sign in sign up
<<
Home , Anthrax toxin, Bacillus anthracis, Cytolysin, IC3b, Ruff (cards)

INTERACTIONS OF ANTHRACIS WITH THE INNATE DURING EARLY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Christopher Premanandan, DVM

The Ohio State University

2007

Dissertation Committee :

Michael D. Lairmore, DVM, PhD, Approved by Advisor

Andrew J. Phipps, DVM, PhD, Advisor Advisor

Jeffrey Lakritz, DVM, PhD Advisor

Larry Schlesinger, MD Veterinary Biosciences Graduate Program ABSTRACT

Bacillus anthracis is a gram-positive, forming facultative anaerobic bacterial organism, which is the etiologic agent for the disease . Infection with this organism traditionally manifests itself in cutaneous, gastrointestinal or pulmonary anthrax. Pathogenesis of the disease is dependent on the ability of the bacterial to evade the immune system, germinate to the vegetative organism and secrete . Pulmonary anthrax is the form associated with the highest mortality in human beings and is considered a significant threat due to its potential as a bioterrorist weapon.

Two receptors have been currently identified, tumor endothelial marker 8 (TEM8) and capillary morphogenesis 2 (CMG2).

Both receptors are thought to be capable of binding and translocating the A-B components of to the of susceptible cells.

The expression of these receptors in human primary , which are considered to be a target of intoxication during infection with B. anthracis, is not well documented. In Chapter 2, we examined the functionality and the expression of mRNA transcripts for both receptors in primary mononuclear and several immortalized lines. We assessed receptor functionality by comparing the ability to translocate edema factor to the cytosol in several immortalized cell

ii lines as well as primary mouse and human macrophages. We concluded that most immortalized cell lines and primary macrophages express functional anthrax toxin receptors; however, the degree of intoxication varied between cell types. In addition, we examined the expression of TEM8 and CMG2 mRNA transcripts in human and mouse cell lines, primary macrophages and mouse tissues by standard and real-time RT-PCR. Our results indicated that CMG2 transcripts are preferentially expressed over TEM8 transcripts in primary human and mouse macrophages.

Alveolar macrophages are thought to play a central role in the pathogenesis of inhalational anthrax. However, the receptors present on macrophages that mediate of Bacillus anthracis have yet to be clearly defined. In Chapter 3, in order to determine if soluble factors that are present in the lung such as immunoglobulin and complement are involved, we characterized the binding of human IgG and the complement protein C3 to the surface of B. anthracis spores at different concentrations in nonimmune human serum. Furthermore, we investigated the importance of nonimmune human serum in the phagocytosis of B. anthracis spores by human -derived macrophages. We showed that B. anthracis spores activate the - dependent classical complement pathway leading to the deposition of C3 fragments on the spore surface. Furthermore, we showed that C3 serves as an for B. anthracis spores resulting in enhanced phagocytosis by human macrophages. These studies provide evidence that nonimmune serum contains

IgG which binds to B. anthracis spores and initiates activation of the classical iii complement pathway but itself is not sufficient to initiate phagocytosis. Thus, C3 opsonization of B. anthracis spores provide a mechanism for enhanced phagocytosis of spores by human macrophages within the context of the lung.

Our findings in Chapter 3 led us to hypothesize that antibody against other

Bacillus species, such as spores, would be cross reactive with B. anthracis spores. In addition, we were interested in determining the role of these cross reactive in complement fixation. We elicited against B. cereus and B. anthracis spores in mice and showed by spore ELISA that this cross reactivity does take place. We subsequently showed

C3b deposition takes place when spores are incubated in antiserum against B. cereus and B. anthracis Sterne. This was higher than the amount of deposition on spores incubated in unvaccinated mouse serum or serum from mice vaccinated with recombinant B. anthracis protective . Phagocytosis of B. anthracis spores by mouse peritoneal macrophages is greater in the presence of serum from mice vaccinated with spores as compared to unvaccinated mouse serum or serum from mice vaccinated with recombinant B. anthracis protective antigen. Phagocytosis in serum from spore vaccinated mice is mediated by both complement and immunoglobulin while phagocytosis in the presence of unvaccinated mouse serum or serum from mice vaccinated with recombinant protective antigen is dependent on complement deposition.

In conclusion, we show that functional anthrax toxin receptors, predominately CMG2, are present in primary mononuclear phagocytes, which provide target for intoxication and possible mechanism for escape during early iv infection. In addition, we show, for the first time, that complement opsonization plays a role in modulating the interaction between B. anthracis spores and mononuclear phagocytes.

v To my parents, Rosalin and Joseph, my sister, Mary and my aunt, Maria

vi ACKNOWLEDGMENTS

I would like to first acknowledge Michael Lairmore. The support he has given me began when I was a veterinary student at the college many years ago.

Andrew Phipps, my primary advisor for the work described in this thesis.

Many unexpected turns took place during the generation of the data presented here and Andrew’s expertise in was invaluable in seeing these projects through to the end.

My committee members, Larry Schlesinger and Jeffrey Lakritz, who were both invaluable resources and provided guidance in both experimental design and writing of manuscripts.

I am indebted to Craig Storozuk and Susie Vogel, who were directly involved in our Bacillus anthracis research and had significant roles in the work described here.

The work presented here and my development as a graduate student would not be possible without the support and the intellectual contributions of the past and present members of the Lairmore lab, Andy Montgomery, Antara Datta,

Bevin Zimmerman, Bindhu Michaels, Krissy Rodgers, Evan Ware, Hajime

Hiraraji, Jennifer Huck, Laurie Millward, Lee Silverman, Nicole Sabo, Raj Nair,

Rashade Haynes, Robyn Haines and Seung Jae Kim. In particular, I would like to

vii thank Rashade, Antara and Bevin, who were the “core” of our lab group in my time here, for their friendship and support. Much of the work presented here required the great technical skills of several individuals. Many thanks go to Anne

Saulsbery for her assistance with the histopathology studies as well as Elizabeth

Wheeler for flow cytometry and confocal work. Also, thanks go to Tim Vojt, for his excellent scientific illustrations and animations and Kate Hayes for her critical evaluation of our manuscripts.

Leonard Tinney and Herbert Betts for getting me started in the veterinary medical field.

And finally to my parents and family, who have supported me through all of this education and training. I would never have made it this far without them.

viii VITA

August 29, 1975 ……………………... Born – Philadephia, Pennsylvania

1997 …………………………………... Bachelor of Science, Zoology Bachelor of Arts, Classical Humanities Miami University, Oxford, Ohio

2001 …………………………………... Doctor of Veterinary Medicine The Ohio State University, Columbus, Ohio

2001-2002 ……………………………. Staff Veterinarian Navajo Nation Veterinary Program Window Rock, Arizona

2002-2004 ……………………………. Graduate Research Associate Department of Veterinary Biosciences The Ohio State University

2004-present …………………………. Post-Doctoral Fellow Department of Veterinary Biosciences The Ohio State University

PUBLICATIONS

1. Premanandan C, Lairmore M, Fernandez S, Phipps A. Quantitative measurement of anthrax toxin receptor messenger RNA in primary mononuclear phagocytes. Microb Pathog. 2006 Jul 17.

2. Phipps A, Premanandan C, Barnewall R, Lairmore M. Rabbit and nonhuman primate models of toxin-targeting human . Microbiol Mol Biol Rev. 2004 Dec;68(4):617-29.

FIELDS OF STUDY

Major Field: Veterinary Biosciences ix TABLE OF CONTENTS

Page

Abstract ...... ii

Dedication ...... vi

Acknowledgements ...... vii

Vita ...... ix

List of Tables ...... xiii

List of Figures ...... xiv

Chapters

1. Literature Review ...... 1

1.1 The History of Anthrax ...... 1

1.1.1 Human and Veterinary Anthrax Vaccines ...... 3

1.2 Bacillus anthracis Microbiology, Ecology and Epidemiology ...... 4

1.3 Anthrax Pathogenesis ...... 7

1.3.1 Cutaneous, Gastrointestinal, and Pulmonary Forms of Anthrax...... 7 1.3.2 Bacillus anthracis Factors ...... 8 1.3.3 Interactions with Bacillus anthracis ...... 13

1.4 Murine Models of Bacillus anthracis Pathogenesis, Immunogenicity and Vaccine Efficacy ...... 18

x 1.4.1 Overview of Non-murine Models of Anthrax ...... 18

1.4.2 Anthrax Pathogenesis in Immune Competent Murine Models...... 20 1.4.2.1 Intraperitoneal Inoculation ...... 21 1.4.2.2 Subcutaneous or Intradermal Inoculation ...... 21 1.4.2.3 Pulmonary Exposure ...... 22 1.4.2.4 Gastrointestinal Exposure...... 23

1.4.3 Associated Systemic Pathologic Findings ...... 24

1.4.4 Anthrax Pathogeneis is Immune Incompetent Murine Models ...... 25 1.4.4.1 C5 Deficient Mice ...... 25 1.4.4.2 Irradiated Mice ...... 27 1.4.4.3 Leukocyte Depleted Mice...... 27

1.4.5 Immunogenicity and Vaccine Efficacy ...... 28 1.4.5.1 Immunogenicity and Vaccine Efficacy of Bacillus anthracis Protective Antigen, Edema Factor and Lethal Factor ...... 28 1.4.5.2 Immunogenicity and Efficacy of Inactivated Spore Vaccines ...... 30 1.4.5.3 Immunogenicity and Vaccine Efficacy of Bacterial Cells and PDG Capsule...... 31 1.4.5.4 DNA based and Viral vectored Vaccines ...... 32

1.5 Conclusions ...... 33

1.6 References ...... 33

2. Expression of Functional Anthrax Toxin Receptors in Mononuclear Phagocytes ...... 59

2.1 Introduction ...... 59 2.2 Materials and Methods ...... 61 2.3 Results ...... 69 2.4 Discussion ...... 72 2.5 References ...... 75

xi 3. Compleme nt Protein C3 Binding to Bacillus anthracis Spores Initiated by the Classical Pathway Enhances Phagocytosis by Human Macrophages ...... 89

3.1 Introduction ...... 89 3.2 Materials and Methods ...... 91 3.3 Results ...... 98 3.4 Discussion ...... 104 3.5 References ...... 108

4. Role of spore antibodies in complement mediated phagocytosis of Bacillus anthracis spores in primary macrophages ...... 118

4.1 Introduction ...... 118 4.2 Materials and Methods ...... 121 4.3 Results ...... 127 4.4 Discussion ...... 133 4.5 References ...... 137

5. Synopsis and Future Directions ...... 148

5.1 Introduction ...... 148 5.2 Further characterization of expression of anthrax toxin receptors and associated in mononuclear phagocytes ...... 148 5.3 Characterization of anti-spore antibodies found in naïve human serum ...... 150 5.4 Comparison of survival of B. anthracis spores in different phagocytic pathways and further characterization of spore ...... 152 5.5 Summary ...... 155 5.6 References ...... 155

Bibliography ...... 158

xii LIST OF TABLES

Table Page

Table 1.1 Mouse strain susceptibility to B. anthracis ...... 53

Table 2.1 Absolute cAMP concentrations per 3 x 104 cells treated with PA, EF or ET ...... 80

Table 2.2 RT-PCR primer sequences and expected amplicon size ...... 81

Table 2.3 TEM8 and CMG2 mRNA expression in immortalized cell lines, primary mononuclear phagocytes, and mouse tissues by RT-PCR ...... 82

xiii LIST OF FIGURES

Figure Page

Figure 1.1 An electron photomicrograph of a spore of Bacillus anthracis Sterne...... 54

Figure 1.2 Schematic detailing the process of toxin ...... 55

Figure 1.3 A/J mouse model of gastrointestinal anthrax ...... 56

Figure 1.4 A/J mouse model of pulmonary anthrax...... 57

Figure 1.5 Photomicrographs of a serial time course study detailing the cutaneous histopathology of A/J mice inoculated subcutaneously with B. anthracis Sterne...... 58

Figure 2.1 Lethal factor cytotoxicity assay representing a decrease in viability in J774A.1 cells...... 83

Figure 2.2 Edema factor cytotoxicity assays...... 84

Figure 2.3 Primer design strategy for TEM8 and CMG2...... 85

Figure 2.4 TEM8 isoform expression by standard RT-PCR...... 86

Figure 2.5 mRNA expression of TEM8 and CMG2 by real time RT-PCR...... 87

Figure 2.6 mRNA expression of TEM8 and CMG2 by real time RT-PCR after stimulation of MDMs with spore infection, LPS or IL-1β...... 88

Figure 3.1 Phagocytosis of B. anthracis spores by human MDMs...... 112

xiv Figure 3.2 Intracellular spores as detected by light microscopy per 50 human MDMs (phagocytic index)...... 113

Figure 3.3 Detection of human IgG which binds to B. anthracis spores by ELISA...... 114

Figure 3.4 Detection of C3b deposition on Bacillus anthracis Sterne spores as shown by spore C3b ELISA...... 115

Figure 3.5 Detection of C3b deposition on Bacillus anthracis Sterne spores as shown by spore immunoprecipitation and western blot (Part 2)...... 116

Figure 3.6 Detection of spore binding antibody and C3b deposition by fluorescence assisted cytometric sorting (FACS)...... 117

Figure 4.1 Mouse anti-spore IgG response to vaccination with Bacillus anthracis Sterne (BAS), Bacillus cereus (BC), and protective antigen (PA)...... 141

Figure 4.2 Detection of complement protein C3b on B. anthracis Sterne spores by FACS analysis...... 142

Figure 4.3 Detection of complement protein C3b on B. anthracis Sterne spores by FACS analysis (Part 2)...... 143

Figure 4.4 Colony forming units of B. anthracis Sterne associated with infected mouse peritoneal macrophages (MPMs)...... 144

Figure 4.5 Phagocytic index of B. anthracis Sterne spores in mouse peritoneal macrophages...... 145

Figure 4.6 Erythrophagocytosis assay in mouse peritoneal macrophages which demonstrates FcγR sequestration...... 146

Figure 4.7 Phagocytic index of B. anthracis Sterne spores in mouse peritoneal macrophages following Fc sequestration...... 147

xv CHAPTER 1

LITERATURE REVIEW

1.1 The history of anthrax

Anthrax is a disease with a history which can be dated back to ancient times. The cause of the fifth and sixth plagues described in the Old Testament of the Bible are believed to have been caused by Bacillus anthracis, while the plague of Athens in 430 B.C. may have been caused by B. anthracis as well1.

The roman poet Virgil described a disease affecting humans and livestock which closely resembled anthrax1,2. The disease’s significance in veterinary history far predates that observed in human medicine1. However, pulmonary anthrax was recorded as a significant occupational disease with the exposure of mill workers during the 18th century1.

Fundamental knowledge of the pathogenesis of anthrax progressed significantly due to the work of and . In the late 1800s,

Koch observed the bacillus in the blood of infected cattle, elucidated the formation of the vegetative organism from the spore form and developed an

1 animal model of infection, which helped form the basis of his famous

postulates3,4. Pasteur, considered a rival in the study of anthrax pathogenesis at

this time, built upon Koch’s work by developing an attenuated strain, with which

he performed the first modified live anthrax vaccine efficacy study in cattle5.

Through the early 20th century, cases of anthrax in the

continued to be predominately a disease of occupational exposure associated

with animal hide workers (primarily goat hair and wool), and was most commonly

the pulmonary form1,6. By the late 20th century, the incidence of the disease had

decreased tremendously and a majority of cases reported in the United States

through the rest of the 20th century were cutaneous . Outside of the

U.S., approximately 100,000 to 300,000 cases were reported during the 20th century, a majority of these being cutaneous anthrax with fewer cases of gastrointestinal anthrax and virtually no cases of pulmonary anthrax7.

By the end of the 20th century, anthrax was considered a relatively rare

disease in the United States. In 2001, the first recorded outbreak of intentional

anthrax exposure in the U.S took place in multiple areas, including New York,

Florida, Connecticut and Washington, DC. These outbreaks were associated with

the opening of envelopes containing processed B. anthracis spores, resulting in

18 confirmed cases and four suspect cases of pulmonary anthrax7,8. Since the

events of 2001, there has been renewed interest in investigating the

pathogenesis of this disease9.

2 1.1.1 Human and veterinary anthrax vaccines

Although the first anthrax vaccination is commonly attributed to Louis

Pasteur’s use of an attenuated strain in sheep, a killed vaccine for the disease

created by the veterinarian John-Joseph Henri Toussaint, was successfully

tested in dogs and sheep in 18803. The vaccine was created by heating to

55 ºC for ten minutes. In 1881, Pasteur announced the development of a

“modified live vaccine”, which was tested in a famous challenge experiment on

May 31st, 1881 in a flock of sheep. Interestingly, Pasteur actually did not use a

modified live vaccine, substituting in killed bacilli for the test. Later the live

vaccine strain was adopted, along with Pasteur’s two dose schedule. Also in

1881, William Greenfield, a British physician, tested an attenuated strain with

similar results10,11. A more effective live attenuated vaccine was developed by

Sterne in the early 1900’s which is still in use today for livestock vaccination12-14.

In terms of human vaccination, most vaccines have been based around toxin components. However, it is reported that an attenuated live strain similar to

Sterne has been used in the former U.S.S.R5. In the mid 1900’s, a cell free

culture filtrate was developed in the and the United States

composed predominately of the toxin component, protective antigen (PA),

adsorbed to aluminum hydroxide5. The currently used PA vaccine is produced by

+ a similar but refined method utilizing B. anthracis V770-NP1-R, which is a pX01 ,

pX02- strain. Recombinant PA vaccines are under development, but not currently

used15.

3 1.2 Bacillus anthracis microbiology, ecology and epidemiology

Bacillus anthracis is a gram positive, non-hemolytic, -positive, spore-forming, facultative anaerobic bacterium. The size of the vegetative organism ranges from 1-1.5 x 3-10 μm and exhibits a thick capsule visible after staining with McFaydean’s methylene blue. The organism is non-motile, non­ hemolytic on sheep’s blood and forms characteristic large white ovoid colonies with a tacky consistency when touched with a microbiological loop. The vegetative cell is also gamma phage sensitive which aids in distinguishing B. anthracis from other species of Bacillus. The spore form of the organism is highly resistant to environmental extremes. The Bacillus anthracis spore is a multi­ layered structure consisting of an outer loose membrane called the , the spore coat located immediately under the exosporium, a compact inner envelope termed the cortex and the centrally located core, which the bacterial nucleic acid is stored (Fig. 1.1). These structures are critical to the spore’s ability to survive harsh environments. In particular, the cortex exerts “pressure” on the spore core in an effort to extrude water from it16. The low amount of water in the core is thought to aid in buoyancy, as well as help protect the nucleic acid from

UV light, heat and other environmental stresses. Although the spore form is considered metabolically inactive, environmental conditions can greatly influence its sturdiness and ability to germinate. Small molecules are able to diffuse through the exosporium, spore coat and cortex to the core. Calcium, in particular

4 is thought to play a major role in spore and structure. It has been shown that the core of the anthrax spore contains 90% of the endospore’s calcium. Also, calcium likely plays a role in protein and nucleic acid stabilization and that sporulation of the organism in calcium deficient media greatly decreases the spore’s ability to germinate16,17.

