Modification and Utilization of Carbohydrates by Streptococcus pneumoniae DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Carolyn Marion M.S. The Integrated Biomedical Science Graduate Program ***** The Ohio State University 2012 Dissertation Committee: Dr. Samantha King, Advisor Dr. Kevin Mason Dr. Robert Munson Jr. Dr. Larry Schlesinger Copyright by Carolyn Marion 2012 Abstract Streptococcus pneumoniae (the pneumococcus) is responsible for over one million deaths each year worldwide. The majority of this mortality is in children and S. pneumoniae is therefore considered the greatest cause of vaccine preventable pediatric deaths. Colonization of the airway is a necessary precursor to disease, but little is known about how the bacterium establishes and maintains colonization. Carbohydrates are required as a carbon source for growth and therefore colonization, however free sugars are not readily available in the airway. Carbohydrates are instead present in the airway as N- and O-linked glycans. S. pneumoniae is adept at manipulating carbohydrates and produces at least ten sugar-cleaving enzymes. We have previously demonstrated the ability of S. pneumoniae to deglycosylate N-linked glycans; however it was unknown whether O- linked glycans could similarly be modified. It was known that pneumococci produced an O-glycosidase, but unknown what gene encoded this and whether it impacted colonization. We identified eng as encoding the O-glycosidase and showed that Eng acts sequentially with neuraminidase NanA to deglycosylate O-linked glycans to release sialic acid and galactose β1-3 N-acetylgalactosamine. An eng mutant was deficient in adherence to airway epithelial cells and was reduced in airway colonization. Deglycosylation of O-linked glycans may contribute to colonization my multiple mechanisms including mediating adherence as well as utilizing released carbohydrates ii for growth. Sialic acid is the most common terminal modification on N- and O-linked glycans and is likely encountered frequently in the airway. We showed that sialic acid can support growth as the sole carbon source. In order to utilize liberated carbohydrates including sialic acid for nutrition, S. pneumoniae must encode a mechanism for import. Out of three candidate transporters, we identified satABC as encoding the substrate binding protein and two permeases of an ATP binding cassette (ABC) importer that recognizes sialic acid. SatABC contributes to growth on a human glycoprotein and to airway colonization. In addition to the substrate binding protein and permeases, two ATPases are also required for ABC transporters; however no predicted ATPase is encoded in the satABC locus. Mutation of a candidate gene encoding a predicted carbohydrate ATPase, msmK, revealed that like satABC, msmK is required for growth on sialic acid. MsmK was able to hydrolyze ATP; this suggests that MsmK energizes SatABC. In addition to satABC there are five additional loci predicted to encode CUT1 carbohydrate ABC transporters that contain a substrate binding protein, two permeases, but lack predicted ATPases. Together, these are the only six loci in the TIGR4 genome with this arrangement, and comprise all predicted CUT1 carbohydrate transporters; thus we hypothesized that MsmK energizes all CUT1 carbohydrate ABC transporters. Indeed, msmK is required for growth on each CUT1 ABC importer substrate identified to date. Unlike many other characterized carbohydrate ABC transporters, msmK is encoded in its own transcript although the reason for this remains unknown. A shared carbohydrate ATPase may have implications in carbohydrate substrate preference or gene regulation iii and creates an opportunity to gain greater understanding of carbohydrate utilization in S. pneumoniae. iv To my family: Wesley, Mom, Dad, and Jessica v Acknowledgements I am humbled by the incredible number of people that have supported me over the past five years. This has been an amazing journey and I’m indebted to all the mentors, colleagues, friends and family who have helped me along my way. I am extraordinarily grateful to my mentor Sam without whom, I would not be the scientist I am. I hope that as I leave this lab, I carry with me Sam’s logical approach to science, articulate storytelling and highest standards. She’s helped me see that I am capable of all these things and I will continue to look up to Sam throughout my career. Sam is only one of the amazing team with Drs. Mason, Munson and Schlesinger who have watched me grow these past five years. I am so fortunate to have had this knowledgeable dissertation committee guiding me and pushing me to achieve more than I thought was possible. It’s difficult for me to think of anyone in the Center for Microbial Pathogenesis that hasn’t helped me in some way. Science is a team sport and I’m thankful to the CMP for all of the technical and intellectual input and for keeping me always looking forward to cake o’clock. I’m grateful to all past and present members of the King Lab, especially my vi coauthors for making my papers better than I could on my own and to Caroline Linke, Greg Bobulsky and Hussam Salhi whose unpublished data are included within. I also thank the Munson and White Labs for technical advice in my experimental designs, and William Barson, Mario Marcon, and Marilyn Hribar for providing clinical isolates. I’m lucky to have Rebecca, Jen, Dee, Matt, Katie, Laura and my sister Jessica to thank, who despite never really understanding what I do, support me endlessly. Lastly, I’m grateful to my parents John and Cathy, who have given me opportunities, confidence and love from day one, and to my husband Wesley, who makes me nicer. This research was supported in part by The American Heart Association pre-doctoral fellowship 10PRE3490014. vii Vita February 22, 1984 ………………………………… Born, Garfield Heights, Ohio May, 2006 ………………………………………… B.S. Biology, Duquesne University May, 2007 ………………………………………… M.S. Forensic Science and Law, Duquesne University June 2007 - present…………………………………Graduate Research Assistant and Fellow, The Ohio State University Publications Marion C, Limoli DH, Bobulsky GS, Abraham JL, Burnaugh AM, King SJ. 2009. Identification of a pneumococcal glycosidase that modifies O-linked glycans. Infect. Immun. 77:1389-1396. Marion C, Burnaugh AM, Woodiga SA, King SJ. 2011. The pneumococcal sialic acid transporter contributes to colonization. Infect. Immun. 79:1262-1269. Marion C, Aten AE, Woodiga SW, King SJ. 2011. Identification of an ATPase, MsmK, which energizes multiple carbohydrate ABC transporters in Streptococcus pneumoniae. Infect. Immun. 79:4193-4200. Marion C, Stewart JM, Tazi MF, Burnaugh AM, Linke CM, Woodiga SA, King SJ. 2012. Streptococcus pneumoniae utilization of hyaluronic acid for growth. Infect. Immun. 80:1390-1398. viii Fields of Study Major Field: Integrated Biomedical Science Area of Emphasis: Microbial Pathogenesis ix Table of Contents Page Abstract ............................................................................................................................... ii Dedication ...........................................................................................................................v Acknowledgements ........................................................................................................... vi Vita .................................................................................................................................. viii List of Tables .................................................................................................................. xiv List of Figures ...................................................................................................................xv Chapter 1: Introduction ......................................................................................................1 1.1. Background ......................................................................................................1 1.1.1. Streptococcus pneumoniae ................................................................1 1.1.2. Colonization .......................................................................................2 1.1.3. Adult disease ......................................................................................3 1.1.4. Pediatric disease .................................................................................5 1.1.5. Virulence determinants ......................................................................6 1.1.6. Antibiotic therapy and resistance .......................................................7 1.1.7. Vaccines .............................................................................................9 1.1.7.1. Pneumococcal polysaccharide vaccine (PPSV) ..................9 1.1.7.2. Protein conjugate vaccine (PCV) ......................................10 1.1.7.3. Vaccine limitations ...........................................................12 1.1.7.4. Serotype-independent vaccines .........................................13 1.2. Molecular aspects of colonization ..................................................................15 1.3. Host glycosylation ..........................................................................................18 1.3.1. N-linked glycans ..............................................................................19 1.3.2. O-linked glycans ..............................................................................20