Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Summer 8-15-2013 The aN tural Variants of the Streptococcus NAD+- glycohydrolase SPN Modulate Divergent Host Cell Signaling Pathways and Death Sukantha Chandrasekaran Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Biology Commons Recommended Citation Chandrasekaran, Sukantha, "The aN tural Variants of the Streptococcus NAD+-glycohydrolase SPN Modulate Divergent Host Cell Signaling Pathways and Death" (2013). Arts & Sciences Electronic Theses and Dissertations. 1023. https://openscholarship.wustl.edu/art_sci_etds/1023 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Molecular Microbiology and Microbial Pathogenesis Dissertation Examination Committee: Michael Caparon, Chair David Haslam Jeff Henderson David Hunstad David Sibley Joseph Vogel The Natural Variants of the Streptococcus NAD+-glycohydrolase SPN Modulate Divergent Host Cell Signaling Pathways and Death by Sukantha Chandrasekaran A dissertation presented to the Graduate School of Arts and Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2013 St. Louis, Missouri TABLE OF CONTENTS List of Figures................................................................................................................................iv List of Tables.................................................................................................................................vi Acknowledgments ........................................................................................................................ vi Abstract ....................................................................................................................................... viii CHAPTER I: Introduction .......................................................................................................... 1 Classification ............................................................................................................................... 2 Diseases and Prevalence and Burden .......................................................................................... 3 Adhesion ..................................................................................................................................... 7 Virulence Factors ........................................................................................................................ 9 Cholesterol-Dependent Cytolysins ........................................................................................... 12 Cytolysin Mediated Translocation ............................................................................................ 15 SPN ........................................................................................................................................... 20 References ................................................................................................................................. 27 CHAPTER II: Analysis of Polymorphic Residues Reveals Distinct Enzymatic and Cytotoxic Activities of the Streptococcus pyogenes NAD+ Glycohydrolase ............................ 39 Summary ................................................................................................................................... 40 Introduction ............................................................................................................................... 41 Results ....................................................................................................................................... 45 Discussion ................................................................................................................................. 60 Materials and methods .............................................................................................................. 70 References ................................................................................................................................. 84 CHAPTER III : SPN activity modulates PARylation in epithelial cells influencing the release of the pro-inflammatory molecule HMGB-1 ............................................................... 89 Summary ................................................................................................................................... 90 Introduction ............................................................................................................................... 91 Results ....................................................................................................................................... 96 ii Discussion ............................................................................................................................... 115 Materials and methods ............................................................................................................ 120 References ............................................................................................................................... 125 CHAPTER IV: SPN activity influences cellular signaling and death in epithelial cells .... 130 Summary ................................................................................................................................. 131 Introduction ............................................................................................................................. 132 Results ..................................................................................................................................... 137 Discussion ............................................................................................................................... 153 Materials and methods ............................................................................................................ 160 References ............................................................................................................................... 164 CHAPTER V: Conclusion and Future Directions ................................................................. 171 Conclusions ............................................................................................................................. 172 Future directions ..................................................................................................................... 175 References ............................................................................................................................... 181 LIST OF FIGURES iii Chapter I: Introduction Figure 1 Map of the emm patterns found in S. pyogenes ....................................................3 Figure 2 Pathogenesis of Streptococcal infections ..............................................................4 Figure 3 The structure of various M proteins grouped by emm pattern ..............................8 Figure 4 Virulence Factors produced Streptococcus pyogenes .........................................12 Figure 5 Structure of PFO and CDC pore formation ........................................................14 Figure 6 Type III secretion system ....................................................................................16 Figure 7 SPN, IFS and SLO operon ..................................................................................17 Figure 8 Cytolysin-Mediated Translocation ......................................................................19 Figure 9 NAD+ cleavage ....................................................................................................20 Figure 10 Crystal Structure of SPN and IFS .......................................................................22 Figure 11 The ARTT like fold of SPN ................................................................................24 Chapter II: Analysis of Polymorphic Residues Reveals Distinct Enzymatic and Cytotoxic Activities of the Streptococcus pyogenes NAD+ Glycohydrolase Figure 1 Distribution of polymorphic residues between two SPN alleles ........................46 Figure 2 Three residues are required to restore NADase activity to SPNH5 ....................47 Figure 3 SPNJ4 single and double swap proteins have NADase activity .........................50 Figure 4 Cluster 4 swaps have low, but detectable NADase activity ................................52 Figure 5 NADase-deficient SPN remains cytotoxic to A549 cells ...................................57 Figure 6 Catalytic bi-glutamic acid residues are not required for cytotoxicity .................56 Figure 7 Catalytic Polymorphic residues are located in the substrate binding pocket ......64 Chapter III: SPN activity modulates PARylation in epithelial cells influencing the release of the pro-inflammatory molecule HMGB-1 Figure 1 PARP is activated in an SLO dependent manner ................................................ 97 Figure 2 Purified SLO is not sufficient to induce PARylation .........................................