Germination into the vegetative cell is a critical step in the life-cycle of the organism. The microscopic changes associated with germination were first described in 1877 by Koch18. Almost a century later the morphologic changes of germination were described in finer detail using electron microscopy. The germination process has been described as three distinct steps19. Activation prepares the spore to germinate in the presence of the required germination signals. Structural changes do not appear to be associated with this stage.

Germination describes the transformation from a dormant non-metabolically active structure to the early vegetative cell. This phase is characterized with swelling of the core and by the disappearance of the inner aspect of the spore coat and the entire cortex. The exosporium remains intact. The nucleic acid elements of the early germinated organism are not segregated within the at this stage. Outgrowth, the last stage of germination, represents the maturation of the bacilli and subsequent division, which requires the typical nutrients necessary for bacterial growth. Multiple amino acids serve as the predominant germination signals for the organism, L-alanine being the most potent signal20. Proteins synthesis begins at this stage. The wall of the core

5 becomes the vegetative organism’s cell wall. The exosporium and spore coat are typically shed although small portions can remain adhered to the cell wall.

Anthrax is considered to be endemic in regions of the United States including regions from central Texas to South Dakota, as well as regions in

California. Multiple theories exist concerning the epidemiology of naturally occurring anthrax in livestock. The oldest theory was put forward by Van Ness in

1971 who described two major components that are factors in propagating the disease21. The first is the establishment of incubator areas, which commonly are rocky regions where water can collect or slow moving water can move through and evaporate (i.e. flash floods, etc). It was thought that the movement of water allows the collection of spores, which are buoyant structures. The second component is the soil conditions in endemic areas. Van Ness hypothesized that these were areas of high alkalinity, high moisture content and high organic matter content. With these two components, the organism could renew its soil population by multiple cycles of germination, replication and sporulation. While the theory of incubator areas may be correct, the cycling theory fell into disfavor due to the fact that the vegetative organism requires very specific conditions to grow and does not exist as a saprophyte. Subsequently a theory was put forward that the host plays a role in soil propagation. The involvement of a host animal would allow for multiple cycles of germination and sporulation, therefore propagation of the organism.

6 1.3 Anthrax pathogenesis

1.3.1 Cutaneous, gastrointestinal, and pulmonary forms of anthrax

Depending on the route of exposure, disease may present as either the cutaneous, gastrointestinal or the pulmonary form in humans. Cutaneous anthrax is the most common manifestation following infection by B. anthracis. The pathogenesis of the disease is initiated by the inoculation of spores into the dermis or the deep epidermis through a previously existing wound or by penetrating contaminated hair fibers. Following inoculation, the organism germinates and replicates in the local dermis and subcutaneous tissue. The lesions associated with cutaneous anthrax are focal pustule formation with furunculosis, necrosis and formation of the classical eschar. Although this form of anthrax is typically associated with the lowest morbidity and mortality, septicemia and toxemia can occur in severe cases9.

Gastrointestinal anthrax is the least studied and understood manifestation of the disease. Gastrointestinal anthrax is primarily associated with the ingestion of meat from livestock carcasses contaminated with the organism and is presumed to be the rarest form of the disease22. However, this may be due to inaccurate diagnosis of this form of the disease22. Clinical signs and symptoms in humans include nausea, bloody diarrhea, vomiting, ascites and hematemesis. It is thought that mucosal ulceration is present at the site of infection23. In addition, in a limited number of human cases, oropharyngeal anthrax has been

7 described22,24-26. This disease is not well recognized, however, the association with contaminated meat appears to be the same. The exact mechanism through which the organism breaches the mucosal barrier and gains access to systemic circulation is unknown.

In terms of world wide cases, pulmonary anthrax is considered the least common form of anthrax. However, the pulmonary form is associated with the highest mortality and has come to public attention due to its use as a bioterrorist weapon. Following deposition into the alveolus, the spore is phagocytosed by resident alveolar macrophages which then move into systemic circulation towards the regional lymph nodes. The organism then escapes by an unknown mechanism and replicates in circulation27. The clinical signs associated with pulmonary anthrax include the onset of “flu-like” symptoms early during infection followed by rapid development (usually within 24-48 hours) of respiratory distress. Untreated, the disease can progress to coma and death. Pulmonary anthrax is associated with hemorrhagic mediastinitis, pleural effusion and often in a majority of cases, hemorrhagic meningitis27.

1.3.2 Bacillus anthracis virulence factors

Once the organism has germinated and entered into circulation, virulence factors are expressed which both aid in the avoidance of the immune system and mediate systemic effects which are detrimental to the infected host. The most well documented factors of B. anthracis are the capsule, composed of poly-D­

8 glutamic acid (PDG) and components of an A-B type toxin, protective antigen

(PA), edema factor (EF) and lethal factor (LF). All of these virulence factors are associated. The capsule of the organism is encoded by the plasmid pX02 (95 kilobases), which contains the capsule synthetic (capBCAD).

Four open reading frames, capA, capB, capC and capE, have been shown to be required for adequate capsule synthesis in B. anthracis28,29. A fifth , capD, is required to anchor the capsule to the peptidoglycan of the bacterial cell wall30.

The poly-D-glutamic acid structure of the B. anthracis is unique to the organism as the glutamic acid used by other bacillary capsules are typical composed of a mixture of L and D isomers31. The capsule is also exhibits anti-phagocytic properties in vitro and synthesis has been shown to be required for effective dissemination in murine pulmonary infection32,33.

The most extensively studied of B. anthracis are the A-B toxins, lethal toxin and edema toxin. The components of lethal toxin are the 83 kDa protective antigen and lethal factor, a 90 kDa zinc metalloprotease. The components of edema toxin are protective antigen and edema factor, a 90 kDa adenylate cyclase. The three toxin components are encoded by the pagA, cya and lef located on the second plasmid, pX0134.

The mechanism in which host cells are intoxicated is complex (Fig. 1.2).

Once the toxin components are in circulation, protective antigen binds to cell surface receptors known as anthrax toxin receptors or ATRs in its 83 kDa form.

After the initial binding, a 20 kDa portion of PA is cleaved on N-terminal end by a

9 -like , leaving the 63 kDa portion of PA and the ATR intact as a complex. This PA-receptor complex associates with six other like complexes to form a heptamer structure. This heptameric structure (also known as a pre-pore) binds LF and EF at a stoichiometry of three molecules for each heptamer. The heptameric complex undergoes raft dependent mediated endocytosis35. The association of an additional co-receptor, LDL receptor related protein 6 (LRP6), is required for this heptamer internalization36. The function of this association is unknown; however, LRP6 is known to stimulate the Wnt signaling pathway, which may be related to a decrease in glycogen synthase kinase-3β36,37. The PA-receptor complex is now considered a pore structure which allows the translocation of EF or LF into the cytosol. Critical to this translocation is the acidification of the endocytic vesicle which promotes its insertion through the endosomal limiting membrane. A new model suggests that the prepore structure inserts into intraluminal vesicular structures that are present in the early which subsequently fuse with the endosomal membrane, releasing toxins to the cytosol35. Edema factor, a calcium/ adenylate cyclase, increases intracellular concentrations of cAMP. These increases in cAMP have been shown to cause intracellular edema and reduce the phagocytic ability of and macrophages38-40. Lethal factor, a zinc metalloprotease, inhibits the transduction activity of mitogen activated protein kinases

(MAPKs) such as p38, ERK and JNK by cleaving MAPK kinases necessary for

10 phosphorylation and activation. Interestingly, some evidence exists suggesting that LF and EF can mediate their effects without the ATR prepore structure41.

There are two currently known anthrax toxin receptors, tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2)42-44. TEM8 was the first protein to be shown to function as an ATR42. Three isoforms of this protein have been described. Isoform 1 is a transmembrane protein with a von

Willebrand factor type A and integrin inserted domains (VWA/I domains). In addition, isoform 1 has a 221 cytoplasmic tail with multiple putative phosphorylation sites, however, this cytoplasmic tail has been shown to be non­ essential for PA binding and toxin internalization45. Isoform 2 has the extracellular and transmembrane domains and isoform 3 is thought to be secreted with a structure identical to the extracellular domain of isoforms 2 and 3. Expression has long been associated with the vasculature of human tumors and has been specifically demonstrated in human vascular endothelium46-49. In addition, expression of TEM8 has been shown in the epithelium of the lung, and gastrointestinal tract47. This expression has been shown to be upregulated in response to IL-1β46. The specific biological function of TEM8 is unknown, however, the natural ligand of TEM8 appears to be the cleaved C5 domain of type VI collagen50. In addition, TEM8 appears to have a function in cellular adherence to extracellular matrix49,51.

CMG2, the second anthrax toxin receptor, shares structural homology with

TEM8, such as the presence of the VWA/I domains and exists in multiple

11 isoforms. CMG2 is expressed in multiple tissues and mRNA transcripts of CMG2

are preferentially expressed over TEM8 in mononuclear phagocytes52-54. CMG2

appears to have a similar adhesion function as TEM8 and has been shown to

bind to collagen IV and laminin. Mutations in the CMG2 gene have been

associated with two related laminin adhesion defects, infantile systemic

hyalinosis and juvenile hyaline fibromatosis, possibly indicating the physiologic

role of this protein53,55.

The distribution of ATR expression in terms of cell types and subcellular

localization is important in the pathogenesis of anthrax. In terms of subcellular

localization, it has been found that TEM8 and CMG2 exhibit different insertional

behavior based on the pH of the endosome56. Cell lines in which the ATRs are

overexpressed are more susceptible to killing after infection with spores52, which suggests that intracellular of toxin may provide a mechanism of escape for the organism following phagocytosis, although reports in the past appear to conflict with these data57-59. Thus, ATRs may have a significant role in terms of

early infection as well as in the late septicemia.

Although the glutamic acid capsule encoded by pX02 and toxin

components encoded by pX01 are considered as the major virulence factors of

the organism, other virulence factors have been discovered. Anthrolysin O, a

dependent , is similar to produced by other

pathogenic gram-positive such as monocytogenes,

Arcanobacterium pyogenes and spp60-62. In a similar manner to

12 the cytolysins of these bacterial species, anthrolysin O has been shown to lyse erythrocytes, neutrophils, macrophages and in vitro in a dose dependant manner63,64 and has been shown to induce a pro-inflammatory response as a toll-like receptor 4 agonist65. Other virulence factors continue to be investigated and their roles in pathogenesis sometimes conflict with previous findings. For instance, the usage of attenuated strains devoid of specific phospholipases are significantly less pathogenic when deposited in murine lungs66. However, Drysdale et al has previous shown that capsule synthesis is necessary for systemic dissemination in the mouse33.

1.3.3 Macrophage interactions with Bacillus anthracis

The macrophage is an important host cell in the pathogenesis of anthrax, particularly in the pulmonary form. It is well established that vegetative B. anthracis is resistant to phagocytosis due to its glutamic acid capsule67.

However, understanding the interaction of the spore with the macrophage is considered critical due to its supposed role in transporting the organism across the alveolar septum. Because of this, many investigators have studied the interaction of the organism with this cell, primarily focusing on two aspects of pathogenesis. The first is the interaction of the spore with the macrophage, reflecting events taking place during early infection. These studies include initial interactions which include phagocytosis of the spore, the behavior of the spore within the macrophage and the possible mechanisms in which organism

13 mediates escape. Phagocytosis is a characteristic trait of innate and an initial step towards adaptive immunity. The process of phagocytosis is characterized by dependence on actin polymerization, unlike pino- and endocytosis, which are clathrin dependent and usually actin independent. Both phagocytosis and endocytosis are receptor mediated. The challenge of highly variable antigenicity is circumvented by phagocytes targeting conserved motifs between termed as “ associated molecular patterns”

(PAMPs)68. Once internalization is complete, the vacuole containing the organism, called the phagosome, undergoes maturation that endows the phagosome with bactericidal properties. Initial maturation deals with interaction of the phagosome and sorting endosomes, which govern rerouting and organizational interaction between phagosomes and early endosomes. Later steps of maturation include fusion with “late endosomes” and lysosomes that impart hydrolytic and rapidly drop the pH of the phagosome, now called the phagolysosome69.

It is generally accepted that the phagocytosis of most bacterial pathogens involves the recognition of molecules inherent to the structure of the organism by cellular “scavenger” receptors or by the deposition of soluble host molecules

(lectins, antibody, complement, etc), which interact with their respective receptors. Little is known about either method for B. anthracis and this is the focus of Chapters 3 and 4 of this thesis. Interestingly, protective antigen has been demonstrated in the outer structures of the spore, which appears to

14 increase phagocytosis in the presence of anti-PA antibody70,71. This may provide

an important, but secondary role of the involvement of anti-PA antibodies during

infection. Multiple studies have demonstrated that spores co-localize in

compartments containing phagolysosomal markers within mouse alveolar

macrophages and macrophage-like cell lines58,72, suggesting that the organism

does not induce phagosomal arrest as in the case of Mycobacterium

tuberculosis73-75 or escape the phagosome before lysosomal fusion occurs like

Listeria monocytogenes 76-79. Phagocytosed spores have been shown to

germinate intracellularly and escape from the macrophage in cell culture 57. The manner in which the organism escapes is controversial and the data probing this mechanism are sometimes conflicting, particularly in reports investigating the role of intracellular toxin secretion. Escape of the vegetative cell has been described as both toxin-dependent or toxin independent, but toxin transactivator dependent57,80. It was recently found that efficient killing of macrophage-like cell

lines overexpressing CMG2 takes place after spore infection, suggesting that

intracellular toxin secretion and intoxication takes place52,81. Because of these

conflicts, it has been suggested that the escape process is more complex and

multiple factors produced by the vegetative organism are required for effective

phagolysosomal and eventual cytoplasmic escape. Virulence factors including

anthrolysin O (ALO) and a specific phospholipase, phosphatidylinositol-specific

phospholipase C (PI-PLC) have been implicated in mediating escape by

disrupting the phagolysosome or by killing the macrophage outright82,83.

15 Superoxide dismutase encoded by B. anthracis appears to reside in the spore coat, which may aid the survival of the spore within the phagolysosome84.

Intracellular spores have a stimulatory effect on pro-inflammatory production through multiple MAPK pathways (ERK, p38, Jun), but the spore component interaction which stimulates this response is unknown85,86.

The second paradigm focuses on the effects of toxin on macrophages which can represent early and late events during infection. One aspect of the early effects of toxin has been touched upon in terms of intracellular secretion.

However, pretreatment of macrophages with LF decreases bactericidal activity when infected with spores in vitro, supporting the theory that externally secreted toxin can propagate infection87. Late in the course of infection, it was thought that of the macrophage due to toxin exposure caused uncontrolled pro- inflammatory cytokine release57,88,89, however, recent reports indicate that intoxication has both stimulatory or inhibitory effects on pro-inflammatory cytokines90-92. The role of macrophages in anthrax pathogenesis may be over represented in the the scientific literature, as we now know that other cell types such as endothelium are target of anthrax toxins93-96. This is also supported by data that indicate that spores deposited in the alveoli can reach systemic circulation in mice depleted of alveolar macrophages, suggesting other mechanisms for spore in the lung96. However, it is generally accepted that the macrophage plays a role in both clearance of the pathogen as well as propagation of the disease97,98.

16 Many of the studies described above utilize a variety of macrophage

types, including primary cells of humans and mice. It is important to take in

account the differences between the primary macrophages of these two species,

particularily in terms of surface receptors, as behavior in mouse macrophages is

often equated to behavior in human cells. Cell receptors involved in phagocytosis

can be roughly divided into the scavenger receptors, which recognize motifs

inherent to the structure of phagocytized structure, or receptors which recognize

opsonins, such as Fc receptors or complement receptors. Mouse and human

macrophages express a variety of scavenger receptors, including Class A and B

scavenger, MARCO and mannose receptors99-101. However, fresh mouse

peritoneal macrophages have been shown to have low scavenger receptor

activity which increases after adherence ex vivo102. Also, scavenger receptor

expression in alveolar macrophages can be variable in different mouse strains100.

Receptors for opsonins, such as FcγR for IgG or complement receptors for complement proteins, are present on both subsets of human and murine primary macrophages. Four classes of FcγR are expressed in the human and mouse, including the inhibitory class FcγRIIB, which contains inhibitory tyrosine motifs.

However, FcγRIIA does not appear to be expressed in mouse macrophages68.

Complement receptors (CR) are also expressed in macrophages of both species.

However, CD11c, a component of the heterodimeric CR4, is expressed at much lower level in resident and stimulated mouse macrophages compared to their

17 human counterparts. CD11b which forms CR3 (CD11b/CD18) is expressed at the same levels in mouse and human macrophages103 .

1.4 Murine models of Bacillus anthracis pathogenesis, immunogenicity and vaccine efficacy

1.4.1 Overview of non-murine models of anthrax

Several animal models of anthrax pathogenesis have been developed to primarily study the pulmonary form and to a lesser extent, the cutaneous form.

Non-human primates served as some of the earliest and most extensively studied models to study the disease, no doubt due to the similarity in clinical signs and pathologic findings in humans. The chimpanzee, the African green monkey, rhesus and cynomolgus macaques are considered the most accurate for studying inhalation anthrax given the similarity of the lesions between humans and these species, although in a inhalation study using cynomolgus macaques only 40% of the animal consistently showed the typical lesions104-106. Non-human primate models have also been of value in studying the immunopathology of infection as well as vaccine efficacy, given the obvious difficulty in studying these two disease aspects in humans. The efficacy of several PA vaccines have been studied in rhesus macaques, with a majority of studies showing high survival percentages (approximately 95%) after challenge107. Despite the advantage of this animal model, the most obvious disadvantage for its widespread use include

18 the high cost for animals and containment infrastructure and equipment

necessary for experiments. Despite these disadvantages, non-human primate

models have provided important information about the response of the immune

system following infection and correlates of immunity following vaccination.

Studies of the pathology of anthrax in non-primate models are infrequent

with a majority using canines, pigs, guinea pigs and rabbits108-111. Because there

can be significant differences in pathogenesis, the data obtained from non-

primate models should be interpreted with caution. For instance, canines and

pigs, in general, are relatively resistant to infection even with fully virulent strains,

while rabbits are remarkably sensitive to fully virulent strains112. In addition,

guinea pigs are relatively resistant to challenge with preformed edema and lethal

toxin but are easy to infect with B. anthracis spores and produce bacteremia,

while rats are far more sensitive to B. anthracis toxins113-115. Current policies of

the United States Food and Drug Adminstration (FDA) requires human vaccine

efficacy data produced from at least two animal models. Thus, non-primate

models including infection of rabbits are useful models based on responses to

PA vaccines (AVA, rPA-aluminum hydroxide) using multiple protocols116-118. The rabbit is a useful model because of the reduced cost of animals, as well as handling and housing expenses. The guinea pig has also been used as a vaccine efficacy model, but has produced inconsistent results in efficacy testing during standard PA vaccine trials. Fellows et al demonstrated this by vaccinating

New Zealand white rabbits and guinea pigs with two doses of AVA four weeks

19 apart, followed by an aerosol challenge to the rabbits and an intramuscular challenge to the guinea pigs with a strain uniquely pathogenic to the guinea pig119. Finally, guinea pigs can be successfully infected transcutaneously by , Stomoxys calcitrans, and two species of Aedes mosquitos, which supports anecdotal evidence of humans and domestic animals developing cutaneous anthrax from fly bites111 .

