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Anthrax Edema Toxin Impairs Neutrophil Actin Based Motility By Stimulating VASP Phosphorylation

Permanent Link: http://ufdc.ufl.edu/UFE0041387/00001

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Title: Anthrax Edema Toxin Impairs Neutrophil Actin Based Motility By Stimulating VASP Phosphorylation
Physical Description: 1 online resource (137 p.)
Language: english
Creator: Szarowicz, Sarah
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: actin, anthrax, bacillus, cytoskeleton, edema, neutrophils, vasp
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Inhalation anthrax can lead to sepsis and death within days. The fulminate nature of illness reveals minimal elevation of peripheral polymorphonuclear (PMNs) counts at time of hospital admissions indicating a profound impairment of the innate immune response. Edema toxin (ET) is one of the major Bacillus anthracis virulence factors and consists of edema factor (EF) and protective antigen (PA). Low concentrations of ET (100-500 ng/ml of PA and EF) significantly impair human PMN chemokinesis, chemotaxis, and the ability to polarize. These changes are accompanied by a significant reduction in chemoattractant-stimulated PMN actin assembly. ET also causes a significant decrease in Listeria monocytogens intracellular actin-based motility within HeLa cells. These defects in actin assembly are accompanied by a > 50-fold increase in intracellular cyclic AMP and a > 4-fold increase in the activation of protein kinase A (PKA). We have previously shown that anthrax lethal toxin (LT) also impairs neutrophil actin-based motility (1), and we now find that LT combined with ET causes an additive inhibition of PMN chemokinesis, polarization, chemotaxis, and FMLP (N-formyl-met-leu-phe)-induced actin assembly. We have previously shown that anthrax LT blocks the Hsp27 phosphorylation cycle, impairing actin assembly and chemotaxis (2). We find that ET impairs neutrophil and Listeria monocytogenes actin-based motility by phosphorylating VASP at serine 157 (S157). Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP) localizes to regions of dynamic actin remodeling and promotes filament formation. Phosphorylation affects the interaction of VASP with actin; however, the crucial role of phosphorylation in the regulation of actin filaments remains to be clarified. Introduction of pseudo-phosphorylated VASP into MVd7 cells lacking VASP, Ena, or Mena slows Listeria-induced actin assembly as compared to MVd7 cells transfected with wild-type VASP, and transfection with pseudo-unphosphorylated VASP accelerates Listeria-induced actin motility. Introduction of these proteins into human neutrophils has identical effects on chemokinesis. We conclude the phosphorylation of VASP at S157 inhibits actin-based motility in living cells.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Sarah Szarowicz.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Southwick, Frederick S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-10-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041387:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041387/00001

Material Information

Title: Anthrax Edema Toxin Impairs Neutrophil Actin Based Motility By Stimulating VASP Phosphorylation
Physical Description: 1 online resource (137 p.)
Language: english
Creator: Szarowicz, Sarah
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: actin, anthrax, bacillus, cytoskeleton, edema, neutrophils, vasp
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Inhalation anthrax can lead to sepsis and death within days. The fulminate nature of illness reveals minimal elevation of peripheral polymorphonuclear (PMNs) counts at time of hospital admissions indicating a profound impairment of the innate immune response. Edema toxin (ET) is one of the major Bacillus anthracis virulence factors and consists of edema factor (EF) and protective antigen (PA). Low concentrations of ET (100-500 ng/ml of PA and EF) significantly impair human PMN chemokinesis, chemotaxis, and the ability to polarize. These changes are accompanied by a significant reduction in chemoattractant-stimulated PMN actin assembly. ET also causes a significant decrease in Listeria monocytogens intracellular actin-based motility within HeLa cells. These defects in actin assembly are accompanied by a > 50-fold increase in intracellular cyclic AMP and a > 4-fold increase in the activation of protein kinase A (PKA). We have previously shown that anthrax lethal toxin (LT) also impairs neutrophil actin-based motility (1), and we now find that LT combined with ET causes an additive inhibition of PMN chemokinesis, polarization, chemotaxis, and FMLP (N-formyl-met-leu-phe)-induced actin assembly. We have previously shown that anthrax LT blocks the Hsp27 phosphorylation cycle, impairing actin assembly and chemotaxis (2). We find that ET impairs neutrophil and Listeria monocytogenes actin-based motility by phosphorylating VASP at serine 157 (S157). Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP) localizes to regions of dynamic actin remodeling and promotes filament formation. Phosphorylation affects the interaction of VASP with actin; however, the crucial role of phosphorylation in the regulation of actin filaments remains to be clarified. Introduction of pseudo-phosphorylated VASP into MVd7 cells lacking VASP, Ena, or Mena slows Listeria-induced actin assembly as compared to MVd7 cells transfected with wild-type VASP, and transfection with pseudo-unphosphorylated VASP accelerates Listeria-induced actin motility. Introduction of these proteins into human neutrophils has identical effects on chemokinesis. We conclude the phosphorylation of VASP at S157 inhibits actin-based motility in living cells.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Sarah Szarowicz.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Southwick, Frederick S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-10-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041387:00001


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1 ANTHRAX EDEMA TOXIN IMPAIRS NEUTROPHIL ACTIN -BASED MOTILITY BY ST IMULATING 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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2 2010 Sarah E. Szarowicz

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3 To all who have believed in me, cultured my intellectual curiosity, academic interest throughout my lifetime, making this milestone possible

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4 ACKNOWLEDGMENTS I thank my chair and members of my supervisory committee for their mentoring. I thank my lov ing 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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5 TABLE OF CONTENTS page ACKNOWLEDGMEN TS ...................................................................................................... 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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6 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 V ASP ......................................................................................... 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 ....................................................... 7 4 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 ActinBased 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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7 LIST OF TABLES Table page 1 -1 Key proteins involved in filopodia formation .......................................................... 38 3 -1 Primers for the generation of VASP mutations by sitedirected 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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8 LIST OF FIGURES Figur e page 1 -1 Anthrax toxins ......................................................................................................... 39 1 -2 cAMP signaling pathway ........................................................................................ 40 1 -3 PKA signaling pathway .......................................................................................... 41 1 -4 Actin structure ......................................................................................................... 42 1 -5 Neutrophil function ................................................................................................. 43 1 -6 Neutrophil actin -based motility ............................................................................... 44 1 -8 VASP structure ....................................................................................................... 45 4 -1 Effects of ET on Intracellular cAMP levels and PKA phosphorylation. ................ 80 4 -2 Effects of anthrax toxins on neutrophil chemokinesis, polarity, and chemotaxis ............................................................................................................. 82 4 -3 Time course of ET on neutrophil chemokinesis and polarity ................................ 83 4 -4 Effects of a nthrax toxins on CD11/CD18 surface expression by neutrophils ...... 84 4 -5 Effects of anthrax toxins on FMLP induced neutrophil actin assembly ................ 85 4 -6 Effects of anthrax toxins on Listeria actin based motility ...................................... 86 4 -7 Effects of ET on P S157 VASP .............................................................................. 87 4 -8 The effects of ET on P S157 VASP localization on neutrophils and Listeria infected HeLa cells ................................................................................................. 88 4 -9 Characterization of pTAT vecto r and the steps involved in protein synthesis ..... 89 4 -10 Effects of P S157 VASP on regulating actin assembly in human neutrophils ..... 90 4 -11 Pseudophosphorylated VASP mimics the effects of ET on Listeria motility ........ 91 4 -12 Effects of P S239 VASP on regulating actin assembly in human neutrophils ..... 92 4 -13 Actin Bundling Assay ............................................................................................. 93 4 -14 Effects of ET+LT on intracellular cAMP levels ...................................................... 95 4 -15 How LT inhibits intracellular cAMP levels induced by ET ..................................... 96

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9 4 -16 Effects of ET+LT on PKA phosphorylation ............................................................ 97 4 -17 Effects of ET+LT on intracellular cAMP levels over time ...................................... 98

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10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ANTHRAX EDEMA TOXIN IMPAIRS NEUTROPHIL ACTIN -BASED MOTILITY BY ST IMULATING VASP PHOSPHORYLATION By Sarah E. Szarowicz May 2010 Chair: Frederick Southwick Major: Medical Sciences Microbiology and Immunology Inhalation anthrax can lead to sepsis and death within days. The fulminate nature of illness reveals minimal elevation of peripheral polymorphonuclear (PMNs) counts at tim e of hospital admissions indicating a profound impairment of the innate immune response. Edema toxin (ET) is one of the major Bacillus anthracis virulence factors and consists of edema factor (EF) and protective antigen (PA). Low concentrations of ET (10 0 -500 ng/ml of PA and EF) significantly impair human PMN chemokinesis, chemotaxis, and the ir ability to polarize. These changes are accompanied by a significant reduction in chemoattractant -stimulated PMN actin assembly. ET also causes a significant decr ease in Listeria monocytogenes intracellular actin based motility within HeLa cells. These defects in actin assembly are accompanied by a >50fold increase in intracellular cyclic AMP and a >4-fold increase in the activation of protein kin ase A (PKA). Our studies have shown that anthrax lethal toxin (LT) also impairs neutrophil actin-based motility, and we now find that LT combined with ET causes an additive inhibition of PMN chemokinesis, polarization, chemotaxis, and FMLP (N -formyl met -leuphe) induced actin assembly. Our studies have shown that anthrax LT blocks the Hsp27 phosphorylation cycle, impairing actin assembly and chemotaxis.

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11 However, the complete molecular pathway leading to ET -induced impairment of actin based motility has not yet been det ermined. Listeria infection of MVd7 cells (lack all members of the VASP protein family) transfected with different VASP constructs, as well as introduction of VASP -TAT recombinant proteins into human neutrophils point to the actin -regulatory protein VASP a s the likely end target of edema toxin, and other cyclic AMP -inducing agents that impair s actin based motility. Phosphorylation of VASP serine -157 impairs actin -based motility, and introduction of a VASP pseudounphosphorylated construct completely neutralizes the ability of ET and forskolin plus IBMX (adenylate cyclase activating compound and cAMP phosphodiesterase inhibitor) to block actin -based motility. These findings reveal the primary pathway by which ET impairs neutrophil motility, and emphasizes th e importance of the cAMP signaling pathway and VASP phosphorylation in the regulation of actin based motility in neutrophils and Listeria

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12 CHAPTER 1 INTRODUCTION Bioterrorism and Bacillus anthracis In October 2001 Bacillus anthracis was used as a bioweapon and this attack has rekindled scientific interest in anthrax pathogenesis (1) Once inhaled, B. anthracis can lead to sepsis, meningitis and death withi n days if not properly diagnosed and treated. Following the bioterrorist attacks analysis of clinical findings revealed patients suffering with systemic anthrax had little to no increase in polymorphonuclear neutrophils (PMNs) in their blood (1) Also, pleural effusions from these patients demonstrated a paucity of white blood cells providing further evidence for poor chemotaxis to the area of infection (2) An understanding of how B. anthracis is able to escape the innate and adaptive immune responses may lead to new therapeutic strategies to improve the host immune response to anthrax. History of Baci llus anthracis Microbiology B. anthracis is a gram positive spore forming rod that is the causative agent of anthrax (3) When grown in culture, B. anthracis forms a distinct boxcar chain. It is nonhemolytic and catalase positive. The thick capsule wall is visible after staining with McFaydeans methylene blue. When cultured on sheep blood agar, the bacteria form characteristic large white ovoid colonies w ith tacky consistency when touched with a loop. Also, B. anthracis is gamma phage-sensitive, distinguishing it from other species of bacilli. Of historical interest anthrax was the first disease for which a m icrobial origin was established (4) Robert Kochs paper in 1876 demonstrated rodlike bacteria in the b lood/tissue of diseased animals, spore formation under starvation conditions, nutrient

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13 induced transformation of the spores back to bacteria, and cult uring of either form would lead to anthrax. Koch called the bacterium B. anthracis which was the name given by Cohn in 1875 (5) The work on anthrax l ed to the formulation of what is commonly referred to as Kochs postulates. Fundamental knowledge of the pathogenesis of anthrax progressed significantly due to the work of Koch and Pasteur. Anthrax is primarily a zoonotic disease that affects herbivores and was believed to be first documented in the B ible as the fifth great plague of Egypt (6) Hippocrates named the disease anthrax (coal in Greek) due to the cutaneous black skin lesions that appear at the site of spore entry (7) Anthrax remained a major killer of livestock until the late 19th century. A striking feature of the disease is the suddenness of clinical pathology. Symptoms usually appear one to two days prior to death. Animals usually look depressed, display signs of cardiac and res piratory failure, mucosa become hemorrhagic, the throat and abdomen become swollen, and blood leaks from the vessels and fails to coagulate (6) B. anthracis spores are highly resistant to killing and remai n viable in soil for many years. The simplici ty for its aerosilization and high mortality rate make it an ideal bioweapon. The CDC classifies it as a category A agent. There are many historical examples of B. anthracis being used in warfare. The Japanese contaminated food and water in the 1930s and 40s (8) The former Soviet Union weaponized B. anthracis during the 1970s in a factory in Sverdlovsk, where in 1979 there was an accidental release of anthrax leading to 68 deaths in surrounding towns (9) Most notable in the US was the attack in 2001 via the US mail that led to 22 cases and 5 deaths (10)

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14 B. anthracis spores are dormant forms of the bacteria. Like seeds, they only germi nate in a fertile environment. If inhaled, larger spores lodge in the upper respiratory tract, where they are less dangerous, but spores between 1 and 5 microns penetrate into the alveoli, tiny sacks in the lung. The LD50 is calculated to be 2 50055, 000 i nhaled spores (10) however recent data suggest s that as few as 13 spores may cause death (11) Once inside the host, the spores are able to germinate and initiate transcription of virulence facto rs to enhance immune evasion and survival within the host. B. anthracis spores are particularly resistant to extreme temperatures, low nutrients and harsh chemical environments. Spores can survive for decades in the environment (6) It is thought that people come into contact with anthrax primarily through inf ected herbivores. The transformation of the spore into a vegetative cell is one of the key steps in the pathogenic cycle of anthrax, enabling bacteria to proliferate and synthesize virulence factors. Alveolar macrophages and dendritic cells are thought t o be primary sites for germination (12 -14) Guinea pigs were used as a model for spore uptake, and it was determined that spores are taken up by lung phagocytes within an hour, within four hours spores were detected in the lymph nodes and within twenty -four hours the bacteria were proliferating in the blood (15) Non -human primates have also been used as a model system for spore uptake and it was determined that it takes six to eighteen hours for the spores to be taken up by lung phagocytes and transported to the lymph nodes (15, 16) Germination is closely followed by the expression of toxin genes. Isolates are extremely uniform in chromosome composition. B. anthracis is phenotypically and

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15 ge notypically similar to B. cereus and B. thuringenesis except for two virulence plasmids px01 and px02. Px01 contains the genes that encode anthrax toxins: lef, pagA and cya (4) Lef encodes for lethal factor (LF), pagA encodes for the protective antigen (PA) and cya encodes for edema factor (EF). The px01 plasmid also encodes the toxin gene transactivator atx a (17) and the plasmid -borne germination genes; ger -xc, ger -a and ger b (18) Migot, et al (19) found that atxa functions as a m aster regulator controlling pxo1 and px02 expression. Atxa is triggered at 37 C under high CO2, which is a condition found in the host Px02 encodes for the poly D -glutamic acid protein capsule. The poly D glutamic acid structure is unique, as glutamic acid used by other bacteria are typically a mixture of L and D isomers (20) The capsule has a negative charge which inhibits macrophages from engulfing and destroying the vegetative cells, impeding the hosts immune response. Thus the capsule allows virulent anthrax bacilli to grow virtually unimpeded in the infected host (21) Little is currently known about the function of pXO2 open reading frames (ORFs) beyond the virulence gene s associated with the capsule ( d ep, capACB and acpA) (4) Although pXO1 and pXO2 are considered major virulence factors, other virul ence factors have been found including: anthrolysin O (22) and cholesterol dependent cytolysin, similar to cytolysins produced by pathogenic gram -positive bacteria such as Listeria and Streptococcus species (23, 24) Anthrolysin O is shown to ly se RBC, neutrophils, macrophages and lymphocytes in vitro in a dose dependent manner (25) and has been shown to induce pro-inflammator y response s as a Toll like receptor (TLR) 4 agonist (26)

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16 Anthrax Infections Anthrax infection can be present in three very distinct forms depending on the site of entry. Symptoms and disease depend on the route of infe ction: cu taneous anthrax results when spores come into contact with an open wound, gastrointestinal anthrax results from the ingestion of contaminated meat or other contaminated products, and inhalation anthrax results from spores being inhaled into the lungs. Generally it is not spread personperson. Anthrax can also be contracted during a lab accident, mishandling of infected animals, and as a bioweapon. Inhalational anthrax is almost 100% fatal if not diagnosed and treated promptly (1) Once spores are inhaled into the lungs, alveolar macrophages phagocytose the spores and carry them to the mediastinal lymph nodes where germination and subsequent rupture of macrophages occurs resulting in the release of vegetative bacteria into the blood stream (7) Initial clinical presentation includes flulike symptoms for several days, followed by sudden respir atory collapse and sepsis, usually occurring with in a 4 5 day period (27) Gastrointestinal anthrax usually occurs when anthrax spores are consumed through ingestion of contaminated meat. Clinical signs are vomit ing blood, severe diarrhea, acute inflammation of the intestinal tract and loss of appetite. If the pathogen invades the bowels it can gain access to the bloodstream allowing for sepsis to occur. Gastrointestinal anthrax can be treated with antibiotics but left untreated 25 -60% of cases will result in death (28) Cutaneous anthrax presents as black boil like lesions on the skin. The lesions are often large, painless necrotic ulcers that usually form at the site of spore entry 25 days

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17 post exposure. Cutaneous anthrax is rarely fatal, but can progress to toxemia and death without proper treatment (28) Anthrax Toxins B. anthracis excretes three exotoxins; protective antigen (PA), edema toxin (ET) and lethal toxin (LT), which account for many of the clinical manifestations PA is responsible for transporting EF and LF into the cytoplasm by a pH dep endent endocytic event As the pH becomes acidic the PA will form a pore allowing the release of toxins into the host cytosol (29, 30) Prote ctive antigen is able to bind two separate receptors, TEM -8 (tumor necrosis factor -8) or CMG 2 (capillary morphogenesis protei n 2) (31, 32) TEM -8 is expressed on tumor endothelial cells and its only known function currently is to bind PA. CMG 2 functions in cellular interactions with laminin and the extracellula r matrix through binding laminin and collagen IV (32) PA is an 83kDa protein that is cleaved by furin (33) present on the c ell surface, to a 63kDa protein that is able to heptamerize and form a pore for the entry of EF and LF into the cell (34) Even though the heptameric PA 63 prepore has radial sevenfol d symmetry, measurements by two independent methods have shown that the heptamer binds only three molecules of ligand under saturating conditions (35) T he toxin -receptor complex is internalized primarily through clathrin-coated pits and is then trafficked to the early endosomes. The low pH conditions of the endosome facilitate the transition of PA 63 from a pre-pore to a pore and the unfolding of LF and EF. LT and ET are then released fr om PA 63 into t he cytosol (Figure 11 ) (36) LT is a zinc -metalloprotease that cleaves the N -terminal seven amino acids of mitogen activated protein kinases 14, 67 (MAPKKs or MEKs) resulting in thei r inactivation (37, 38) MEK1/2 is in the ERK pathway that functions in cellular

