FUNCTION AND REGULATION OF THE POLYSACCHARIDE UTILIZATION LOCUS, DON, IN THE GUT SYMBIONT BACTEROIDES FRAGILIS By Yanlu Cao August, 2014 Director of Dissertation: C. Jeffrey Smith, Ph.D. Major Department: Department of Microbiology and Immunology Abstract Bacteroides fragilis is the most common anaerobe isolated from clinical infections and in this report we demonstrate a novel feature of the species that is critical to their success as an opportunistic pathogen. Among the Bacteroides spp. in the gut, B. fragilis has a unique ability to efficiently harvest complex N-linked glycans from the glycoproteins common to serum and serous fluid. This activity is mediated by a Sus-like outer membrane protein complex designated as Don. Using the abundant serum glycoprotein transferrin as a model it was shown that B. fragilis alone can rapidly and efficiently deglycosylate this protein in vitro and that transferrin glycans can provide the sole source of carbon and energy for growth in defined media. We then showed that transferrin deglycosylation occurs in vivo when B. fragilis is propagated in the rat tissue cage model of extraintestinal growth and that this ability provides a competitive advantage in vivo over strains lacking the don locus. Thus, Don functionally is an extraintestinal growth factor that may contribute to B. fragilis opportunistic infection. The regulation of don expression is controlled by two independent pathways. The first one was shown to be a typical ECF sigma/anti-sigma factor switch, commonly found in Sus-like Polysaccharide Utilization Loci (PULs), which responds to the presence of specific substrate. In the ECF sigma factor deletion mutant, ΔdonA, expression of the don PUL was completely abolished in the presence of substrate glycans, while the cognate anti-sigma deletion strain, ΔdonB, expressed the don genes even in the absence of substrate glycans. The donA overexpressing strain highly expressed the don PUL regardless of the substrate glycan presence. The second regulatory pathway is involved with a cis-encoded antisense sRNA which is associated within the don locus, DonS. DonS was shown to negatively regulate don expression. In contrast, expression of the don genes was induced two- to six-fold in the donS silencing mutant and highly repressed in the donS overexpressing strain. Notably, this sRNA controlled regulatory pathway is not commonly found associated with B. fragilis PULs. Only 14 of more than 50 PULs in B. fragilis possess DonS-like sRNAs, but at the present time their roles in commensal colonization and opportunistic infections is not understood. FUNCTION AND REGULATION OF THE POLYSACCHARIDE UTILIZATION LOCUS, DON, IN THE GUT SYMBIONT BACTEROIDES FRAGILIS A Dissertation Presented to The Faculty of the Department of Microbiology and Immunology Brody School of Medicine at East Carolina University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Microbiology and Immunology By Yanlu Cao August 15th, 2014 ©Yanlu Cao, 2014 FUNCTION AND REGULATION OF THE POLYSACCHARIDE UTILIZATION LOCUS, DON, IN THE GUT SYMBIONT BACTEROIDES FRAGILIS by Yanlu Cao APPROVED BY: DIRECTOR OF DISSERTATION C. Jeffrey Smith, Ph.D. COMMITTEE MEMBER Everett C. Pesci, Ph.D. COMMITTEE MEMBER R. Martin Roop, II, Ph.D. COMMITTEE MEMBER Rachel L. Roper, Ph.D. COMMITTEE MEMBER Warren Knudson, Ph.D. CHAIR OF THE DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY C. Jeffrey Smith, Ph.D. INTERIM DEAN OF THE GRADUATED SCHOOL Paul J. Gemperline, Ph.D. ACKNOWLEDGEMENTS First I would like to thank my mother Hong Cao, who had encouraged and supported me to pursue an education and career in the United States, for her constant love. I would also like to thank all my family members from my maternal side, especially my grandparents, for their support and help since my childhood. For the past six years during my high education in ECU, I foremost have to give my thanks to my advisor, Dr. C. Jeffrey Smith. For his dedication and mentorship, I can accomplish the work in this study and be qualified to have a doctoral degree in microbiology and immunology. I also must thank Dr. Edson R. Rocha, Dr. Ivan Ndamukong, Anita C. Parker and Michael Betteken for their support and cooperation, both as co-workers and great friends. I also need to acknowledge Dr. Clayton C. Caswell for his technique support during the small RNA study. I would also like to thank my committee members, Dr. Everett C. Pesci, Dr. R. Martin Roop, II, Dr. Rachel L. Roper and Dr. Warren Knudson for their contribution to my educational development. For many years I always want, but have not had a chance, to say thanks to the Biology Department of Indiana University of Pennsylvania, especially to Dr. Carl Luciano and Dr. Narayanaswamy Bharathan, for their help which are not just limited to education. There are many people in my life that I sincerely appreciate but not be able to mention every one individually here. To them, I just want to say: thank you all and God bless. TABLE OF CONTENTS LIST OF TABLES .............................................................................................................................vi LIST OF FIGURES ...........................................................................................................................vii CHAPTER ONE: INTRODUCTION ...................................................................................................1 1.1 Bacteroides taxonomy, structure, physiology and metabolism ......................................1 1.2 The mutualistic relationship between Bacteroides and their hosts ................................7 1.3 Bacteroides as opportunistic pathogens .........................................................................10 1.4 Virulence Mechanisms .....................................................................................................11 1.5 The polysaccharide utilization systems in Bacteroides....................................................14 CHAPTER TWO: EFFICIENT UTILIZATION OF COMPLEX N-LINKED GLYCANS IS A SELECTIVE ADVANTAGE FOR BACTEROIDES FRAGILIS IN EXTRAINTESTINAL INFECTIONS ...........................21 2.1 Abstract ............................................................................................................................21 2.2 Significance ......................................................................................................................22 2.3 Introduction .....................................................................................................................22 2.4 Materials and methods ....................................................................................................24 2.4.1 Bacterial strains and growth .................................................................................24 2.4.2 Construction of a don operon deletion mutant ....................................................27 2.4.3 Animal model of infection .....................................................................................27 2.4.4 Total RNA extraction .............................................................................................28 2.4.5 cDNA synthesis, qRT-PCR, and microarray analysis ..............................................29 2.4.6 Whole cell deglycosylation assay ..........................................................................30 2.4.7 Glycomics analyses of B. fragilis treated human transferring ..............................30 2.5 Results ..............................................................................................................................31 2.5.1 In vivo, extraintestinal PUL expression .................................................................31 2.5.2 In vitro induction of the donCDEFG operon ..........................................................35 2.5.3 Deglycosylation of transferrin is mediated by donCDEFG ....................................38 2.5.4 Growth on transferrin as the sole carbon and energy source ..............................43 2.5.5 In vivo role of the don PUL ....................................................................................46 2.5.6 Efficient transferrin deglycosylation is unique to B. fragilis .................................50 2.6 Discussion.........................................................................................................................53 2.7 Supplementary Figures ....................................................................................................58 2.8 Addendum........................................................................................................................67 CHAPTER THREE: BACTEROIDES FRAGILIS PRIMARY TRANSCRIPTOME ANALYSIS ......................68 3.1 Introduction .....................................................................................................................68 3.2 Materials and Methods ....................................................................................................69 3.2.1 Total RNA Extraction .............................................................................................69 3.2.2 Preparation of cDNA libraries and 454 pyrosequencing ......................................69 3.2.3 Data Visualization ..................................................................................................70 3.3 Results ..............................................................................................................................71 3.3.1 Identification of B. fragilis TISs