1.4.2 Anthrax pathogenesis in immune competent murine models

The availability of various mouse strains is a unique factor which sets the mouse model apart from other animal models of pathogenesis. A distinct advantage is the elucidation of immune system components responsible for control of infection by the use of knock-out strains or strains in which a natural deficiency of an immune system component is present. As demonstrated in

Table 1.1, many commonly used mouse strains demonstrate varying susceptibility to fully virulent and attenuated strains. A distinct disadvantage of the mouse model is the dissimilarity in the pathogenesis between mouse and human anthrax. Atoxigenic encapsulated strains of B. anthracis can be highly virulent in the mouse120-122. Although this sensitivity can be strain dependent, it appears that either the capsule or some of the more recently discovered virulence factors (anthrolysin O, phospholipases) play a more significant role in murine disease than in humans. But despite these disadvantages, studies investigating different aspects of pathogenesis in the mouse have become

20 widespread. Early studies in the mouse examined the pathology and dissemination of bacteria in multiple organs by light microscopy and electron microscopy123,124, survival times, LD50s and strain susceptibility125. Welkos et al., demonstrated the marked sensitivity of certain strains (A/J, DBA) to the attenuated Sterne strain126. In addition, valuable pharmacologic studies were performed in BALB/C mice, which evaluated the efficacy of various through the course of infection127.

1.4.2.1 Intraperitoneal inoculation of B. anthracis

The intraperitoneal challenge method in the mouse model of anthrax is the most common method reported in the literature128,128-130. Inoculation by the intraperitoneal route can be accomplished with minimal operator training, but does not mimic any natural exposure route and is generally useful only in determining the efficacy of a treatment or vaccine in a model of full septicemia.

1.4.2.2 Subcutaneous or intradermal inoculation of B. anthracis

Inoculation of B. anthracis spores cutaneously has been performed using multiple methods. The most common injection route of spores is intradermally or subcutaneously. Although these methods do not imitate naturally occurring exposures completely, they are the most reliable and least technically challenging. Another method of inoculation involves the abrasion of the epidermis and application of the spores epicutaneously131. This method accurately mimics

21 natural infection cutaneous infection. The pathologic findings in cutaneous anthrax have been evaluated by multiple investigators including our laboratory.

Mice typically do not form the traditional eschar seen in human cutaneous anthrax. This may be due to the short clinical course of the disease compared to humans. The inoculation site may be mildly to moderately erythematous and edematous. The lesions associated with cutaneous infection of A/J mice (an immune incompetent strain) are described in a later section.

1.4.2.3 Pulmonary exposure

Experimental pulmonary anthrax in the mouse is well documented.

Pulmonary inoculation is typically achieved by intranasal and intratracheal inoculation or aerosolization of spores132. Intranasal inoculation is technically straightforward, but much of the inoculum can be lost by swallowing.

Intratracheal inoculation allows for direct exposure of pulmonary parenchyma without confounding factors such as gastrointestinal or oropharyngeal exposure.

This method can be technically challenging due to the requirements of general anesthesia and surgical technique. Aerosol exposure is most representative of natural infection but requires special equipment and expertise in aerosol biology.

True experimental aerosol exposures of mice are rare in the literature. The pulmonary lesions described in mice are minimal. Lyons et al describes mild vacuolar degeneration and sloughing of bronchiolar epithelium with intravascular bacilli132. However, Duong et al.,133 describes marked alveolar infiltration with

22 neutrophils and macrophages in mice inoculated subcutaneously. Our findings in

A/J mice inoculated with B. anthracis Sterne subcutaneously and by aerosol show minimal lesions in the lungs, mostly congestion, mild hemorrhage and mild to moderate intravascular accumulations of bacilli (Fig. 1.3, unpublished data).

1.4.2.4 Gastrointestinal exposure

Studies using the gastrointestinal route of inoculation are relatively rare and have not focused on the pathogenesis of gastrointestinal anthrax. The only reported oral inoculations used Bacillus species expressing protective antigen in the context of oral vaccination134,135. Our laboratory attempted to determine the gastrointestinal LD50 in A/J mice using B. anthracis Sterne in pathology studies of gastrointestinal anthrax (Fig.1.4. unpublished data). Our findings indicate that the colony forming units required to reach a 50% lethal dose are much higher than the established LD50 for cutaneous and pulmonary inoculation (Table

1.1)128. Inoculated spores pass through the gastrointestinal tract and it is possible that this is why the LD50 is increased. Histologic examination of the gastrointestinal shows minimal lesions however bacilli are present in small amounts in the laminae propria. This preliminary work suggests that Bacillus anthracis does not cause epithelial necrosis of the GI tract to gain access to the circulation. However, more extensive studies will be required to elucidate host pathogen interactions of gastrointestinal anthrax.

23 1.4.3 Associated systemic pathologic findings

Lesions in animal models exposed by cutaneous and pulmonary routes are similar. The most dramatic findings are typically in the spleen. The periarteriolar lymphoid shealths often contain marked lymphocytolysis with marked bacillary proliferation. The red pulp often exhibits necrosis and neutrophilic inflammation132,133. Kidneys often contain foci of bacteria within the glomerular tufts. Other sources have described mild neutrophilic in kidneys of affected animals and bacterial proliferation over the renal capsule suggesting retroperitoneal spread133. Liver lesions are either absent or range from accumulation of bacilli in sinusoids with mild neutrophilic inflammation in periportal and centrolobular regions133. Fibrinoid necrosis of vessels in the portal trial can be observed. Fibrin thrombi and fibrinoid necrosis of vessels in the parenchyma of cerebrum are also occasionally observed133.

Cytokine production in response to toxins contributes to systemic anthrax in mouse models136. Not surprisingly, the cytokine profiles of infected mice can vary based on strain. BALB/C mice, a relatively resistant strain, show increased

IL-6 and IL-1β mRNA and protein expression compared to similarly infected and more susceptible C57B/6 mice, suggesting these may help control infection136.

Lethal toxin was well studied as a key of B. anthracis in early studies. These reports defined the relative lack of lethality in mice when infected with EF- deficient mutants compared to LF- and/or PA- mutants137,138 and

24 systemic cytokines responses and tissue recruitment134. Interestingly,

Drysdale et al.,139 demonstrated that EF may play a larger role in leukocyte recruitment than lethal toxin. Moayeri et al.,140 demonstrated that C57/B6 mice are less susceptible to intoxication to lethal toxin alone than BALB/C mice which is interesting considering the is true in terms of infection with the organism. Firoved et al.,141 described the histopathologic findings, and changes in blood chemistry and cytokine profiles of BALB/CJ mice exposed to concomitant doses of PA and EF which are extensive and suggest that EF also contributes significantly to disease progression.

1.4.4 Anthrax pathogenesis in immune incompetent murine models

1.4.4.1 C5 deficient mice

The use of the A/J mice in challenge studies is a prominent example of a mouse strain with an immune component deficit. A/J mice are deficient in complement component C5, a protein required for the formation of the membrane attack complex142. A/J mice have been shown to be remarkably susceptible to infection with attenuated B. anthracis Sterne strain as well as fully virulent B. anthracis suggesting that the membrane attack complex plays a significant role in clearance of bacteria lacking a capsule. This is supported by the increased susceptibility of complement depleted C57/B6 mice, a strain normally somewhat resistant to B. anthracis Sterne143. The use of A/J mouse

25 strain as a model is particularly useful for investigators without access to biosafety level 3 facilities. The strain has been used to study anthrax pathology133,144, systemic cytokines responses136 and vaccine response and efficacy against spore, bacillus and toxin challenge145-152. A/J mice infected cutaneously with B. anthracis Sterne with a dose several times over the published LD50 typically show a clinical course to death of approximately 10 days or less. Our laboratory has examined the lesions in infected A/J mice in a time course study over 4 days (Fig. 1.5, unpublished data). Eight A/J mice were inoculated subcutaneously on the dorsum with B. anthracis Sterne spores (6 x

4 10 CFU = 60 X LD50). Each day post-infection, two mice were euthanized and necropsied. Formalin fixed tissues were processed for routine histopathology.

Day 1 post-infection shows very little changes in the skin although the organism can be cultured from the inoculation site. Day 2 post-infection, shows the vascular margination and infiltration of polymorphonuclear leukocytes into the subcutis of the inoculation site as well as bacterial colonization. By day three, the inoculation sites had marked edema and pronounced bacterial proliferation, however the inflammatory cell infiltrate remains relatively unchanged similar to published studies133. At day four, the bacterial proliferation is pronounced enough to be visible subgrossly and correlated with the colony forming units cultured from the skin. Marked acute hemorrhage can be observed in some specimens.

Vascular endothelial loss, presumably due to necrosis, was observed as well as platelet aggregation. Interestingly, mice exposed by the application of high doses

26 to unabraded intact skin showed accumulation of organism in the hair follicles, suggesting that the follicle may provide a route of entry153 .

1.4.4.2 Irradiated mice

Irradiated mice have been used extensively in modeling bacterial pathogenesis in an immune suppressed host154. Typically these animals develop sepsis following translocation of gram-negative bacteria across the gastrointestinal mucosa due to loss of enterocytes and bone marrow suppression155. Often these models are used to determined treatment efficacy of pharmaceuticals during infection156-161. Studies of anthrax pathogenesis have been performed in irradiated mice. B6D2F1/J mice (a ordinarily resistant strain) show nearly 100% mortality following intratracheal inoculation of B. anthracis

Sterne after exposure to a 4 Gy dose of 60Co162,163. This model has primarily been used to study the efficacy of antimicrobials. For the most part, various quinolone and macrolide therapies have been shown to increase survival although certain combinations of therapy such as clindamycin and increase mortality rates163-165.

1.4.4.3 Leukocyte depleted mice

The use of leukocyte depleted mice has been valuable in studying the significance of particular cells in the pathogenesis of anthrax. Macrophage depletion has been performed using silica treatment or more effectively with

27 clodronate loaded liposomes96. Interestingly, macrophage depletion leads to decreased survival of BALB/C challenged with B. anthracis Ames spores, suggesting that macrophages play a significant role in clearing infection.

However, this study utilized an intraperitoneal challenge which did not allow the investigators to make inferences concerning different populations of tissue macrophages166. The same group performed intranasal and intratracheal inoculations of BALB/C mice depleted of macrophages using clodronate or neutrophils using cyclophosphamide97. Macrophage depleted mice showed decreased survival following exposure, while neutrophil depletion had no effect, suggesting that macrophages are more significant in clearing the infection.

1.4.5 Immunogenicity and vaccine efficacy

1.4.5.1 Immunogenicity and vaccine efficacy of Bacillus anthracis protective antigen, edema factor and lethal factor

Efficacy data of protective antigen based vaccines in mouse models is strain, adjuvant and delivery dependent. CBA/J mice are fully protected by immunization with PA producing Bacillus subtilis strains, while BALB/c mice show only partial protection against fully virulent strains such as B. anthracis Vollum.

PA vaccines alone have been shown to be less efficacious in these strains167,168 ranging from no protection to approximately 80% survival depending on the PA vaccine/adjuvant169. A/J mice are protected after vaccination with PA or PA

28 producing Bacillus species and passive transfer of anti-PA antibody when

challenged with B. anthracis Sterne. Domain four of protective antigen has been

shown to be the immunodominant peptide in mice170. Protective antigen

vaccination without adjuvants is often not sufficient to develop protective

immunity and adjuvants have been utilized to boost an antibody response.

Aluminum hydroxide, the same adjuvant used in the human AVA (Anthrax

Vaccine Adsorbed)171, used in conjunction with rPA is the most commonly used

PA/adjuvant combination172. Other adjuvant systems have been studied. These

include lipopeptides, synthetic CpG oligonucleotides with or without polylactide­

co-glycolide, and lipid products from various species of Mycobacterium169,173-176.

Perhaps the most innovative method of PA/aluminum hydroxide vaccination strategy is the integration of the pagA into the chloroplast genome followed by administration of the purified chloroplast protein with aluminum hydroxide177. It is important to note that eliciting a high immunogenic response to PA in mice does not always correlate with protection, thus the need for challenge data following immunogenicity studies168,169,178,179.

Various methods of PA delivery to mice have been investigated. PA

formulations administered nasally are effective in eliciting IgG and IgA responses

as well as a TH2 CD4 response180,181. Immunogenicity against PA by nasal

administration has been augmented by utilizing carriers such as liposome-

protamine-DNA particles and adjuvants such as polyriboinosinic-polyribocytidylic

acid182,183. Microsphere associated PA formulations are effective in protecting A/J

29 mice when administered mucosally or parenterally184 as are transcutaneous administration of PA185,186.

Compared to the information available demonstrating PA immunogenicity and vaccine efficacy, similar information regarding EF and LF is limited.

Administering an adenoviral vector encoding a 254 amino acid N-terminal sequence of EF to A/J mice induced multiple IgG isotypes, but protected only

187 57% mice challenged with B. anthracis Sterne (100 X LD50) . Interestingly, the role of EF as an adjuvant for other peptides has been explored188,189. Similarly,

LF has not been a priority in mouse vaccine immunogenicity and efficacy studies.

Immunizing BALB/C mice with a plasmid encoding the N-terminal region of LF is protective against lethal toxin challenge190. In addition, Zhao et al.,191 has demonstrated significant immunogenicity of LF administered to BALB/C mice in complete Freud’s adjuvant in addition to generating monoclonal antibodies against LF, which protect nude mice from lethal toxin challenge following passive transfer.

1.4.5.2 Immunogenicity and efficacy of inactivated spore vaccines

There are few reports of spore-based in mouse models.

Spore component vaccines would be predicted to initiate an adaptive TH2 response against spores in the early stages of infection. Immunization of Swiss outbred mice with formalin inactivated spores augments the protective effect of

PA following a subcutaneous challenge with fully virulent B. anthracis192,

30 however, the dose of spores required to achieve this effect in mice is rather large

(ranging from 1x108 to 5x108 spores/mouse). Although it is known that a collagen

like exosporial protein, BclA is considered the immunodominant of the

spore, other immunogenic proteins have been identified on the spore by matrix

assisted laser desorption time of flight (MALDI-TOF), which interestingly, some of

which are shared between Bacillus cereus and Bacillus thuringiensis193. In

addition, PA may be present in the exosporium or spore coat of the spore71,194,

which may contribute to spore immunogenicity.

1.4.5.3 Immunogenicity and vaccine efficacy of bacterial cells and the poly-

D-glutamic acid (PDG) capsule

Components of the vegetative cell wall would be predicted to elicit

antibodies important in various stages of anthrax. Vegetative cell component

vaccines, predominately directed at the capsule, would develop a response

directed at the organism following germination. In support of this, anti-PDG

antibodies are formed subsequent to infection, however the capsule itself is

poorly immunogenic and resists agglutination195. Some groups have investigated

acquired immune responses to vegetative cell walls using T-cell costimulatory

molecules or conjugating the capsule to more immunogenic proteins196­

199. Although the capsule is traditionally thought of as highly non-immunogenic,

glutamic acid with a chain length of 15 amino acids conjugated to toxoid

or PA elicits a clearly recognized immunogenic response200. The ability of these

31 antibodies to protect against disease progression has been described by multiple

groups recently199. Joyce, et al.,199 described a method of activating free alpha

carboxyl groups on glutamic acid chains which elicited a specific anti-capsule

response protective in BALB/c mice. Kozel et al.,201 has shown that coupled

purified PDG with a CD40 agonist , which stimulates

proliferation, results in the generation of significant anti-PDG titers202. This group

succeeded in generating a panel of anti-PDG monoclonal antibodies that protects

90% of BALB/C mice infected intratracheally after passive administration.

1.4.5.4 DNA based and viral vectored vaccines

Studies of DNA-based anthrax vaccines encompass many of the epitope

strategies described previously145,203-205. Several reports demonstrated increased

survival following spore challenge in mice immunized with the pagA gene

inserted into a variety of plasmid vectors145,203,204,206. Topical immunization with

PA encoding also induce PA antibody responses in mice204. DNA

vaccines have also been used to test the immunogenicity of LF and EF187,190.

The use of attenuated or engineered viral vectors as vaccines has been used for both infectious disease prevention and cancer immunotherapy207-210 .

This strategy has been applied to the prevention of anthrax. Similar to the other

previously discussed vaccine strategies in mice, most revolve around the delivery

of protective antigen. McConnell et al.,211 elicited PA antibodies after single

intramuscular injection of an adenoviral vector expressing protective antigen

32 domain 4. Two administrations of this vaccine were sufficient to protect 67% of the animals challenged with lethal toxin. Other viral vectors used to deliver protective antigen include parvovirus B19, Venezuelan equine encephalitis virus, influenza and vaccinia viruses152,168,212-215.

1.5 Conclusions

The data presented in this thesis provides valuable information concerning the pathogenesis of anthrax, particularly during early infection. Our data presented in Chapter 1 demonstrate the pattern of mRNA expression of anthrax toxin receptors and their functional properties in primary macrophages supporting their role in the early escape of the organism following phagocytosis. Our studies in Chapter 3 demonstrate that antibody-complement spore interactions modulate anthrax spore interaction with the macrophage. Finally, in Chapter 4, we expand on this interaction by eliciting spore specific antibodies and examining their significance in vitro and ex vivo. Collectively, these data are the first to demonstrate specific macrophage interactions with anthrax spores that likely influence early pulmonary infection by this deadly organism.

1.6 References

1. Sternbach G. (2003) The history of anthrax. J Emerg.Med 24, 463-467.

2. Dirckx JH (1981) Virgil on anthrax. Am J Dermatopathol 3, 191-195.

3. Smith K.A. (2005) Wanted, an Anthrax vaccine: Dead or Alive? Med Immunol. 4, 5.

33 4. Morens D.M. (2003) Characterizing a "new" disease: epizootic and epidemic anthrax, 1769-1780. Am J Public Health 93, 886-893.

5. Turnbull P.C. (1991) Anthrax vaccines: past, present and future. Vaccine 9, 533-539.

6. Metcalfe N. (2004) The history of woolsorters' disease: a Yorkshire beginning with an international future? Occup.Med (Lond) 54, 489-493.

7. Brachman P.S. (2002) Bioterrorism: an update with a focus on anthrax. Am.J Epidemiol. 155, 981-987.

8. Bales M.E., Dannenberg A.L., Brachman P.S., Kaufmann A.F., Klatsky P.C., & Ashford D.A. (2002) Epidemiologic response to anthrax outbreaks: field investigations, 1950-2001. Emerg.Infect.Dis. 8, 1163-1174.

9. Spencer R.C. (2003) Bacillus anthracis. J Clin.Pathol. 56, 182-187.

10. Tizard I. (1999) Grease, anthraxgate, and kennel cough: a revisionist history of early veterinary vaccines. Adv.Vet.Med 41, 7-24.

11. Tigertt W.D. (1980) Anthrax. William Smith Greenfield, M.D., F.R.C.P., Professor Superintendent, the Brown Animal Sanatory Institution (1878-81). Concerning the priority due to him for the production of the first vaccine against anthrax. J Hyg.(Lond) 85, 415-420.

12. Sterne M. (1939) The use of anthrax vaccines prepared from avirulent (uncapsulated) variants of Bacillus anthracis. Onderstepoort J.Vet.Sci.An.Ind. 13, 307-312.