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18 proliferation, differentiation and survival. MEK3/6 is in the p38 pathway that functions in environmental stresses, cell growth, differ entiation, cell -cycle arrest and apoptosis. The MEK4/7 is in the JNK pathway that functions in stress stimuli, T -cell differentiation and apoptosis. Inactivation of the MEK pathway allows for LT to affect many different cell types. LT is able to inhibit actin polymerization and thus chemotaxis in the neutrophil (39) and inhibits the pr oliferation and differentiation of monocytes (40) In neutrophils LT not only impairs chemotaxis but also induces neutrophils to release factors that are hemolytic triggering the lysi s of red blood cells (RBCs) (39, 41) LT is able to impair dendritic cells (D Cs) by inhibiting the proinflammatory cytokines and co -stimulatory molecules necessary for stimulating antigen specific T cells (4 2) L T also induces apoptosis of macrophages (40) and inhibits phagocytic production of phospholipase A2 (43) and t he glucocorticoid receptor responsible for inflammation (44) ET functions as an adenylate cyclase that converts adenosine triphosphate (ATP) to cAMP (45) ET requires the uptake of extracellular calcium for generation of optimal cAMP response in neutrophils and macrophages (46) A rise in cAMP in neutrophils has been shown to inhibit phagocytosis of the avirulent Sterne strain of B. a n thracis (47) cAMP is a second messenger, and its purposes include activation of protein kinases and regulating the effects of glucagon and adrenaline (48) By accumulating cAMP within the host cell, ET disr upts bacterialcidal functions of immune effector cells disabling the host defense mechanism. This may help facilitate the survival and replication of B. anthracis within the host. Unlike LT, ET has not been extensively studied, so not much is known about the effects on the host. ET not only has specific activity 1000 fold higher than mammalian adenylate cyclase (AC) but also

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19 is also able to disrupt water homeostasis and other crucial pathways responsible for edema seen during infection (49) ET has been shown to block phagocytosis in neutrophils thereby increasing susceptibility to infection and impairing the innate immune respons e (49, 50) ET has also been shown to increase toxin receptor expression, resulting in increased rates of toxin internalization (51) ET and LT activities, cAMP and MEK cleavage, are likely to act synergistically to enhance pathogenesis. cAMP Signaling cAMP is an ancient and extremely important signaling molecule that is preserved from bacteria to humans (52) In eukaryotes concentrations of cAMP are up regulated in response to activation of adenylate cyclase ( AC ) through G protein coupled receptors (GPCRs) (53) cAMP or 3 -5 -cyclic adenosine monophosphate is a second messenger that is important for many different biological processes. Earl Sutherland discovered cAMP during studies of hormonal regulation of metabolism in mammalian heart and liver samples in 1958 (54) cAMP is derived from ATP and used for intracellular signal transd uction, such as transferring the effects of hormones, which cannot pass through the cell mem brane. cAMP is also used by cells to regulate metabolism, gene expression, cell cycle, cytoskeletal function, and proliferation (Figure 1-2) (53) cAMP signaling involves the synthesis of cAMP by membrane bound AC isoforms and subseque nt diffusion through the cell to points of spatially restricted cAMP effector proteins like protein kinase A (PKA) (55) Hormones or neurotransmitters binding the GPCR leads to the increase in AC, which then catalyzes ATP conversion into cAMP and inorgani c pyrophosphate cAMP is able to form discrete and often minute gradients within cells. Three different downstream effectors sample cAMP gradients: 1) cyclic

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20 nucleotide gated ion channels, 2) EPAC and 3) PKA. Each of these effectors is solely activated by cAMP, and proteinprotein interactions that maint ain spatial resolution are essential for cellular signaling (56) There are several different classes of ACs. Heterogeneity exists between many organisms and cell types demonstrating there are a multitude of cellular processes that are regulated by cAMP. Mammals normally utilize class III AC, a f amily of transmembrane AC (tAC): tAC is directly regulated by heterotrimeric G -coupled proteins and generates cAMP in response to hormones and neurotransmitters, which signal through GPCR (57) The AC has been divided into six different classes based on sequence homology within the catalytic portions. AC s from E. coli and other G ram -negative pathogens are classified as class I. Class II are the toxins. These A C are secreted and translocated into the host and disrupt the intracellular signaling by flooding the cell with supraphysiological levels of cAMP. This class contains ET from B. anthracis ExoY of Pseudomonas aeruginosa, Yersinia pestis and Bordetella pertussis (58) Eukaryotic AC belongs to class III, which are the broadest class. Class IV, V a nd VI are relatively newly identified classes and only contain a handful of prokaryotic members (57) Phosphodiesterases (PDEs) are the only known route of cAMP degradation (59) PDEs control the magnitude and duration of cAMP regulated events (58) If AC is left unchecked it will rapidly increase in a uniform manner, which will have dire consequences for normal cellular function. To this end, cells express def i ned subsets of PDEs from eight different families of cAMP phosphodiesterases (60) The in ability to generate and shape int racellular cAMP gradients depends on PDEs activity to degrade cAMP to 5 -AMP (55)

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21 The concept of cAMP signaling compartmentalization was first introduced in 1981 by Brunton and is thought to allow for various effects on cellular targets (61) This idea came about with the discovery of A -Kinase anchoring proteins (AKAP). AKAP bind directly to the regulatory subunit of PKA to target the kinase to defined cellular locations (62) In creating a signaling network, enzymes are needed to act ivate and hydrolyz e cAMP. AKAPs are uniquely equipped to modulate spatial and temporal PKA signaling (63) PKA An important role of P KA is in the cAMP dependent regulation of the cytoskeleton and cell mov ement PKA is promiscuous with targets found in the membrane, cytoplasm, nucleus and nearly all families of cytoskeletal networks (64) The requirement for tight control and regulation of PKA activity during cell migration is supported by reports of both stimulatory and inhibitory effects of PKA on cell migration (6567) PKA is heterotetrameric enzyme comprising two regulatory (R) subunits and two catalytic subunits (C). Four R subunits are encoded by four separate genes (R1 RI RII and RII ), while thre e genes encode for the C unit ( and ) (68) There are two types of PKA holoenzymes, Type I and Type II, which are de fined by their R subunits. T he R subunit has four functions: 1) maintain holoenzyme in an inactive state, 2) releases an active C subunit upon the cooperative binding of two cAMP per R subunit, 3) the N -terminus mediates dimerization, and 4) interacts wit h AKAPs (63) The C subunit can be down-regulated by phosphorylation (69) Down -regulation of PKA

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22 occurs by a feedback mechani sm: one of th e substrates activated by PKA is a PDE that converts cAMP to 5 AMP (63) Inactive PKA holoenzyme exists as a homodimer made up of two R and two C subunits. In the inactive state, C subunits are bound to R subunits. In the active state the R sub units are released and the C subunits are free to phosphorylate substrates they come into contact with (Figure 1-3). Protein phosphorylation allows eukaryotic cells to translate extracellular signals into a bio-response. AKAPs have been linked not only to PKA compartmentalization but also to the cytoskeleton. AKAP Lbc has been linked with the actin cytoskeleton through its interaction with the Rho GTPases and is able to promote GPCR dependent stress fiber formation (70, 71) AKAP13 has been linked with the actin cytoskeleton by gravin (72) and a close relative SSeCKs (Src -suppressed C -kinase substrate) (73) as well as WASp and verprolin homology protein1 (WAVE1) (74) Members of the functionally related but structurally diverse AKAP family compartmentalize PKA. AKAP typically compartmentalizes RII to various subcellular regions of structures and has been used as an valuable tool for measuring the impact of cAMP/PKA signaling (70, 75) Also, AKAPs direct the assembly of multi enzyme complexes that are capable of integrating multiple input signals to control phosphorylation of a target with great specificity (75) PKA has positive and negative effects on cytoskeletal organization. These effects are not due to a cAMP/ PKA blanket effect but rather to a balance of cAMP/PKA in space and time that is crucial for its effects on the cytoskeleton and cell migration (66, 70)

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23 Actin Structure and Function The actin cytoskeleton is a dynamic structure that plays an important role in many ongoing processes like immune response, homeostasis and blood vessel preservation. Actin is a globular 42kDa protei n with an ATP binding site in the center of the molecule. Monomeric actin, t ermed "G actin" will dimerize or form a trimer, which serves as a site for nucleation and f urther growth of the actin filament. ATP is hydrolyzed immediately after the molecule is incorporated into an actin filament. The ADP is trapped in the actin fi lament until it depolymerizes, t hen an exchange can occur. G actin forms F actin efficiently (the filament) in the presence of ATP, Mg and K Actin behaves like other polymers, above critical concentration of G actin the molecules will polymerize into filaments. Below the critical concentration, actin filaments will depolymerize. Actin filaments are polar structures that have two distinct ends: the plus end (barbed end) and the minus end (pointed end) (76) Monomers can add or leave the barbed end at much faster rates then the p ointed end of the filament (77) Actin polymerization has three distinct stages: nucleation, elongation and steady state. The nucleation stage is the rate limiting step in actin assembly and requires the binding of three actin monomers to initiate actin assembly. The elongation phase is the period of rapid addition of monomers to the filaments. The steady state phase occurs when the addition of a ctin monomers onto filaments is equal to the disassembly of monomers from the filament. The concentration of m onomers left in solution at steady state is called the critical concentration (Figure 14) (76) Actin Binding Proteins There are several classes of actin-binding proteins that are required to control actin assembly. Thymosin 4 is a small protein which binds actin monomers and inhibits

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24 the association of actin with either the plus or minus end of the actin filament and prevents exchange of the bound nucleotides (78) Recruitment of actin monomers depends on another monomer -binding protein called profilin. Profilin binds to the face of t he actin monomer opposite the ATP binding cleft, blocking the side of the monomer that associates with t he minus end of the filament. The profilin actin complex can readily access the free plus end, which causes a change in conformation that reduces actin s affinity for profilin, subsequently profilin falls off leaving the actin filament one subunit longer. Profilins ability to move sequestered actin subunits onto the growing end of the filament is critical for filament assembly at the plasma membrane. B esides binding to actin and phospholipids, profilin binds to various other intracellular proteins that help to localize profilin to sites of rapid actin assembly (79, 80) Capping proteins, such as CapZ and CapG, bind filament ends and prevent further polymerization (81) Once the actin filament is capped it cannot continue to grow nor can it be disassembled. CapG is sensitive to both calcium levels in the cell and PI(4,5)P2 (81) Actin severing and cross linking proteins, such as gelsolin and ADF -cofilin, bind sides of filaments and then s ever the filaments (82, 83) These actin regulatory proteins enhance the depolymerization of filaments. Gelsolin binds preexisting actin filaments and then severs the filaments. This severing is important for remodeling of the actin cytoskeleton by creating new barbed ends for filament growth (84) Nucleation is the rate limiting step in actin assembly and is template driven by the Arp2/3 complex (85) Arp2/3 binds the pointed ends of actin monomers and nucleates actin assembly. Arp2/3 complex can also bind the sides of actin filaments t o create a

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25 Y shape that branches at a 67 angle (85) The Arp2/3 complex is act ivated by small GTPases, Rac, Rho and Cdc42 (86) Rho activation is responsible for formation of stress fibers and focal contacts. Rac activation leads to the formation of lamellipodia while Cdc42 forms filopodia. (Filopodia are long finger -like projections of tightly bundled actin filaments .) Small GTPases bind and activate N -WASP, WASp (87) and ActA components of Listeria monocytogene s (88) which then binds to Arp2/3 allowing for the nucleation of actin filaments. Neutrophil Function Neutrophils are the most abundant type of whit e blood cell (WBC) in mammals and form an essential part of the innate immune response. The main physiological activities of neutrophils are adherence and migration, degranulation and release of inflammatory mediators, phagocytosis and apoptosis (89) Neutrophils arise in the bone marrow from myeloid progenitor cells, which differentiate in response to specific growth factors (90) Neutrophils enter into circulation in an immature band state. Unlike mature neutrophils, band cells possess few cytoplasmic granules and lack the segmented nucleus characterist ic of a mature neutrophil. Mature neutrophils synthesize minimal amounts of protein. Instead, much of what the neutrophil requires for its response is found pre-packaged in the cells cytoplasmic granules and thus is immediately available for cellular functions (90, 91) After release into the blood, the life span of the neutr ophil is limited to five -six hours (92) During a disease process functionally mature neutrophils are released from the bone marrow, due to mature neutrophils exploiting functional attributes The level of circulating neutrophils remains fairly constant in healthy adults, although the levels will

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26 quickly rise during infection. Neutrophils are extremely active and maintain blood levels of 10,000 cells/ m l in a healthy adult (91) Neutrophils respond to factors secreted by bacteria during the host response, as well as to factors generated during activation o f the coagulation cascade, including bioactive lipids released by platelets during clotting. The cell also responds vigorously to host derived substances, which accumulate at sites of previously damaged tissues (93) Microbial factors that initiate the neutrophil to respond are secreted by the invading pathogen or activate chemokines and cytokines of the host s innate immune response, which activates the hosts complement system resulting in the generation of potent neutrophil agonists like C5a. Neutrophils are also able to respond to the synthetic tripeptide, formylated methi onyl -leucyl phenylanine (FMLP), wh ich is derived from bacteria, in much the same way it responds to microbial pathogens. In addition, cytokines such as tumor necrosis factor (TNF) and IL -8 (94) as well as many growth factors, including granulocyte macrophage colony -stimulating factor (GM -CSF) (91, 95, 96) help to fine -tune the neutrophil signaling pathway resulting in enhanced neutrophil response. The mamm alia n TLR family is known to consist of thirteen members and play a crucial role in innate immunity. As microbe -recognition receptors, TLRs mediate cellular responses to a large array of microbial ligands, including LPS, proteins and nucleic acid s. Activ ation of different TLR s leads to various downstream responses that help tailor the immune response to be effective against specific pathogens (97) Human neutrophils express all currently known TLRs. TLR stimulation on neutrophils can result

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27 in the shedding of L-selectin, reduction in chemotaxis increased phagocytosis, priming of superoxide, and the production of a number of chemokines and cytokines (91) Chemokine s are leukocyte attractants divided into two major groups based upon the positions of the first two cysteine residues in their primary sequence the C X-C and the C -C (93) RANTES (acronym for regulated upon activation of normal T cell expressed and secreted), macrophage i nflammatory protein-1 (MIP 1 ), MIP -1 and MIP 1 are members of the CC chemokine subfamily produced by neutrophils. By contrast the CXC subfamily exerts effects towards neutrophils (91) Cytokines and chemokines can be produce by neutrophils in three ways. Cytokines are stored in granu les until stimulated by inflammatory mediators like IL-8 causing the neutrophil to degranulate and release cytokines (98) Neutrophils also have the ability to cleave surfacebound inactive cytokines from their plasma membrane (99) Human neutrophils have the capacity to produce a number of CXC chemokines, including IL-8, growthregulated oncogene alpha (GRO ), IFN and cytokine induced neutrophil chemoattractant (CINC) (91) However, when compared to macrophages, neutrophils are responsible for less than 2% of the total cytokine array (100) The containment and killing of bacteria are the major functions of neutrophils in host defense. After penetrating the body, factors rel eased by the pathogen activate complement or other inflammatory cells which attract neutrophils to the site of invasion. After the pathogen attaches to the neutrophil surface recept or, they are rapidly sequestered into p hagolysosomes and then killed through t he release of granules and from the production of oxygen radicals. Neutrophils undergo degranulation to release proteases and reactive oxygen species into the surrounding tissue to kill invading

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28 pathogens. N eutrophil possess at least three types of granules: primary granules cont aining the enzyme myeloperoxidase, secondary granules contain ing hydrolytic enzymes and other antibacterial components, and less dense tert iary granules which fuse with the plasma membrane and alter the cytoskeletal architecture and location of the oxidative generating NADPH oxidase by translocating components to the plasma membrane (101) It is important that once the infection is cleared that neutrophils are removed, an d there is a fine line that separates beneficial from detrimental tissue damage or inflammation. The primary role of the neutrophil is to phagocytose and kill the pathogen that has invaded the host, limiting the infection. To accomplish this task the neutrophil has the ability to undergo dynamic actin remodeling to allow for chemotaxis to the site of infection (102) Neutrophils are among the first cells to arrive at the site of infection and are important contributors to the acute inflammatory response (103, 104) The neutrophil can sense as small as a 5 -10% difference in the concentration of a ch emoattractant. As the neutrophil rolls along the blood vessel wall, the L -selectin on its surface binds to carbohydrate structures such as sialyl -Lewis found on the adhesion molecules of the vas cular endothelium, and rolling is eventually halted. As th e neutrophil becomes activated, it replaces L-selectin with other cell -surface adhesion molecules, such as integrins; t hese molecules bind E -selectin, expressed on the surface of the vascular endothelium in response to inflammatory mediators such as bacterial lipopolysaccharides (LPS) a nd the cytokines IL1 and TNF The activated neutrophil then enters the tissues through diapedisis where it is attracted to the infection site by a

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29 number of chemoattractants. The neutr ophil can then phagocy tose and kill the pathogen (Figure 15) (105) Our lab has previously shown that both LT and ET impair the ability of neutrophils to assemble and disassemble actin filaments (39, 106) Unstimulated neutrophils contain 6070% of total actin in monomeric form (~200 M) (10 7) It is therefore important for the cell to synthesize proteins that directly regulate actin monomer function. Neutrophils are the first line of defense during bacter ial infections and are able to quickly crawl, or chemotax, to the site of infection, and defects in the ability of PMNs to chemotax severely compromises the innate immune response (108, 109) Neutrophils migrate by membrane protrusions at the leading edge of the cell, which are formed by the assembly of branched actin filaments and the continued polymerization of actin allows for the physical movement of the cell (110) The mechanism (s) coordinating these even ts are poorly understood, however, it is well known that cAMP, via activation of PKA, can inhibit neutrophil migration (111113) Neutrophil Chemotaxis Cell motility is a fundamental cellular process that is essential to many biological processes such as embryonic development, immune system function and wound healing (63) For cells to move they must be able to sustain shape and interact with the surrounding environment. As a cell moves on a substrate (the extracellular matrix if the cell moves inside an organism or a cover slide if it moves outside an organism), it experiences external forces, which include the viscous force or resistance from the surrounding medium and cell -substrate interaction forces, and internal forces that are generated by the cytoskeleton. In most animal cells, the cytoskeleton is the essential

