IDENTIFICATION AND CHARACTERIZATION OF FACTORS CONTRIBUTING TO THE PATHOGENICITY OF MORAXELLA CATARRHALIS by SEAN WILLIAM BUSKIRK (Under the Direction of Eric Lafontaine) ABSTRACT Moraxella catarrhalis is a human-specific pathogen and causative agent of otitis media in children and respiratory tract infections in adults. During the course of pathogenesis, M. catarrhalis is able to colonize and persist in the human host through processes such as adherence to the mucosal epithelium and resistance to complement-mediated killing. The first part of this dissertation describes the identification and characterization of the protein responsible for cardiolipin synthesis in M. catarrhalis (MclS) and the contribution of cardiolipin to bacterial adherence to human epithelial cells. To study the role of MclS, we constructed an mclS insertion mutant strain that lacks expression of MclS and an mclS point mutant strain that expresses an enzymatically-inactive version of MclS. Both mutant strains are unable to synthesize cardiolipin and are defective in their adherence to human epithelial cells in vitro, thereby suggesting that MclS mediates adherence in an indirect manner. We hypothesize that cardiolipin contributes to adherence through its interaction with surface-displayed adhesins. Examination of outer membrane vesicles revealed that cardiolipin is present in the outer membrane of M. catarrhalis. However, five major proteinaceous adhesins (UspA1, Hag, McaP, OMP CD, MhaB1/MhaB) were all expressed and displayed on the surface of the cardiolipin- deficient strains. Conversely, expression of lipooligosaccharide, which has previously been implicated in adherence of M. catarrhalis to epithelial cells, may depend on the presence of cardiolipin. The second part of this dissertation describes a series of experiments designed to identify factors that contribute to complement resistance of M. catarrhalis. Several factors are currently known to contribute to complement resistance including UspA2 which mediates the majority of resistance exhibited by M. catarrhalis. Using experimental evolution, we demonstrate that an M. catarrhalis strain lacking expression of UspA2 is able to develop complement resistance through repeated exposure to human serum. We intend to identify the mutations that confer complement resistance in the evolved lineages using whole-genome sequencing. Ultimately, we hope to gain a better understanding of the factors that impact complement resistance in M. catarrhalis. INDEX WORDS: Moraxella catarrhalis, Virulence, Adherence, Cardiolipin, Complement resistance, Experimental evolution IDENTIFICATION AND CHARACTERIZATION OF FACTORS CONTRIBUTING TO THE PATHOGENICITY OF MORAXELLA CATARRHALIS by SEAN WILLIAM BUSKIRK B.S. The Pennsylvania State University, 2008 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2014 © 2014 Sean William Buskirk All Rights Reserved IDENTIFICATION AND CHARACTERIZATION OF FACTORS CONTRIBUTING TO THE PATHOGENICITY OF MORAXELLA CATARRHALIS by SEAN WILLIAM BUSKIRK Major Professor: Eric Lafontaine Committee: Robert J. Hogan Vincent Starai Frederick Quinn Mark Farmer Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2014 iv DEDICATION While I acknowledge that countless friends and family members have contributed to my accomplishments, I have decided to dedicate this dissertation to my late grandfathers, Arlington “Ty” Frutchey and Richard “Dick” Buskirk. I have always cherished the time that we spent together as I was growing up, and both of you have made a lasting impact in my life. From you I have learned so many life lessons. I know you would be proud of me today. v ACKNOWLEDGEMENTS The completion of my degree would not have been possible without contributions from numerous individuals including: My amazing wife Katie who has displayed unwavering support and encouragement throughout our relationship. She has made several personal sacrifices over the years so that I could further my education. Most of all, I am grateful for the love that she shows me each and every day. My parents Stanley and Julie Buskirk who have been my foundation. I am so thankful for their love and support throughout my life. I owe them for all of my accomplishments and the person I am today. My advisor Dr. Eric Lafontaine from whom I have received countless of hours of scientific training. The advice I have received from Eric has been invaluable. He has continually pushed my boundaries so that I could grow and develop as a researcher, and I could not be more thankful for this. My committee members Dr. Vincent Starai, Dr. Jeff Hogan, Dr. Mark Farmer, and Dr. Fred Quinn who have allowed me to pursue both my research and