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  • 1

    ANTHRAX EDEMA TOXIN IMPAIRS NEUTROPHIL ACTIN-BASED MOTILITY BY STIMULATING VASP PHOSPHORYLATION

    By

    SARAH E. SZAROWICZ

    A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

    OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

    UNIVERSITY OF FLORIDA

    2010

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    © 2010 Sarah E. Szarowicz

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    To all who have believed in me, cultured my intellectual curiosity, academic interest

    throughout my lifetime, making this milestone possible

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    ACKNOWLEDGMENTS

    I thank my chair and members of my supervisory committee for their mentoring. I

    thank my loving husband for all of his support and love, which motivated me to complete

    my studies. I also thank my parents for their loving encouragement and support

    throughout my academic career.

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    TABLE OF CONTENTS page

    ACKNOWLEDGMENTS ...................................................................................................... 4

    LIST OF TABLES ................................................................................................................ 7

    LIST OF FIGURES .............................................................................................................. 8

    ABSTRACT........................................................................................................................ 10

    CHAPTER

    1 INTRODUCTION ........................................................................................................ 12

    Bioterrorism and Bacillus anthracis ............................................................................ 12 History of Bacillus anthracis Microbiology ................................................................. 12 Anthrax Infections ....................................................................................................... 16 Anthrax Toxins ............................................................................................................ 17 cAMP Signaling........................................................................................................... 19 PKA ............................................................................................................................. 21 Actin Structure and Function ...................................................................................... 23 Actin Binding Proteins ................................................................................................ 23 Neutrophil Function..................................................................................................... 25 Neutrophil Chemotaxis ............................................................................................... 29 Listeria monocytogenes.............................................................................................. 31 VASP ........................................................................................................................... 33 Filopodia ...................................................................................................................... 34 ET Inhibits Neutrophil Motility by VASP Phosphorylation of S157 ........................... 36

    2 LITERATURE REVIEW ON NEUTROPHILS AND ANTHRAX ................................ 46

    Anthrax Clinical Data .................................................................................................. 46 Previous Studies with ET and Neutrophils................................................................. 47

    3 MATERIALS AND METHODS ................................................................................... 49

    Toxin Purification ........................................................................................................ 49 Chemicals Used .......................................................................................................... 49 Neutrophil Isolation and Toxin Treatment .................................................................. 50 Cell Culture ................................................................................................................. 50 Annexin V Staining, Analysis for Necrosis and Nitroblue Tetrazolium Assay .......... 51 Neutrophil Chemokinesis, Chemotaxis and Polarity ................................................. 51 Whole Cell cAMP Levels ............................................................................................ 52 PMN Phalloidin and CD11/CD18 Staining and Fluorescence-Activated Cell

    Sorting (FACS) Analysis ......................................................................................... 52 Measurement of Phosphorylated PKA ....................................................................... 53

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    Listeria monocytogenes and Shigella flexerni Infection and Phalloidin Staining ..... 53 Plasmid and Protein Purification ................................................................................ 54 Cloning and Expression of TAT-VASP Isoforms ....................................................... 55 PMN Isolation and Treatment with pTAT: .................................................................. 56 Quantitative Western Blots ......................................................................................... 57 Low Speed Actin Bundling Assay .............................................................................. 58 Pull-Down Assay ......................................................................................................... 58 Statistical Analysis ...................................................................................................... 58

    4 RESULTS.................................................................................................................... 61

    Bacillus anthracis Edema Toxin Impairs Neutrophil Actin-Based Motility ................ 61 Anthrax ET is Active in Human Neutrophils ........................................................ 61 Effects of ET on Apoptosis, Necrosis, and Nitroblue Tetrazolium Reduction .... 62 PMN Chemokinesis and Chemotaxis .................................................................. 63 CD11/CD18 Expression in Neutrophils ............................................................... 65 Effects of ET Alone and ET Combined with LT on Neutrophil Actin

    Assembly ........................................................................................................... 65 Effects of ET Alone and Combined with LT on Listeria monocytogenes and

    Shigella flexeneri Actin-Based Motility ............................................................. 66 Effects of ET on VASP ......................................................................................... 68 Effects of Changes in VASP S157 Phosphorylation on Human Neutrophils ..... 68 Effects of ET Induced VASP Phosphorylation on L. monocytogenes Actin-

    Based Motility .................................................................................................... 71 Effects of VASP S239 Phosphorylation on Human Neutrophils......................... 73 Effects of VASP Isoforms on Actin Bundling....................................................... 74 Pull-Down Assay .................................................................................................. 74

    Dual Toxin Affects on Human Neutrophils ................................................................. 75 ET and LT are Able to Enter Human Neutrophils ............................................... 75 Effects of ET+LT Interactions on cAMP Levels .................................................. 75 Signaling Pathway Utilized by LT to Decrease ET cAMP Production ................ 76 Effects of ET+LT Exposure to cAMP Downstream Target PKA ......................... 77 Timing of Toxin Entry ........................................................................................... 78

    5 DISCUSSION .............................................................................................................. 99

    ET and ET+LT Inhibit Neutrophil Actin-Based Assembly and Impair Listeria monocytogenes Intracellular Motility ...................................................................... 99

    VASP Phosphorylation Impairs Neutrophil and Listeria Actin-Based Motility ........ 103 Cell Entry and Manipulation of Host Signaling Pathways ....................................... 107

    6 FUTURE WORK ....................................................................................................... 110

    Vascular Sepsis Caused During Inhalation Anthrax................................................ 110 The Role of VASP in Filopodia Formation ............................................................... 112

    BIOGRAPHICAL SKETCH.............................................................................................. 136

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    LIST OF TABLES

    Table page 1-1 Key proteins involved in filopodia formation .......................................................... 38

    3-1 Primers for the generation of VASP mutations by site-directed mutagenesis ..... 60

    4-1 Effects of ET on cell necrosis and apoptosis ........................................................ 81

    4-2 VASP S157D Interacts with CKAP4 ...................................................................... 94

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    LIST OF FIGURES

    Figure page

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