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30 component in creating these motility -driving forces All spatial and mechanical functions are performed by a system of filaments called the cytoskeleton, particularly the protein actin. The cytoskeleton provides support to the cell but must also undergo dynamic remodeling to facilitate cell movement (114) The neutrophil actin cytoskeleton is a highly dynamic structure that rapidly adapts to the cell s demands in order to successfully utilize two of its most important features: motility and phagocytosis. Directional F actin poly merization in the lamellipodia of a migrating human neutrophil is signaled by GPCRs It is known that the ligation of chemoattractant receptors tri ggers the release of G (115) Downstream fr om the GPCRs, is phosphatidylinositol 3-kinase (PI3K ) which regulates F actin polymerization (116) A ctivation the Rho family GTPases has also been shown to be instru mental in orchestrating membrane protrusion and retraction in neutrophils (115) The Rho family GTPases have been shown to activate the nucleation promoting factor WAVE/Scar, one activator of the actin -related protein 2/3 (Arp2/3) and to promote the formation of free barbed ends (117) that in turn initiates cytoskeletal actin polyme rization in the lamellipodia (118) The most prominent Rho GTPases in neutrophils are of the Rac subfamily, Cdc42 and Rac (119) which are capable of promoting actin polymerization. After chemoattractant GPCR stimulation, G activation leads to the accumulation of lipid products of PI3K, such as phosphatidlyinositol -3,4,5triphosphate (PIP3), at the leading edge and the activat ion of Cdc42 and Rac, which promotes actin polymerization (120, 121) The sma ll GTPases are thought to activate N WASP (Wiscott -Aldrich syndrome protein), which results in Arp2/3 activation (122, 123) Arp2/3 nucleates new actin assembly (Figure 16). Unfortunately, most reviews

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31 on neutrop hil actin based motility focus on G -proteins and phosphoinositides as key signaling molecules in actin assembly and do not review other potentially important molecules. Listeria monocytogen e s L. monocytogen e s is used extensively as a model system to identify key proteins involved in c ell motility. L. monocytogene s is a G ram -positive intracellular bacterium that invades mammalian epithelial cells and propagates in the intestinal, feto-placental and blood brain barriers (124) Listeria is an intracellular pathogen, thus it avoi ds host defenses, immune response, and complex signaling (125) During intracellular growth Listeria polymerizes the host actin at one pole of the bacteria, allowing the bacteria to move through the cytoplasm via actinbased motility (126) When Listeria encoun ters the plasma membrane it form s a membrane protrusion (filopodia) from the infected cell into an adjacent cell leading to the formation of a double membrane vacuole in the uninfected cell (127) Bacteria escape the double membrane vacuole and resume intracytosolic growth within the newly infected cell (128) Listeria actin based motility relies on ActA (126) a bacterial factor that mimics the nucleation promoting activity of WASp/WAVE family members (129) and by recruiting the Arp2/3 complex to the surface of the bacteria (130) WASp is activated by small GTPases Rac and Cdc42 (131) Listeria uses internalin A (InlA), a cell wall covalently anchored protein, to bind to the cellular adhesion molecule E -cadherin and induce bacterial entry into polarized epithelial cells (132) Listeria also utilizes internalin B (InlB), which int eracts with gC1qR and hepatocyte growth factor receptor Met to allow for entry into the polarized epithelial cell (133) Listeria hijacks the molecular machinery associated with the tail portion of E -

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32 cadherin (132) and -catenins are required to link the adherence junction (E cadherin) to the underlying actin cytoskeleton (134) Listeria recruits these catenins to the entry site for actin remodeling to allow the bacterium to enter into the cell. After internalization, Listeria lyse the membrane compartm ent through bacteria phospholipases A and B as well as listerolysin O (LLO) (135) In the host cytoplasm bacteria begin to multiply and use the cellular actin machinery of the host. Bacteria are initially coated with an actin cloud, consisti ng of new actin filaments that will rearrange themselves to form a comet shaped tail at the bacterial pole (136) Nucleation and elongation of actin filaments will provide the necessary propulsion to force the bacteria through the cell cytoplasm (137) The characteristic actin rocket tail at one pole of the bacteria is formed by actin polymerization, which is initiated by a protein complex containing asymmetrical distributed bacterial surface protein ActA and several host proteins like VASP and Arp2/3 complex. ActA is necessary for Listeria motility (130) The central region of ActA has four proline rich repeats of FPPPP or FPPPIP motifs (138, 139) These proline rich ar eas mimic the host cytoskeleton proteins vinculin and zyxin, which are associated with focal adhesions and stress fibers (138) The proline rich sequence in these proteins and ActA bind Ena/VASP family of proteins (139) En a/VASP proteins localize to actin stress fibers, filopodia tips, the leading edge of cells, and are implicated in the actinbased processes of cell migration and axonal guidance (140) Ena/VASP is recruited to Listeria actin comet tails and increases bacterial speed and directional per sistence (141, 142)

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33 In extracts, Listeria actin based motility requires very few host proteins to generate movement. Arp2/3 is required to nucleate actin assembly, while actin -depolymerizing facto r (ADF) helps recycle monomers from filaments, and CapZ blocks the barbed ends of filaments (125) Listeria motility is enhanced by actin regulatory proteins such as profilin, VASP and the actin bundling protein actinin (141) VASP Ena/VASP proteins are a structurally conserved family found in vertebrates, invertebrates and Dictyostelium discoideum cells (143) VASP was originally discovered and initially characterized as a substrate for c AMP and cGMP dependent protein kinases in human platelets (144146) VASP is found not only in platelets but also in many other cell types, including: leukocytes, T cells, smooth muscle, fibroblasts and cardiomyocytes (144, 147-149) Evidence for the role of Ena/VASP proteins in motility comes from the analysis of VASP deletion/overexpression on the motile behavior of living cells (140, 143, 150) and on actinbased motility of Listeria (151 -153) VASP is a 46 kDa membrane associated protein that is a member of the Enabled (Ena) family of proteins (111, 154) Two other mammalian family m embers, Mena (mammalian enabled) and EVL (Ena -VASP like) were identified by sequence similarity (155) VASP has a tripartite domain containing an N ter minal Ena/VASP homology 1 (EVH1) domain, a central proline-rich domain (PRR), and an EVH2 domain at the C terminus (Figure 1 -8) (156, 157) The EVH1 domain aids in the binding of Ena/VASP to proline rich ligands like vinculin and zyxin (158) The PRR domain contains binding sites for the actin monomer binding protein profilin (159) Src homology 3 (SH3) and WW domain con taining proteins (160) suggesting that VASP is a key element in

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34 cytoskeletal r eorganization. The EVH2 domain contains F and G actin binding sites and a coiled -coiled motif impor tant fo r tetramerization (161) VASP promotes actin filament elongation by delivering profilin G actin complexes to the barbed end of a growing filament VASP harbors three serine/threonine phosphorylation sites. Serine 157 (S157) is located N -terminally of the central PRR. Serine 239 (S239) and threonine 278 (T278) are within the EVH2 domain, adjacent to the G and F actin b inding sites (156) The equivalents of VASP S157 and S239 but not T278 are found in Mena, whereas S157 is the only site found in EVL (Figure 1-8) (155) In vivo S157 is preferentially phosphorylated by PKA whereas S239 and T278 are targeted by cGMP dep endent protein kinase (PKG) or AMP activated protein kinase (AMPK) (162) Phosphorylation of VASP at S157 is thought to regulate its affinity for F actin (163, 164) and play an important role in F actin assembly at the lamellipodia, filopodia and focal adhesions (165) Phosphorylation affects the interaction of VASP with actin; however, the crucial phosphorylation site(s) involved remain unclear. Several key factors support Ena/VASP proteins as important regulators in the actin cytoskeleton. They associate with the surface of motile Listeria in an asymmetric manner (155) and are necessary for efficient Listeria motility (139, 141, 151, 152) Ena/VASP proteins localize to area s of actin remodeling such as the front of spreading lamellipodia in motile cells (166) tips of growth cone filopodia, focal adhesions and cell junctions (155, 166 -168) Despite the relevance of Ena/VASP proteins to dynamic actin based processes their exact molecular functions remain elusive. Filopodia Two alternative forms of actin machinery coexist at the leading edge of most motile cells: lamellipodia which seem to be designed for persistent protrusion over a

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35 surface, and filopodia which appear to perform sensory and exploratory functions to steer cel ls depending on cues from the environment (169) Lamellipodia have been investigated since the 1970s, however, filopodia have only recently emerged as imp ortant structures in wound healing, immune cell function and cell invasion (170) Actin based filopodia protrusions, first described by Porter, Claude and Fullam as early as 1945 (171) are composed of tightly bundled parallel filaments 5-50 m long and 0.10.5 m thick (172) Despite the importance of filopodia in wound healing, metastasis, neuronal growth cone formation and embryonic development, the exact mechanism governing their initiation and formation has yet to be fully elucidated (173176) Generally, filopodia are described as antennae or tentacles that cells use to probe their microenvironment, serving as pioneers during protrusion. However, the roles of filopodia seem diverse and, in many c ases remain vague. Small GTPases of the Rho superfamily are linked to the regulation of cell morphology and the actin cytoskeleton. The best -studied mammalian Rho GTPases are Rac1, Cdc42 and RhoA. RhoA is implicated in the formation of st ress fibers and focal adhesions, while Rac1 promotes lamellipodium formation and Cdc42 functions in the formation of filopodia (177) A large number of proteins that regulate the actin cytoskeleton have been shown to localize to filopodia and/or to regulate filopodia formation. Although many of these pr oteins are specific to only certain cell types, key sets of proteins seem to contribute to the generation of filopodia in diverse cell types and organisms (Table 11). There are several model systems for filopodia formation ; however, each model has a tip complex of proteins thought to mediate: lateral interactions between barbed

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36 ends, bundling and filopod extensions (178) It is important to note that some controversy still exists concerning the activities of individual proteins, and is likely that the relative importance of each in f ilopodia varies depending on the organism and cell type. A subset of uncapped actin filaments of the Arp2/3 network are targeted for continued elongation by formins and/or by ENA/VASP proteins (179) The barbed ends of these elongating ac tin filaments converge together through the motor activity of myosin X, leadi ng to the initiation of filopodia (180) The forces resulting from polymerization of tightly packed filaments are thought to result in the extension of the plasma membrane, and insulin-receptors substrate p53 (IRSp53) or inverse (I) -BAR domain containing proteins might further facilitate plasma membrane protrusion by directly deforming the membrane Alternatively, IRSp53 might sense the negative membrane curvature that is induced by pushing forces of elongation filaments and recruit other components to the site of filopodia initiation (181) Ena/VASP proteins can also function as initial F actin cross-linking proteins in the tip of the elongat ing filopodia. The incorporation of the actin cross -linking protein fascin in the shaft of the filopodia generates a stiff actin filament bundle (182) However, the exact role of Ena/VA SP during filopodia initiation and elongation still needs to be characterized along with understanding the function of myosinX and its cargo binding domain in the tips of filopodia (180) A full understanding will only come with a complete inventory of all the proteins and their specific functions within the dynamics of filopodia initiation and elongation (178) ET Inhibits Neutrophil Motility by VASP Pho sphorylation of S157 The downstream effects of ET activity have not been extensively studied and it is only recently that we have begun to understand how ET impairs the hosts innate

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37 immune response. Other agents that elevate cAMP within the neutrophil r esult in the inhi bition of a number of functions such as: phagocytosis, migration, spreading, oxidative burst and bacterial killing (183) VASP localizes to regions of dynamic actin remodeling and promotes filament formation (155, 166, 184) Phosphorylation affects the interaction of VASP with actin; however, the crucial role of phosphorylation in the regulation of actin filaments remains to be clarified (163, 164, 185) Listeria infection of MVd7 cells (lack all membe rs of the VASP protein family) transfected with different VASP constructs, as well as introduction of VASP-TAT recombinant proteins into human neutrophils point to the actin-regulatory protein VASP as the likely end target of edema toxin and other cyclic AMP inducing agents that impair actin-based motility. Phosphorylation of VASP serine 157 impairs actin based motility, and introduction of a VASP pseudo-unphosphorylated construct completely neutralizes the ability of ET and forskolin plus IBMX (adenylate cyclase activating compound and cAMP phosphodiesterase inhibitor) to block actin-based motility. These findings reveal the primary pathway by which ET impairs neutrophil motility, and emphasizes the importance of the cAMP signaling pathway and VASP phosphorylation in the regulation of actin -based motility.

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38 Table 1 1. Key proteins involved in filopodia formation Protein Proposed activities and functions Cdc42 Small GTPase that induces filopodia formation RIF Small GTPase that promotes the formation of long filopodia through the forming protein Dia2 Arp2/3 Complex Actin filament nucleator that generates the formation of a branched lamellipodial F actin network WASP/WAVE Proteins that activate the F actin nucleation activity of the Arp2/3 downstream of Rho family GTPases Dia2 Protein that induces the formation of unbranched actin filaments in filopodia Ena/VASP Factors that promote actin filament elongation, anti branching and/or bundling to induce filopodia formation Myosin X Motor prot ein that promotes filopodia formation by converging filament barbed ends together and by transporting proteins to filopodial tips Fascin Major F actin cross linking protein of filopodia IRSp53 Scaffolding protein that also deforms membranes to promote th e formation of plasma membrane protrusions LPR1 Lipid phosphatase related protein that induces filopodia formation through a currently unidentified mechanism This table has been modified from Faix et al. (182)

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39 Figure 11. Anthrax toxins. The tox ins produced by Bacillus anthracis are derived from three proteins : edema factor (E F), lethal factor (LF), and protective antigen (PA). The entry of toxin into cells begins wit h the reco gnition of cellular receptor s in the plasma membrane by PA. Proteolytic cleavage of cell -bound PA creates a smaller fragment that then multimerizes into a pore like structure in the plasma membrane. The LF and EF proteins bind to the PA pre pore, followed by internalization of the entire structure through receptor mediated endocytosis. In the endosomal compartment, the acidic pH causes a conformational change that inserts PA fragments and releases LF and EF into the cytoplasm. In the cytoplasm, LF acts as a p rotease that cleaves MAP kinase, inhibiting pathways that rely on this kinase family and causing cell death. EF is an adenylate cyclase th at inhibits the immune response and increases swelling and edema due to increased cAMP levels (186). Reproduced with permission of BioCarta and author Glenn Croston.

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40 Figure 12. cAMP signaling pathway An amine receptor is activated by binding to a specific ligand. The ligand -bound receptor then activates a stimulatory G protein (Gs), which leads to an increase in th e enzymatic activity of adenylate cyclase (AC). Adenylate cyclase catalyzes the conversion of ATP to cAMP. As the intracellular concentration of cAMP increases, cAM Pdependent PKA is activated and phosphorylates different target proteins on ser ine and threonine residues. Phosphodiesterases (PDEs) break down cAM P into 5 AMP (63)

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41 Figure 13. PKA signaling pathway. A) PKA signaling is a widely used intracellular pathway and is the major route for channeling the second messenger cAMP signal. cAMP binding to the PKA complex leads to dissociation of the catalytic subunits from the holoenzyme. PKA cross -talks or integrates with other signaling pathways. The PKA signaling pathway has been implicated in many cellular and physiological processes, from metabolism and reproduction, to cardiac function, memory and learning. B) Each regulatory subunit has two distinct sites for binding cAMP (called A and B), located in different domains. cAMP binding to B induces conformational change, unmasking A allowing for cAMP to bind to A releasing the catalytic subunit (63)

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42 Figure 14. Actin structure. All of the diverse actin structures found in cells are assembled from the same basic building blocks (globular actin monomers) into filaments, which are then organized by actinassociated proteins into specialized arrays with distinct arch i tectures and dynamic properties Reproduced with permission of Dr. Bruce Goode from his website: bio.brandeis.edu.

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43 Figure 15. Neutrophil function. Neutrophil delivery in the postcapillary venule. ICAM, intercellular adhesion molecule. 1) Neutrophils are found rolling through the vascular endothelium; 2) neutrophils begin to slow down by the use of Lselectin; 3) neutrophils adhere to the endothelium; 4) neutrophils diapedisis through the vascular endothelium; 5) neutrophils chemotax to the site of infection; and 6) neutrophils undergo phagocytosis of the bacteria/pathogen. Reproduced with permission from Critical Care press and authors.

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44 Figure 16. Neutrophil actinbased motility. A polarized, migrating neutrophil requires rapid assembly and disassembly of actin filaments, which is controlled by the heterotrimeric GPCR pathway. The G subunit activates small GTPases such as Rac, Rho, and Cdc42. The small GTPases are thought to activate N WASP, which results in Arp2/3 activation. Arp2/3 can nucleate new actin assembly (122, 187)

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45 Figure 18. VASP structure. (A) Domain organization of Ena/VASP proteins. EVH1: Ena/VASP Homology 1, PRR: proline-rich region, LERER: region harboring LERER repeats, Q -rich: glutamine-rich region, EVH2: Ena/VASP Homology 2. Serine and Threonine phosphoryl ation sites are indicated as orange stars (B) Major binding partners for the EVH1, EVH2 and PRR regions of Ena/VASP proteins (157)

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46 CHAPTER 2 LITERATURE REVIEW ON NEUTROPHILS AND ANTHRAX Anthrax Clinical Data Inhalation anthrax can lead to sepsis and death within days if not diagnosed early and treated effec tively (27) Epidemiological analyses of the anthrax bioterrorist attacks in 2001 indicated a mean duration of 4.5 days between exposure and symptom onset in the 6inhalation anthrax cases for whom the exposure dates could be determined. Analyses of the clinical findings from 10 of the 11 -inhalation anthrax cases revealed normal or minimally elevated peripheral PMN counts at the time of hospital admission, despite high level B. anthracis bacteremia (1) Furthermore, heavily infected pleural fluid demonstrated a paucity of white blood cells. In the fatal cases, mediastinal infection was associated with marked edema and hemorrhage, but minimal infiltration by acute inflammatory cells (2) Similarly, experimental ly induced inhalation anthrax in monkeys was associated with edema and hemorrhage o f the mediastinum and pulmonary interstitium with absent or modest infiltration by neutrophils (188) These findings suggest an impaired delivery of neutrophils to the sites of infection during the early stages of systemic B. anthracis infect ion. Neutrophils constitute the first line of defense against bacterial infections. These phagocytic cel ls are able to quickly chemotax to the site of infection, and defects in neutrophil chemotaxis compromise the innate immune response. Chemotaxis is a ccompanied by shape changes that are mediated by rapid assembly and disassembly of actin filaments (189) Actin polymerization is dependent on elongation of existing filaments catalyzed through uncapping and/or severing of existing filaments and catalysis of their elongation through members of the Ena/VASP and formin family of proteins.