13. Sterne M., Nichol J., & Lambrechts M.C. (1942) The effect of large scale active immunization against anthrax. J.S.African Vet.Med.Assoc. 13, 53-63.

14. Sterne M. (1988) Anthrax vaccines. J Am.Vet.Med.Assoc. 192, 141.

15. Gu M., Hine P.M., James J.W., Giri L., & Nabors G.S. (2007) Increased potency of BioThrax((R)) anthrax vaccine with the addition of the C-class CpG oligonucleotide adjuvant CPG 10109. Vaccine 25, 526-534.

16. Dragon D.C. & Rennie R.P. (1995) The ecology of anthrax spores: tough but not invincible. Can.Vet.J 36, 295-301.

17. Dragon D.C., Elkin B.T., Nishi J.S., & Ellsworth T.R. (1999) A review of anthrax in Canada and implications for research on the disease in northern bison. J Appl.Microbiol. 87, 208-213.

34 18. Koch R. (1876) Die Aetiologie der Milzbrand-Krankheit, begründet auf die Entwicklungsgeschichte des Bacillus anthracis. Beiträge zur Biologie der Pflanzen 2, 277-310.

19. Keynan A.H.H. (1965) In: Spores III In L. L. Campbell and H. 0. Halvorson [ed.], edn, p. 174-179 American Society for Microbiology, Ann Arbor, Michigan.

20. Fisher N. & Hanna P. (2005) Characterization of Bacillus anthracis germinant receptors in vitro. J Bacteriol. 187, 8055-8062.

21. Van Ness G.B. (1971) Ecology of anthrax. Science 172, 1303-1307.

22. Sirisanthana T. & Brown A.E. (2002) Anthrax of the gastrointestinal tract. Emerg.Infect.Dis. 8, 649-651.

23. Chensue S.W. (2003) Exposing a killer: pathologists angle for anthrax. Am J Pathol. 163, 1699-1702.

24. Navacharoen N., Sirisanthana T., Navacharoen W., & Ruckphaopunt K. (1985) Oropharyngeal anthrax. J Laryngol.Otol. 99, 1293-1295.

25. Sirisanthana T., Nelson K.E., Ezzell J.W., & Abshire T.G. (1988) Serological studies of patients with cutaneous and oral-oropharyngeal anthrax from northern Thailand. Am.J Trop.Med.Hyg. 39, 575-581.

26. Sirisanthana T., Navachareon N., Tharavichitkul P., Sirisanthana V., & Brown A.E. (1984) Outbreak of oral-oropharyngeal anthrax: an unusual manifestation of human infection with Bacillus anthracis. Am.J Trop.Med.Hyg. 33, 144-150.

27. Oncu S., Oncu S., & Sakarya S. (2003) Anthrax--an overview. Med Sci Monit. 9, RA276-RA283.

28. Candela T. & Fouet A. (2005) Bacillus anthracis CapD, belonging to the gamma-glutamyltranspeptidase family, is required for the covalent anchoring of capsule to peptidoglycan. Mol.Microbiol 57, 717-726.

29. Ezzell J.W. & Welkos S.L. (1999) The capsule of Bacillus anthracis, a review. J Appl.Microbiol. 87, 250.

30. Candela T., Mock M., & Fouet A. (2005) CapE, a 47-amino-acid peptide, is necessary for Bacillus anthracis polyglutamate capsule synthesis. J Bacteriol. 187, 7765-7772.

31. Schneerson R., Kubler-Kielb J., Liu T.Y., Dai Z.D., Leppla S.H., Yergey A., Backlund P., Shiloach J., Majadly F., & Robbins J.B. (2003) Poly(gamma-D­ glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of 35 Bacillus anthracis: a potential addition to the anthrax vaccine. Proc.Natl.Acad.Sci U.S.A 100, 8945-8950.

32. Makino S., Uchida I., Terakado N., Sasakawa C., & Yoshikawa M. (1989) Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis. J Bacteriol. 171, 722-730.

33. Drysdale M., Heninger S., Hutt J., Chen Y., Lyons C.R., & Koehler T.M. (2005) Capsule synthesis by Bacillus anthracis is required for dissemination in murine inhalation anthrax. EMBO J 24, 221-227.

34. Baldari C.T., Tonello F., Paccani S.R., & Montecucco C. (2006) Anthrax toxins: A paradigm of bacterial immune suppression. Trends Immunol. 27, 434­ 440.

35. Abrami L., Liu S., Cosson P., Leppla S.H., & van der Goot F.G. (2003) Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin­ dependent process. J Cell Biol. 160, 321-328.

36. Wei W., Lu Q., Chaudry G.J., Leppla S.H., & Cohen S.N. (2006) The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin. Cell 124, 1141-1154.

37. Tucker A.E., Salles I.I., Voth D.E., Ortiz-Leduc W., Wang H., Dozmorov I., Centola M., & Ballard J.D. (2003) Decreased glycogen synthase kinase 3-beta levels and related physiological changes in Bacillus anthracis lethal toxin-treated macrophages. Cell Microbiol 5, 523-532.

38. Ulmer T.S., Soelaiman S., Li S., Klee C.B., Tang W.J., & Bax A. (2003) Calcium-dependence of the interaction between calmodulin and anthrax edema factor. J Biol.Chem.

39. Kumar P., Ahuja N., & Bhatnagar R. (2002) Anthrax edema toxin requires influx of calcium for inducing cyclic AMP toxicity in target cells. Infect.Immun. 70, 4997-5007.

40. Crawford M.A., Aylott C.V., Bourdeau R.W., & Bokoch G.M. (2006) Bacillus anthracis toxins inhibit human neutrophil NADPH oxidase activity. J Immunol. 176, 7557-7565.

41. Ribot W.J., Panchal R.G., Brittingham K.C., Ruthel G., Kenny T.A., Lane D., Curry B., Hoover T.A., Friedlander A.M., & Bavari S. (2006) Anthrax lethal toxin impairs innate immune functions of alveolar macrophages and facilitates Bacillus anthracis survival. Infect.Immun. 74, 5029-5034.

36 42. Bradley K.A., Mogridge J., Mourez M., Collier R.J., & Young J.A. (2001) Identification of the cellular receptor for anthrax toxin. Nature 414, 225-229.

43. Bradley K.A. & Young J.A. (2003) Anthrax toxin receptor proteins. Biochem.Pharmacol. 65, 309-314.

44. Scobie H.M., Rainey G.J., Bradley K.A., & Young J.A. (2003) Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc.Natl.Acad.Sci U.S.A 100, 5170-5174.

45. Liu S. & Leppla S.H. (2003) Cell surface tumor endothelium marker 8 cytoplasmic tail-independent anthrax toxin binding, proteolytic processing, oligomer formation, and internalization. J Biol.Chem. 278, 5227-5234.

46. Rmali K.A., Al Rawi M.A., Parr C., Puntis M.C., & Jiang W.G. (2004) Upregulation of tumour endothelial marker-8 by interleukin-1beta and its impact in IL-1beta induced angiogenesis. Int.J Mol.Med 14, 75-80.

47. Rmali K.A., Puntis M.C., & Jiang W.G. (2005) Prognostic values of tumor endothelial markers in patients with colorectal cancer. World J Gastroenterol. 11, 1283-1286.

48. Bonuccelli G., Sotgia F., Frank P.G., Williams T.M., de Almeida C.J., Tanowitz H.B., Scherer P.E., Hotchkiss K.A., Terman B.I., Rollman B., Alileche A., Brojatsch J., & Lisanti M.P. (2005) ATR/TEM8 is highly expressed in epithelial cells lining Bacillus anthracis' three sites of entry: implications for the pathogenesis of anthrax infection. Am J Physiol Cell Physiol 288, C1402-C1410.

49. Hotchkiss K.A., Basile C.M., Spring S.C., Bonuccelli G., Lisanti M.P., & Terman B.I. (2005) TEM8 expression stimulates endothelial cell adhesion and migration by regulating cell-matrix interactions on collagen. Exp.Cell Res. 305, 133-144.

50. Nanda A., Carson-Walter E.B., Seaman S., Barber T.D., Stampfl J., Singh S., Vogelstein B., Kinzler K.W., & St Croix B. (2004) TEM8 interacts with the cleaved C5 domain of collagen alpha 3(VI). Cancer Res. 64, 817-820.

51. Werner E., Kowalczyk A.P., & Faundez V. (2006) Anthrax toxin receptor 1/tumor endothelium marker 8 mediates cell spreading by coupling extracellular ligands to the actin cytoskeleton. J Biol.Chem. 281, 23227-23236.

52. Banks D.J., Barnajian M., Maldonado-Arocho F.J., Sanchez A.M., & Bradley K.A. (2005) Anthrax toxin receptor 2 mediates Bacillus anthracis killing of macrophages following spore challenge. Cell Microbiol. 7, 1173-1185.

37 53. Hanks S., Adams S., Douglas J., Arbour L., Atherton D.J., Balci S., Bode H., Campbell M.E., Feingold M., Keser G., Kleijer W., Mancini G., McGrath J.A., Muntoni F., Nanda A., Teare M.D., Warman M., Pope F.M., Superti-Furga A., Futreal P.A., & Rahman N. (2003) Mutations in the gene encoding capillary morphogenesis protein 2 cause juvenile hyaline fibromatosis and infantile systemic hyalinosis. Am J Hum.Genet. 73, 791-800.

54. Premanandan C., Lairmore M.D., Fernandez S., & Phipps A.J. (2006) Quantitative measurement of anthrax toxin receptor messenger RNA in primary mononuclear phagocytes. Microb.Pathog. 41, 193-198.

55. Dowling O., Difeo A., Ramirez M.C., Tukel T., Narla G., Bonafe L., Kayserili H., Yuksel-Apak M., Paller A.S., Norton K., Teebi A.S., Grum-Tokars V., Martin G.S., Davis G.E., Glucksman M.J., & Martignetti J.A. (2003) Mutations in capillary morphogenesis gene-2 result in the allelic disorders juvenile hyaline fibromatosis and infantile systemic hyalinosis. Am J Hum.Genet. 73, 957-966.

56. Rainey G.J., Wigelsworth D.J., Ryan P.L., Scobie H.M., Collier R.J., & Young J.A. (2005) Receptor-specific requirements for anthrax toxin delivery into cells. Proc.Natl.Acad.Sci U.S.A 102, 13278-13283.

57. Dixon T.C., Fadl A.A., Koehler T.M., Swanson J.A., & Hanna P.C. (2000) Early Bacillus anthracis-macrophage interactions: intracellular survival survival and escape. Cell Microbiol. 2, 453-463.

58. Guidi-Rontani C., Levy M., Ohayon H., & Mock M. (2001) Fate of germinated Bacillus anthracis spores in primary murine macrophages. Mol.Microbiol. 42, 931-938.

59. Guidi-Rontani C., Weber-Levy M., Labruyere E., & Mock M. (1999) Germination of Bacillus anthracis spores within alveolar macrophages. Mol.Microbiol. 31, 9-17.

60. Kayal S. & Charbit A. (2006) : a key protein of with multiple functions. FEMS Microbiol Rev. 30, 514-529.

61. Alouf J.E. (2000) Cholesterol-binding cytolytic protein toxins. Int.J Med Microbiol 290, 351-356.

62. Alouf J.E. (2001) Pore-forming bacterial protein toxins: an overview. Curr.Top.Microbiol Immunol. 257, 1-14.

63. Cocklin S., Jost M., Robertson N.M., Weeks S.D., Weber H.W., Young E., Seal S., Zhang C., Mosser E., Loll P.J., Saunders A.J., Rest R.F., & Chaiken I.M. (2006) Real-time monitoring of the membrane-binding and insertion properties of

38 the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis. J Mol.Recognit. 19, 354-362.

64. Mosser E.M. & Rest R.F. (2006) The Bacillus anthracis cholesterol- dependent cytolysin, Anthrolysin O, kills human neutrophils, and macrophages. BMC.Microbiol 6, 56.

65. Park J.M., Ng V.H., Maeda S., Rest R.F., & Karin M. (2004) Anthrolysin O and other gram-positive cytolysins are toll-like receptor 4 agonists. J Exp.Med 200, 1647-1655.

66. Heffernan B.J., Thomason B., Herring-Palmer A., Shaughnessy L., McDonald R., Fisher N., Huffnagle G.B., & Hanna P. (2006) Bacillus anthracis phospholipases C facilitate macrophage-associated growth and contribute to virulence in a murine model of inhalation anthrax. Infect.Immun. 74, 3756-3764.

67. Scorpio A., Chabot D.J., Day W.A., O'Brien D.K., Vietri N.J., Itoh Y., Mohamadzadeh M., & Friedlander A.M. (2007) Poly-{gamma}-Glutamate Capsule-Degrading Treatment Enhances Phagocytosis and Killing of Encapsulated Bacillus anthracis. Antimicrob.Agents Chemother. 51, 215-222.

68. Aderem A. & Underhill D.M. (1999) Mechanisms of phagocytosis in macrophages. Annu.Rev.Immunol. 17, 593-623.

69. Vieira O.V., Botelho R.J., & Grinstein S. (2002) Phagosome maturation: aging gracefully. Biochem.J 366, 689-704.

70. Cote C.K., Rossi C.A., Kang A.S., Morrow P.R., Lee J.S., & Welkos S.L. (2005) The detection of protective antigen (PA) associated with spores of Bacillus anthracis and the effects of anti-PA antibodies on spore germination and macrophage interactions. Microb.Pathog. 38, 209-225.

71. Welkos S., Friedlander A., Weeks S., Little S., & Mendelson I. (2002) In-vitro characterisation of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody. J Med.Microbiol. 51, 821-831.

72. Ruthel G., Ribot W.J., Bavari S., & Hoover T.A. (2004) Time-lapse confocal imaging of development of Bacillus anthracis in macrophages. J Infect.Dis. 189, 1313-1316.

73. Chua J., Vergne I., Master S., & Deretic V. (2004) A tale of two : Mycobacterium tuberculosis phagosome maturation arrest. Curr.Opin.Microbiol 7, 71-77.

74. Vergne I., Chua J., Singh S.B., & Deretic V. (2004) Cell biology of mycobacterium tuberculosis phagosome. Annu.Rev.Cell Dev.Biol. 20, 367-394. 39 75. Hestvik A.L., Hmama Z., & Av-Gay Y. (2005) Mycobacterial manipulation of the host cell. FEMS Microbiol Rev. 29, 1041-1050.

76. Kayal S. & Charbit A. (2006) Listeriolysin O: a key protein of Listeria monocytogenes with multiple functions. FEMS Microbiol Rev. 30, 514-529.

77. Dubail I., Autret N., Beretti J.L., Kayal S., Berche P., & Charbit A. (2001) Functional assembly of two membrane-binding domains in listeriolysin O, the cytolysin of Listeria monocytogenes. Microbiology 147, 2679-2688.

78. Lety M.A., Frehel C., Dubail I., Beretti J.L., Kayal S., Berche P., & Charbit A. (2001) Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes. Mol.Microbiol 39, 1124-1139.

79. Portnoy D.A., Auerbuch V., & Glomski I.J. (2002) The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell- mediated immunity. J Cell Biol. 158, 409-414.

80. Guidi-Rontani C. (2002) The alveolar macrophage: the Trojan horse of Bacillus anthracis. Trends Microbiol. 10, 405-409.

81. Cleret A., Quesnel-Hellmann A., Mathieu J., Vidal D., & Tournier J.N. (2006) Resident CD11c+ lung cells are impaired by anthrax toxins after spore infection. J Infect.Dis. 194, 86-94.

82. Wei Z., Schnupf P., Poussin M.A., Zenewicz L.A., Shen H., & Goldfine H. (2005) Characterization of Listeria monocytogenes expressing anthrolysin O and phosphatidylinositol-specific phospholipase C from Bacillus anthracis. Infect.Immun. 73, 6639-6646.

83. Mosser E.M. & Rest R.F. (2006) The Bacillus anthracis cholesterol- dependent cytolysin, Anthrolysin O, kills human neutrophils, monocytes and macrophages. BMC.Microbiol 6, 56.

84. Passalacqua K.D., Bergman N.H., Herring-Palmer A., & Hanna P. (2006) The superoxide dismutases of Bacillus anthracis do not cooperatively protect against endogenous superoxide stress. J Bacteriol. 188, 3837-3848.

85. Pickering A.K., Osorio M., Lee G.M., Grippe V.K., Bray M., & Merkel T.J. (2004) Cytokine response to infection with Bacillus anthracis spores. Infect.Immun. 72, 6382-6389.

86. Pickering A.K. & Merkel T.J. (2004) Macrophages release tumor necrosis factor alpha and interleukin-12 in response to intracellular Bacillus anthracis spores. Infect.Immun. 72, 3069-3072. 40 87. Ribot W.J., Panchal R.G., Brittingham K.C., Ruthel G., Kenny T.A., Lane D., Curry B., Hoover T.A., Friedlander A.M., & Bavari S. (2006) Anthrax lethal toxin impairs innate immune functions of alveolar macrophages and facilitates Bacillus anthracis survival. Infect.Immun. 74, 5029-5034.

88. Cordoba-Rodriguez R., Fang H., Lankford C.S., & Frucht D.M. (2004) Anthrax lethal toxin rapidly activates caspase-1/ICE and induces extracellular release of interleukin (IL)-1beta and IL-18. J Biol.Chem. 279, 20563-20566.

89. Hanna P.C., Acosta D., & Collier R.J. (1993) On the role of macrophages in anthrax. Proc.Natl.Acad.Sci.U.S.A 90, 10198-10201.

90. Erwin J.L., DaSilva L.M., Bavari S., Little S.F., Friedlander A.M., & Chanh T.C. (2001) Macrophage-derived cell lines do not express proinflammatory cytokines after exposure to Bacillus anthracis lethal toxin. Infect.Immun. 69, 1175-1177.

91. Pellizzari R., Guidi-Rontani C., Vitale G., Mock M., & Montecucco C. (2000) Lethal factor of Bacillus anthracis cleaves the N-terminus of MAPKKs: analysis of the intracellular consequences in macrophages. Int.J Med.Microbiol. 290, 421­ 427.

92. Pellizzari R., Guidi-Rontani C., Vitale G., Mock M., & Montecucco C. (1999) Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNgamma-induced release of NO and TNFalpha. FEBS Lett. 462, 199-204.

93. Pandey J. & Warburton D. (2004) Knock-on effect of anthrax lethal toxin on macrophages potentiates cytotoxicity to endothelial cells. Microbes.Infect. 6, 835­ 843.

94. Kirby J.E. (2004) Anthrax lethal toxin induces human endothelial cell apoptosis. Infect.Immun. 72, 430-439.

95. Steele A.D., Warfel J.M., & D'Agnillo F. (2005) Anthrax lethal toxin enhances cytokine-induced VCAM-1 expression on human endothelial cells. Biochem.Biophys.Res.Commun. 337, 1249-1256.

96. Warfel J.M., Steele A.D., & D'Agnillo F. (2005) Anthrax lethal toxin induces endothelial barrier dysfunction. Am J Pathol. 166, 1871-1881.

