Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences Structural and Functional Analysis of Moraxella catarrhalis Adhesins MCAP and OMPCD Submitted by: Chritine Akimana In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences Examination Committee Major Advisor: Eric Lafontaine, Ph.D. Academic Robert Blumenthal, Ph.D. Advisory Committee: R. Mark Wooten, Ph.D. Ray Larsen, Ph.D. Venkatesha Basrur, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: April 16, 2007 Structural and Functional Analysis of Moraxella catarrhalis Adhesins McaP and OMPCD Christine Akimana University of Toledo Health Science Campus 2007 DEDICATION This dissertation is dedicated to my son, Bennett Kiza, who has filled my life with untold blessings. I would like to thank my husband, Nicolas Kiza, and my mother-in-law, whose support and encouragement has enabled me to complete this work. I also would like to thank my parents for their love, inspiration, and support, especially for their hard work that has enabled me to achieve so much in life. There are several people, family members, friends, and mentors, whose support and inspiration has helped me throughout my academic years. For those as well, I offer my full gratitude. ii ACKNOWLEDGEMENTS I would like to thank my major advisor Dr. Eric Lafontaine for giving me the opportunity to work on this very exciting project. I thank him for training me, for sharing his expertise, and for providing me with the opportunity to attend several scientific conferences. I also thank him for his kindness, support, and patience. I would like to thank my committee members, Dr. Mark Wooten, Dr. Robert Blumenthal, Dr. Ray Larsen, and Dr. Venkatesha Basrur for taking time out of their busy schedule, and for their helpful suggestions and discussions during our meetings. I would like to thank Dr. Mark Wooten for agreeing to become my administrative advisor when Dr. Eric Lafontaine moved to the University of Georgia and for bleeding mice in the antibody production experiments. I would like to thank Serena Lipski, Rachel Balder, and Brian Bullard, the former members of Dr. Eric Lafontaine’s laboratory, for their help in lab and for their friendship. I would like to acknowledge Serena Lipski for her contribution to manuscript 1. I want to thank all the members of the Club Coli Conclave for their friendship, support, and for their help during our weekly laboratory meeting. iii I would like to thank Dr. Louise Smithson for proofreading the drafts of this dissertation. I also would like to thank the Medical Microbiology and Immunology Department, especially the secretaries for making this department an enjoyable place to work. I would also like to thank the graduate school and the Molecular Basis of Diseases Program for giving me the opportunity to obtain a Ph.D. degree at the University of Toledo – Health Science Campus. iv TABLE OF CONTENTS Page DEDICATION-------------------------------------------------------------------------------------ii ACKNOWLEDGMENTS-----------------------------------------------------------------------iii TABLE OF CONTENTS------------------------------------------------------------------------v INTRODUCTION---------------------------------------------------------------------------------1 LITERATURE -------------------------------------------------------------------------------------5 History and Taxonomy--------------------------------------------------------------------5 Characterization and identification-----------------------------------------------------7 Respiratory tract colonization------------------------------------------------------------9 Diseases caused by M. catarrhalis----------------------------------------------------9 Otitis media--------------------------------------------------------------------------9 Exacerbation of chronic obstructive pulmonary disease----------------13 Sinusitis and other diseases---------------------------------------------------14 Treatment of M. catarrhalis infections and antibiotic resistance M. catarrhalis surface antigens--------------------------------------------------------16 Porin proteins----------------------------------------------------------------------17 Autotransporter proteins--------------------------------------------------------20 UspA1 and UspA2---------------------------------------------------------------22 UspA2H----------------------------------------------------------------------------29 Hag/MID---------------------------------------------------------------------------30 Lipooligosacharide--------------------------------------------------------------34 v Pili------------------------------------------------------------------------------------35 