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47 This process of elongation is followed by the appearance of new branch es through activation of the Arp2/3 complex. Stimulation of Arp2/3 may be controlled through Rac dependent acti vation of the Scar/WAVE and WASp complexes (190) B. anthracis acts to limit chemotaxis by targeting any of the aforementioned mechanisms by which the neutrophil creates new actin. Ther e is substantial evidence to support the possibility of a defect in neutrophil chemotaxis as evidenced by the autopsy results and hospital admissions finding from the inhalation anthrax cases during the bioterrorist anthrax scare of 2001 (1) Previous Studies with ET and Neutrophils The effects of ET on neutrophil chemotaxis has not been visited in nearly twenty years (191) Little is known about the effects of ET on cel l motility. Only recently have investigators been able to express ET in E. coli and have shown that this purified recombinant protein has binding affinity and biological activity comparable to those of the purified toxins from B. anthracis (192, 193) Twenty years ago the target of ET, an adenylate cyclase, was determined to be increased intracellular cAMP levels (194) A few years later it was determined that both ET and LT increased PMN chemotaxis. However, this previous study was performed without being able to determine the activity of the toxins and the researchers did not detect a significant increase in intracellular cAMP levels (191) During et al. (39) revisited effects of LT on neutrophil chemotaxis and fo und that purified toxin inhibited neutrophil chemotaxis and this effect required two hours to achieve maximal inhibition. When taken together, the clinica l and research data c all for a re evaluation of effects of ET on neutrophils. Only recently have researchers found that both LT and ET exhibit distinct inhibitory effects on FMLP and C5a receptor -mediated superoxide production (49) Anthrax

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48 toxins are able to effect ively suppress human neutrophil -mediated innate immunity by inhibiting their ability to generate superoxides for bacterial killing (49) During cases of anthrax -related meningitis it has been found that ET suppresses the blood brain barrier s recruitment of neutrophils due to a downregulati on in neutrophil chemoattractants IL -8, CXCL -1 and CXCL -2 (195) I have chosen to study the effects of anthrax ET and ET+LT on neutrophils actin -based motility, as well as determine the exact mechanism of ETs action on actin assembly.

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49 CHAPTER 3 MATERIALS AND METHODS Toxin Purification Edema factor (EF) was expressed and purified from E. coli as previously described (generous gift from Dr. Wei -Jen Tang, University of Chicago) (193) Briefly, for highlevel gene expression, pPN -EF was transformed in E. coli SG13009 (pREP4) competent cells. Cultures were induced for four hours with IPTG and subsequent analysis confirmed EF was mainly localized to the cytosol. EF was purified from the cytosol with a 50% Ni -nitrilotriacetic acid resin. Affinity chromatography resulted in 456fold purification o f EF. EF was further purified by Sepharose cationexchange column, concentrated down and stored at -80 C in aliquots (192) Protective antigen (PA) and lethal factor (LF) were purified from B. anthracis as previously described (generous gift from Dr. Co nrad Quinn, CDC) (196) Briefly, culture media w ere filtered through a 0.22 M filter, followed by diethylaminoethyl cellulose (DEAE) anion exchange chromatography. The resulting toxin components were then subjected to gel filtration and hydrophobic interaction fast protein liquid chromatography (FPLC) as previously described. Cultures of 15 liters were found to generally yield 8 mg of PA, 13 mg of LF, and 8 mg of EF with purity assessed by coomassie blue staining to be 90% (196) Chemicals Used Positive controls for cAMP are Forskolin (Fsk, Sigma-Aldrich), 3isobutyl -1 methylaxthine (IBMX, Sigma -Al drich) and 6db-cAMP (Biolog, Life Sciences). Three different MEK inhibitors were used: p38 inhibitor is SB203580 (Cal biochem), Erk1/2 inhibitor is PD98059 (Calbio chem), and the JNK inhibitor is SP600125 (Sigma). Antibodies used: ant i -MEK antibody (Cell Signaling) P S157 VASP (Cell Signaling) and

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50 anti -VASP (BD Bioscience). Neutrophil Isolation and Toxin Treatment Human neutrophils were purified using a Ficoll -Hypaque gradient media (ICN Biomedical, Irvine, CA). The study followed US Department of Health and Human Services guidelines and was approved by the Institutional Review Board at the University of Florida. Healthy volunteer donors (total of 7 subjects) ranged in age from 2458 years, and included both males and females of Caucasian and Asian desce nt. Purified neutrophils were resuspended in RPMI with Lglutami ne (Mediatech), and adjusted to 1 X 106 cells/ml. Neutrophils were treated with varying concentrations of EF + PA, LF + PA or PA + LF + EF for two hours at 37C while gently rotating the cell s to prevent clumping or cell activation. For experiments with ET and LT, a 1:1 mass ratio of PA to EF or LF was used. We found that a weight ratio PA to EF to 2:1 had identical effects to a 1:1 ratio. For experiments combining all three toxins, the PA co ncentration was increased to 1 g/ml to assure sufficient binding sites for both EF and LF. For the majority of experiments control cells were incubated with buffer alone. To assure the specificity of our findings for each experimental condition cells were incubated with EF, LF and PA alone. Neutrophils were studied immediately after the two hours of incubation and experiments were completed within four to five hours of blood drawing. Cell Culture HeLa cells were grown in Dulbeccos modified eagles media (DMEM) with 4.5 g/L glucose supplemented with 10% fetal bovine serum (FBS) and 5% penic illin and streptomycin (Cellgro.) Cells were grown to 70-80% confluence. Cells were used at passage 5 -20. MVd7 (rat fibroblast cell -line devoid of Ena/Mena/VASP) MVd 7 egfpVASP, and MVd7 egfp VASP S157A stablely transfected cell lines were grown in

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51 Immorto media (DME with 15% FBS, 5% penicillin and streptomycin, 5% L-Glutamine and 50 U/ml of mouse interferon gamma {Gibco}). MVd7 cells were transiently transfected wit h Lipofectamine 2000 (Invitrogen) and analyzed 2436 hours later. Annexin V Staining, Analysis for Necrosis and Nitroblue Tetrazolium Assay Annexin V staining was performed on neutrophils using the annexin V -Fluos Staining Kit (Roche) combined with propidi um iodide to assess necrosis and 10,000 cells were analyzed by FACS as previously described (39) To further assess necrosis in separate experiments cells were mixed in a one to one ratio with 0.4% Trypan Blue (Sigma). Samples were then loaded onto a hemacytometer, allowed to sit for two minutes, and the intracellular content of tryp an blue was determined by light microscopy. Two hundred cells were analyzed for each condition. The NBT test was performed before and after stimulation with a final concentration of 200 ng/mL of phorbol myristate acetate (Sigma), in accordance with the manufacturers protocol. One hundred cells were analyzed for each condition. Neutrophil Chem o kinesis, Chemotaxis and Polarity Neutrophil chemokinesis, chemotaxis and polarization were performed as previously described (39) Briefly, untreated ET, LT and ET+LT treated PMNs (1X105 cells in 2ml of RPMI) were added to a 35mm glass bottom mic rowell dish (Matek) coated in 0.1% fibronectin (Sigma). For chemokinesis experiments 1 M FMLP (N formyl met leuphe) was added to each plate five minutes prior to time-lapse phase contrast video imaging. Images were captured at 10s intervals using an inv erted Zeiss microscope and a cooled charge-coupled device camera (model C5985; Hamamatsu) The v elocity of PMNs was determined using the Metamorph software track image program (Universal Imaging). The percent of polarized PMNs (a dist inct lamellipod and

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52 u ropod) was assessed 15 minutes after the addition of FMLP. Chemotaxis toward a gradient was assessed using a FemtoJet needle (0.5um tip, Eppendorf) containing a conce ntration of 10 M FMLP. The tip of the needle was placed just inside the visual field a nd chemoattractant was infused into buffer solution at a pressure of 15 psi using an Eppendorf micromanipulator. The velocity of movement toward the needle was measured by time-lapse video microscopy at 10s intervals. In addition to anthrax toxins, in sel ected experiments cells were treated with 10 M of Forskolin (Sigma Aldrich) and 100 M 3 -isobutyl -1 -methylaxthine (IBMX, Sigma -Aldrich) for 15 minutes at 37 C. Whole Cell cAMP Levels c AMP levels in human neutrophils and HeLa cells were determined using an enzyme -linked immunoassay (Amersham Biosciences) as previously described (49) Briefly cAMP levels in human neutrophils were determined using the cAMP Biotrak Enzymeimmunoassay System (Amersham Biosciences). Human neutrophils were suspended at 1 x 10 ^ 6 cells/ml in DMEM and were either left untreated or were treated with the indicated amounts of toxins for various time points at 37C. Positive controls were treated with forskolin (10 M, final) plus 3-isobutyl -1 -methylxanthine (IBMX) (100 M, final) for 15 min at 37C. Neutrophils (1 x 10 ^ 6) from each group were assayed in duplicate on 96well tissue culture plates and lysed using the supplied lysis buffer. Supernatants were then collected and cAMP concentrations were determined according to the manufacturers instructions. PMN Phalloidin and CD11/CD18 Staining and Fluorescence Activated Cell Sorting (FACS) Analysis Phalloidin staining was performed as previously described (39) Briefly, after

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53 incubation for two hours at 37 C with PA + EF (ET), PA + LF (LT) or both toxins (PA + LF + EF) neutrophils were exposed to 1 M FMLP (Sigma -Aldrich) for 0, 5, 10, 15, 30, 60, and 120 seconds. Cells were fixed at these time points with a final concentration of 3.7% formaldehyde followed by pe rmeabilization with 0.2% Triton and staining with Alexa 488 phalloidin stain for 30 minutes (InvitrogenMolecular Probes). In separ ate experiments live PMN were stained using CD11/CD18 primary antibody (Abcam) at 2.5 g/ml followed by secondary antibody mouse IgG H&L conjugated with HRP at a 1:100 dilution in PBS (Invitrogen). Immediately following staining, cells were subjected to F ACS analysis. Measurement of Phosphorylated PKA PKA phosphorylation was determined using the PKA Assay Kit (Upstate Cell Signaling Solutions) with [ -32,P] ATP (PerkinElmer.) Neutrophils were treated with 500 ng/ml of ET and incubated at 37 C for 2h or tr eated with the positive control 6 dbcAMP at 100 M for 1h (Biolog, Life Science Institute.) Cells were lysed using non denaturing lysis buffer (1% Triton X -100, 50 mM TrisCl, 150 mM KCl, 50 mM EDTA, 0.2% NaA 200 mM imidazole, 100 mM NaFl, 100 mM Na3VO4) containing a complete mini protease inhibitor cocktail tablet (Roche). As previously described (197) phosphorylation of PKA was determined using 25 g of radiolabeled protein lysate by measuring the transfer of radioactive phosphate from ATP into Kemptide. Listeria monocytogen e s and Shigella flexerni Infection and Phalloidin Staining Listeria monocytogenes infection and phalloidin staining were performed as previously described (88) Listeria motility and tail lengths were determined four to six hours after the initiation of Listeria infection, as pre viously described (198) Listeria

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54 infections were allowed to proceed for 2 hours before the addition of various concentrations of ET or LT from 50ng/ml to 500ng/ml or Fsk/IBMX (10 M/100 M). Listeria 10403S (1 X 10^6 cells/ml) were used to infect 1 X 10^5 HeLa cells, followed by the addition of 50 g/ml gentamicin sulfate 60 min after infection to prevent extracellular growth. Listeria motili ty was observed three to four hours after infection by using phase microscopy as described above. Velocity measurements were determined using the Axiovert imaging software. Shigella flexerni infection was performed as pr eviously described (199) and velocities were determined two and a half hours after infection. Briefly, Shigella (1 X 10^6) were used to infect 1 X 10 ^5 HeLa cells, followed by the addition of 50 g/ml gentamicin sulfate 60 min after infection to prevent extracellular growth. Shigella motility was observed 1.5 to two hours after infection using phase microscopy as described above. Velocity measurement s were determined using the Axiovert imaging software. Neutrophils were fixed in 3.7% formaldehyde, permeabilized with 0.4% Triton, and incubated with Rhodamine -Phalloidin (Molecular Probes -Invitrogen). After treatment with anti -fading agent Fluoromont -G (Southern Biotech), the signal was visualized by Zeiss Confocal imaging system with a 63X oil -immersion lens (Zeiss). Plasmid and Protein Purification VASP point mutations were generated by site-directed mutagenesis using the QuikChange Multi Kit (Stratag ene). Human VASP in the pEGFP -C1 vector (Clonetech) and primers (see Table 1) were used to exchange S157 and S239 with alanines (S157A, S239A) or acidic amino acids (S157D, S239D). Human VASP in the pQE 30 plasmid (kind gift of Dr. Dorothy Schafer Assoc iate Professor of Biology at the

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55 University of Virginia) and primers (see Table 1) was used to exchange S157 and S239 for alanines and acidic amino acids. All constructs were verified by sequence analysis. VASP protein was purified as previously described (200) VASP isoforms were transformed in BL21DE3 ( Stratagene) cells. Cultures were grown at 37 C until OD600 was between 0.50.8, then induced with 0.1 nM of isopropryl -d thiogalactopyranoside (IPTG) for five hours followed by centrifu gation at 5,000 G x 15 min at 4C. Bacterial pellets were resuspended in cold PBS with protease inhibitors (Roche), 2 ME and sonicated. Cell lysates wer e centrifuged at 17000 rpm for one hour at 4 C and the His -tagged constructs purified using a cobalt column using standard methods. Purity w as assessed by commassie blue stained SDS -PAGE gels. Protein concentrations were determined using the Bradford Assay (Thermo -Scientific) Cloning and Expression of TAT -VASP Isoforms High -fidelity DNA Polymerase-Based PCR of the pEGFP C1 VASP, pEGFP-C1 VASP S1 57A and pEGFP -C1 VASP S157D was used to amplify the various VASP isoforms using the primers listed in Table 1. The pTAT vector (generous gift of Dr. Steven Dowdy Washington University, St. Louis, MO ) has an N -terminal His6 leader followed by the 11 a mino acid TAT protein transduction domain and a polylinker. The different products we re cloned into the KpnI/SphI sites on the pTAT vector. The resulting constructs were confirmed by sequence analysis. The pTAT -VASP isoforms were transformed in BL21D E3 ( Stratagene), followed by ind hours of induction at 37 C, cells were harvested by centrifugation (5,000 X G for 15 min at 4 C). The pellet was resuspended

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56 in ice -cold Buffer A (20 mM Tris, pH 7.5, 600 mM NaCl, 20 mM imida zole, 100 lysozyme and protease inhibitors {Roche}) followed by sonication and centrifugation (40,000 X g for 30 min at 4 C). Cell lysate was applied to a pre equilibrated cobalt column followed by washes with Buffer A and elution in 40 ml of Buf fer B (20 mM T ris, pH 7.5, 1 M NaCl and 250 mM Imidazole) and collected in 3-5 ml fractions. A 420% Tris SDS-PAGE gel was run on the fractions and samples were concentrated. S200 size exchange column was run using Buffer F (10 mM Na, K PO4, pH 6.8 {Fish er Scientific}, 6 00 mM NaCl, 5% glycerol, 1 mercaptoethanol ) and collected in 3 ml fractions. A 4-20% Tr is SDS -PAGE gel was run and fractions were concentrated down. The Bradford assay (Thermo -Scientific) was used to determine the protein concentrat ion and 1 mg/mL of bovine serum albumin was added prior to storage of samples at -80 C. PMN Isolation and Treatment with pTAT : Human neutrophils were purified using a Ficoll -Hypaque Gradient as previously described (39) The study followed US Department of Health and Human Services guidelines and was approved by the Institutional Rev iew Board at the University of Florida. Purified neutrophils were resuspended in RPMI with L-glutamine (Mediatech), and adjusted for 1 X 106 cells/ml. TAT fusion proteins were added to the final concentration of 5.2 ng/ml and placed at 37 C for 30 min wh ile gently rotating the cells. Neutrophils were centr ifuged at 1500 rpm for 5 min and extensively washed. Neutrophils were subsequently treated with 500 ng/ml of ET for two hours at 37C while gently rotating the cells to prevent clumping or cell activat ion. Control cells were incubated with buffer alone. Neutrophils were studied immediately after the two hours

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57 of incubation and experiments were completed within four to five hours of blood drawing. To determine the efficiency of protein delivery, neutr ophils with TAT fusion protein were analyzed by immunoblotting. To assess actin filament localization, neutrophils were fixed in 3.7% formaldehyde and permeabilized with 0.4% Triton and incubated with RhodaminePhalloidin (Molecular Probes Invitrogen). Af ter treatment with anti -fading agent Fluoromont -G (Southern Biotech), the signal was visualized by Zeiss Confocal imaging system with a 63X oil immersion lens (Zeiss). Cell images were processed with Adobe Photoshop Software. Quantitative Western Blots C ells were lysed in non-denaturing lysis buffer (1% Triton X 100, 50 mM Tris Cl ph 7.4, 150 mM KCl, 50 mM EDTA and 0.02% NaA2) with PhosSTOP phosphatase Inhibitors (Roche) and EDTA free protease tablets were added (Roche). BCA assay (Pierce) was performed to determine protein concentration. Neutrophils were lysed in 2X SDS sample buffer. Samples were sonicated, iced and centrifuged to remove cellular debris. Fifty micrograms of lysate w as separated by 12.5% SDS -PAGE, transferred to Polyvinylidene fluoride (PVDF) membrane, incubated with anti VASP (BD Bioscience) at 1:1000 overnight at 4C, washed in TBS -T and incubated wit h 1:10,000 goat anti mouse for one hour at room temperature. The wash was repeated and VASP and phosphorylated VASP at S157 was detect ed by using the SuperSignal Chemiluminescence detection system (Pierce) and quantitated using a FluorImager and ImageQuant Software (Molecular Dynamics). Phosphorylation of VASP on S157 or pseudophosphorylated mutant S to D 157results in a slowing of elec trophoretic mobility

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58 migrating as 50kD polypeptide; while unphosphorylated and pseudounphosphorylated VASP migrate as 46kD polypeptide (111, 144, 145) Low Speed Actin Bundling Assay G actin was first polymerized at room temperature in 1X Bundling Buffer (2.5 mM HEPES pH 7.4, 10 mM KCl, 0.2 mM MgCl2, 0.2 mM EGTA) for 3 0 min. F actin was incubated with 1 M of bundling protein in 1X Bundling Buffer for one hour. The samples were centrifuged at low speed (10,000 X g for 15 min), the supernatant and pellet fractions were separated on 816% SDS -PAGE gel, fixed and stained in coomassie blue. PullDown Assay For his -tag pull -down assays, MVd7 -/ cells were grown to confluence and lysed in 40mM Hepes NaOH, pH 7.4, 150 mM NaCl, 1% NP 40, PhosSTOP phosphatase Inhibitors (Roche) and EDTA free protease tablets were added (Roche), and incubated with 42.5 ng TAT -VASP constructs. Sample analysis was performed at the University of Florida Interdisciplinary Center for Biotechnology Research. Briefly, s amples were run on 10% Tris -Glycine SDS gel. Gel plugs were washed, equilibrated, and treated with trypsin, as previous ly described (201) Generated peptides w ere analyzed using LC MS/MS. Three database search engine and quantitative proteomic software; Mascot 2.1, Scaffold 1.6, and ProQuant 1.1. Statistical Analysis The Kruskal Wallis test for multiple comparisons and the Fischers exact test were used to determine statistical significance. In all experiments except for FACS

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59 analysis, n refers to the number of cells analyzed. Results are the average from three separate experiments unless otherwise noted.