97. Cote C.K., Van Rooijen N., & Welkos S.L. (2006) Roles of macrophages and neutrophils in the early host response to Bacillus anthracis spores in a mouse model of infection. Infect.Immun. 74, 469-480.

41 98. Cote C.K., Rea K.M., Norris S.L., van Rooijen N., & Welkos S.L. (2004) The use of a model of in vivo macrophage depletion to study the role of macrophages during infection with Bacillus anthracis spores. Microb.Pathog. 37, 169-175.

99. Peiser L. & Gordon S. (2001) The function of scavenger receptors expressed by macrophages and their role in the regulation of inflammation. Microbes.Infect. 3, 149-159.

100. Palecanda A. & Kobzik L. (2001) Receptors for unopsonized particles: the role of alveolar macrophage scavenger receptors. Curr.Mol.Med 1, 589-595.

101. de Villiers W.J. & Smart E.J. (1999) Macrophage scavenger receptors and foam cell formation. J Leukoc.Biol. 66, 740-746.

102. Kim J.G., Keshava C., Murphy A.A., Pitas R.E., & Parthasarathy S. (1997) Fresh mouse peritoneal macrophages have low scavenger receptor activity. J Lipid Res. 38, 2207-2215.

103. Gordon S. & Taylor P.R. (2005) Monocyte and macrophage heterogeneity. Nat.Rev.Immunol. 5, 953-964.

104. Albrink W.S. & Goodlow R.J. (1959) Experimental inhalation anthrax in the chimpanzee. Am J Pathol. 35:1055-65., 1055-1065.

105. Fritz D.L., Jaax N.K., Lawrence W.B., Davis K.J., Pitt M.L., Ezzell J.W., & Friedlander A.M. (1995) Pathology of experimental inhalation anthrax in the rhesus monkey. Lab Invest 73, 691-702.

106. Vasconcelos D., Barnewall R., Babin M., Hunt R., Estep J., Nielsen C., Carnes R., & Carney J. (2003) Pathology of inhalation anthrax in cynomolgus monkeys (Macaca fascicularis). Lab Invest 83, 1201-1209.

107. Phipps A.J., Premanandan C., Barnewall R.E., & Lairmore M.D. (2004) Rabbit and nonhuman primate models of toxin-targeting human anthrax vaccines. Microbiol.Mol.Biol.Rev. 68, 617-629.

108. Gleiser C.A., Gochenour W.S., Jr., & Ward M.K. (1968) Pulmonary lesions in dogs and pigs exposed to a cloud of anthrax spores. J Comp Pathol. 78, 445­ 448.

109. Gleiser C.A., Berdjis C.C., Hartman H.A., & Gochenour W.S. (1963) Pathology of experimental respiratory anthrax in macaca mulatta. Br.J Exp.Pathol. 44:416-26., 416-426.

42 110. Kobiler D., Gozes Y., Rosenberg H., Marcus D., Reuveny S., & Altboum Z. (2002) Efficiency of protection of guinea pigs against infection with Bacillus anthracis spores by passive immunization. Infect.Immun. 70, 544-560.

111. Turell M.J. & Knudson G.B. (1987) Mechanical transmission of Bacillus anthracis by stable flies (Stomoxys calcitrans) and mosquitoes (Aedes aegypti and ). Infect.Immun. 55, 1859-1861.

112. Zaucha G.M., Pitt L.M., Estep J., Ivins B.E., & Friedlander A.M. (1998) The pathology of experimental anthrax in rabbits exposed by inhalation and subcutaneous inoculation. Arch.Pathol.Lab Med 122, 982-992.

113. Klein F., Dearmon I.A., Jr., Lincoln R.E., Mahlandt B.G., & Fernelius A.L. (1962) Immunological studies of anthrox. II. Levels of immunity against Bacillus anthracis obtained with protective antigen and live vaccine. J Immunol. 88, 15­ 19.

114. Scorpio A., Blank T.E., Day W.A., & Chabot D.J. (2006) Anthrax vaccines: Pasteur to the present. Cell Mol.Life Sci 63, 2237-2248.

115. Keppie J., Smith H., & Harris-Smith P.W. (1955) The chemical basis of the virulence of Bacillus anthracis. III. The role of the terminal bacteraemia in death of guinea-pigs from anthrax. Br.J Exp.Pathol. 36, 315-322.

116. Belton F.C. & Strange R.E. (1954) Studies on a protective antigen produced in vitro from Bacillus anthracis: medium and methods of production. Br.J Exp.Pathol. 35, 144-152.

117. Wright G.G., Green T.W., & Kanode R.G., Jr. (1954) Studies on immunity in anthrax. V. Immunizing activity of alum-precipitated protective antigen. J Immunol. 73, 387-391.

118. Auerbach S. & Wright G.G. (1955) Studies on immunity in anthrax. VI. Immunizing activity of protective antigen against various strains of Bacillus anthracis. J Immunol. 75, 129-133.

119. Fellows P.F., Linscott M.K., Ivins B.E., Pitt M.L., Rossi C.A., Gibbs P.H., & Friedlander A.M. (2001) Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine 19, 3241-3247.

120. Drysdale M., Heninger S., Hutt J., Chen Y., Lyons C.R., & Koehler T.M. (2005) Capsule synthesis by Bacillus anthracis is required for dissemination in murine inhalation anthrax. EMBO J 24, 221-227.

43 121. Heninger S., Drysdale M., Lovchik J., Hutt J., Lipscomb M.F., Koehler T.M., & Lyons C.R. (2006) Toxin-deficient mutants of Bacillus anthracis are lethal in a murine model for pulmonary anthrax. Infect.Immun. 74, 6067-6074.

122. Heffernan B.J., Thomason B., Herring-Palmer A., Shaughnessy L., McDonald R., Fisher N., Huffnagle G.B., & Hanna P. (2006) Bacillus anthracis phospholipases C facilitate macrophage-associated growth and contribute to virulence in a murine model of inhalation anthrax. Infect.Immun. 74, 3756-3764.

123. COMINSKY N.C. (1959) Histological changes in mice injected with Bacillus anthracis. Tex.Rep.Biol.Med 17, 85-93.

124. Kramer M.J. & Roth I.L. (1970) Electron Microscopy of the Phagocytosis of Capsulated Bacillus anthracis. Infect.Immun. 2, 216-221.

125. Seto K. & Takizawa T. (1969) Dose-response relationship between inoculum dose of anthrax spores and survival time of mice. Natl.Inst.Anim Health Q.(Tokyo) 9, 129-135.

126. Welkos S.L., Keener T.J., & Gibbs P.H. (1986) Differences in susceptibility of inbred mice to Bacillus anthracis. Infect.Immun. 51, 795-800.

127. Jones W.I., Jr., Klein F., Lincoln R.E., Walker J.S., Mahlandt B.G., & Dobbs J.P. (1967) treatment of anthrax infection in mice. J Bacteriol. 94, 609­ 614.

128. Kalns J., Morris J., Eggers J., & Kiel J. (2002) Delayed treatment with has limited effect on anthrax infection in BLK57/B6 mice. Biochem.Biophys.Res.Commun. 297, 506-509.

129. Enkhtuya J., Kawamoto K., Kobayashi Y., Uchida I., Rana N., & Makino S. (2006) Significant passive protective effect against anthrax by antibody to Bacillus anthracis inactivated spores that lack two virulence plasmids. Microbiology 152, 3103-3110.

130. Perkins S.D., Flick-Smith H.C., Garmory H.S., Essex-Lopresti A.E., Stevenson F.K., & Phillpotts R.J. (2005) Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax. Genet.Vaccines.Ther. 3, 3.

131. Hahn B.L., Sharma S., & Sohnle P.G. (2005) Analysis of epidermal entry in experimental cutaneous Bacillus anthracis infections in mice. J Lab Clin.Med 146, 95-102.

132. Lyons C.R., Lovchik J., Hutt J., Lipscomb M.F., Wang E., Heninger S., Berliba L., & Garrison K. (2004) Murine model of pulmonary anthrax: kinetics of

44 dissemination, histopathology, and mouse strain susceptibility. Infect.Immun. 72, 4801-4809.

133. Duong S., Chiaraviglio L., & Kirby J.E. (2006) Histopathology in a murine model of anthrax. Int.J Exp.Pathol. 87, 131-137.

134. Aloni-Grinstein R., Gat O., Altboum Z., Velan B., Cohen S., & Shafferman A. (2005) Oral spore vaccine based on live attenuated nontoxinogenic Bacillus anthracis expressing recombinant mutant protective antigen. Infect.Immun. 73, 4043-4053.

135. Duc l.H., Hong H.A., Atkins H.S., Flick-Smith H.C., Durrani Z., Rijpkema S., Titball R.W., & Cutting S.M. (2007) Immunization against anthrax using Bacillus subtilis spores expressing the anthrax protective antigen. Vaccine 25, 346-355.

136. Popov S.G., Popova T.G., Grene E., Klotz F., Cardwell J., Bradburne C., Jama Y., Maland M., Wells J., Nalca A., Voss T., Bailey C., & Alibek K. (2004) Systemic cytokine response in murine anthrax. Cell Microbiol 6, 225-233.

137. Pezard C., Duflot E., & Mock M. (1993) Construction of Bacillus anthracis mutant strains producing a single toxin component. J Gen.Microbiol. 139 ( Pt 10), 2459-2463.

138. Pezard C., Berche P., & Mock M. (1991) Contribution of individual toxin components to virulence of Bacillus anthracis. Infect.Immun. 59, 3472-3477.

139. Drysdale M., Olson G., Koehler T.M., Lipscomb M.F., & Lyons C.R. (2007) Murine innate immune response to virulent toxigenic and non-toxigenic Bacillus anthracis strains. Infect.Immun.

140. Moayeri M., Haines D., Young H.A., & Leppla S.H. (2003) Bacillus anthracis lethal toxin induces TNF-alpha-independent hypoxia-mediated toxicity in mice. J Clin.Invest 112, 670-682.

141. Firoved A.M., Miller G.F., Moayeri M., Kakkar R., Shen Y., Wiggins J.F., McNally E.M., Tang W.J., & Leppla S.H. (2005) Bacillus anthracis edema toxin causes extensive tissue lesions and rapid lethality in mice. Am J Pathol. 167, 1309-1320.

142. Welkos S.L. & Friedlander A.M. (1988) Pathogenesis and genetic control of resistance to the Sterne strain of Bacillus anthracis. Microb.Pathog. 4, 53-69.

143. Harvill E.T., Lee G., Grippe V.K., & Merkel T.J. (2005) Complement depletion renders C57BL/6 mice sensitive to the Bacillus anthracis Sterne strain. Infect.Immun. 73, 4420-4422.

45 144. Welkos S.L., Trotter R.W., Becker D.M., & Nelson G.O. (1989) Resistance to the Sterne strain of B. anthracis: phagocytic cell responses of resistant and susceptible mice. Microb.Pathog. 7, 15-35.

145. Hahn U.K., Alex M., Czerny C.P., Bohm R., & Beyer W. (2004) Protection of mice against challenge with Bacillus anthracis STI spores after DNA vaccination. Int.J Med Microbiol 294, 35-44.

146. Kenney R.T., Yu J., Guebre-Xabier M., Frech S.A., Lambert A., Heller B.A., Ellingsworth L.R., Eyles J.E., Williamson E.D., & Glenn G.M. (2004) Induction of protective immunity against lethal anthrax challenge with a patch. J Infect.Dis. 190, 774-782.

147. Stokes M.G., Titball R.W., Neeson B.N., Galen J.E., Walker N.J., Stagg A.J., Jenner D.C., Thwaite J.E., Nataro J.P., Baillie L.W., & Atkins H.S. (2006) Oral administration of a Salmonella-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis. Infect.Immun.

148. Williamson E.D., Hodgson I., Walker N.J., Topping A.W., Duchars M.G., Mott J.M., Estep J., Lebutt C., Flick-Smith H.C., Jones H.E., Li H., & Quinn C.P. (2005) Immunogenicity of recombinant protective antigen and efficacy against aerosol challenge with anthrax. Infect.Immun. 73, 5978-5987.

149. Perkins S.D., Flick-Smith H.C., Garmory H.S., Essex-Lopresti A.E., Stevenson F.K., & Phillpotts R.J. (2005) Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax. Genet.Vaccines.Ther. 3, 3.

150. Flick-Smith H.C., Waters E.L., Walker N.J., Miller J., Stagg A.J., Green M., & Williamson E.D. (2005) Mouse model characterisation for anthrax vaccine development: comparison of one inbred and one outbred mouse strain. Microb.Pathog. 38, 33-40.

151. Garmory H.S., Titball R.W., Griffin K.F., Hahn U., Bohm R., & Beyer W. (2003) Salmonella enterica Serovar Typhimurium Expressing a Chromosomally Integrated Copy of the Bacillus anthracis Protective Antigen Gene Protects Mice against an Anthrax Spore Challenge. Infect.Immun. 71, 3831-3836.

152. Lee J.S., Hadjipanayis A.G., & Welkos S.L. (2003) Venezuelan equine encephalitis virus-vectored vaccines protect mice against anthrax spore challenge. Infect.Immun. 71, 1491-1496.

153. Hahn B.L., Sharma S., & Sohnle P.G. (2005) Analysis of epidermal entry in experimental cutaneous Bacillus anthracis infections in mice. J Lab Clin.Med 146, 95-102.

46 154. Brook I., Elliott T.B., Ledney G.D., Shoemaker M.O., & Knudson G.B. (2004) Management of postirradiation infection: lessons learned from animal models. Mil.Med 169, 194-197.

155. Alper T. (1979) Cellular Radiobiology., Cambridge University Press, New York.

156. Albrink W.S., Brooks S.M., Biron R.E., & Kopel M. (1960) Human inhalation anthrax. A report of three fatal cases. Am.J Pathol. 36:457-71., 457-471.

157. Brook I. & Ledney G.D. (1992) Quinolone therapy in the management of infection after irradiation. Crit Rev.Microbiol 18, 235-246.

158. Brook I. & Ledney G.D. (1994) Quinolone therapy in the prevention of endogenous and exogenous infection after irradiation. J Antimicrob.Chemother. 33, 777-784.

159. Brook I. & Ledney G.D. (1994) Effect of antimicrobial therapy on the gastrointestinal bacterial flora, infection and mortality in mice exposed to different doses of irradiation. J Antimicrob.Chemother. 33, 63-72.

160. Brook I., Tom S.P., & Ledney G.D. (1993) Development of infection with and Aspergillus sp. in irradiated mice after glycopeptide therapy. J Antimicrob.Chemother. 32, 705-713.

161. Brook I. & Ledney G.D. (1992) Short and long courses of ofloxacin therapy of Klebsiella pneumoniae sepsis following irradiation. Radiat.Res. 130, 61-64.

162. Brook I., Elliott T.B., Harding R.A., Bouhaouala S.S., Peacock S.J., Ledney G.D., & Knudson G.B. (2001) Susceptibility of irradiated mice to Bacillus anthracis sterne by the intratracheal route of infection. J Med.Microbiol. 50, 702­ 711.

163. Elliott T.B., Brook I., Harding R.A., Bouhaouala S.S., Shoemaker M.O., & Knudson G.B. (2002) Antimicrobial therapy for Bacillus anthracis-induced polymicrobial infection in (60)Co gamma-irradiated mice. Antimicrob.Agents Chemother. 46, 3463-3471.

164. Brook I., Giraldo D.E., Germana A., Nicolau D.P., Jackson W.E., Elliott T.B., Thakar J.H., Shoemaker M.O., & Ledney G.D. (2005) Comparison of clarithromycin and ciprofloxacin therapy for Bacillus anthracis Sterne infection in mice with or without (60)Co gamma-photon irradiation. J Med Microbiol 54, 1157­ 1162.

165. Brook I., Germana A., Giraldo D.E., Camp-Hyde T.D., Bolduc D.L., Foriska M.A., Elliott T.B., Thakar J.H., Shoemaker M.O., Jackson W.E., & Ledney G.D. 47 (2005) Clindamycin and quinolone therapy for Bacillus anthracis Sterne infection in 60Co-gamma-photon-irradiated and sham-irradiated mice. J Antimicrob.Chemother. 56, 1074-1080.

166. Cote C.K., Rea K.M., Norris S.L., Van Rooijen N., & Welkos S.L. (2004) The use of a model of in vivo macrophage depletion to study the role of macrophages during infection with Bacillus anthracis spores. Microb.Pathog. 37, 169-175.

167. Welkos S.L. & Friedlander A.M. (1988) Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice. Microb.Pathog. 5, 127-139.

168. Iacono-Connors L.C., Welkos S.L., Ivins B.E., & Dalrymple J.M. (1991) Protection against anthrax with recombinant virus-expressed protective antigen in experimental animals. Infect.Immun. 59, 1961-1965.

169. Ivins B.E., Welkos S.L., Little S.F., Crumrine M.H., & Nelson G.O. (1992) Immunization against anthrax with Bacillus anthracis protective antigen combined with adjuvants. Infect.Immun. 60, 662-668.

170. Flick-Smith H.C., Walker N.J., Gibson P., Bullifent H., Hayward S., Miller J., Titball R.W., & Williamson E.D. (2002) A recombinant carboxy-terminal domain of the protective antigen of Bacillus anthracis protects mice against anthrax infection. Infect.Immun. 70, 1653-1656.

171. Berthold I., Pombo M.L., Wagner L., & Arciniega J.L. (2005) Immunogenicity in mice of anthrax recombinant protective antigen in the presence of aluminum adjuvants. Vaccine 23, 1993-1999.

172. Sastry K.S., Tuteja U., & Batra H.V. (2003) Generation and characterization of monoclonal antibodies to protective antigen of Bacillus anthracis. Indian J Exp.Biol. 41, 123-128.

173. Klinman D.M. (2006) CpG oligonucleotides accelerate and boost the immune response elicited by AVA, the licensed anthrax vaccine. Expert.Rev.Vaccines. 5, 365-369.

174. Klinman D.M., Xie H., Little S.F., Currie D., & Ivins B.E. (2004) CpG oligonucleotides improve the protective immune response induced by the anthrax vaccination of rhesus macaques. Vaccine 22, 2881-2886.

175. Xie H., Gursel I., Ivins B.E., Singh M., O'Hagan D.T., Ulmer J.B., & Klinman D.M. (2005) CpG oligodeoxynucleotides adsorbed onto polylactide-co-glycolide microparticles improve the immunogenicity and protective activity of the licensed anthrax vaccine. Infect.Immun. 73, 828-833.

48 176. Huber M., Vor Dem E.U., Grunow R., & Bessler W.G. (2005) Generation of mouse polyclonal and human monoclonal antibodies against Bacillus anthracis toxin. Drugs Exp.Clin.Res. 31, 35-43.

177. Koya V., Moayeri M., Leppla S.H., & Daniell H. (2005) -based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge. Infect.Immun. 73, 8266-8274.

178. Ivins B.E., Pitt M.L., Fellows P.F., Farchaus J.W., Benner G.E., Waag D.M., Little S.F., Anderson G.W., Jr., Gibbs P.H., & Friedlander A.M. (1998) Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques. Vaccine 16, 1141-1148.

179. Welkos, S, D.Becker, A.Friedlander, and R.Trotter. Pathogenesis and host resistance to Bacillus anthracis: a mouse model. J.R.H.Pinkerton. Proceedings of the International Workshop on Anthrax [68], 49-52. 1990. Salisbury, England, Salisbury Medical Society.

