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  • CONTRIBUTION OF COMPLEX FORMATION IN THE IN VITRO AND IN VIVO ACTION OF CLOSTRIDIUM PERFRINGENS ENTEROTOXIN

    by

    Justin Angelo Caserta

    BS Biology, University of Dayton, 2004

    Submitted to the Graduate Faculty of

    The School of Medicine in partial fulfillment

    of the requirements for the degree of

    Doctor of Philosophy

    University of Pittsburgh

    2010

  • ii

    UNIVERSITY OF PITTSBURGH

    SCHOOL OF MEDICINE

    This dissertation was presented

    by

    Justin Angelo Caserta

    It was defended on

    July 9, 2010

    and approved by

    Billy W. Day, Ph.D., Professor, Pharmaceutical Sciences

    Neal A. DeLuca, Ph.D., Professor, Microbiology and Molecular Genetics

    Michael A. Parniak, Ph.D., Professor, Microbiology and Molecular Genetics

    Russell D. Salter, Ph.D., Professor, Immunology

    Dissertation Advisor: Bruce A. McClane, Ph.D., Professor, Microbiology and Molecular

    Genetics

  • iii

    Copyright by Justin A. Caserta

    2010

  • iv

    Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin that is responsible for causing

    the symptoms of type A food poisoning, a leading cause of bacterial foodborne illness in the US.

    CPE-induced pore formation on intestinal epithelial cells results in ion permeability alterations

    leading to Ca2+ influx and activation of cell death pathways. Upon binding to its receptor,

    certain claudins, the interactions between CPE and the target membrane result in the formation

    of a series of toxin complexes (CH-1 and CH-2) that represent the formation of the functional

    CPE pore. Many bacterial toxins, particularly, pore-forming toxins, hijack cholesterol-rich lipid

    raft domains in the target cell membrane to aid in their virulence. Lipid rafts serve as platforms

    to cluster receptor proteins to allow for more efficient binding and oligomerization. Due to the

    pore-forming activity of CPE, we wished to determine if membrane rafts play a role in the

    mechanism of action of CPE. Interestingly, CPE was found to be a novel pore-forming toxin

    that does not require raft domains for its action in that CPE complexes do not form within lipid

    rafts and cholesterol depletion had no effect on CPE-induced cytotoxicity. These findings

    illustrate the unique interactions between CPE and target cells. Despite recent research findings

    indicating the presence of claudins in the various CPE complexes, these intricate interactions

    have not been fully elucidated, and the exact composition of the toxin complexes is unknown.

    Therefore, the research presented here describes the development of a two-step method of

    electroelution/immunoprecipitation that allows for the isolation and purification of the CPE

    CONTRIBUTION OF COMPLEX FORMATION IN THE IN VITRO AND IN VIVO

    ACTION OF CLOSTRIDIUM PERFRINGENS ENTEROTOXIN

    Justin Angelo Caserta, PhD

    University of Pittsburgh, 2010

  • v

    complexes for compositional analysis by mass spectrometry. Finally, a mouse model has been

    developed and characterized to show that the molecular interactions that occur in cell culture

    models, such as complex formation and inflammatory cell death, also occur in vivo.

    Furthermore, the mouse model mimics the lethality that is occasionally seen in humans that

    suffer from type A food poisoning-related deaths.

  • vi

    TABLE OF CONTENTS

    PREFACE ................................................................................................................................... XV

    1.0 INTRODUCTION ........................................................................................................ 1

    1.1 GENERAL BACTERIOLOGY OF CLOSTRIDIUM PERFRINGENS ......... 1

    1.2 TOXINS AND DISEASES ASSOCIATED WITH C. PERFRINGENS ......... 2

    1.2.1 Toxinotyping .................................................................................................... 2

    1.2.2 Non-typing Toxins ........................................................................................... 4

    1.3 CLOSTRIDIUM PERFRINGENS ENTEROTOXIN ....................................... 5

    1.3.1 Role in Type A Food Poisoning ...................................................................... 5

    1.3.1.1 Clinical and Epidemiological Aspects of Type A Food Poisoning .... 5

    1.3.1.2 In vivo Effects of Type A Food Poisoning ........................................... 6

    1.3.1.3 Direct Evidence for CPEs Role in Food Poisoning ........................... 8

    1.3.1.4 What is the Reservoir for cpe+ Isolates? ............................................. 8

    1.3.2 Role of CPE in Non-foodborne Disease ....................................................... 10

    1.3.3 CPE Genetics.................................................................................................. 11

    1.3.3.1 The cpe Gene and Locus ..................................................................... 11

    1.3.3.2 Transcriptional Regulation of CPE ................................................... 12

    1.3.4 Mechanism of Action ..................................................................................... 13

    1.3.4.1 Binding ................................................................................................. 13

  • vii

    1.3.4.2 Formation of the Small Complex ....................................................... 18

    1.3.4.3 Formation of the Pre-Pore and Large Complexes ........................... 19

    1.3.4.4 Mechanism of CPE-induced Cell Death............................................ 21

    1.3.5 CPE Structure/Function ............................................................................... 23

    1.3.5.1 N-terminal Activation Domain .......................................................... 23

    1.3.5.2 N-terminal Cytotoxicity Domain ....................................................... 24

    1.3.5.3 C-terminal Binding Domain ............................................................... 26

    1.3.5.4 Putative Transmembrane Stem Domain........................................... 28

    1.3.6 Practical Applications of CPE and CPE Derivatives ................................. 29

    1.3.6.1 CPE as a Cancer Therapeutic ............................................................ 29

    1.3.6.2 CPE Derivatives in Drug Delivery ..................................................... 31

    1.4 SPECIFIC AIMS ............................................................................................... 33

    1.4.1 Specific Aim 1................................................................................................. 33

    1.4.2 Specific Aim 2................................................................................................. 33

    1.4.3 Specific Aim 3................................................................................................. 34

    2.0 THE ROLE OF LIPID RAFTS IN THE ACTION OF C. PERFRINGENS

    ENTEROTOXIN......................................................................................................................... 35

    2.1 INTRODUCTION AND RATIONALE .......................................................... 35

    2.1.1 Bacterial Pore-Forming Toxins .................................................................... 35

    2.1.2 Lipid Rafts and Their Role in Bacterial Pathogenesis ............................... 36

    2.1.3 Rationale ......................................................................................................... 38

    2.2 MATERIALS AND METHODS ...................................................................... 38

    2.2.1 Materials ......................................................................................................... 38

  • viii

    2.2.2 Cell Culture .................................................................................................... 39

    2.2.3 Small Complex Formation ............................................................................ 39

    2.2.4 CH-1 and CH-2 Complex Formation........................................................... 39

    2.2.5 Ib Treatment .................................................................................................. 40

    2.2.6 Cholesterol Depletion .................................................................................... 40

    2.2.7 Cholesterol Quantitation ............................................................................... 40

    2.2.8 TX-100 Extraction of Caco-2 cells and Sucrose Density Gradient

    Centifugation .............................................................................................................. 41

    2.2.9 Western Blotting ............................................................................................ 41

    2.2.10 Trypan Blue Staining ................................................................................... 42

    2.2.11 Formation of CH-1 in Absence of CH-2 ..................................................... 42

    2.3 RESULTS ........................................................................................................... 43

    2.3.1 Detergent Solubility and Raft Localization of CPE Small Complex ........ 43

    2.3.2 Detergent Solubility and Raft Localization of CH-1 and CH-2 Complexes

    44

    2.3.3 Effect of Cholesterol Depletion on CPE Complexes ................................... 46

    2.3.4 Effect of Cholesterol Depletion on CPE Cyt

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