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60 Table 3 1. Primers for the generation of VASP mutations by site-directed mutagenesis Point Mutation Primer Sequence S157A 5 C ATA GAG CGC CGG GTC GCC AAT GCA GGA GGC 3 S157D 5 C ATA GAG CGC CGG GTC GAC AAT GCA GGA GGC 3 S239A 5 AA CTC AGG AAA GTC GCC AAG CAG GAG GAG GCC T 3 S239D 5 AA CTC AGG AAA GTC GAC AAG CAG GAG GAG GCC T 3

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61 CHAPTER 4 RESULTS Bacillus anthracis Edema Toxin Impairs Neutrophil ActinBased Motility Anthrax ET is Active in Human Neutrophils To determine if ET entered both human neutrophils and HeLa cells we performed an ELISA based assay to measure intracellular cAMP levels. Neutrophils and HeLa cells exposed to increasing concentrations of ET (501000 ng/ml of a 1:1 weight ratio of EF and PA, see Fig. 4 -1A figure legend) for two hours demonstrated a progressi ve rise in intracellular c AMP, reaching a maximum of > 50 fold above untreated cells p<0.00 01 for 300 ng/ml to 1000 ng/ml plus Fsk/IBMX compared with control (Fig. 4 -1 A -D). In neutro phils this marked increase in c AMP was similar in magnitud e to th at induced by the cAMP agonists Fsk combined with IBMX (Fig. 4 1A). (Fsk resensitizes cell receptors by activating the enzyme adenylyl cyclase and increasing the intracellular levels of cAMP IBMX is a c ompetitive nonselective phosphodiesterase inhibitor, which raises intracellular cAM P. This makes the combination of Fsk and IBMX an ideal positive control.) In HeLa cells ET caused a greater rise in c AMP levels than these agonists (Fig 4 1C ). These effects were shown to be time dependent in neutrophils and HeLa cells since prolonged exposure was associated with a progressive accumulation of c AMP. (Fig. 4 1B, 4 1D). cAMP levels in n eutrophils treated with 500 ng/ml of ET for a minimum of one hour and Fsk/IBMX were considered statistically significant at p<0.0001 as compared to control. Similarly the levels of cAMP in HeLa cells treated with 500 ng/ml of ET for a minimum of one hour and Fsk/IBMX were considered statistically signi ficant at p<0.0001 compared to control. Exposure of human neutrophils and HeLa cells to PA, EF, or LF alone had n o effect on

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62 c AMP levels (data not shown). Cellular c AMPs main downstream effe ctor protein is PKA (202) For assessment of PKA phosphorylation, cell extract s from untreated neutrophils were compared to extracts from cells treated with 6db-cAMP (positive control) or treated with 500 ng/ml of ET. ET treatment of neutrophils resulted in a four -fold increase P32 incorporation into PKA (p< 0.001) (Fig. 4 1E). This is the first time that ET -induced P32 incorporation in PKA has been demonstrated in neutrophils. Effects of ET on Ap optosis, Necrosis, and Nitroblue Tetrazolium Reduction To ensure that the effects of ET on motility were not the result of apoptosis, we co mpared Annexin V staining in ET -treated cells to those exposed to buffer. Propidium iodide exclusion was also measured to assess necrosis. As observed previously with LT (39) exposure of concentrations up to 500 ng/ml of ET did not significant ly increase neutrophil apoptosis and resulted in only low levels of necrosis as compared to buffer alone (Ta ble 4 1 ). Similarly these concentrations of ET minimally affected HeLa cel l apoptosis and necrosis (Ta ble 4 -1 ). To further assess cell viability, the ability of control, ET, and ET + LT treated cells to exclude trypan -blue was assessed. Under control conditions 99% of PMNs excluded trypan -blue. After ET treatment (100500 ng/ml, 1:1 weight ratio), 9697% o f cells excluded trypan -blue, and following ET + LT treatment (50 ng500 ng/ml of ET and LF combined with 1 g of PA), 9394% of cells excluded the dye. To further assure that PMN functions unrelated to actin-based motility remained intact, we compared the ability of control and ET -treated, as well as ET + LT treated PMNs to reduce NBT. The reduction of this dye to a blue precipitate reflects the generation of superoxide. We found a comparable percentage of NBT positive cells in phorbol myristate acetate st imulated PMN: control, 91%; ET treated 89% (PA 500

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63 ng/ml + EF 500 ng/ml) and ET + LT treated, 91% (PA 1 g/ml + EF 500 ng/ml + LF 500 ng/ml) (p= 1.0, Fishers exact test.) PMN Chemokinesis and Chemotaxis We have previously shown that exposure to LT significantly impairs neutrophil chemotaxis and chemokinesis (39) ; h owever, during systemic anthrax infection, neutrophils are exposed to both LT and ET. Therefore, we examined the effects of ET alone, as well as the combination of ET and LT on human neutrophil motility. The velocity of chemokinesis was significantly reduced by treatm ent with ET, a reduction in velocity being observed at concentrations of 50 ng/ml at a p<0.0001 with maximal reduction in velocity of 40% being observed at 300-500 ng/ml (Fig. 4 2A). Dual treatment with ET and LT had additive inhibitory effects on the velo city of chemokinesis, causing a maximal inhibition of 80% at a concentration of 250 ng/ml for both toxins (Fig. 4 -2B). When the concentration of ET combined with LT was raised to 300 ng/ml, neutrophils no longe r adhered to the surface; therefore, our chemokinesis and chemotaxis experiments combining both toxins, and the individual concentrations of EF and LF did not exceed 250 ng/ml. ET and LT alone at concentrations up to 1000 ng/ml had no measurable effect on adherence, indicating that these effects required the activities of both toxins (data not shown). In control neutrophils, exposure to FMLP resulted in a distinct polarized morphology, a high percentage of cells forming broad lamellipodia at the leading edge and small uropodia at the rear (Fig. 4 -2C E). Exposure to ET alone markedly impaired the ability of neutrophils to polarize, maximum inhibition being seen at the same concentrations that maximally slowed chemokinesis, 300500 ng/ml (p<0.0 01) (Fig. 4 2 E). This difference is highly significant (p <0.001) except at 50 ng/ml (p>0.5). Exposure

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64 of neutrophils to dual toxin treatment resulted in greater inhibition than either toxin alone, while maximum reductions in the per centage of polarized cells was observed at 250 ng/ml (p<0.0001, Fishers exact t est) (Fig. 4 -2D and F). ET treatment also reduced the speed of neutrophil dir ected migration or chemotaxis. T reatment with 300 ng/ml or hig her concentration of ET reduced mean chemotactic velocity by 50% (p<0.0001) (Fig. 4 -2G ). Under these same conditions, LT also caused a concentration-dependent reduction in chemotaxis velocity, and a maximum inhibition of nearly 50% was observed at concentrations between 250500 ng/ml (p<0.0001 ) (Fig. 4 -2H). Dual toxin treatment also inhibi ted chemotaxis, maximal inhibition of 80% being observed at 250 ng/ml of the combination (p<0.0001) (Fig. 4 -2I). Thus as observed for chemokinesis and polarity, the combination of LT and ET had additive inhibitory effects on chemotaxis. PA, EF, or LF alone at concentrations of 500 ng/ml had no significant effec t on chemokinesis or chemotaxis; velocities were identical to control cells (data not shown). We sought to determine the time of maximum effects on neutrophils exposed to ET. Neutrophils were treated with buffer only (Ctl), 10 mM Fsk or 500 ng/ml of ET for 1 hour to two hours followed by time-lapse video imaging. The significant increase in cAMP levels is first noted one hr after ET treatment (p<0.0001) Even a slight increase in cAMP leads to in hibition of phagocytosis (203) superoxide killing (204) and bacterial killing of cells (49) The mean velocity of ET treated neutrophils was significantly decreased at one and two hours compared with buffer only (Fig. 4-3A). Neutrophils were treated with 500 ng/ml of ET for one or two hours and were not significantly different (p=0.96 ), leadin g us to conclude the increase in cAMP at one hour is sufficient to cause maximal effects.

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65 In control neutrophils, exposure to FMLP resulted in a distinctly polarized morphology, a high percentage of cells forming broad lamellipodia at the leading edge and small uropodia at the rear (Fig. 4 -3B). Exposure to 500 ng/ml of ET markedly impaired the ability of neutrophils to polarize (p<0.0001 ) with no statistical difference between one and two hours of exposure (p=1.0). CD11/CD18 Expression in Neutrophils Sign aling via the adhesion molecules of the 2 integrin family, CD11b/CD18 plays an essential role in PMN recruitment and activation during inflammation (205) ET induced reductions in chemotaxis and chemokinesis could in p art be mediated by a change in the surface expression of these adherence molecules. Therefore, we examined the effects of treatment with 500 ng/ml ET and Fsk /IBMX on the PMN surface marker expression of CD11b/CD18. No significant differences in surface ex pression were observed as compared to control neutrophils (p=0.143) (Fig. 4 4 A). Similarly LT at concentrations up to 500 ng/ml had no effect on the surface expression of CD11b/CD18 (Fig. 4 4 A). However, when EF, LF and PA were combined a concentration dependent decrease in receptor surface expression was observed (Fig. 4 4 B). Minimal effects were seen at 100 ng/ml (p=0.9857 not statistically significant) ; however a 50% reduction in expression was observed at 300 ng/ml (p<0.001) PA, LF, and EF alone (500 ng/ml) had no effects on surface expression (data not shown). Effects of ET Alone and ET Combined with LT on Neutrophil Actin Assembly Neutrophil chemotaxis requires rapid assembly of actin filaments at the leading edge, and ET -induced impairment of chemotaxis could be mediated by inhibition of neutrophil actin assembly. As assessed by Alexa-phalloidin staining of filamentous actin and FACS analysis, ET treatment resulted in a delay in FMLP -stimulated actin

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66 assembly a reduction in the maximum actin filament content (p<0.001) (Fig 4 5 A open circles), and the extent of inhibition was comparable to treatment with 500ng/ml of LT (p<0.001) (Fig. 4 5 A, open squares) (39) The combination of PA + EF + LF (ET + LT) resulted in an additive reduction in F actin content (p<0.001) suggesting that EF and LF impair neutrophil actin assembly by different signaling pathways (Fig. 4 -5 A, closed squares). PA, EF, or LF alone at 500 ng/ml had no significant effect on FMLP stimulated actin assembly, where F actin content was nearly identical to neutrophils incubated in buffer (data not shown). To further explore the effects of anthrax toxins on neutrophil actin assembly we, compared fluorescence micrographs of Alexa-phalloidin stained adherent neutrophils following exposure to 1 M FMLP. Control neutrophils were polarized and demonstrated a high content of F actin at the leading edge in lamellipodia (Fig. 4 -5 B). LT -treated adherent neutrophils appeared rounded with considerably reduced F actin content (Fig. 4 -5 C). ET and ET + LT -treated neutrophils demonstrated a distinctly differ ent distribution of filamentous actin as compared to control cells or cells treated with LT alone (Fig. 4 5 D and 4 -5 E). Small discrete regions of increased F actin content were noted throughout the horizontal plane of the cells. By shifting the Z -plane of focus in 0.5 m steps beginning at the top of the cell, these F actin structures were found to be in the lowest focal plane, adjacent to the slide surface (Fig. 4 -5 F). Effects of ET Alone and Combined with LT on Listeria monocytogen e s and Shigella flexen eri Actin Based Motility Listeria monocytogenes (88, 128) and Shigella flexneri (206) both highjack the actin regulatory system of host cells to induce the assembly of actin filaments. Both organisms bypass many of the signal transduction mechanisms required for receptor

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67 mediated actin assembly, and we have used these model systems to fur ther assess the effects of ET and the combination of PA + EF + LF on in vivo actin assembly. The velocity of bacterial movement directly correlates with the rate of actin assembly and assuming that the rate of actin disassembly is constant, the length of each actin filament tail also directly correlates with the assembly rate of actin fil aments within the tail (137, 2 07) Therefore, we examined the effects of these toxins on bacterial intracellular velocities and on actin tail lengths. Exposure of HeLa cells to 500 ng/ml ET resulted nearly 50% reduction in Listeria velocity (p<0.001) and treatment with Fsk/IBMX also reduced Listeria velocity (p<0.001) (Fig. 4 -6 A). Listeria actin tail lengths were comparably reduced (p<0.001) (Fig 4 6 B -D). The combination of PA + E F + LF (ET + LT) also impaired Listeria actin-based motility, with maximum inhibition occurring at a concentration of 50 -100 ng/ml (Fig. 4 6 E, F). The maximal inhibition of the combined toxins was similar to that of ET or LT alone. PA, EF, or LF alone at 500 ng/ml had no significant effect on Listeria intracellular actin based motility, velocity or tail length, being identical to cells treated with buffer alone (data not shown). Treatment of S. flexerni infected HeLa cells with 500 ng/ml of ET minimally s lowed the velocity of intracellular movement (mean control velocity: 0.125 0.007 m/s SEM, n= 270 vs. 0.109 0.007 m/s SEM, n = 315 for ET treated cells, p = 0.07). This finding is consistent with previous observations that Shigella and Listeria actin based motility utilize different actin-regulatory proteins and signal transduction pathway (208) Shigella requires bacterial surface protein IcsA. IcsA directly attracts and activates the host cell protein N WASP, and this protein in turn activates the Arp2/3 complex. Also, Shigella, unlike Listeria does not require VASP for intracellular actinbased motility

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68 (125) VASP Phosphorylation Impairs Neutrophil and Listeria Actin Based Motility Effects of ET on VASP Neutrophils were treated wi th 500ng/ml of ET for two hours and extracts were subject to quantitative western blot analysis. Equal amounts of neutrophils were analyzed for total VASP. Phosphorylation of VASP at S157 resulted in a change in electrophoretic mobility allowing relative quantification of both unphosphorylated and S157 phosphorylated VASP. Based on scanning densitometry ET treatment for two hours resulted in a five-fold increase in phosphorylation on S157 as compared to cells in cubated in buffer (p<0.001)(Fig. 4 -7C, 47D ). Forskolin, a cAMP signaling activator, mimiced the effects o f ET treatment, resulting in a one -fold increase in VASP phosphorylation at S157 (p=0.1036, not significant) Phospho -S157 anti -VASP antibody was used to detect localization phosphorylated VASP in human neutrophils, and in Listeria infected HeLa cells. Phospho -S157 localized to the lamellipodia and focal adhesions of neutrophils with greater intensity of staining in ET treated as compared to neutrophils incubated with buffer alone. Some d iffuse staining throughout the neutrophil was also apparent (Fig. 48A ). Phospho S157 was shown to localize around intracellular bacteria with much greater intensity than cells inc ubated in buffer alone (Fig. 48B ). Effects of Changes in VASP S157 Phosp horylation on Human Neutrophils VASP phosphorylation at S157 may be regulating actin turnover during ET infection, thereby inhibiting the ability of the neutrophil to chem otax to the site of infection; h owever, neutrophils are refractory to transfection, l imiting the number of approaches that can be used to gain insight into how this protein functions. A method

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69 that has been successfully used t o introduce proteins into primary human neutrophils while maintaining normal functional responses involves the addition of a sequence derived from the HIV TAT protein linked to the protein of interest, which can then be introduced into intact neutrophils (209) The various VASP isoforms were cloned into the pTAT -HA vector at the KpnI/SphI restriction sites (Fig. 4 9 A). The plasmids were transformed in BLD21 cells, subsequently cultured in Luria-Broth ( LB ) and induced for five hours with IPTG. S157A and S157D both wer e induced at three hours, whereas wild -type VASP was continuously produced without the need for induction (Fig. 49 B). VASP is 46 -kDa and with TAT we saw a band at ~54kDa. Western blot analysis probing for anti VASP confirmed VASP production (data not shown). The samples were applied to a Cobalt column (Qiagen) and c olumn fractions were analyzed by Coomassie blue staining of an SDSpolyacryl amide gel (Fig. 4 9 C, representative of all three isoforms). The appropriate fractions were pooled together run through a S200 (Qiagen) size exchange column to remove smaller protein contaminates and again analyzed by Coomassie blue staining of an SDSp olyacrylamide gel (Fig. 4 -9 D). We first estimated the concentration of VASP present in human neutrophils (Fig. 4 -10 A). Densitometric scans of the recombinant protein were plotted and the amount of VASP protein in the lysate was interpolated from the linear portion of the curve. Based on this approach, we were able to estimate that neutrophils contain 0.1 ng of VASP per cell or approximately 1.5% of the total protein found in hum an neutrophils The TAT VASP fusion proteins were added to a final concentration o f 5.2 g (Fig. 4 -9E). To determine the efficiency of protein delivery, neutrophils with and without TAT -

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70 VASP protein treatment w ere subject to extensive washes and analyzed by Western blot with anti VASP antibody. After 30 min of TAT -VASP incubation approxi mately 8590% of the TAT entered into the neutrophil and no longer appeared to be bound to the surface of the neutrophil after western blot analysis of cytoplasmic exacts devoid of cell membrane (data not shown). After TAT -VASP protein treatment neutrophils were incubated in buffer only or in 500ng/ml of ET for two hours at 37 C. The effects of TAT -VASP wild type, TAT-VASP S157A, TAT -VASP S157D and control neutrophils treated incubated in buffer alone or ET were examined for effects on motility (Fig. 4 -10B ). As we have pr eviously shown (106) ET significantly impaired neutrophil chemokinesis by 25 30% in control cells and TAT -VASP wild -type treated cells (p<0.001) ET treatment had no effect on chemokinesis of TAT -VASP S157A treated cells (p>0.05 ); h owever, TAT -VASP S157D treated cells incubated with buffer or ET demonstrated a significant reduction in neutrophil chemokinesis (2530% decrease) (p<0.01 as compared to untreated neutrophils and neutrophils treated with wild -type TAT -VASP). These findings strongly suggest that ET -ind uced phosphorylation of VASP at S157 is responsible for ET mediated impairment of neutrophil motility. In addition to chemokinesis, the effects of FMLP on neutrophil polarity was also examined. Untreated neutrophils, those treated with TAT -VASP wild type protein and ET -treated TAT -VASP S157A neutrophils, demonstrated a distinctly polarized morphology, with a high percentage of cells forming broad lamellipodia at the leading edge and small uropodia at the rear (Fig. 410C ). Exposure of cells with wild-type VASP to ET or cells containing TAT -VASP S157D incubated with buffer or ET