teaching interests. My committee has always been very supportive of my research as it has progressed. Thank you for the helpful recommendation all along the way; I know you have always had my best interest in mind. Current and past members of the Lafontaine Lab, namely Teresa Shaffer, Dr. Shawn Zimmerman, Dr. Rachel Balder, Dr. William Grose, and Laura Wiese. You all have vi contributed in many different ways and have always been a source of advice, support, and entertainment. A countless number of students, faculty, and staff within numerous departments at UGA. I am lucky to be surrounded by so many talented and helpful individuals. I consider myself blessed to be able to call so many of you friends. I am extremely thankful for the financial assistance I have received from the Department of Infectious Diseases, the Department of Microbiology, the Interdisciplinary/Integrated Life Sciences Program, and the Graduate School at UGA. Without this support, I simply would not have been able to complete my degree. vii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS………………………………………………………………………. v CHAPTER 1 INTRODUCTION……………………………………………………………………... 1 2 LITERATURE REVIEW ……………………………………………………………..3 Taxonomy of Moraxella catarrhalis…………………………………………...… 3 Microbiology of Moraxella catarrhalis…………………………………………... 3 The Moraxella catarrhalis Genome……………………………………………… 4 Moraxella catarrhalis Carriage and Disease….………………………………….. 5 Virulence Traits of Moraxella catarrhalis………………………………………... 9 Animal Models for Moraxella catarrhalis……………………………………….20 Phospholipids in Bacteria………………………………………………………...24 3 MORAXELLA CATARRHALIS EXPRESSES A CARDIOLIPIN SYNTHASE THAT IMPACTS ADHERENCE TO HUMAN EPITHELIAL CELLS………………………. 32 Abstract………………………………………………………………………….. 33 Introduction……………………………………………………………………… 34 Materials and Methods…………………………………………………………... 38 Results…………………………………………………………………………… 49 Discussion……………………………………………………………………….. 58 Acknowledgements……………………………………………………………… 64 viii Tables……………………………………………………………………………. 65 Figures……………………………………………………………………………67 4 CHARACTERIZATION OF MCLS AND CARDIOLIPIN IN MORAXELLA CATARRHALIS………………………………………………………………………….. 79 Introduction……………………………………………………………………… 79 Section I: Lipooligosaccharide Expression may be Dependent on Cardiolipin in Moraxella catarrhalis………………………………………..………………….. 80 Section II: MCORF 821 is not a Positive Effector of Adherence in Moraxella catarrhalis……………………………………………………………………….. 85 Section III: MclS does not Require Lipidation of Cysteine for Cardiolipin Synthase Activity…………………………………………….............................. 90 Section IV: Cardiolipin is Present in the Outer Membrane of Moraxella catarrhalis……..................................................................................................... 95 Section V: Genetic Approaches to Determine Substrates of MclS are Unsuccessful due to Requirement of Enzymes involved in Phosphatidylethanolamine Synthesis................................................................... 100 Conclusions…………………………………………………………………….. 105 Tables…………………………………………………………………………... 107 5 IDENTIFICATION OF FACTORS CONTRIBUTING TO THE DEVELOPMENT OF COMPLEMENT RESISTANCE OF MORAXELLA CATARRHALIS THROUGH EXPERIMENTAL EVOLUTION……………………………………….…………….. 109 Abstract……………………………………………………………………...…. 110 Introduction…………………………………………………………………….. 111 ix Materials and Methods…………………………………………………….…… 113 Results………………………………………………………………………….. 117 Discussion……………………………………………………………………… 122 Tables…………………………………………………………………………... 127 Figures…………………………………………………………………………..129 6 CONCLUSIONS……………………………………………………………………..136 REFERENCES………………………………………………………………………………….140 APPENDIX…………………………………………………………………………………..… 163 1 CHAPTER 1 INTRODUCTION Moraxella catarrhalis is a Gram negative diplococcus and human-specific pathogen of the mucosa. It is a causative agent of otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. Once considered simply a commensal, today M. catarrhalis is recognized as a significant public health concern. Treatment of M. catarrhalis infection has become troublesome due to the wide-spread resistance to β-lactam antibiotics. The introduction of vaccines against non-typeable Haemophilus influenzae and Streptococcus pneumoniae is predicted to shift the landscape of the human mucosa resulting in an increase in the incidence and prevalence of M. catarrhalis carriage. Disease caused by M. catarrhalis imparts a large financial burden on the United States, in part due to the lack of a licensed vaccine to combat M. catarrhalis infection. To promote the development of effective therapeutics, research is being conducted into how M. catarrhalis is able to cause disease