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A STUDY of SURFACE MOTILITY and BIOFILM FORMATION in Pseudomonas

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A STUDY of SURFACE MOTILITY and BIOFILM FORMATION in Pseudomonas A STUDY OF SURFACE MOTILITY AND BIOFILM FORMATION IN Pseudomonas aeruginosa: QUORUM SENSING AND PHOTODYNAMIC ANTIMICROBIAL CHEMOTHERAPY Dissertation Submitted to The College of Arts and Sciences of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in Biology By Tracy Lynn Collins UNIVERSITY OF DAYTON Dayton, Ohio December 2010 A STUDY OF SURFACE MOTILITY AND BIOFILM FORMATION IN Pseudomonas aeruginosa: QUORUM SENSING AND PHOTODYNAMIC ANTIMICROBIAL CHEMOTHERAPY Name: Collins, Tracy Lynn University of Dayton APPROVED BY: ___________________________ Jayne B. Robinson Ph.D. Major Advisor Chair Department of Biology ___________________________ Carissa Krane Ph.D. Committee Member ___________________________ Mark Nielsen Ph.D. Committee Member ___________________________ John Rowe Ph.D. Committee Member ___________________________ Shawn Swavey Ph.D. Committee Member ii ABSTRACT A STUDY OF SURFACE MOTILITY AND BIOFILM FORMATION IN Pseudomonas aeruginosa: QUORUM SENSING AND PHOTODYNAMIC ANTIMICROBIAL CHEMOTHERAPY Name: Collins, Tracy Lynn University of Dayton Advisor: Dr. Jayne B. Robinson Pseudomonas aeruginosa is an opportunistic pathogen that commonly causes infection in immunocompromised individuals. This bacterium forms complex communities known as biofilms. Biofilm formation is dependent on motility as well as quorum-sensing. In this study, we show that P. aeruginosa surface motility is inhibited in the presence of the quorum-sensing molecule E,E-farnesol. In the presence of E,E-farnesol, there is an 4- fold increase in rhamnolipid production. Because swarming motility is dependent on rhamnolipid production, this increase could account for the inhibition in swarming motility observed in the presence of E,E-farnesol. In addition, the effect of the cationic porphyrin 5,10,15,20-tetrakis(1-methyl-pyridino)-21H,23H-porphine, tetra-p-tosylate salt (TMP) on P. aeruginosa biofilms was examined. Exposure to 225 µM TMP and photoactivation resulted in almost complete killing of biofilm associated cell as well as the detachment of wild-type PAO1 biofilms. In contrast, pqsA mutant biofilms that contain less extracellular DNA remained intact. Our results suggest that the action of iii photoactivated TMP on P. aeruginosa biofilms is two-fold: direct killing of individual cells within biofilms and detachment of the biofilm from the substratum. There was no evidence of porphyrin toxicity in the absence of light; however, biofilms pretreated with TMP without photoactivation were substantially more sensitive to tobramycin than untreated biofilms. iv DEDICATION This is dedicated to my mother, Linda Collins, and to the memory of my grandparents. v ACKNOWLEDGEMENTS Nothing I ever accomplish is done entirely of my own merit. The Lord has blessed me in my life with the help and support of a number of people. I would like to first thank the biology department staff: Karen Bahr, Lynda Routley, Cathy Wolfe, Jay Lee, and Sue Trainum. Each of them does a wonderful job making sure the department runs smoothly. I’d especially like to acknowledge Karen Bahr because she has always looked out for me and is my guardian in the department. I am very thankful to my committee members John Rowe, Mark Nielsen, Carissa Krane, and Shawn Swavey. I appreciate the time and input each of them has contributed to my research project. Because I have been at UD for ten years, there are a number of labmates that I’d like to thank. From “Robinson Generation I”, I’d to acknowledge Mary Connolly (for scraping me off the bathroom floor that one time), Paul DeLange (for loaning me $4 when I forgot my purse), Michelle Yingling (for our “complaint” walks), Amy Beumer (for always listening), Chris Moler (for not drowning at sea), Lisa Kaiser (for your superlatives), Rachel Ducharme (for being “The Roach”), Bethany Yager (for the pub story), Kevin Wheeler (for not playing Jazz music), and Ian Kearns (for the techno music). “Robinson Generation I” was truly made up of a great group of intelligent and enjoyable individuals who were a pleasure to work with in the lab, even when Environmental Safety busted us. vi I would also like to thank my labmates from “Robinson Generation II”: Sulabha Chaganaboyana (for being my indian sister), Mike Goodson (for tea and chimney sweeps), Muhamad Shakhatrah (for the baklava), Laura Gueltzow (for your dimples), Jen Lang (for being “Jen Lange!”), Liz Markus (for being my partner in crime), Liz Raphael (for the word “ridonculous”), Mariah Roller (for serenading me), Brittany Demmitt (for the “Thursday night cry”), and honorary lab member Alison DesJardins (for Britney Spears). This group of people has been so much fun to work with