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71 demonstrated marked impairment in the ability to polarize in response to the chemoattractant FMLP (p<0.01) These findings suggest that ET impairs neutrophil actin based assembly by phosphorylating VASP S157. Recent studies have implicated VASP in promotion of filopodial formation from the lamellipodi um mesh network (210) Filopodia have been implicated in a number of diverse cellular pr ocesses including growth-cone formation, wound healing and metastasis (178, 211) ; t herefore, we also explored the effects of various isoforms of VASP proteins on neutrophil filopodia formation (Fig. 4 10D, 410 E). In response to phosphorylation of VASP as a consequence of ET exposure or by treatment with TAT VASP S157D, the number of filopodia per cell was significantly increased as compared to control neutrophils (p<0.00 1) TAT -VASP wild -type and TAT -VASP S157 Atreated neutrophils (p>0.5) (Fig.4 10D, 4-10E ). Effects of ET Induced VASP Phosphorylation on L. monocytogene s Actin Based Motility To unambiguously study the role of VASP protein in Listeria actin -based motility it was necessary to utilize a cell line that fails to express any of Ena/VASP proteins. This condition eliminates interference from the endogenous proteins (184, 212) MVd7 cells fulfill this condition, lacking all Ena/VASP proteins. As previously shown in the absence of transf ection with VASP or VASP mutant s MVd7 cells supported only very slow intracell ular Listeria velocities (Fig. 4 11A CT). (MVd7 cells still have a small level of Mena remaining allowing for some Listeria motility.) This decrease in velocity was comparable to the slow velocities seen in cells transfected with S157D (p>0.5) MVd7 cells transfected with wild-type VASP were able to significantly increase Listeria velocities as compared to untransfected cells (p<0.001) ; however ET treat ed MVd7

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72 cells rescued with wild -type VASP reversed this effect, velocities being comparable to untransfected cells and cells rescued with S157D VASP. Rescue with S157A VASP resulted in a marked increase in the mean intracellular Listeria velocity and was s ignificantly faster than MVd7 cells rescued with wildtype (p<0.001) Furthermore cells rescued with this mutant construct were resistant to the effects of ET treatment. Because a significant increase in filopodia formation was observed in neutrophils containing S157 phosphorylated VASP, we sought to determine how the various isoforms of VASP proteins contribute to Listeria induced membrane projec tions using MVd7 cells (Fig. 4-11 B). Bacterial projection formation was significantly increased in MVd7 cells containing phosphorylated S157 VASP (p<0.046 ). The number of bacteria containing projections per c ell was increased in MVd7 cells or cells rescued with wildtype VASP, treated with ET (p=0.093, not quite significant) and transfected with VASP S157D buffer (p<0.046 ) as compared to untransfected MVd7 cells or cells rescued with wild -type and S157A VASP. These findings suggest that VASP phosphorylation at S157 facilitates the ability of Listeria to form membrane projections imp ortant for cell -to cell spread of the bacteria. These findings were contrasted in MVd7 cells infected with S. flexerni No significant difference in bacteria containing membrane projections was noted when comparing null cells to cells rescued with the three VASP constructs, MVd7 nul l cells forming 0.32 0.032 (projections per cell standard error of the mean [SEM], MVd7 WT VASP: 0.25 0.026, MVd7 S157A: 0.245 0.027 and MVd7 S157D: 0.35 0.05). Our findings are consistent with previous observations that Shigella and Listeria actin -

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73 based motilities utilize different actin-regulating proteins and signal trans duction pathways (2 08) Effects of VASP S239 Phosphorylation on Human Neutrophils Harbeck, et al. (185) previously showed in mouse VASP extracts that phosphorylation of S239 significantly impairs actin polymerization. S239 is preferentially phosphorylated by PKG but it can also be phosphorylated by PKA; however the preferred site for PKA is S157. Neutrophils were treated for two hours with buffer only, 10mM Fsk or 500ng/ml of ET. Immunoblotting using anti -Phospho S239 VASP antibody (Cell Signaling) confirmed slight phosphorylation in Fsk and ET treated cells (data not shown). To determine if the decrease in neutrophil a ctin assembly was the result not only of S157 phosphorylation, but also S239 phosphorylation, we transfected MVd7 cells with various VASP isoforms, followed by infection with Listeria and incubation in buffer only or 500 ng/ml of ET (Fig. 412). Intracel lular motility of Listeria was determined in MVd7 VASP wild-type (WT), S157A, S157D, S239A, S239D and MVd7 null cells treated with buffer only (CT) or ET. As we have pr eviously shown (106) ET significantly impaired Listeria motility in wild -type cells. Control and ET treated S157A cells significantly increased Listeria motility over wildtype control (p<0.001) ; h owever, S157D and MVd7 null cells treated with buffer only or ET all showed a significant impair ment in overall Listeria motility (p<0.001) Transfection of MVd7 null cells wit h S239A and S239D constructs were only able to rescue the wild type Listeria phenotype to approximately 85%. (p<0.0091 and p<0.0011 respectively.) This set of experiments res cued with the S157 VASP construct reproduced our previous findings, while S239A and S239D had no effect on Listeria

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74 velocity. These findings suggest that S239 does not play a significant role in ET mediated impairment of Listeria motility Effects of VASP Isoforms on Actin Bundling In addition to examining the VASP function in cells and extracts, we examined the effects of recombinant VASP proteins on actin based bundling. Filopodia are highly dynamic finger -like cell protrusions filled with parallel bundl es of actin filaments. A key player in the formation of filopodia across many species is VASP (143, 168) It has been proposed that the essential role of VASP for formation of filopodia is its ability to bundle actin filaments. Therefore to better understand the function o f VASP in filopodium formation we examined the ability of purified native VASP, VASP S157A, VASP S157D, as well as alpha actinin (positive control) to bundle purified skeletal muscle actin filaments. As shown in Fig. 4 13 alpha actinin enhanced the concentration of F actin that sedimented after slow speed centrifugation (p=0.002) All three isoforms of VASP also enhanced F actin sedimentation und er the same conditions (Fig. 413 A). No significant differences in supernatant and pellet were observed between the three proteins (P values be tween 0.13 -0.59 ) (Fig. 4 -13 B). These findings suggest that all three isoforms are equally capable to efficiently bundle actin filaments and suggest that the differences in their ability to induce filopodia formation is lik ely to be the consequence of additional proteinprotein interactions. PullDown Assay To explore the possibility of an additional protein binding partner being recruited by VASP S157 phosphorylation, MVd7 cells were treated with 6XHis tagged fusion

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75 prote ins followed by lysis and incubated with His -binding beads. Using this methodology we were unable to identify any proteins of interest (See Table 4 2). Dual To xin Affects on Human Neutrophils ET and LT are Able to Enter Human Neutrophils ET enters human neutrophils and HeLa cells as evidenced by a concentration dependent rise in cAMP levels (Fig. 4 1A -D). cAMP levels increase in a time and dose dependent manner. Previous work from our lab (39) assess ed LT activity in neutrophils. Neutrophil lysate extracts were subject ed to western blot analysis using an antibody to detect the amino-terminus of MEK. The antibody identifies epitopes on the first seven amino acids of MEK, the region cleaved by LF, and loss of antibody cross -reactivity indicates pro teolysis by LF. MEK cleavage in neutrophils and HeLa cells required two hours of treatment at 37 C with 50 ng/ml of LT (39) Effects of ET+LT Interactions on cAMP Levels To better understand the potential synergism of ET and LT we exposed neutrophils and HeLa cells to increasing concentrations of ET and LT (50500 ng/ml of a 1:1:1 weight ratio of PA, EF and LF ). Two hours of dual toxin exposure significantly lowered the cAMP levels in neutrophils (Fig. 414A ) and HeLa cells (Fig. 414C ) than treatment with ET alone. ET or LT binds with high affinity to the exposed domain sites on PA. It is expected that the binding of the alternative moiety to this site would make the site unavailable for the other moiety (192) To address a potential issue of steric constraints we exposed neutrophils for two hours to increasing concentrations of ET and LT (50500 ng/ml) o f a 2:1:1 weight ratio of PA, EF and LF. The cAMP response

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76 again remained at a low level (data not shown). Next we exposed neutrophils and HeLa cells to a 5:1:1 weight ratio of PA, EF and LF, which leaves PA molecules free and assures that any effects se en were not the consequence of LF competing with EF for PA binding sites. Again, cAMP levels remained low in neutrophils (Fig. 4-14B) and HeLa cells treated with ET+LT (data not shown). Increasing concentrations of ET and LT did not increase cAMP in a dose dependent manner as observed with ET alone nor did we find the >50-fold increase seen with ET treatment. Increasing the ratio of moieties to overcome any steric c onstraints did not improve the four -fold increase in cAMP levels noted with dual toxin treatment. These findings indicate the low levels of cAMP associated with dual toxin treatment are not the consequence of competitive inhibition of EF by LF for their binding partner PA. We sought to determine if the concentration of the individual toxins pl ayed a role in cAMP inhibition. N eutrophils (Fig. 414D) and HeLa cells (Fig. 4-14E ) were treated with 100 ng/ml of LT in addition to increasing concentrations of ET (5:1:1 weight ratio used for PA, EF and LF). A 100 ng/ml concentration of LT was able to impair the ability of ET to increase cAMP levels in both neutrophils and HeLa cells. Signaling Pathway Utilized by LT to Decrease ET cAMP Production W e next sought to examine if LT cleavage and inactivation of the MEK pathway played a role in decreased cAMP response. We treated neutrophils (Fig. 415A) and HeLa cells (Fig. 4 15B) with 500 ng/ml of ET (5:1 weight ratio) for one hour followed by the addition of MEK inhibitors for the second hour. MEK inhibitors were : SB203580 (inhibits the p38 pathway ), SP600125 ( inhibits the JNK pathway ) and PD98059 (inhibits the ERK pathway ). All three inhibitors significantly impaired the ability of ET to increase intracellular cAMP levels (p<0.001)

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77 cAMP levels are down-regulated by phosphodiesterases. To rule out LT as a potential phosphodiesterase, neutrophils (Fig. 416A) and HeLa cells (Fig. 4 -16B) were again exposed to 500 ng/ml of ET and 100 ng/ml of LT (5:1:1 weig ht ratio of PA, EF and LF) for one hour followed by an additional hour of treatment with 50 mM of t heophylline. Theophylline is an inhibitor of phosphodiesterase enzymes and would be expected to allow for increased cAMP levels after LT exposure if LT had phosphodiesterase cha racteristics. The addition of theophylline still le d to a dramatic decrease in intracellular cAMP levels with dual toxin treatment (p<0.001). These results (Fig. 416) le d us to conclude that LT does not activate phosphodiesterases and appears to impair increased cAM P levels by inhibiting one or more of the MEK pathways. We also confirmed that the c hemical inhibitors did not react wit h the assay itself (data not shown ). Effects of ET+LT Exposure to cAMP Downstream Target PKA Previous wor k from our lab (106) has shown that ET -induced rise in cAMP is accompanied by the phosphorylation of PKA and downstream phosphorylation of VASP at S157 (in press ). After ET+LT treatment is ET still able to activate its downstream signaling pathway? U ntreat ed neutrophils were compared to neutrophils treated for two hours with 6-db -cAMP (positive control), 100 ng/ml of LT, 500 ng/ml of ET and increasing concentrations of ET with 100 ng/ml of LT (5:1:1 weight ratio) (p<0.001) (Fig. 4 -16 A). LT does not appear to inhibit the ability o f ET to phosphorylate PKA as a two fold increase in P -PKA was detected. Neutrophils were next treated with a 100 ng/ml of LT and 500 ng/ml of ET (5:1:1 weight ratio) in a time-dependent manner to determine if the 2 -fold increase in PKA was still able to phosphorylate VASP at S157. VASP phosphorylation at S157A is noted one

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78 hour after dual toxin treatment (Fig. 4 16 B). The two -fold increase in PKA noted after ET+LT treatment is sufficient to activate its downstream effector molecule VASP. Timing of Toxin Entry Even though LT impairs increases in cAMP levels at two hours post exposure, PKA phosphorylation is still occurring. This led us to speculate that what we were seeing was related to the timing of toxin a ctivity. It is highly likely that both EF and LF enter at the same time; however LF takes time to cleve the MEKs. Neutrophils were exposed to buffer only, 500 ng/ml of ET or 500 ng/ml of ET and 100 ng/ml of LT (5:1:1 weight ratio) over a time course rang ing from 30 minutes to two hours (Fig. 417 A). ET begins increasing intracellular cAMP levels at 30 minutes post exposure and steadily i ncreases cAMP levels up to the two-hour time point. The additio n of 100 ng/ml of LT caused cAMP levels to increase up to the one and a half hour time point and at two hours LT impaired the increase in cAMP, brin g ing the levels back to physiologically normal levels. We are able to conclude that ET is able to significantly affect intracellular cAMP levels by 30 minutes. Previous work from our lab found that experiments conducted on neutrophils exposed to LT needed a minimum twohour incubation period before impairing neutrophil actinbased motility (39) To assess LT activity we exposed neutrophils to 100 ng/ml of LT over a two-hour time course (Fig. 4-17 B). Neutrophil lysates were subjected to western blot analysis using an antibody against the amino-terminus o f MEK. MEK cleavage begins at one hour but does not reach its maximal cleavage unti l two hours post exposure. During, et al (39) showed that neutrophils treated with 50 ng/ml of LT at one hour had no e ffect on polarity or chemotaxis; however, two hours p ost exposure caused a 50% reduction in the ability of neutrophils not only to polarize

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79 but also to chemotax, which corresponds to the two hours necessary for complete MEK cleavage. We conclude that ET acts quickly within 30 minutes to increase cAMP and ph osphorylate PKA, and then its activity is shut off by LTs at 1.5 to 2 hours after toxin exposure. Sequential toxin effects serves to limit the duration of cAMP.

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80 Figure 41. Effects of ET on Intracellular cAMP levels and PKA phosphorylation. A) C oncentration dependent increase in intracellular cAMP levels in human neutrophils treated with ET The far right bar shows neutrophils treated with the cAMP agonists Fsk (10 mM) and IBMX (100 mM) for 15 min at 37C. B) Effects of incubation time on ET induced rise in intracellular cAMP levels. Neutrophils were treated with 500 ng/ml of ET C) Concentration dependence of ET induced intracellular cAMP levels in HeLa cells. Experimental conditions are identical to A D) Effects o f incubation time on ET induced rise in intracellular cAMP levels in HeLa cells. Conditi ons are identical to B. E) Phosphor ylation of PKA was determined using 25 g of radiolabeled protein lysate and measuring the transfer of radioactive phosphate from ATP into Kemptide. A > 4 -fold increase in phosphorylated PKA was obser ved after ET treatment. Standard errors indicate the SEM of 3 experiments. = p<0.001 and ** = p<0.0001 compared to Ctl treated cells.

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81 Table 4 1. Effects of ET on cell necrosis and apop tosis Neutrophils % Viable Cells % Necrosis % Apoptosis Control 96.7 3.3 0 50ng/ml ET 89.9 10.1 <1 300ng/ml ET 95.8 4.2 0 500ng/ml ET 92.0 8.0 <1 HeLa Cells Control 93.9 6.1 <1 50ng/ml ET 98.5 1.5 <1 300ng/ml ET 96.3 3.7 <1 500ng/ml ET 96.0 4.0 <1 Cells were treated with ET for 2h and stained with annexin V and propidium iodide, followed by FACS analysis (see Materials and Methods). HeLa cells were scraped from tissue culture dishes, explaining the relatively high percentage of necrosis in control HeLa cells (p=1.0, Fishers exact test)

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82 Figure 42. Effects of anthrax toxins on neutrophil chemokinesis, polarity, and chemotaxis. A) Mean relative velocity of human neutrophil chemokinesis after treatment with buffer (Ctl), i ncreasing concentrations of ET or Fsk /IBMX (10 nM/100 nM). Error bars indicate the SEM of 7 experiments. B) Effects of PA, EF, and LF on neutrophil chemokinesis. Standard errors indicate the SEM of 3 experiments. C) Phase cont rast micrograph of control PMNs 5 min after the addition of 1 M FMLP. The arrows point to the direction of polarity and movement of each cell. D) Phase contrast micrograph of PA + EF +LF treated ( PMN 5 min after the addition of 1 M FMLP. E) P ercentage of polarized PMNs after FMLP ex posure. Standard errors = SEM of 7 experiments. For each condition, 130 cells were analyzed per experiment F) Effects of PA, EF, and LF on neutrophil polar ity. Error bars indicate the SEM of 3 experiments. G) R elative velocity of human neutrophil chemotax is after treatment with buffer (Ctl), i ncreasing concentrations of ET or Fsk /IBMX (10 nM/100 nM). Error bars indica te the SEM of 3 experiments H) R elative velocity of human neutrophil chemotaxis after treatment with buffer (Ctl) or increasing concentrati on of I) Effects of PA, EF, and LF on neutrophil chemotaxis. Error bars indicate the SEM of 3 experiments. Note the additive inhibition of chemotaxis as compared to ET and LT alone (G and H). = p>0.05, ** = p<0.001 and *** = p<0.0001.

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83 Figure 43 Tim e course of ET on neutrophil chemokinesis and polar ity. A) M ean relative velocity of human neutrophils after treatment with buffer only (Ctl), 500 ng/ml of ET (weight ratio of 5:1) f or 1 to 2 h and 10 n M of Fsk for 20 min The differences in relative vel ocity of ET at 1 and 2 h are not statisti cally different (p>0.05) Error ba rs are indicative of SEMs of 2 experiments (n=210). ET significantly impairs the velocity of neutrophils 1 h post exposure. B) Percentage of polarized neutrophils 5 min after the addition of 1 mmol/L of FMLP. Error bars indicat e the SEMs of 2 separate experiments (n=100) = p<0.001.

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84 Figure 44. Effects of anthrax toxins on CD11/CD18 surface expression by neutrophils. A) Neutrophils were treated with buffer, 500ng/ml ET or LT (500 ng/ml PA + 500 ng/ml of EF or LF) for 2 h, or with Fsk/IBMX (10 mM/100 mM) for 15 min followed by surface staining and FACS analysis of 10,000 cells. As compared to buffer alone, no significant differences in receptor expression were observed i n toxin or Fsk/IBMX -treated cells. B) Neutrophils were treated with 1 g/ml of PA plus increasing concentrations of a 1:1 weight ration of EF and LF (identical to conditions in Fig 2 F) for 2 h followed by staining and sorting as described above. A 50% r eduction in the CD11/CD18 surface receptor expression was observed with 300 ng/ml (1 g/ml PA + 300 ng/ml EF + 300 ng/ml LF). Error bars indicat e the SEM of 3 experiments *= p<0.001 compared with Ctl.