180. Gaur R., Gupta P.K., Banerjea A.C., & Singh Y. (2002) Effect of nasal immunization with protective antigen of Bacillus anthracis on protective immune response against anthrax toxin. Vaccine 20, 2836-2839.

181. Boyaka P.N., Tafaro A., Fischer R., Leppla S.H., Fujihashi K., & McGhee J.R. (2003) Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen. J Immunol. 170, 5636-5643.

182. Sloat B.R. & Cui Z. (2006) Nasal immunization with anthrax protective antigen protein adjuvanted with polyriboinosinic-polyribocytidylic acid induced strong mucosal and systemic immunities. Pharm.Res. 23, 1217-1226.

183. Sloat B.R. & Cui Z. (2006) Strong mucosal and systemic immunities induced by nasal immunization with anthrax protective antigen protein incorporated in liposome-protamine-DNA particles. Pharm.Res. 23, 262-269.

184. Flick-Smith H.C., Eyles J.E., Hebdon R., Waters E.L., Beedham R.J., Stagg T.J., Miller J., Alpar H.O., Baillie L.W., & Williamson E.D. (2002) Mucosal or parenteral administration of microsphere-associated Bacillus anthracis protective antigen protects against anthrax infection in mice. Infect.Immun. 70, 2022-2028.

185. Matyas G.R., Friedlander A.M., Glenn G.M., Little S., Yu J., & Alving C.R. (2004) Needle-free skin patch vaccination method for anthrax. Infect.Immun. 72, 1181-1183.

186. Kenney R.T., Yu J., Guebre-Xabier M., Frech S.A., Lambert A., Heller B.A., Ellingsworth L.R., Eyles J.E., Williamson E.D., & Glenn G.M. (2004) Induction of 49 protective immunity against lethal anthrax challenge with a patch. J Infect.Dis. 190, 774-782.

187. Zeng M., Xu Q., Hesek E.D., & Pichichero M.E. (2006) N-fragment of edema factor as a candidate antigen for immunization against anthrax. Vaccine 24, 662-670.

188. Duverger A., Jackson R.J., van Ginkel F.W., Fischer R., Tafaro A., Leppla S.H., Fujihashi K., Kiyono H., McGhee J.R., & Boyaka P.N. (2006) Bacillus anthracis edema toxin acts as an adjuvant for mucosal immune responses to nasally administered vaccine antigens. J Immunol. 176, 1776-1783.

189. Quesnel-Hellmann A., Cleret A., Vidal D.R., & Tournier J.N. (2006) Evidence for adjuvanticity of anthrax edema toxin. Vaccine 24, 699-702.

190. Price B.M., Liner A.L., Park S., Leppla S.H., Mateczun A., & Galloway D.R. (2001) Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein. Infect.Immun. 69, 4509-4515.

191. Zhao P., Liang X., Kalbfleisch J., Koo H.M., & Cao B. (2003) Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model. Hum.Antibodies 12, 129-135.

192. Brossier F., Levy M., & Mock M. (2002) Anthrax spores make an essential contribution to vaccine efficacy. Infect.Immun. 70, 661-664.

193. Delvecchio V.G., Connolly J.P., Alefantis T.G., Walz A., Quan M.A., Patra G., Ashton J.M., Whittington J.T., Chafin R.D., Liang X., Grewal P., Khan A.S., & Mujer C.V. (2006) Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and . Appl.Environ.Microbiol 72, 6355-6363.

194. Cote C.K., Rossi C.A., Kang A.S., Morrow P.R., Lee J.S., & Welkos S.L. (2005) The detection of protective antigen (PA) associated with spores of Bacillus anthracis and the effects of anti-PA antibodies on spore germination and macrophage interactions. Microb.Pathog. 38, 209-225.

195. Goodman J.W. & D.E.Nitecki (1966) Immunochemical studies on the poly- gamma-D-glutamyl capsule of Bacillus anthracis. I. Characterization of the polypeptide and of the specificity of its reaction with rabbit antisera. 5, 657-663.

196. Wang J.Y. & Roehrl M.H. (2005) Anthrax vaccine design: strategies to achieve comprehensive protection against spore, bacillus, and toxin. Med Immunol. 4, 4.

50 197. Kozel T.R., Murphy W.J., Brandt S., Blazar B.R., Lovchik J.A., Thorkildson P., Percival A., & Lyons C.R. (2004) mAbs to Bacillus anthracis capsular antigen for immunoprotection in anthrax and detection of antigenemia. Proc.Natl.Acad.Sci U.S.A 101, 5042-5047.

198. Rhie G.E., Roehrl M.H., Mourez M., Collier R.J., Mekalanos J.J., & Wang J.Y. (2003) A dually active anthrax vaccine that confers protection against both bacilli and toxins. Proc.Natl.Acad.Sci U.S.A 100, 10925-10930.

199. Joyce J., Cook J., Chabot D., Hepler R., Shoop W., Xu Q., Stambaugh T., Aste-Amezaga M., Wang S., Indrawati L., Bruner M., Friedlander A., Keller P., & Caulfield M. (2006) Immunogenicity and protective efficacy of Bacillus anthracis poly-gamma-D-glutamic acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy. J Biol.Chem. 281, 4831-4843.

200. Kubler-Kielb J., Liu T.Y., Mocca C., Majadly F., Robbins J.B., & Schneerson R. (2006) Additional conjugation methods and immunogenicity of Bacillus anthracis poly-gamma-D-glutamic acid-protein conjugates. Infect.Immun. 74, 4744-4749.

201. Kozel T.R., Thorkildson P., Brandt S., Welch W.H., Lovchik J.A., AuCoin D.P., Vilai J., & Lyons C.R. (2007) Protective and immunochemical activities of monoclonal antibodies reactive with the Bacillus anthracis polypeptide capsule. Infect.Immun. 75, 152-163.

202. Banchereau J., Bazan F., Blanchard D., Briere F., Galizzi J.P., van Kooten C., Liu Y.J., Rousset F., & Saeland S. (1994) The CD40 antigen and its ligand. Annu.Rev.Immunol. 12, 881-922.

203. Hahn U.K., Boehm R., & Beyer W. (2006) DNA vaccination against anthrax in mice-combination of anti-spore and anti-toxin components. Vaccine 24, 4569­ 4571.

204. Cui Z. & Sloat B.R. (2006) Topical immunization onto mouse skin using a microemulsion incorporated with an anthrax protective antigen protein-encoding plasmid. Int.J Pharm. 317, 187-191.

205. Galloway D.R. & Baillie L. (2004) DNA vaccines against anthrax. Expert.Opin.Biol.Ther. 4, 1661-1667.

206. Perkins S.D., Flick-Smith H.C., Garmory H.S., Essex-Lopresti A.E., Stevenson F.K., & Phillpotts R.J. (2005) Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax. Genet.Vaccines.Ther. 3, 3.

207. Harrop R., John J., & Carroll M.W. (2006) Recombinant viral vectors: cancer vaccines. Adv.Drug Deliv.Rev. 58, 931-947. 51 208. Harrop R. & Carroll M.W. (2006) Viral vectors for cancer immunotherapy. Front Biosci. 11, 804-817.

209. van Oers M.M. (2006) Vaccines for viral and parasitic diseases produced with baculovirus vectors. Adv.Virus Res. 68, 193-253.

210. Xing Z., Santosuosso M., McCormick S., Yang T.C., Millar J., Hitt M., Wan Y., Bramson J., & Vordermeier H.M. (2005) Recent advances in the development of adenovirus- and poxvirus-vectored tuberculosis vaccines. Curr.Gene Ther. 5, 485-492.

211. McConnell M.J., Hanna P.C., & Imperiale M.J. (2006) Cytokine response and survival of mice immunized with an adenovirus expressing Bacillus anthracis protective antigen domain 4. Infect.Immun. 74, 1009-1015.

212. Ogasawara Y., Amexis G., Yamaguchi H., Kajigaya S., Leppla S.H., & Young N.S. (2006) Recombinant viral-like particles of parvovirus B19 as antigen carriers of anthrax protective antigen. In Vivo 20, 319-324.

213. Riemenschneider J., Garrison A., Geisbert J., Jahrling P., Hevey M., Negley D., Schmaljohn A., Lee J., Hart M.K., Vanderzanden L., Custer D., Bray M., A., Ivins B., Bassett A., Rossi C., & Schmaljohn C. (2003) Comparison of individual and combination DNA vaccines for B. anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus. Vaccine 21, 4071-4080.

214. Iacono-Connors L.C., Schmaljohn C.S., & Dalrymple J.M. (1990) Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants. Infect.Immun. 58, 366-372.

215. Li Z.N., Mueller S.N., Ye L., Bu Z., Yang C., Ahmed R., & Steinhauer D.A. (2005) Chimeric influenza virus hemagglutinin proteins containing large domains of the Bacillus anthracis protective antigen: protein characterization, incorporation into infectious influenza viruses, and antigenicity. J Virol 79, 10003­ 10012.

216. Abrami L., Reig N., & van der Goot F.G. (2005) Anthrax toxin: the long and winding road that leads to the kill. Trends Microbiol 13, 72-78.

52 Fully Virulent Bacillus C57/B6 A/J BALB/C DBA/2 anthracis S.C. 19 5 19 5 I.N. 1.24x104 6250 3.7x104 <6250 I.T. 500 NR 870-1000 35 I.P NR <1x102 5x102 NR Aerosol NR 5x102 NR NR Attenuated Bacillus C57/B6 A/J BALB/C DBA/2 anthracis S.C. 8.6x10 5 1x103 6.8x107 2.0x103 I.N. NR NR NR NR I.T. NR NR NR 8.3x103 I.P. NR 1.0x104 NR 3.0x106 Aerosol NR NR NR NR

Table 1.1 – LD50 values of commonly used mouse strains exposed to B. anthracis33,122,126,132,136,197. S.C. - subcutaneous, I.N. - intranasal, I.T. – intratracheal, I.P – intraperitoneal, NR – no references available.

53 Exosporium Spore Coat

Cortex Core

Figure 1.1 – An electron photomicrograph of a spore of Bacillus anthracis Sterne.

54 Figure 1.2 – Schematic detailing the process of toxin endocytosis. The protective antigen (PA) binds to the anthrax toxin receptor which associates with LDL receptor related protein 6 (LRP6). Protective antigen is processed by a furin-like protease to its 63 kDa form. Heptamerization of the PA receptor complex allows the binding of edema factor (EF) or lethal factor (LF) followed by clathrin mediated endocytosis of the receptor complex. The endocytic vesicle progresses through phagosomal maturation along microtubules. The receptor complex inserts and releases EF and LF molecules into intra-endosomal vesicles called endosomal carrier vesicles (ECV) or multivesicular bodies (MVB). At the termination of the pathway, the vesicle fuses with the endosomal membrane, releasing its contents into the cytosol. Adapted from Abrami, et al and Wei et al36,216.

55 100 A. Control B. 90 80 1.8 x 103 70

l 60 a v i

v 50 r

Su 40 6.3 x 104 30 20 10 1.6 x 106 0 1 2 3 4 5 6 7 8 9 10 Days

Group Dose MNDD Survival Control n/a n/a 5/5 Exp. 1 1.8 x 103 10** 9/10 Exp. 2 6.3 x 104 6** 3/10 Exp. 3 1.6 x 106 4.5** 0/10 ** p<0.001 Kruskal-Wallis test (nonparametric ANOVA)

Figure 1.3 – A/J mouse model of pulmonary anthrax. A. Survival curve and aerosol dosages of B. anthracis Sterne. The LD50 in this experiment was 2.2 x 104 CFU (calculated by the Spearman-Karber method). B. Photomicrograph of lung showing chains of bacilli in the alveolar septae.

56 A. 100 B. 90 1 x 107 80 70

l 60 a 8 v 1 x 10 i

v 50 r

Su 40 30 20 10 0 0 1 2 3 4 5 6 7 8 9 10 Days

Group Dose (CFU) MNDD Survival Control n/a n/a 10/10 Exp. 1 1 x 104 n/a 10/10 Exp. 2 1 x 106 n/a 10/10 Exp. 3 1 x 107 9 9/10 Exp. 4 1 x 108 5.5 6/10

Figure 1.4 – A/J mouse model of gastrointestinal anthrax. A. Survival curve of A/J mice inoculated with Bacillus anthracis Sterne by gavage. Only a dose of 1 x 108 spores approaches 50% lethality in the inoculated group of mice. B. Photomicrograph of a small intestinal villus showing bacilli in the laminae propria.

57 A. B.

C. D.

Figure 1.5 – Photomicrographs of a serial time course study detailing the cutaneous histopathology of A/J mice inoculated subcutaneously with B. anthracis Sterne. A. Day 1 post inoculation. B. Day 2 post inoculation. Minimal inflammation is present in the subcutaneous tissue. C. Day 3 post inoculation. The subcutaneous tissue is expanded by edema, however the inflammatory cell infiltrate remains minimal to moderate. Mats of bacillus are visible in the deep subcutis. D. Marked hemorrhage with intravascular platelet aggregation. A large mat of bacteria is present in the deep subcutis.

58 CHAPTER 2

EXPRESSION OF FUNCTIONAL ANTHRAX TOXIN RECEPTORS IN MONONUCLEAR PHAGOCYTES

2.1 Introduction

Bacillus anthracis is a gram positive, non-hemolytic, catalase-positive, spore-forming, facultative anaerobic bacterium. Depending on the route of exposure, B. anthracis can cause inhalational, cutaneous, or gastrointestinal anthrax1,2. Spores introduced into the body are typically phagocytosed by macrophages and carried to regional lymph nodes. The current model of infection proposes that B. anthracis spores germinate inside the macrophages and become vegetative cells, which may briefly replicate intracellularly and then escape from the macrophage to multiply extracellularly3,4.

Virulent B. anthracis strains harbor two plasmids designated pX01 and pX025. The pX01 plasmid contains a 44.8 kb that encodes for the three toxin genes, cya, lef, pagA6,7. The pX02 plasmid encodes five capsule genes, capA, capB, capC, capD and capE and a gene associated with depolarization of the capsule, dep8. The pX01 gene pagA encodes for an 83 kDa

59 protein known as protective antigen (PA, PA83). PA binds to one of the anthrax toxin receptors (ATRs) as a 83 kDa form (PA83)9-11. Following binding to the

ATR, PA83 is cleaved by furin-like protease on the cell surface into the 63 kDa receptor-bound molecule (PA63) and a 20 kDa fragment that is released into the extracellular milieu9,12-14. Two ATRs have been identified to date, tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2

(CMG2)10,11,15-17. Both ATRs are type 1 transmembrane proteins with an extracellular domain that is highly related to von Willebrand factor type A and integrin inserted domains (VWA/I domains)17. The function of TEM8 or CMG2 in normal cell physiology has not been identified, however there are at least two potential phosphorylation sites located in the cytoplasmic portion of TEM8 and

CMG2, this strongly suggests that TEM8 and CMG2 can transduce signals from the cell surface to the cytoplasm16. The binding of PA83 to the ATR is a critical step in the process by which the catalytic domains of the exotoxins are translocated to the cytoplasm.

Lethal toxin (LT) is formed by the binding of the lef gene product, lethal factor (LF), to receptor-bound heptamerized PA6318. By analogy, edema toxin

(ET) is formed by the binding of the cya gene product, edema factor (EF), to receptor-bound PA6318. Lethal factor is a zinc metalloprotease that inactivates mitogen activated protein kinase kinases (MAPKK)19-21. The cleavage of MAPKK prevents activation of p38 MAPKs which subsequently prevents induction of certain NF-κB target genes including genes necessary to prevent apoptosis of

60 activated macrophages22. B. anthracis LT may contribute to the evasion of innate immunity by inducing apoptosis in activated macrophages that leads to a massive release of IL-1 and TNF- α23,24. ET is a calcium-calmodulin dependent adenylate cyclase that increases intracellular cyclic AMP (cAMP) in susceptible cells25-31. ET alters water homeostasis and may be responsible for the edema that frequently occurs with B. anthracis infection1,32,33. ET also differentially regulates macrophage responsiveness to -induced production of TNF-α and IL-634.

Although TEM8 and CMG2 function as receptors for LT and ET, their expression in mononuclear phagocytes has not been reported. Expression data concerning these receptors has been limited to mouse and human tissues and a subset of cells, mostly endothelial cell lines35. In the present study, we describe the presence of functional anthrax toxin receptors in primary human and mouse mononuclear phagocytes, as well as the expression patterns of mRNA transcripts of TEM8 and CMG2 in these important cells of the .

2.2 Materials and methods

Isolation of mouse mononuclear phagocytes and tissues

Mouse peritoneal macrophages (MPM) and alveolar macrophages (AM) were harvested by lavage from 32 – 52 day old female C57BL/6 mice (Charles

River Laboratories, Wilmington MA). The mice were euthanatized using CO2 and

61 the peritoneal cavity was lavaged with RPMI Media 1640 (Gibco Invitrogen Corp,

Grand Island, NY). The peritoneal lavage samples were centrifuged and resuspended in RPMI 1640 medium supplemented with 20 mM HEPES, 20 mM

L-glutamine (Gibco Invitrogen Corp.), and 10% FBS (Gibco Invitrogen Corp.).

The peritoneal macrophages were adhered directly to tissue-culture flasks for 2 hours and then the non-adherent cells were removed by washing. Samples of liver, lung, and spleen were collected, immediately frozen in liquid nitrogen, and stored frozen at -80 °C. The Ohio State University Institutional Laboratory Animal

Care and Use Committee (ILACUC) reviewed and approved all procedures involving the use of animal research models.

Isolation and culture of human monocyte derived macrophages

Concentrated human leukocytes from anonymous blood donors were purchased from the American Red Cross. Peripheral blood mononuclear cells

(PBMCs) were separated on a Ficoll (Amersham Biosciences, New York NY) density gradient and any contaminating platelets were removed by centrifugation at 100 x g. Monocyte derived macrophages (MDMs) were differentiated by culturing the PBMCs for 5 to 7 days in Teflon culture dishes (Savillex,

Minnetonka, MN) containing RPMI 1640 medium supplemented with 20 mM

HEPES, 20 mM L-glutamine, and 10% pooled serum as previously described36.

MDM differentiation was verified by flow cytometric analysis and light microscopy. Differentiated MDMs were washed and then adhered to tissue

62 culture vessels (flasks or culture plates) containing serum-free RPMI-1640 for at least four hours. The non-adherent cells were removed by washing with RPMI­

1640 and the MDMs were incubated in RMPI-1640 containing 20mM HEPES, 20 mM L-glutamine, and 10% human serum. For some experiments human MDMs were stimulated with lipopolysaccharide (LPS) purified from at a concentration 100 ng/ml (Sigma, St. Louis, MO) or IL-1β (Roche labs, Nutley, NJ) at a concentration of 50 IU/ml for four hours at 37 °C.

Immortalized human and mouse cell lines

HeLa (American Type Culture Collection, Catalog # CCL-248 ), Raw 264.7

(ATCC Catalog # TIB-71), J774A.1 (ATCC Catalog # TIB-67) and THP-1 (ATCC

Catalog #TIB-202) cells were propagated and sub-cultured using medium and supplementary reagents as recommended by ATCC. THP-1 cells were differentiated in media containing 3 ng/ul of phorbol myristate acetate (PMA)

(Sigma chemicals, St. Louis, MO) for 3 days at 37 °C.

