giving my CV Paula Harrington, which in turn, led me to first lab mentor, Dr. Jeffery Frelinger. You “grew hair” on my scientific chest; thanks for giving me my first shot in the Show. I’d also like to think Cindy Hensley who was my first lab mom. These relationships led me to Dr. Barbara Vilen who encouraged me to apply to the IBMS program in which I met the program director, Dr. Sharon Milgram. Thanks for the letters of recommendation and scoring me that sweet talk at the NIH. Thanks to my first IBMS family, specifically Kate, Bret, Deb, Rose, Deepak, Erica, Alaina and Pam, you all were my class before I had a class. I’d like to give a big thanks to my current research mentor, Dr Matthew Redinbo. I still remember our first interview in which we talked about Duke basketball and you told me to a rotation in your lab. I don’t know if I could describe a better interview. Since joining your lab, you have given me a lot of scientific rope and I have been made a better researcher because of it. I hope you can get a grant out of it. While in the Redinbo lab, my apartment burned down. Thank you to all the past and present members of the lab as well as my IBMS family that helped me move and helped with donations. vi There are a number of people who are not in the Redinbo lab that have been very helpful. Here I would like to thank Kevin and Michelle for your friendship and Leah for your realism. I have also had the opportunity to mentor people on this journey, Justin, Stephani, Angela, and Brian, you all have taught me so much about myself and who I want to be. Thank you to my collaborators; Dr. Matthew Wolfgang, Kimberly, Dr, Joe St. Geme, Eric, Dr. Christopher Thomas, Jeff, and Christine. I would like to thank a number of people in the Redinbo lab. Joe, you were always like a big brother/mentor to me and I will always appreciate that, Rebekah, for showing how to think on the other side of the road, and Monica, the other half of the original happy corner, my lab wife, and just a good overall friend, thanks for not making lab seem like work all the time; and yes, the plans are still set. I’d also like to thank Keith, the walking UNC encyclopedia, Yuan, and all the past and present members of the Redinbo Lab for great scientific and mindless conversations. So much of what I accomplished in graduate school would not have been possible without Pat Phelps. Your Midas touch on my graduate school life has been much appreciated. Thanks for the outreach opportunities, giving me the tools to succeed, and for being extremely helpful in me getting a postdoc at St. Jude. Meeting you made me a better person. Most of all, I couldn’t have completed this work without the support of my loving family. Thank you Mom, for all you have done for me throughout my life. Words cannot describe how much I appreciate what you have done for me. Most of all, thank you to my wife for talking me up, down, and whatever other direction I needed thought out my grad school life. Thank you for being my other half. vii Table of Contents List of Tables ......................................................................................................................... xiii List of Figures........................................................................................................................ xiv List of Abbreviations and Symbols ...................................................................................... xviii Chapter 1: Introduction to Pseudomonas aeruginosa Type IV Pili ......................................... 1 1.1 Pseudomonas aeruginosa Overview ................................................................. 1 1.2 P. aeruginosa Infection ......................................................................................... 2 1.3 Type IV Pili ......................................................................................................... 3 1.4 Proteins in the Type IV Pilus ................................................................................ 3 1.5 The fimUpilEVWXY1Y2E Operon .................................................................... 5 1.6 PilY1 .................................................................................................................. 5 1.7 References ........................................................................................................ 7 Chapter 2: Crystal Structure Analysis Reveals Pseudomonas PilY1 as an Essential Calcium-Dependent Regulator of Bacterial Surface Motility ................................ 12 2.1 Summary .......................................................................................................... 13 2.2 Introduction ...................................................................................................... 14 2.3 Results ............................................................................................................ 15 2.3.1 PilY1 Exhibits a Modified β-Propeller Fold .............................................. 15 2.3.2 PilY1 Contains a Unique Calcium Binding Loop ...................................... 17 viii 2.3.3 Calcium Binding and Release by PilY1 are Essential for Functional Pili . 18 2.4 Discussion ....................................................................................................... 20 2.5 Methods ............................................................................................................... 22 2.6 Credits ................................................................................................................. 24 2.7 Figure Legends .................................................................................................. 25 2.8 Supplemental Figure Legends ........................................................................... 26 2.9 Additional Figure Legends .................................................................................. 28 2.10 References Additional ...................................................................................... 45 Chapter 3: Pseudomonas aeruginosa PilY1 Binds