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85 Figure 45. Effects of anthrax toxins on FMLP -induced neutrophil actin assembly. A) Effects of ET ( open circles), LT (open squares) as well as the combination of ET and LT (closed squares) on actin filament content of neutrophils as compared to cells incubated in buffer (Control, closed circles). The median fluorescence intensity was determined by FACS analysis of 10,000 cells for each time point. Both ET and LT alone and in combination slowed the onset of actin assembly and the combination resulted in an additive reduction in peak F actin content, 34% as compared to ET (15% reduction) and LT (15% redu ction) alone (p<0.001). Error bars = SEM of 3 experiments. B) Fluorescence micrograph of human neutrophils incubated with buffer for 2 h, allowed to attach to fibronectin coated glass slides Bar = 10 m C) Fluorescence micrograph of human neutrophils incubated with 500 ng/ml LT D) Fluorescence micrograph of human neutrophils incubated with 500 ng/ml ET E) Fluorescence micrograph of human neutrophils incubated with ET +. = p<0.001 compared to control.

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86 Figure 46. Effects of anthrax toxins on Listeria actin based motility. A) R elative mean velocities of intracellular Listeria in HeLa cells after treatment for 2 h with buffer (Ctl), and increasing concentrations of ET or 15 min with Fsk/IBMX. Error bars indicate the SEM, n=730. Measuremen ts were made 5-6 hours after initial Listeria infection. B) Comparing mean lengths of Listeria -induced actin filament tails. Error bars indicates the SEM, n=100. C) Fluorescent micrographs of buffer -treated HeLa cells infected with Listeria for 6 h fixed and stained with Alexa488 phalloidin. Note the long Listeria actin rocket tails (arrows: point to the beginning and end of selected tails). Bar = 10 m D) Fl uorescent micrograph of ET -treated HeLa cells infected with Listeria Cells were fixed and stained as described in C. Note the shorter Listeria actin tails. E) Effects of increasing concentrations of LF, EF, and PA in combination on the velocity of Liste ria Error bars indicate the SEM, n=50. F) M ean Listeria tail lengths under the same conditions as E. Error bars indicate the SEM, n=50. = p<0.001 compared to control (Ctl)

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87 Figure 47. Effects of ET on P S157 VASP. A) Purified neutrophils were treated from left to right with buffer only, Fsk (10 mM) or 500 ng/ml of ET for 2 h at 37 C. Equal protein amounts of each treatment were analyzed for P S157 by western blot analysis. A >5-fold increase in P S157 was observed a fter ET treatment p<0.001. Blots are representative of 2 separate experiments. The lower blot is beta actin for load control. Numbers on the right side represents molecular weight markers in kilodaltons. Ctl=control (buffer only); Fsk=forskolin. B) De nsitometry was performed, and the densities of the lower 46kDa and upp e r 50kDa bands were measured. = p<0.001.

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88 Figure 48 The effects of ET on P S157 VASP localization on neutrophils and Listeria infected HeLa cells. A ) Fluorescent micrograph of human neutrophils incubated with buffer for 2 h, allowed to adhere to fibronectin coated glass slides, and then exp osed to 1 M FMLP for 10 min followed by formalin fixation, permeabilization, and staining with Alexa-Rhodamine (red) and P S157 VASP antib ody (green). Note the high concentration of filamentous actin at the leading edge with small amount of P S157 VASP staining towards the rear of the neutrophil. Fluorescent micrograph of human neutrophils incubated with 500 ng/ml of ET for 2 h have a reduc tion in fluorescence intensity, indicative of reduced filamentous actin with increased amounts of P S157 VASP staining at the plasma membrane. B) Fluorescent micrograph of buffer treated HeLa cells infected with Listeria for 6 h and treated as described in panel A. Note not only the long Listeria actin rocket tails but also the P S157 staining around the back portion of the bacteria. Fluorescent micrograph of ET treated HeLa cells infected with Listeria for 6 h. Cells were fi xed and stained as described i n panel A Note the shorter Listeria actin rocket tails and the increased staining intensity of P S157 around the back portion of the bacteria. Ctl=control (buffer treated); Fsk=forskolin.

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89 Figure 49 Characterization of pTAT vector and the steps involved in protein synthesis A) pTAT expression vector B) Induction of pTAT -VASP S157A expression. pTAT -VASP S157A was induced with 500 mM IPTG. Black arrow points to the induction of the pTAT -VASP protein of interest. C ) Purifi cation of pTAT VASP S157A fusi on protein over a cobalt resin (Frac, fractions 1 -8; S, sample prior to purification; W, washes) were resolved by SDS -Page and stained with Coomassie blue. D) P urification of pTAT -VASP S157A fusion protein over an S20 0 si ze exchange column (Fraction 1 -8 ).

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90 Figure 410 Effects of P S157 VA SP on regulating actin assembly in human neutrophils. A) Concentration curve of VASP present in human neutrophils. A defined amount of native VASP (0.1 ng, 0.25 ng, 0.5 ng, 0. 75 ng and 1 ng) was resolved by SDS -Page and probed with anti -VASP antibody. Protein lysate s from human neutrophi ls at various concentrations were also resolved to determine the concentration of VASP in human neutrophils (A=2.5X103, B=2.5X104, C=2.5X105 and D=2.5X106 cells/mL) B) Concentration curve of pTAT -VASP wild type fusi on protein. C) R elative velocity of neutrophils transduced with buffer only, pTAT -VASP wild -type (VASP), pTAT -VASP S157A (S157A) and pTAT -VASP S15D (S157D) followed by treatment f or 2 h wi th buffer only (Ctl), Fsk (10 mM) or 500 ng/ml of ET. Error bars indicate the SEMs of the results of 4 experiments. D ) P erc entage of polarized neutrophils. Error bars show SEMs of 2 experiments. For each condition, 50 cells per experiment were analy zed. E ) N umber of filopodia per cell formed in neutrophils Error bars indic ate the SEMs 2 experiments, n=50 F) Fluorescent micrograph of human neutrophils transduced with buffer only, pTAT -VASP wild type, pTAT -VASP S157A or pTAT -VASP S157D for 30 m in then treated with buffer only (Ctl) or 500ng/ml of ET. These images are representative of neutrophils observed during polarity analysis. = p<0.001.

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91 Figure 411. Pseudophosphorylated VASP mimics the effects of ET on Listeria motility. A) T he relativ e velocity of intracellular Listeria in MVd7 cells expressing no Ena/Mena/VASP ( -/ -), wild -type VASP (VASP), VASP S157A (S157A) or VASP S157D (S157D) after treatment for 2 h with buffer only, Fsk/IBMX (10 mM /100 mM ) or 500 ng/ml of ET. Phosphorylation of VASP at S157 decreases Listeria motility. Error bars indicate the SEMs n=115. B) N umber of filopodia per cell formed by intracellular infection of Listeria in MVd7 cells expressin g -/ -, WT VASP, S157A or S157D VASP after treatment for 2 h with buffer or 500 ng/ml of ET. Phosphorylation of VASP at S157 increases Listeria induced filopodia formation. Error bars indicate the SEMs n=50. = p<0.001 and ** = p<0.046.

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92 Figure 412 Effects of P S239 VASP o n regulating actin assembly in human neut rophils. R e lative velocity of intracellular Listeria in MVd7 cells e xpressing no Ena/Mena/VASP (-/ null), wild -type VASP (VASP), S157A, S157D, S239A and S239D after treatment for 2 h with buffer only (CT ) or 500 ng/ml of ET. Phosphorylation of VASP at S157 signific antly decreased Listeria motility while ET appeared to only have a slight effect on Listeria motility. Error bars indicate the SEMs (n=73). = p<0.0001, ** = p<0.0091 and *** = p<0.0011.

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93 Figure 413. Actin B undling Assay. A) B undled F actin was pelleted by 10,000 X g centrifugation, and pellets (P) and supernat ants (S) were run on an SDS PAGE gel. Only in the presence of the F actin bundling protein alpha actinin and all three isoforms of VASP (wil d -type (WT), S157A and S157D) was actin pelleted at this centrifugation speed. B) Pellet to supernantant ratios were determined A ll three VASP isoforms bundle the same pellet/supernatant ratios. P<0.001 for actinin and all VASP isoforms compared to actin alone. *, p<0.001.

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94 Table 4 2. V ASP S157D Interacts with CKAP4 Protein Wild type S157A S157D VASP EIF5A SFPQ CKAP4 Similar to non POU domain EIF5A: eukaryotic initiation translocation factor 5A; SFPQ: isoform long of splicing factor, proline glutamine rich; CKAP4: isoform of cytoskeleton associated protein 4; similar to non POU: octomer binding is oform 1

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95 Figure 414. Effects of ET+LT on intracellular cAMP l ev els. A) D ecreased ability of ET to increase cAMP levels while in the presence of LT. Neutrophils were treated with increasing amounts of ET and LT ( 1:1:1 of PA, EF and LF) Error bars indicate the SEM results of 3 separate exper iments run in duplicate. B) D ecreased ability of ET to increase cAMP levels while in the presence of LT. Neutrophils were treated with increasing amounts of ET and LT (5:1:1 of PA, EF and LF). Error bars indicate the SEM results of 2 separate experiments run in duplicate. C) D ecreased ability of ET to increase cAMP levels while in the presence of LT. HeLa c ells were treated with increasing amounts of ET and LT (5:1:1 of PA, EF and LF). Error bars indicate the SEM results of 2 separate experiments run in duplicate. D ) C omplete inhibition of ET to increase cAMP levels in the presence of 100 ng/ml of LT. Error bars indicat e the SEM results of 2 experiments run in duplicate. E) C omplete inhibition of ET to increase cAMP levels in the presence of 100 ng/ml of LT. Error bars indicate the SEM results of 2 experiments run in duplicate. = p<0.001.

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96 Figure 415 Ho w LT inhibits intracellular cAMP levels induced by ET. A) C omplete inhibition of cAMP levels in neutrophils after ET and LT treatment, which appears to be due to LT inhibiting one or more MEK pathways Neutrophils were treated with: buffer only (Ctl), 500 ng/ml of ET (weight ratio 5:1), 100 ng/ml of LT (weight ratio of 5: 1), cAMP agonist Fsk at 10 mM, t heophylline (50 mM) with 500 ng/ml of ET and then with 500 ng/ml of ET and 100 ng/ml of LT (weight ratio of 5:1:1). Neutrophils were also treated with 500 ng/ml of ET (weight ratio of 5:1) and an individual MAPK inhibitor: SB203880 (100 mM), SP600125 (10 mM) and PD98059 (10 mM). Neutrophils were treated with toxins for 2 h at 37 C. 1 h into the toxin exposure inhibitors were added. Error bars indicate th e SEMs results of 2 experiments run in duplicate. All three MEK inhibitors were able to decrease cAMP levels to physiological normal levels while t he ophylline had no effect on the effects of LT on cAMP B) C omplete inhibition of cAMP levels in HeLa cells after ET and LT treatment. HeLa cells were treated as described in panel A above. Error bars indicate the SEM results of 2 experiments run in duplicate. = p<0.0001 compared to control treated cells.

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97 Figure 416 Effects of ET+LT on PKA phosphorylation. Bar graph showing the effects of ET+LT on Protein Kinase A (PKA) phosphorylation. Neutrophils were exposed to buffer only (Ctl), treated with 6-db -cAMP (100 mM) for 1 h and treated with 100 ng/ml LT (weight ratio of 5:1), 500 ng/ ml of ET (weight ratio of 5:1) and 100 ng/ml of LT with 500 ng/ml of ET (weight r atio of 5:1:1 of PA, EF and LF) for 2 h at 37 C. A >2 -fold increase in phosphorylated PKA was observed not only with ET treatment but also with ET+LT treatment. Control vers us 100 ng/ml of LT was not statistically significant (p>0.05) nor was 500 ng/ml of ET versus the ET+LT dose response (p>0.05). There was no st atistical difference between ET and the various concentrations of ET+LT on PKA phosphorylation independent of LT c oncentrations. Erro r bars indicate the SEM results of 3 separate experiments. B) Western Blot depicting a time course of VASP phosphorylation at S157 in HeLa cells after ET+LT treatment at 500 ng/ml of each toxin. As with PKA phosphorylation, VASP phosph orylation has been initiated prior to LT entry into the HeLa cell. = p<0.001 compared to control treated cells.

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98 Figure 417 Effects of ET+LT on intracellular cAMP levels ov er time. A) T ime dependent response of ET and ET+LT induced intracellular cAMP levels in human neutrophils. Neutrophils were treated with 500 ng/ml of ET (weight ratio of 5:1) or 500 ng/ml of ET and 100 ng/ml of LT (weight ratio of 5:1:1) and incubated at 37 C. Error bars indicate the SEMs of the results of 2 separate experim ents run in duplicate. B) Western bl ot analysis of extracts from buffer only treated (Ctl) and 100 ng/ml LT treated HeLa cells over a 2 h time course using an anti amino terminal MEK antibody. Under the conditions of our experiment MEK was almost fully cl eaved by LT at the 2 h time point. Western blot image is representative of 2 separate experiments. D) D ensitometry readings of band intensity from MEK cleavage following LT exposure. = p<0.001 and ** = p<0.0001.

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99 CHAPTER 5 DISCUSSION ET and ET+LT Inhibit Neutrophil ActinBased Assembly and Impair Listeria monocytogen e s Intracellular Motility Neutrophils are a primary component of the innate immune response and are the earliest responder s to invasion by bacterial pathogens (213) Recently, we investigated the effects of lethal toxin (LT) on neutrophil motile function and discovered that relatively low concentrations of lethal toxin (50100 ng/ml) impair neutrophil chemotaxis and chemoattractant -induced actin assembly (39) Both LT and ET have been shown to markedly impair activation of neutrophil NADPH oxidase activity ; thus disarming the powerful superoxide bactericidal system (49) Less is known about the effects of edema toxin on cell motility. Over two decades have passed since the effects of ET on human neutrophil chemotaxis were l ast examined (214) Recently investigators have been able to express edema toxin in E. coli and have shown that this purified recombinant protein has comparable binding affinity and biological activity to toxin purified f rom B. anthraci s (192, 193) This advance has allowed us to reexamine the biological effects of this calcium -sensitive calmodulindependent adenylate cyclase on neutrophil motility. Unlike the original study that noted a doubling of directed neutrophil migration in response to ET as well as to the combination of PA + EF + LF (191) w e fi nd that ET treatment resulted in a concentration dependent reduction in neutrophil chemotaxis (Fig. 4 -2G). We utilized a different assay for chemotaxis, video microscopy of neutrophils adherent to a fibronectin-coated surface, rather than migration throug h agarose, and our utilization of this assay may account for our contradictory findings. As further support for ET mediated impairment of chemotaxis, we find that ET -treatment also reduces

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100 chemokinesis (Fig. 4 -2A) and the ability of neutrophils to polarize in response to the chemoattractant FMLP (Fig 4 -2E). These findings suggested that ET may globally impair neutrophil actin assembly, and our assessment of filament assembly kinetics using A lexa phalloidin staining revealed that ET slows both the onset and extent of chemoattractant -stimulated neutrophil actin assembly (Fig. 4 5 A). The ET -mediated reduction in actin filament content is accompanied by distinct changes in the actin filament localization. Rather than homogeneously concentrating at the leading edge in lamellipodia, as observed in untreated neutrophils exposed to FMLP, actin filaments in ET -treated neutrophils concentrate in discrete small foci dispersed throughout the cell near the adherent membrane surface. This staining pattern is reminiscent of actin filament clusters associated with focal contacts in neutrophils adhered to uncoated plastic surfaces, a condition shown to stimulate actin assembly in the absence of FMLP (154, 187) and suggests that ET may act through similar signal transduction pathways. Because systemic anthrax infection would be expected to expose neutrophils to the combination of lethal factor, edema factor, and protective antigen we also examined the effects of this combination. We found that inhibition of chemotaxis, chemokinesis, polarization, as well as FMLP -induced actin assembly are additive. These findings suggest that LF and EF act by different pathways to impair actinbased motility. In addition, the combination of both toxins can reduce the surface expression of the adherence receptor CD11/CD18 (Fig. 4 -4 ) and this effect may contribute to the poor delivery of neutrop hils to the sites of infection. It has previously been reported that cAMP elevating agents lead to decreased respiratory burst of neutrophils due to decreased CD18 and impaired actin filament assembly (215) Given the close

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101 association between the cytoskeleton and integrins it is likely th at the marked reduction in actin filament assembly induced by dual toxin treatment may contribute the reduction in CD11/CD18 surface expression. ET -mediated activation of PKA would be expected to phosphorylate and inactivate the actin regulatory protein VA SP, as well as activate the G -proteins CDC42 and Rac (216) while LT would be expected to block the activation of ERK a necessary step in early focal contact formation (217) Future experiments will focus on assessing the contributions of these pathways to adherence and actin assembly. It is of interest that in HeLa cells this same combination does not result in an additive reduction in Listeria actin based motility, each indiv idual toxin, as well as the combination resulting in similar inhibition (Fig. 4 6 ). However, Listeria bypasses many of the signal transduction pathways required for FMLP -induced actin assembly, and therefore, the pathways by which these two toxins inhibit Listeria may differ from receptor -induced actin assembly. The shape changes associated with chemotaxis, cel l spreading, and phagocytosis are complex and make analysis of rates and directionality of actin assembly difficult. Furthermore, these events invol ve several steps of receptor mediated signal transduction. Listeria intrace llular infection and movement are a particularly useful model for examining the parameters of in vivo actin assembly, allowing discrete temporal and spatial resolution of actin filament formation (218) We have recently discovered that one of the primary dow nstream targets for LT is the actin monomer sequestering protein heat -shock protein 27 (198) The i nability of LT -treated cells to phosphorylate Hsp27 prevents the shuttling of actin monomers to the leading edge of motile cells (198) We are presently beginning to explore the pathway

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102 or pathways b y which ET interferes with actin assembly. As previously reported (49, 191) we find t hat ET induces a rise in c AMP levels, and under the conditions of our experiments we observe a >50 fold rise of c AMP in neutrophils. Our findings are consistent with previous observations in neutrophils and T lymphocytes showing that a gents inducing a rise in c AMP impair actin assembly and chemot axis (67, 189, 214, 219-221) However, a simple quantitat ive relationship between c AMP levels and alterations in chemotaxis has not been observed, suggesting that additional signal transduction pathways can modify the effects of c AMP (67, 214, 219) For the first time we document tha t the ET -induced rise in c AMP is accompanied by the phosphory lation of PKA in human neutrophils. Our findings are consistent with previous observations that ET treatment increases c AMP levels and activates PKA in many other cell types (196, 222224) Our experiments utilizing the intracellular bacteria Listeria help to narrow the potential downstream targets for ET and activated PKA. Li steria requires the bacterial surface protein ActA, and this protein directly activates the host cell Arp2/3 complex (225) ActA also attract s the actin -reg ulatory protein VASP (139) On the other hand, Shigella requires the bacterial surface protein IcsA (226) IcsA directly attracts and activates the host cell prot ein N -WASP, and this protein in-turn activates Arp2/3 complex (227) Also Shigella unlike Listeria does not require VASP for intracellular ac tin based motility (208) Finally, Listeria requires PI3 kinase activity, while Shigella does not (218) Thus the ability of ET to impair Listeria but not Shigella actin b ased motility, points to three potential mechanisms of ET action: 1. impairment of ActA -induced activation of the Arp2/3 complex, 2. inhibition of VASP binding or activation by ActA, or 3. inhibition of PI3 kinase activity. It is also possible that an

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103 addi tional previously unappreciated difference in the pathways by which Listeria and Shigella direct the actin -regulatory protein pathways of the host cell accounts for our observations. VASP Phosphorylation Impairs Neutrophil and Listeria Actin Based Motility Inhalation anthrax is a rapidly progressive disease that is associated with high levels of mortality with death occurring shortly after the onset of symptoms (228) Genomic analysis has revealed that, as compared with its close phylogenetic neighbor Bacillus cereus, B. anthracis is unique in possessing edema and lethal toxin (229) These toxins primarily enhance anthrax virulence by paralyzing the hosts innate immune response. Neutrophils are a primary component of the innate immune response and are considered the first r esponders durin g infection. Recently, we showed that ET impairs neutrophil chemotaxis and chemoattractant induced actin assembly (106) Previous work from our lab and others (106, 222224, 230) has shown that ET induces a marked rise in cAMP and this rise is accompanied by the phosphorylation of PKA, a condition that would be expected to activate this kinase. In addition to reducing actin filament assembly and chemotaxis in human neutrophils, ET slows Listeria monocytogenes actin based intracellular motility in HeLa cells, but has no effect on another actinmediated process, intracellular motility of Shigella flexerni (1). Listeria is known to require VASP for efficient intracellular motility, while Shigella does not (231) Furthermore, VASP S157 is known to be a substrate for PKA phosphorylation (63, 111, 144, 145, 148, 160, 184, 232) These observations point to VASP as a potential downstream target for ET -mediated neutrophil paralysis.