McaP--------------------------------------------------------------------------------36 OMPE-------------------------------------------------------------------------------38 OMPCD-----------------------------------------------------------------------------39 Iron regulated proteins----------------------------------------------------------43 OMPG1a and OMPG1b--------------------------------------------------------49 OMP J-------------------------------------------------------------------------------48 McmA--------------------------------------------------------------------------------48 OMP M35 and OlpA--------------------------------------------------------------49 Animal models for M. catarrhalis infections-----------------------------------------50 Adherence mechanisms of bacterial proteins--------------------------------------52 CHAPTER 3 - MANUSCRIPT 1 -------------------------------------------------------------56 Moraxella catarrhalis McaP Autotransporter Adhesin CHAPTER 4 – MANUSCRIPT 2------------------------------------------------------------102 Moraxella catarrhalis OMPCD adherence epitopes CHAPTER 4 ADDENDUM-------------------------------------------------------------------133 CHAPTER 5 – DISCUSSION and SUMMARY-----------------------------------------140 REFERENCES---------------------------------------------------------------------------------149 ABSTRACT--------------------------------------------------------------------------------------180 vi INTRODUCTION Moraxella catarrhalis (M. catarrhalis) is an aerobic, non-encapsulated Gram-negative bacterium that colonizes the human nasopharynx and causes up to 20% of otitis media and approximately 20% cases of sinusitis in children (Karalus and Campagnari, 2000; Verduin et al., 2002). Furthermore, M. catarrhalis causes up to 30% of respiratory tract infection cases in adults with chronic obstructive pulmonary diseases (COPD) (Sethi and Murphy, 2001). In addition, some cases of invasive infections by M. catarrhalis have been reported (Karalus and Campagnari, 2000). Other bacteria that cause otitis media and exacerbation of COPD along with M. catarrhalis are Streptococcus pneumoniae and non-typeable Haemophilus influenzae (H. influenzae). In addition to these, Pseudomonas aeruginosa (P. aeroginasa) and H. influenzae are involved in COPD secondary infections. Of importance, 80% of infants in developed countries will experience otitis media by age three (Murphy, 1996). Otitis media is a highly prevalent pediatric disease worldwide and the primary cause of emergency room visits (Klein, 2000). Direct and indirect treatment costs associated with ear infections exceed $5 billion annually in the United States (US) alone (Klein, 2000). In addition, repeated episodes of otitis media may result in complications such as hearing loss or to a lesser extent behavioral, educational and language development (Klein, 2000). Chronic obstructive pulmonary disease is the fourth-leading cause of death in the US and in the world, (Sethi and Murphy, 2001) and direct cost for treatment of COPD is 1 estimated at $18 billion each year in US (McCrory et al., 2001). Thus, M. catarrhalis is a significant health problem. Currently M. catarrhalis remains susceptible to some antibiotic treatments such as amoxicillin-clavulanic acid, however there has been an increase in antibiotic resistance in the past 30 years that has led to the need for improved treatments (Verduin et al., 2002). Since M. catarrhalis is not encapsulated, a vaccine targeting its surface exposed outer membrane proteins may result in a protective immune response. Most efforts towards designing a vaccine against M. catarrhalis infections are directed at studying surface antigens, especially outer membrane proteins of M. catarrhalis, for their vaccine potential. The Vaccine Panel Report from the 1999 meeting on otitis media specified desirable characteristics of bacterial antigens to be included in a vaccine formulation to prevent this disease. Such antigens should (i) have conserved surface epitope(s), and (ii) be conserved among strains of the same bacterial species. Furthermore in vivo, these antigens should (iii) be good immunogens (i.e., generate protective antibodies and/or cellular responses) in the target population, (iv) generate antibodies that prevent otitis media, and (v) not increase middle ear inflammation via immune complex formation with other otitis media etiological agents; i.e., viral proteins (Giebink et al., 2005). Adhesins are desirable vaccine candidates because they likely fulfill 2 the first two requirements of being conserved within the bacterial species, and contain conserved surface exposed epitope(s); e.g., the receptor binding domain (Ofek et