Lethal toxin cytotoxicity assay

J774A.1 cells were seeded into a 96-well flat-bottom plate at a concentration of 3 x 104 cells/well 17 – 19 hours prior to each experiment. The original media was removed from each well and replaced with serum-free media prior to starting each experiment. Recombinant protective antigen (rPA) was obtained from List Biological Laboratories (Campbell, CA) and the National

63 Cancer Institute (National Institutes of Health-NCI)37. Recombinant lethal factor

(rLF) (List Biological Laboratories) was diluted in serum-free RPMI-1640 media to a final concentration of 1.066 μg/ml. Serial 1:2 dilutions of rPA were prepared in serum-free media in quadruplicate starting at 205 ng/well and ending at 1.6 ng/well. rLF was added to each well at a final concentration of 80 ng/well.

Quadruplicate control wells were treated with serum-free media alone, rPA alone

(205 ng/well) or rLF alone (80 ng/well). All wells contained a final volume of 150

μl. The cells were incubated at 37 ºC for three hours. Following the incubation period, 20 μl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4­ sulfophenyl)-2H-tetrazolium, inner salt (MTS) from Promega (Madison, WI) was placed to each well. The plates were incubated at 37 ºC for three hours and the absorbance at 480 nm was determined using a 96-well plate reader (Coulter

Microplate Reader, Beckman-Coulter, Fullerton, CA). The average absorbance and relative standard deviation was determined for each set of quadruplicate wells.

Edema toxin cAMP assay

The cells were seeded into a 96-well flat-bottom plate at a concentration of

3 x 104 cells/well 17 – 19 hours prior to each experiment. The original media was removed from each well and replaced with serum-free media prior to starting each experiment. Recombinant edema factor (rEF) (List Biological Laboratories) was diluted in serum-free RPMI-1640 media at a final concentration of 1.0 μg/ml.

64 Recombinant PA (rPA) (List Biological Laboratories) was diluted in serum-free

RPMI-1640 media at a final concentration of 2.7 ug/ml. Each cell line was incubated in 75 ul of PA and 75 ul of EF for 30, 60, 120 and 180 minutes

Following stimulation, the cells were washed with sterile PBS. Intracellular cAMP levels were measured using cAMP Direct Biotrak EIA (Amersham Biosciences,

Piscataway, NJ) and the absorbance at 480 nm was determined using a 96-well plate reader (Coulter Microplate Reader, Beckman-Coulter).

B. anthracis propagation and spore preparation

Nutrient agar plates were inoculated with B. anthracis Sterne strain (34F2) and incubated for 24 to 48 hours. The degree of sporulation was determined by

Gram’s stain. B. anthracis spores were removed from the plates by rinsing the plates with sterile water. The B. anthracis spore suspension was heat treated at

60 º C for 30 min to lyse vegetative cells. The spores were pelleted by centrifugation at 10,000 x g in a HB-104 rotor (Beckman Coulter, Inc.) and resuspended in sterile water. The spores were pelleted again at 10,000 x g and resuspended in sterile water for injection containing 1% phenol (v/v) and stored at 4 °C. The purity of the spores was determined by malachite green staining and scanning electron microscopy.

65 Infection of macrophages with B. anthracis spores

Human MDMs were seeded at a density of 1 x 106 cells/well into six-well flat-bottom tissue culture plates 17 to 19 hours before infection. The media was replaced with 750 μl of serum-free RPMI-1640 containing 20mM HEPES, 20 mM

L-glutamine. Washed B. anthracis spores were added to each well at a multiplicity of infection (MOI) of 3 in a volume of 100 μl. The plates were centrifuged at 400 x g for 3 min and incubated at 37 °C for 30 minutes. The wells were washed five times with serum-free RPMI-1640 media. One ml of serum-free

RPMI-1640 media containing 20 mM HEPES was placed in each well and the plates were incubated for an additional 60 minutes Following incubation for one hour at 37 ºC, the media was removed from 2 wells and the cells were lysed in

2.5 % saponin (Fisher Scientific). The media and lysate from one well was subjected to heat treatment in a 60ºC water bath for 30 minutes before culture.

Serial dilutions of each sample were performed before plating. Bacterial culture was performed on nutrient agar plates overnight. Colony counts were performed and the actual MOI was determined by back calculation.

RNA isolation

RNA from cells was isolated using commercial kits (RNA mini-prep,

Qiagen, Valencia, CA), and Trizol (Invitrogen, Carlsbad, CA) and stored in –70

°C in RNAase free water. RNA integrity was determined by the presence of 18S and 28S ribosomal RNA bands on a formaldehyde gel as well as the 260/280

66 ratio by spectrophotometry. RNA quantitation was performed with spectrophotometry and a Ribogreen fluorometry assay (Invitrogen).

Standard RT-PCR

TEM8564 (isoform 1), TEM8368 (isoform 2), and TEM8333 (isoform 3) specific primers (Table 2.2) were designed based on the predicted mRNA splicing variants (Fig. 2.3). In addition, a pan-TEM8 primer set which recognizes all three splice variants in mice and humans was utilized. A CMG2 primer set was designed to amplify the fragment between exons 2 and 4 in all isoforms (Fig.

2.3). Isolated RNA was converted to cDNA using a reverse transcription kit

(Promega, Madison, WI) The reaction was performed in a 20 μl volume containing 5 mM MgCl2, 1X reverse transcription buffer, 1 mM dNTPs, 15 U of

RNAsin Ribonuclease inhibitor, 15 U of AMV reverse transcriptase and 0.5 μg of oligo(dT)15 primers. Prior to the reverse transcription reaction, 1 μg of RNA was incubated at 70 °C for 10 minutes and then cooled on ice. The reverse transcription reaction steps were performed as follows: 42 °C for 60 minutes, 95

°C for 5 minutes and 5 °C for 5 minutes. The cDNA was amplified in a 50 ul volume containing 2 U FastStart-Taq DNA polymerase (Roche, Indianapolis IN),

1X PCR buffer, 2 mM MgCl2, 200 μM dNTP, 0.5 μM downstream primer, and 0.5

μM upstream primer. The reaction mix was cycled for 35 cycles (95 °C for 50 seconds, 48 °C for 50 seconds, 72 °C for 50 seconds). Amplified PCR products were analyzed by horizontal agarose gel electrophoresis and visualized by

67 ethidium bromide staining. Product bands were excised, purified and sequenced followed by software alignment with GenBank accession numbers (Table 2.2).

Real time RT-PCR assay

Real time quantitative PCR was performed using the Lightcycler system

(Roche labs, Nutley, N.J). Standards were made by cloning the TEM8 and CMG2

PCR products into the TOPO 4.1 vector (Invitrogen, Carlsbad, CA) and generating in-vitro mRNA transcripts using T7 RNA polymerase and the

Maxiscript™ in vitro transcription kit (Ambion, Austin, TX). The PCR reaction was performed in Lightcycler capillary tubes at a total volume of 20 μl, with a primer concentration of 0.5 μM and reagents from the Sybr Green RT-PCR kit (Qiagen,

Valencia, CA). Copy number was calculated from the standard curve and shown as copies per 500 ng of RNA. The TEM8 RT-PCR was performed using the following reaction conditions : 50 ºC for 40 minutes and 95 ºC for 15 minutes for one cycle followed by 94ºC for 15 seconds, 49 ºC for 20 seconds, 72 ºC for 10 seconds and a five second acquisition at 79 ºC for 50 cycles. The cycling conditions for the CMG2 RT-PCR were as follows: 50 ºC for 40 minutes and 95

ºC for 15 minutes for one cycle followed by 94 ºC for 15 seconds, 49 ºC for 20 seconds, 72 ºC for 10 seconds and a five second acquisition at 75 ºC for 50 cycles.

68 2.3 Results

Response of human and mouse macrophage cell lines and primary mononuclear phagocytes to anthrax toxins

To determine if functional anthrax toxin receptors were present in human and mouse macrophage cell lines and primary mononuclear phagocytic cells, we measured intracellular cAMP concentration following treatment with ET.

Intoxication requires binding of PA to the ATR followed by heptamerization of the

ATR-PA complex and the internalization of EF (adenylate cyclase) by receptor- mediated endocytosis18,38,39. EF can translocate to the cytoplasm following acidification of the endocytic vesicle38. Prior to these experiments, a LT cytotoxicity assay was performed in J774A.1 cells to determine the activity and optimal dose of PA to use in subsequent experiments. A dose dependent decrease in cell viability was seen in these cells after incubation with PA from two independent sources and LF for three hours (Fig. 2.1). Based on this data, a concentration of ≈205 ng PA and 80 ng LF or EF per 3 x 104 cells was selected.

A dose dependent increase in cAMP concentration (fmols/well) was seen in

J774A.1 cells after 180 minutes of incubation with increasing concentrations of

PA (0 ng to 205 ng per 3 x 104 cells) and a concentration of 80 ng of EF per 3 x

104 cells (Fig. 2.2A). Forskolin was utilized as a positive control with all cell types.

The fold increase in cAMP for forskolin-treated cells was cell line-dependent

(range 1.6 to > 30) (Fig. 2.2B). In addition, a time-dependent increase in cAMP was seen in the J774A.1 cells but not THP-1 cells after incubation with edema

69 toxin (Fig. 2.2C). The maximum increase in cAMP occurred at 180 minutes in

J774A.1 cells.

To determine if other macrophage-like cell lines and primary cells respond in a similar manner as J774A.1 cells, ET intoxication experiments were performed using Raw 264.7, THP-1, human monocyte derived macrophages

(MDM) and mouse peritoneal macrophages (MPM). All primary mononuclear phagocytic cells and cell lines except THP-1 had an increase in cAMP at the 180 minute time point (Fig. 2.2D). RAW 264.7 cells showed a time dependent increase in cAMP concentration but the magnitude of this increase was reduced as compared to J774A.1 cells. Both MDMs and MPMs responded to ET exposure, producing an increase in intracellular cAMP, albeit less than ET- treated J774A.1 cells (Table 2.1).

TEM8 isoform and CMG2 mRNA expression in human and mouse cell lines and primary cells

We developed a standard RT-PCR assay to detect TEM8 and CMG2 transcripts in the chosen cell lines, mononuclear phagocytes, and tissues (Fig 2.3 and Table 2.2). TEM8 mRNA expression was demonstrated in the human and mouse immortalized cell lines, mouse liver, lung and spleen (Fig. 2.4B and Table

2.3). TEM8 mRNA could not be detected in human MDMs and PBMCs as well as

MPM (Fig. 2.4 and Table 2.3). Transcripts from the three isoforms of TEM8 were detected in HeLa cells (Fig. 2.4A) but not in human PBMCs or MDMs.

70 Transcripts from isoform 1 were detected in the mouse macrophage-like cell lines and mouse liver, lung, and spleen but not in MPM. However, CMG2 transcripts were detected in all immortalized cell lines as well as primary mononuclear phagocytes (Table 2.3).

Quantitative expression of TEM8 and CMG2 in human and mouse cell lines and primary mononuclear phagocytes

We developed a real-time RT-PCR assay that utilized in vitro mRNA transcripts of the TEM8 or CMG2 amplicon as a standard to determine the copy number of TEM8 and CMG2 transcripts in these cell lines and primary cells.

Primers were designed to anneal to cDNA containing all published isoforms of

TEM8 and CMG2 (Table 2.2). Basing the standard curve on RNA transcripts eliminates error introduced by the variable efficiency of the RT step in the RT­

PCR reaction. The TEM8 and CMG2 real time RT-PCR data reflected the standard RT-PCR data (Fig. 2.5). TEM8 mRNA copy number was below the level of detection (<103 copies/500 ng RNA) in MDMs derived from six unrelated human donors. The copy number of TEM8 transcripts per 500 ng of RNA was greater than 107 in HeLa, THP-1 and PMA-differentiated THP-1 cells. The copy number of CMG2 transcripts varied in the immortalized cell lines. CMG2 mRNA expression paralleled our standard RT-PCR results, indicating that primary human monocytes and MDMs expressed CMG2 at a mean of 1.78 x 106 copies per 500 ng of RNA (> 10 copies/cell).

71 We next exposed MDMs to LPS, B. anthracis spores, or IL-1β to determine if the expression of TEM8 or CMG2 mRNA expression could be modulated. The MDMs were infected with B. anthracis Sterne spores at a MOI of

3 and total RNA was isolated at 2 hour post-infection. Phagocytosis of spores was confirmed by transmission electron microscopy (Fig. 2.6B). Germination of spores occurs very rapidly following phagocytosis and the replication of intracellular B. anthracis begins at 1.5 hours post-infection (Fig. 2.6A). The number of intracellular bacilli continues to increase over the next seven hours eventually leading to the of MDMs and the release of vegetative cells.

Stimulation of MDMs with LPS, spores or IL-1β had no effect on TEM8 or CMG2 mRNA expression as compared to the untreated control (Fig. 2.6C). Average copy number of CMG2 in the stimulated MDMs were compared to the control and found to be not significant (p = 0.537) using one way analysis of variance

(ANOVA).

2.4 Discussion

Monocytes and macrophages are considered to be targets of intoxication during B. anthracis infection4,22,40-44. While the expression of ATRs has been examined in endothelial cells and tissues, the patterns of TEM8 and CMG2 mRNA expression in primary macrophages has not been reported. The knowledge of expression and functionality of these receptors in primary cells is also important as most in vitro intoxication studies rely on macrophage-like cell

72 lines such as J774A.1 and RAW 264.7. In this report we examined the expression of the two identified ATRs in primary mononuclear phagocytes, in terms of functionality and mRNA expression. To elucidate the presence of functional ATRs in mouse and human macrophages, we utilized an ET intoxication assay which measures cAMP production from cells exposed to PA and EF. EF is a calcium-calmodulin dependent adenylate cyclase. Intoxication of host cells introduces the enzyme into the cytoplasm where it is activated by calmodulin following an increase in intracellular Ca2+27,28. Activated EF catalyzes the synthesis of cAMP from ATP in the host cells28. Our results from ET intoxication assay suggest that primary mononuclear phagocytes have functional

ATRs and introduction of EF into the cytoplasm resulted in increased intracellular cAMP.

In addition, we determined the mRNA expression of the two known ATRs in primary macrophages. TEM8 transcripts were detected in HeLa cells, THP-1 cells, mouse macrophage-like cell lines and selected mouse tissues. However,

TEM8 mRNA expression was not detected in primary human or mouse macrophages. In addition, the expression of isoform specific TEM8 was examined by standard RT-PCR in a subset of cell types. Although HeLa cells express all three isoforms of TEM8, primary cells did not express any isoforms of

TEM8, consistent with our results using the universal TEM8 primers. In contrast,

CMG2 mRNA was detected in primary macrophages, implicating this receptor as

73 an important ATR in primary macrophages. The function of CMG2 in cells of the immune system remains to be determined.

The degree of ET intoxication did not correlate with the level of ATR mRNA expression. Although THP-1 cells and MDMs express mRNA transcripts of at least one of the two known ATRs at similar copy numbers as the macrophage-like cell lines, cAMP production was not elicited to a commensurate level. Theoretically, a cell containing functional ATRs will translocate three molecules of EF for every PA-ATR heptermerized complex45. Potential factors that could prevent an increase in cAMP in the presence of ATR mRNA include:

1) inefficient translation of the ATR mRNA or altered post-translational processing, 2) deficient protease activity at the plasma membrane, 3) non­ functional receptors (i.e. unable to heptamerize and bind EF), 4) defects in the endocytic pathway (e.g. actin polymerization, endosomal acidification) in which the prepore structure cannot insert into the limiting membrane of the , or 5) insufficient increases in the intracellular Ca2+ level.

Our data indicate that primary cells differ significantly in response to exposure to anthrax toxins compared to human and mouse immortalized cell lines. These results are consistent with previous studies that have demonstrated a difference between responses of human and mouse macrophages following exposure to LT46. However, our studies extend these comparisons through the use of primary cells, as well as using EF as the B component of the toxin. Since many cells may differ in their response to the actions of LT (cleavage of MAPK

74 kinases), we chose to evaluate ATR functionality in primary mononuclear phagocytes by exploiting the adenylate cyclase activity of EF.

Although we have shown that primary cells do express CMG2 transcripts, the direct proof that CMG2 is responsible for internalization of toxin was beyond the scope of this study. In addition, the lack of correlation between CMG2 mRNA expression and cAMP production in primary cells intoxicated with ET suggests that other factors are involved in determining the susceptibility of primary cells to intoxication with B. anthracis exotoxins. Further studies comparing the mechanisms of toxin internalization, heptamerization, receptor-mediated endocytosis, and Ca2+ influx will be important in determining the factors related to vulnerability of host macrophages to intoxication and the role of exotoxins in anthrax pathogenesis.

2.5 References

1. Dixon T.C., Meselson M., Guillemin J., & Hanna P.C. (1999) Anthrax. N.Engl.J Med. 341, 815-826.

2. Pile J.C., Malone J.D., Eitzen E.M., & Friedlander A.M. (1998) Anthrax as a potential agent. Arch.Intern.Med 158, 429-434.

3. Hanna P.C. & Ireland J.A. (1999) Understanding Bacillus anthracis pathogenesis. Trends Microbiol. 7, 180-182.

4. Dixon T.C., Fadl A.A., Koehler T.M., Swanson J.A., & Hanna P.C. (2000) Early Bacillus anthracis-macrophage interactions: intracellular survival survival and escape. Cell Microbiol. 2, 453-463.

5. Mikesell P., Ivins B.E., Ristroph J.D., & Dreier T.M. (1983) Evidence for plasmid-mediated toxin production in Bacillus anthracis. Infect.Immun. 39, 371­ 376.

75 6. Okinaka R., Cloud K., Hampton O., Hoffmaster A., Hill K., Keim P., Koehler T., Lamke G., Kumano S., Manter D., Martinez Y., Ricke D., Svensson R., & Jackson P. (1999) Sequence, assembly and analysis of pX01 and pX02. J Appl.Microbiol. 87, 261-262.

7. Okinaka R.T., Cloud K., Hampton O., Hoffmaster A.R., Hill K.K., Keim P., Koehler T.M., Lamke G., Kumano S., Mahillon J., Manter D., Martinez Y., Ricke D., Svensson R., & Jackson P.J. (1999) Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J Bacteriol. 181, 6509-6515.

8. Koehler T.M. (2002) Bacillus anthracis and virulence gene regulation. Curr.Top.Microbiol.Immunol. 271, 143-164.

9. Petosa C., Collier R.J., Klimpel K.R., Leppla S.H., & Liddington R.C. (1997) Crystal structure of the anthrax toxin protective antigen. Nature 385, 833-838.

10. Bradley K.A., Mogridge J., Mourez M., Collier R.J., & Young J.A. (2001) Identification of the cellular receptor for anthrax toxin. Nature 414, 225-229.

11. Bradley K.A. & Young J.A. (2003) Anthrax toxin receptor proteins. Biochem.Pharmacol. 65, 309-314.

12. Klimpel K.R., Molloy S.S., Thomas G., & Leppla S.H. (1992) Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc.Natl.Acad.Sci.U.S.A 89, 10277­ 10281.