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104 Ena/VASP was originally discovered in platelets as a substrate for PKA and PKG (233) VASP localizes to sites of dynamic actin remodeling, such as filopodia, lamellipodia and focal adhesions (147, 159) VASP has several effects on actin including enhancing actin assembly (164, 234) and bundling of actin filaments (153, 164, 179, 234-236) Phosphorylation of VASP at S157 (VASP P157) has been shown to lower the affinity of VASP for actin filaments (163, 237) as well as the SH3 domains of Abl (63) Src and II-spectrin (237) However, the in vivo contribution of VASP phosphorylation to the regulation of actin filament assembly and architecture remains unclear. Chemoattractant stimulation of human neutrophils induces a rapid and transient PKA -dependent phospho rylation of VASP Ser 157, the percentage of total VASP phosphorylated at S157 increasing from 40 to 60% within 30 s of exposure to FMLP, and returning to baseline within 20 minutes. In ET -treated PMN, VASP phosphorylation persists for greater than 2 h prev enting the cycling of VASP between the phosphorylated and dephosphorylated forms (See Fig. 4 -7 ). ET treatment also results in an increase VASP P157 content at the leading edge of neutrophils associated with impaired chemotaxis, chemokinesis and neutrophil polarity, suggesting that persistent VASP phosphorylation may impair neutrophil actin -based motil ity. In addition to neutrophils, accumulation of phosphorylated VASP S157 at sites of high actin dynamics has been observed in the cell periphery of forskolintreated endothelial cells (165) ET treatment also results in an increase in VASP P157 content around intracellu lar Listeria (Fig. 4 -8 ) and is associated with a decrease in Listeria actin based motility (106)

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105 The above findings suggest that persistent phosphorylation of VASP S157 impairs actin based motility. To substantiate this possibility we utilized recombinant pseudophosphorylated and unphosphorylated VASP constructs and introduced them into neutrophils utilizing a TAT -chimera proteins and rescued ENA/VASP null MVd7 cells with VASP wild -type and mutant proteins Consistent with ET treatment, introduction of pseudophosphorylated VASP (S157D) impaired neutrophil chemokinesis and polarity and slowed Listeria motility in MVd7 cells (Figs. 4 9 4 -11 and 4 -12 ). While introduction of pseudounphosphorylated VASP (S157 A) accelerated Listeria motility and rendered both MVd7 cells and neutrophils resistant to the inhibition of actin -based motility by ET. Using these same methods we found that pseudophosphorylated and unphosphorylated VASP S239 had no effect on actinbas ed motility in our cells. We conclude that persistent VASP S157 phosphorylation is the primary mechanism by which anthrax edema toxin impairs actin based motility. An unexpected additional dividend of our investigations was the discovery of an association between VASP phosphorylation and filopodia formation. Filopodia have been implicated in a number of cellular processes including neuronal growth cone path finding, embryonic development, wound healing, and metastasis. Recent work in neuronal cells demonst rates the crucial role Ena/VASP proteins have in filopodia formation (211) Similarly, in Dictyostelium genetic ablation of dVASP, the sole Ena/VASP member present in the organism, decreases filopodia formation and causes chemotactic defects (143) Hyperphosphorylation of VASP S157 in human platelets is associated with hyper -production of filopodia and increased platelet aggregation in response to collagen (166, 238) Much to our initial surprise, we discovered that ET

PAGE 106

106 treatment of human neutrophils significantly increased the number of filopodia per cell. Similarly introduction of pseudophophoshorlated VASP S157D increased while pseudo unphosphor lated VASP S157A reduced filopodia number (Fig. 411). Listeria are also known to form membrane projections that mimic the actin architecture of filopodia (157) and we found that introduction of pseudophophorylated VASP significantly increased the number of membrane projections containing bacteria (Fig. 49 ). Such membrane projections push into adjacent cells and promote Listeria spread to adjacent cells while avoiding the extracellular environment. Thus VASP phosphorylation not only accounts for ET effects on actin based motility, and is also likely to pla y an important role in Listeria cell -to -cell spread. It has been proposed that Ena/VASP protein promotes actin filament bundling (179, 235, 239, 240) bringing filaments in to closer proximity to allow fascin to strengthen filopodia bundles, and fascin as well as VASP are found in both filopodia and Listeria membrane projections (241-243) It is of interest that filopodia fail to form in Dictyostelium lacking VASP and are markedly reduced in neural cells lacking Ena/Mena/VASP (143, 244, 245) To test the possibility that VASP S157 phosphorylation enhances VASP actin filament bundling; we compared the bundling func tion of purified wild-type, pseudophosphorylated and pseudo-unphosphorylated VASP, but found no differences in their abilities to bundle purified actin filaments (Figure 6). We suspect that enhanced binding of phospho-VASP to Abl, -spectrin (163, 237) may f acilitate filopodia formation, a nd these possibilities warrant future investigation. The profound effects of ET on VASP S157 phosphorylation, cell motility and filopodia formation emphasize the importance of cAMP/PKA pathway for actin -based

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107 motility and filopodia formation. These findings not only help to explain how anthrax impairs the innate immune system and Listeria spreads from cell -to cell, but also m a y have relevance for neurite outgrowth, platelet shape change, wound healing, embryonic development and tumor metastasis. Cell Entry and Manipulation of Host Signaling Pathways The i nnate immune response is the first line of defense against invading pathogens and it plays a central role in acute anthrax infection. B. anthracis trigger s a strong inflammatory response by activating TLR4 on neutrophils and macrophages at the entry site into the host. LT and ET interfere with this process, as the MEK pathway cascade is essential for full induction of the oxidative burst and pro inflammatory cytokine expression (246) for cell migration (32) T he strong increase in cAMP induced by ET is a potent inhibitor of these processes (247) In general, bacterial toxins that increase cAMP levels mostly effect immune cells and dampen the immune system to facilitate the survival of bacteria, contributing to the establishment of infection (58) Of the two toxins, LT has been more extensively investigated, with little attention has been paid to ET and ET+LT During systemic anthrax infection, neutrophils are exposed to both LT and ET. Therefore, we examined the effects of ET+LT on human neutrophils. It was of great interest to find that exposure of neutrophils to ET+ LT inhibits the rise in intracellular cAMP levels (Fig. 4-14). cAMP levels are down-regulated by phosphodiesterases (63) Human neutrophils were treated with ET and LT in concert with t heophylline, a known phosphodiestease inhibitor to determine if LT had phosphodiesterase like characteristics (Fig. 4 -15). These results led us to conclude that LT does not act b y activating phosphodiesterases and appears to inhibit the increased cAMP levels by

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108 impairing one or more MEK pathways. Even though LT impaired the intracellular increase in cAMP levels there was still sufficient time for ET to enter the cell and cause its effects because there was no effect on PKA phosphorylation between ET and ET+LT and these effects were independent of the concentration of LT (Fig. 4 -17). ET is still able to cause a two-fold increase in PKA phosphorylation after LT treatment. Timing of toxin affects came to the forefront as an important factor. A time course of ET infection showed that cAMP levels began to increase at 30 minutes post exposure and continu ed to increase throughout the two hour time-course (Fig. 4-18). Neutrophils were subject ed to the same time-course but were exposed to ET and LT. Neutrophils exposed to both toxins were able to increase int racellular cAMP levels until 1.5 hours post exposure when the levels began to decrease and by two hours were at physiological ly normal levels. This led us to speculate that ET may only require an hour to affect cell physiology increase intracellular cAMP levels and activate downstream effector molecules. Previous work done by our lab (39) has shown that LT requires a min imum of two hours for near complete cleavage of MEKs (Fig. 418), and this MEK cleavage at two hours correlates with maximal inhibition of neutrophil chemotaxis after LT treatment. A similar time course of chemokinesis and polarity was done with neutrophils exposed to ET (Fig. 43). We found that the mechanism of LT action takes longer due to the proteolytic cleavage of the MEKs, while once ET is in the cell I can immediately serve as an adenylate cyclase. There must be a biological basis for the difference in the way ET and LT are processed. ET and LT are unique toxins in that they both share the common receptors of TEM8 and CMG2 for binding moieties and subsequent entry into the cell (31, 32) An

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109 increase in surface expression of receptors has consequences for both toxins. It has recently been shown by Maldonado Arocho, et al (51) that ET is able to increase the mRNA levels of both receptors in murine macrophages. Due to the early entry of ET, we speculate t his allows ET to increase receptors during the first 2060 minutes allowing increased entry of LT. Perhaps the concentration requirements of LT to effectively cleave all the MEKs are higher necessitating ET to aid in increased LT entry. This would be of technical benefit for allowing more complete paralysis of the innate immune function allowing anthrax to readily survive in the host. ET is a key component utilized by B. anthracis to thwart the host immune defenses and potentially helps to establish a s uccessful infection. Having continuously high levels of cAMP may be deleterious to the bacteria itself. With Bordetella pertussis, an adenylate cyclase producing bacteria generates high levels of cAMP for a short period of time and then down-regulates cA MP for the duration of infection (249) LT appears to decrease ETs increase in intracellular cAMP similar to what B. pe rtussis does once it has established an infection within the host. Similar to B. pertussis ET is important in establishment of infe ction but prolonged exposure to supraphysiological levels of cAMP could hamper the bacteria in the long run. It appears tha t allowing LT to enter the cell later gives ET time to establish its downstream mechanism of action. The exact mechanism of action is the bases of future research in our lab.

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110 CHAPTER 6 FUTURE WORK Vascular Sepsis Caused During Inhalation Anthrax Hemorrhage and pleural effusion are prominent pathological features of systemic anthrax infection. There is growing evidence for LT having a direct effect on endothelial cell function during infection ultimately leading to septic shock. Primary human end othelial cells exposed to LT have previously shown that the loss of MEK function inhibits the ability of p38 phosphorylation and its s ubsequent downstream effects on phosphorylating the actin monomer binding protein H sp27 (198) Blockage of hsp27 phosphorylation by LT prevents hsp27 from releasing sequestered ac tin monomers for new a ctin filament assembly (198) LT induces a time and concentration dependent decrease in transendothelial electrical resistance (impedance) and an increase in permeability of fluorescently labeled albumin (unpublished work) (250) These changes in permeability following LT treatment are accompanied by altered distribution o f actin fibers and the adherence junctional protein VE -cadherin, which are both necessary fo r bar rier function (250) These findings support a role for LT induced barrier dysfunction in the vascular permeability chang es associated with systemic anthrax infection. To date the effects of ET on vascular endothelial cell junctions have not been studi ed. Previous research (220) has shown that cAMP inducing agents like Fsk inhibit F actin assembly in stimulated T cells via the direct activation of the cAMP PKA pathway. We have shown that ET, LT and ET+LT at low concentrations impair neutrophil chemotaxis and chemotactic movement, which is associated with the ability of neutrophils to undergo actin assembly (251) ET a ffects actin assembly by increasing cA MP, which activates PKA causing the phosphorylation of VASP at S157 to

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111 impair F actin polymerization (in press). Our prior research suggests that in addition to VASP phosphorylation and inhibition of H sp27 phosphorylation ET and LT are likely to alter the functions of one or more additional actin regulatory proteins. The vascular system is integrated with endothelial cells that serve as a barrier and join junctions together to retain fluid and red blood cells within the intravascular space. Breakdown of cell -cell junctions can lead to extravasations of intravascular fluid resulting in shock One mechanism by which pathogens can weaken the endothelial cell -cell junctions is by altering the underlying actin cytoskeleton. There are two types of junctions, adherens junctions and a more exclusive barrier called the tight junction (Figure 61 ). VE-cadherin is the outer most protein that serves to glue the vascular cells together in the adherens junctions. The intracellular portion of VE -cadherin binds to ca tenin, -catenin, ac tinin and vinculin. -catenin, actinin and vinculin can all bind to actin filaments and serve to attract large bundles of filamentous actin to this region to help stabilize the cell junction (252) However, the mechanisms underlying actin filament assembly and disassembly in the region of cell -cel l junctions are poorly understood. We do know that disassembly of actin filaments impairs cell junctions indicating that actin filaments are vital for the integrity of the cell junctions. Once adherens junctions have formed tight junctions can be cr eated. Tight junctions prevent small molecules from leaking through the vascular endothelial junctions. Two transmembrane proteins occludin and claudin are the primary components (253) Phosphorylation of occludin is required for proper membrane localization. Claudin -5 possesses a binding sequence for ZO -1 (254) which associates

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112 with the cytoskeleta l protein -spectrin. -spectrin in tu rn binds VASP (237) which provides a link between tight junctions to the underlying actin cytoskeleton. The ultimate consequence of inhalation anthrax is septic shock, which is 80100% lethal even with proper treatment. To date, the only treatment for sepsis is supportive care because we do not understand the underlying physiological condition to provide better care. One of the major components responsible for maintaining the integrity of vascular junctions is filamentous actin. The signal transduction mechanisms that regulate a ctin found at cell junctions are poorly understood. Future work will analyze septic shock and promises to lead to new therapeutic approaches for all forms of septic shock associated with vascular leakage. A better understanding of biochemical pathways regulating vascular cell junctions could also provide new therapies to alter the permeability of the blood brain barrier and allow for more efficient delivery of chemotherapeutic agents to the cerebral cortex. The Role of VASP in Filopodia Formation The actin cytoskeleton plays a central role in cell -cell junctions. Over time the actin cytoskeleton is able to remodel itself along with remodeling th e cell -cell junctional proteins (255) The exact mechanism underlying this rearrangement has yet to b e fully elucidated. Several reports suggest that small GTPases of the Rh o family may be involved (256) Recent work on adherence junctions has found that when primary cells are stimulated, they form intercellular junctions by an active process dr iven by actin polymerization. The process requires the production of filopodia, which penetrate neighboring cells. The physical force exerted by the filopodia draws the opposing cells together to form adherence junctions. Vasioukhin, et al. (168) were able to show that

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113 production of filopodia with adherence proteins at the tip yielded adherence junction, whose stabilization depended on -catenin, VASP, and actin polymerization and reorganization. To date, limited research on epithelial cells and the formation of adherence junctions has been performed, an d virtually no studies have examined the formation of both adherence and tight junctions in endothelial cells In our work with neutrophils and MVd7 cells we found that VASP isoform S157D increased filopodia formation (Fig. 4-14 and Fig. 416 ). This is in direct correlation with what was recently reported in macrophages. Kim et al. (224) found that the filopodia increased in quantity after ET treatment in macrophages but they also found an increase in chemokinesis where as we have found a decrease in neutrophil ch emokinesis. We attr ibute the diffe rence in chemotaxis to the different nature of cell motility in neutrophils and macrophages. The forward movement of the macrophage requires the extension of the filopodia and lamellipodia. In neutrophils filopodia formation is not common and the neutrophil relies on the extension of the lamellipodia for forward movement (257) It has been shown that when primary endothelial cells are stimulated, they form intercellular junctions by an active and dynamic process of filopodial formation, which penetrates and embeds into neighboring cells. The physical force of drawing two opposing membranes together is maximized at the tips of the filopodia, by cl usters of ad herence junctional proteins, VASP, vinculin and zyxin. The stabilization of the junctions depends on VASP and actin polymerization and reorganization (168) We hypothesize that ETs enhanced filopodia formation will serve to at least partly reverse LTs effects on vascular tight junctions.

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114 Figure 61. Schematic Diagram of Vascular Endothelial Junctions. There are two types of junctions: 1) Adherens junctions (lower junction) made up of VE -cadherin, the catenins, alpha actinin, and vinculin. 2) Tight Junctions (upper junction) made up of oc cluding and claudins, ZO 1, spectrin and VASP (black). Both junctions link to the actin cytoskeleton (red helical structures).

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136 BIOGRAPHICAL SKETCH Sarah Elizabeth Szarowicz was born Sarah Elizabeth Guilmain of Massachusetts. Sarah grew up the only daughter and middle child of two hard working middle class parents. Sarah began her higher educational career by attending Becker College in Leicester, M A where in May of 2000 she received her AS in Veterinary Technology. Her desire and passion for education sent her that fall to the University of Maine to attain her BS in May 2002 in animal and veterinary science. Sarah then decided to try her hand at r esearch and was accepted into the University of Maines masters program in animal science. During her masters program, Sarah had the great fortune of working with a group of passionate professors who gave her the drive to ask questions and further spa rked her interest in research. After completing her masters on a project of hypothyroidism on hyt/hyt mice Sarah received a job working at the National Institutes of Health (NIH) working with Dr. Jim Pickel in the mental health mouse transgenic core faci lity. Here, Dr. Pickel pushed Sarah to learn to work independently on various research projects she could call her own and he encouraged her to continue her education. Also, while working at NIH Sarah met her husband Mark Szarowicz. Sarah was accepted i n the summer of 2006 to the Inter -Disciplinary Program at the University of Florida. It was here that she found her mentor Dr. Frederick Southwick and worked to obtain her PhD in immunology and microbiology. Sarah has taken advantage of several opportuni ties to teach not only undergraduate students but also high school students in infectious diseases and the great opportunities that working in a lab on research can provide for undergraduate students. While attaining her PhD, Sarah

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137 married her husband on O ctober 04, 2009 in an intimate wedding held at the Bauman Center located on the campus of the University of Florida overlooking Lake Alice. Sarah obtained a post doctoral position at the United States Army Medical Research Institute of Infectious Disease ( USAMRIID) where she will continue striving for excellence in infectious disease research.