and I can’t begin to express the joy that each of them have brought to my life. When I think back to “Robinson Generation II”, I will always have fonds memories involving tie dying, dancing to Lady Gaga, and “Pseudomonas in…aeruginosa out.” Thank you especially to Liz Markus for allowing me to be part of the “tetrakis takeover” team, for our countless hours in the confocal room, and adventures involving fishnapping, hello kitty, and albino squirrels. You’re an amazing person and great friend. In addition, I would like to thank all the grad students for their camaraderie and friendship, especially Vandana Sharma, Chris Noriega, and Meagan Roddy. Meagan, thank you for always being a loyal, supportive friend who gets my movie references. It’s great to be able to talk about “the two Corey’s” or “the Brat Pack” and someone know precisely who I’m referring to. Ten years ago, I had no idea what an amazing journey I was about to enter. In retrospect, I know now that it was part of a divine plan that I was supposed to come to UD, be surrounded by wonderful people, and most of all work under a truly great mentor. vii The scientist that I am today is a reflection of the dedication and guidance of my advisor Jayne B. Robinson. Thank you for making me part of your lab and giving me the greatest education I could ever ask for. I have the highest respect for you, but please know I will never surrender to you on Pearl Harbor Day. Most of all, I want to thank my family for their constant love and support. I have four of the funniest, smart brothers: Paul, Phil, Mike, and Robbie. They have always been good to me and are great men. Also, I would like to recognize my sister-in-law, Erica, for being my friend and blessing the family with two beautiful children, Anna and Sam. And, lastly, I would like to acknowledge my mother, Linda, who deserves the most credit of all. She is the reason I am a microbiologist. My mother has always stressed the importance of not settling and striving for the very best. You’ve pushed and disciplined me when I don’t feel like trying. Thank you for providing for me physically and emotionally. You are the best mother. I love you. viii TABLE OF CONTENTS DEDICATION………………………………………………………………………….... v ACKNOWLEDGEMENTS............................................................................................... vi TABLE OF CONTENTS................................................................................................... ix LIST OF FIGURES........................................................................................................... xi LIST OF TABLES............................................................................................................ xv CHAPTERS I. Literature Review…………………………………………………………………1 II. E,E-farnesol inhibits surface motility in Pseudomonas aeruginosa through rhamnolipid production......................................................................................... 45 a. Abstract..................................................................................................... 46 b. Introduction............................................................................................... 48 c. Methods..................................................................................................... 53 d. Results....................................................................................................... 60 e. Discussion................................................................................................. 84 f. Summary................................................................................................... 93 g. Future Studies........................................................................................... 95 III. The effect of a cationic porphyrin on Pseudomonas aeruginosa biofilms........... 97 a. Abstract..................................................................................................... 98 b. Introduction............................................................................................. 100 ix c. Methods................................................................................................... 102 d. Results..................................................................................................... 106 e. Discussion............................................................................................... 116 LITERATURE CITED................................................................................................... 120 x LIST OF FIGURES Chapter I Figure 1. The bacterial flagellum......................................................................... 29 Figure 2. Model of the structure of type IV pilin monomer in Pseudomonas aeruginosa............................................................................................
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