13. Beauregard K.E., Collier R.J., & Swanson J.A. (2000) Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization. Cell Microbiol. 2, 251-258.

14. Beauregard K.E., Wimer-Mackin S., Collier R.J., & Lencer W.I. (1999) Anthrax toxin entry into polarized epithelial cells. Infect.Immun. 67, 3026-3030.

15. Bell S.E., Mavila A., Salazar R., Bayless K.J., Kanagala S., Maxwell S.A., & Davis G.E. (2001) Differential gene expression during capillary morphogenesis in 3D collagen matrices: regulated expression of genes involved in basement membrane matrix assembly, cell cycle progression, cellular differentiation and G- protein signaling. Journal of Cell Science 114, 2755-2773.

16. Carson-Walter E.B., Watkins D.N., Nanda A., Vogelstein B., Kinzler K.W., & St Croix B. (2001) Cell surface tumor endothelial markers are conserved in mice and humans. Cancer Res. 61, 6649-6655.

76 17. Scobie H.M., Rainey G.J., Bradley K.A., & Young J.A. (2003) Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc.Natl.Acad.Sci.U.S.A 100, 5170-5174.

18. Leppla S.H. (1995) Anthrax Toxins. In: Bacterial Toxins and Virulence Factors in Disease (ed. J. Moss, B. Iglewski, M. Vaughan, & A. T. Tu), p. 543­ 572 Dekker, New York.

19. Duesbery N.S., Resau J., Webb C.P., Koochekpour S., Koo H.M., Leppla S.H., & Vande Woude G.F. (2001) Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways. Proc.Natl.Acad.Sci.U.S.A 98, 4089-4094.

20. Duesbery N.S. & Vande Woude G.F. (1999) Anthrax lethal factor causes proteolytic inactivation of mitogen-activated protein kinase kinase. J Appl.Microbiol. 87, 289-293.

21. Duesbery N.S., Webb C.P., Leppla S.H., Gordon V.M., Klimpel K.R., Copeland T.D., Ahn N.G., Oskarsson M.K., Fukasawa K., Paull K.D., & Vande Woude G.F. (1998) Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 280, 734-737.

22. Park J.M., Greten F.R., Li Z.W., & Karin M. (2002) Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science 297, 2048-2051.

23. Hanna P. (1999) Lethal toxin actions and their consequences. J Appl.Microbiol. 87, 285-287.

24. Kim S.O., Jing Q., Hoebe K., Beutler B., Duesbery N.S., & Han J. (2003) Sensitizing anthrax lethal toxin-resistant macrophages to lethal toxin-induced killing by tumor necrosis factor-alpha. J Biol.Chem. 278, 7413-7421.

25. Drum C.L., Yan S.Z., Bard J., Shen Y.Q., Lu D., Soelaiman S., Grabarek Z., Bohm A., & Tang W.J. (2002) Structural basis for the activation of anthrax by calmodulin. Nature 415, 396-402.

26. Drum C.L., Yan S.Z., Sarac R., Mabuchi Y., Beckingham K., Bohm A., Grabarek Z., & Tang W.J. (2000) An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis. J Biol.Chem. 275, 36334-36340.

27. Kumar P., Ahuja N., & Bhatnagar R. (2002) Anthrax edema toxin requires influx of calcium for inducing cyclic AMP toxicity in target cells. Infect.Immun. 70, 4997-5007.

77 28. Leppla S.H. (1984) Bacillus anthracis calmodulin-dependent adenylate cyclase: chemical and enzymatic properties and interactions with eucaryotic cells. Adv.Cyclic.Nucleotide.Protein Phosphorylation.Res. 17, 189-198.

29. Leppla S.H. (1982) Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc.Natl.Acad.Sci.U.S.A 79, 3162-3166.

30. Masure H.R., Shattuck R.L., & Storm D.R. (1987) Mechanisms of bacterial pathogenicity that involve production of calmodulin-sensitive adenylate cyclases. Microbiol.Rev. 51, 60-65.

31. Mock M. & Ullmann A. (1993) Calmodulin-activated bacterial adenylate cyclases as virulence factors. Trends Microbiol. 1, 187-192.

32. Tippetts M.T. & Robertson D.L. (1988) Molecular cloning and expression of the Bacillus anthracis edema factor toxin gene: a calmodulin-dependent adenylate cyclase. J Bacteriol. 170, 2263-2266.

33. O'Brien J., Friedlander A., Dreier T., Ezzell J., & Leppla S. (1985) Effects of anthrax toxin components on human neutrophils. Infect.Immun. 47, 306-310.

34. Hoover D.L., Friedlander A.M., Rogers L.C., Yoon I.K., Warren R.L., & Cross A.S. (1994) Anthrax edema toxin differentially regulates lipopolysaccharide- induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP. Infect.Immun. 62, 4432-4439.

35. Bonuccelli G., Sotgia F., Frank P.G., Williams T.M., de Almeida C.J., Tanowitz H.B., Scherer P.E., Hotchkiss K.A., Terman B.I., Rollman B., Alileche A., Brojatsch J., & Lisanti M.P. (2005) Anthrax Toxin Receptor (ATR/TEM8) is Highly Expressed in Epithelial Cells Lining the Toxin's Three Sites of Entry (Lung, Skin, and Intestine). Am J Physiol Cell Physiol.

36. Gaynor C.D., McCormack F.X., Voelker D.R., McGowan S.E., & Schlesinger L.S. (1995) Pulmonary surfactant protein A mediates enhanced phagocytosis of Mycobacterium tuberculosis by a direct interaction with human macrophages. J Immunol. 155, 5343-5351.

37. Leppla S.H. (1988) Production and purification of anthrax toxin. Methods Enzymol. 165, 103-116.

38. Gordon V.M., Leppla S.H., & Hewlett E.L. (1988) Inhibitors of receptor- mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of adenylate cyclase toxin. Infect.Immun. 56, 1066-1069.

78 39. Liu S. & Leppla S.H. (2003) Cell surface tumor endothelium marker 8 cytoplasmic tail-independent anthrax toxin binding, proteolytic processing, oligomer formation, and internalization. J Biol.Chem. 278, 5227-5234.

40. Hanna P.C., Acosta D., & Collier R.J. (1993) On the role of macrophages in anthrax. Proc.Natl.Acad.Sci.U.S.A 90, 10198-10201.

41. Guidi-Rontani C. & Mock M. (2002) Macrophage interactions. Curr.Top.Microbiol.Immunol. 271, 115-141.

42. Guidi-Rontani C. (2002) The alveolar macrophage: the Trojan horse of Bacillus anthracis. Trends Microbiol. 10, 405-409.

43. Little S.F. & Ivins B.E. (1999) Molecular pathogenesis of Bacillus anthracis infection. Microbes.Infect. 1, 131-139.

44. Pellizzari R., Guidi-Rontani C., Vitale G., Mock M., & Montecucco C. (1999) Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNgamma-induced release of NO and TNFalpha. FEBS Lett. 462, 199-204.

45. Mogridge J., Cunningham K., & Collier R.J. (2002) Stoichiometry of anthrax toxin complexes. Biochemistry 41, 1079-1082.

46. Kassam A., Der S.D., & Mogridge J. (2005) Differentiation of human monocytic cell lines confers susceptibility to Bacillus anthracis lethal toxin. Cell Microbiol 7, 281-292.

79 Intracellular cAMP (fmol/well) a in cells treated with: Cell type PA alone EF Alone PA+EF

RAW 264.7 77.9 ± 21.7 35.9 ± 3.7 270.5 ± 49.1

J774A.1 71.0 ± 32.6 49.7 ± 8.9 452.8 ± 138.1

MDM 131.4 ± 49.9 38.4 ± 1.1 209.9 ± 68.6

MPM 96.0 ± 57.9 71.7 ± 47.9 427.3 ± 69.5

THP-1 56.4 ± 4.7 39.8 ± 10.4 145.8 ± 79.4

Table 2.1 - Absolute cAMP concentrations per 3 x 104 cells treated with PA, EF or ET. aValues are means ± standard deviations (three sample wells per treatment group) for intracellular cAMP in cells treated with PA (203 mg per 3 x 104 cells), EF (75 mg per 3x 104 cells), or PA+EF.

80 Accession Size Target Primer sequences / location # (bp) 307 329 a -TGCTGCACCACTGGAATGAAATC- TEM8 (all isoforms) NM_032208.1 470-CCAGAAAGTTCTGCCAGGAGGAG-492 186 1329-GTTCGTTGGGGAGAAAAG-1346 TEM8 (Isoform 1) NM_032208.1 1496-GGAAAACTCGATGCTTGTG-1515 187 1180-GCTGCACTGTGATTATCAAGG-1200 TEM8 (Isoform 2) NM_053034.1 1283-CAGATAACCTAACACAGCC-1301 122 980-GGCATGAAAGCTGCACTCCAGG-1001 TEM8 (Isoform 3) BC012074.1 1088-CCCACAACAGCTGCTTGCATGG-1109 130 781-CTTTCATTGTGTTTTCTTCTCAAGCAAC-808 CMG2 (all isoforms) NM_058172.3 930-ATTCAGAAAGCAGGAGGCTTGAAAAC-955 175 305 324 b -ACCCCCACTGAAAAAGATGA- β2M (human) BC032589.1 399-CATCATGGAGGTTTGAAGAT-418 114 325-ACCCCCACTGAGACTGATAC-344 β2M (mouse) NM_009735.2 420-CATCATGATGCTCTGAAGAT-439 115 5 23 b -GGAAGGTGAAGGTCGGAGT- GAPDH (human) BT006893.1 212-GAAATCCCATCACCATCTTC-231 227 550-AACTTTGGCATTGTGGAAGG-569 GAPDH (mouse) BC083080.1 753 772 223 -TGTTCCTACCCCCAATGTGT- Table 2.2 - RT-PCR primer sequences and expected amplicon size. aNational Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health accession numbers. bβ2M – beta-2-microglobulin, GAPDH – glyceraldehyde-3-phosphate dehydrogenase

81 mRNA transcript Cell type / tissue TEM8 CMG2 β2M HeLa +a + + THP-1 + + + RAW 264.7 + + + J774A.1 + + + MPMb - + + MDMb - + + Liver (mouse) + + + Kidney (mouse) + + + Spleen (mouse) + + +

Table 2.3 - TEM8 and CMG2 mRNA expression in immortalized cell lines, primary mononuclear phagocytes, and mouse tissues by RT-PCR. a(+) denotes amplification, (-) denotes no amplification, bMPM – mouse peritoneal macrophage, MDM – human monocyte-derived macrophage.

82 160 List 140 List NCNCII 120 y t i 100 * 80 * * 60 * * % Viabil 40 20 0 PA(ng/well) 0 6.4 26.0 205.0 LF(ng/well) 0 80.0 80.0 80.0

Figure 2.1 - Lethal factor cytotoxicity assay representing a decrease in viability in J774A.1 cells with increasing concentrations of PA (■ List Biological Laboratories, ■ NCI) and a constant concentration of LF (1.066 μg/ml). *, p < 0.05 when compared to 0 ng/well. ** p < 0.01 when compared to viability with 0 ng/well. P-values were determined by an unpaired one-tailed t-test.

83 A.A 12 0 B.B 3000 J774A.1 27.0 )

) 2500

l 100 l

el THP-1 w wel /

80 / 2000 ol ol 60 1500 (fm

40 1000 cAMP cAMP (fm 20 500 1.6

0 0 PA(ng/well) 205 0 26 51 102 205 Control Forskolin LF(ng/well) 0 80 80 80 80 80 Fold Increase 1.1 0.9 0.7 3.1 3.5 6.2

C.C 12 00 D.D 10 J774A.1 9 * ) l 1000 8 a

THP- 1 e

wel 7

/ 800

eas 6 ol r + * c 5

600 n

I 4 d * 400 3 Fol 2 cAMP (fm 200 1 0 0 30 60 120 180 MDM MPM THP-1 RAW J774A.1 Time (min.)

Figure 2.2 - A. Edema factor cytotoxicity assay showing a dose dependent increase in cAMP in J774A.1 cells. B. J774A.1 and THP-1 cells unstimulated and stimulated with 100 mmoles/ml of forskolin for 20 minutes C. Edema factor cytotoxicity assay showing a time dependent increase in cAMP in J774A.1 cells but not THP-1 cells. D. cAMP production in multiple cell lines after incubation with PA (2.7 μg/ml) and EF 1.066 μg/ml) at 180 minutes. *, p < 0.05 when compared no response (Fold change = 1). +, p = 0.0595, indicating marginal significance. P-values were determined by a 1 sample, 1 tailed T-test.

84 A.

B.

Figure 2.3 Primer design strategy for TEM8 (A) and CMG2 (B). Primers were designed to cross exon splice sites common to all isoforms of TEM8 and CMG2. * indicate the last exon in each isoform. †CMG2 isoform is a projected splice variant depicted in the Ensembl Genome Browser developed by the Sanger Institute and the European Bioinformatics Institute (Ensembl Gene ID ENSG00000163297)

85 2 1 n r r r e o o 7 e g e A.A n n B.B . v n l o o 4 i u p d d 1 6 L L S . 2 e e e C C s s s a M A M L M M 4 W u u u e D 7 A P o o o B B 7 H P P M J R M M M M PAN-TEM PAN-TEM TEM8 isoform 1 TEM8 isoform 1 TEM8 isoform 2 GAPDH TEM8 isoform 3 GAPDH

Figure 2.4 - TEM8 isoform expression by standard RT-PCR. A. Expression in a human cell line (HeLa) and human primary cells. B. Expression in mouse cell lines, primary cells and tissues. Product size for the universal TEM8 is approximately 186 bp. Product size for human isoforms 1, 2 and 3 are 189 bp, 122 bp and 130 bp, respectively. Purified PCR products were visualized by agarose gel electrophoresis.

86 109 * TEM8 108 CMG2 NA p > 0.5

R * 107

)/500ng 6 10 10 g

o * L 105

Copies ( 104

103 ** ** ** ** ** ** a 1 2 3 4 5 6 1 A eL - M P H HP T -1 MDM (donors 1-6) P H T

Figure 2.5 - mRNA expression of TEM8 and CMG2 by real time RT-PCR. Product size for TEM8 and CMG2 are approximately 186 bp and 191 bp, respectively. ** represents below the level of detection (<1 x 104 copies/500 ng of RNA). *, p < 0.05 when compared with the same receptor expression in Hela cells (non-parametric Kruskal-Wallis test). Comparison of CMG2 expression in donors was performed using one way analysis of variance (ANOVA). Differences CMG2 expression between donors was insignificant.

87 60 B AA. 120 B. Ms 100 50

/ 50 MD 80 40

30

60 i / 50 MDMs l

40 20 acil B inated Spores m 20 10 Ger

0 0 0.5 1.0 1.5 2.5 4.5 8.5 Time (hrs. post-infection) CC. 10 9 TEM8 8 10 CMG2 A N 7 R 10 g

6

500n 10 / es 5

opi 10 C 104

103 ** ** ** ** l s B o re 1 tr PS o - n L p L o S I C

Figure 2.6 - A. Purified Sterne spore preparation stained with malachite green (100X). B. MDM culture uninfected (100X). C. MDM culture infected with purified B. anthracis Sterne spores (100X). D. mRNA expression of TEM8 and CMG2 in MDMs after stimulation with LPS (100 ng/ml) for 1 hours, spore exposure (MOI = 5) for 2 hours or IL-1β (50 IU/ml) for 1 hours.

88 CHAPTER 3

COMPLEMENT PROTEIN C3 BINDING TO BACILLUS ANTHRACIS SPORES INITIATED BY THE CLASSICAL PATHWAY ENHANCES PHAGOCYTOSIS BY HUMAN MACROPHAGES

3.1 Introduction

Bacillus anthracis is a gram positive, non-hemolytic, catalase-positive, spore-forming, facultative anaerobic bacterium. Depending on the route of exposure, B. anthracis can cause inhalational, cutaneous, or gastrointestinal anthrax 1,2. Phagocytosis of B. anthracis spores by macrophages is particularly relevant to the pathogenesis of anthrax. Several investigators have proposed that

B. anthracis spores can germinate within macrophages and become vegetative cells that then briefly replicate intracellularly leading to escape of the bacteria which can multiply in the lymphatic system3,4. Inhalational anthrax, which is of particular concern, begins with the deposition of B. anthracis spores in the alveoli, where the spores are phagocytosed by resident alveolar macrophages that may then migrate from the lungs to regional lymph nodes carrying the bacteria thereby allowing the bacteria to gain access to the systemic circulation3,5. Receptors on mononuclear phagocytes that trigger phagocytosis of

89 B. anthracis spores have yet to be identified and are likely to play an essential

role in disease pathogenesis. Several previously identified macrophage receptors

that are abundantly expressed could be involved.

Alveolar macrophages express complement receptors (CR) CR1, CR3,

and CR4 as well as Fcγ receptors (FcγRs) but the level of expression on these

cells differs from macrophages in other sites and the level of expression depends

on the activation state of the cell6. CRs primarily interact with complement protein

C3b and its degradation product C3bi to mediate phagocytosis. The interaction of

FcγRs on macrophages with immunoglobulin bound to can

trigger complement activation and C3 deposition as well as mediate

phagocytosis by macrophages, the later usually as a result of IgG in immune

serum. The signaling cascade(s) activated following ligation of CRs and FcγRs

differ, leading to differences in the nature of the microbicidal and inflammatory

responses generated by these cells.

Components of the within the lung are produced by

macrophages and epithelial cells7-9. Furthermore, functional complement proteins

can be recovered from bronchoalveolar lavage fluid (BALF)10-14; however,

proteolytic cleavage of C3 in BALF is reduced when compared to C3 in serum13.

Complement may be activated by both the classical and alternative pathways but the low level of factor B in BALF appears favor the classical pathway14,15.

Since opsonin-dependent phagocytosis has been shown to play an important

role in the pathogenesis of other inhaled bacterial pathogens16,17, we

90 hypothesized that soluble factors such as immunoglobulin and C3 interact with B. anthracis spores in the lung to facilitate phagocytosis. To begin to test this hypothesis, we characterized the ability of human macrophages to phagocytose

B. anthracis spores in the presence of nonimmune human serum. We also analyzed the binding of human IgG to B. anthracis spores and the fixation of C3 fragments to the spore surface. We show that nonimmune human serum contains immunoglobulin that triggers activation of the classical complement pathway. The resulting bound C3 fragments mediate phagocytosis of B. anthracis spores by human macrophages. Thus, our data indicate that C3 opsonization of B. anthracis spores facilitates the early steps in the pathogenesis of anthrax. Very little is known about the interactions of the soluble innate immune system with B. anthracis spores. To our knowledge, this is the first documented evidence of the influence of complement interaction with spores of

Bacillus anthracis.

3.2 Materials and methods

Isolation and culture of human monocyte derived macrophages

Concentrated human leukocytes from anonymous blood donors were purchased from the American Red Cross. Peripheral blood mononuclear cells

(PBMCs) were separated on a Ficoll (Amersham Biosciences, New York NY) density gradient and any contaminating platelets were removed by centrifugation at 100 x g. Monocyte derived macrophages (MDMs) were differentiated by

91 culturing the PBMCs for 5 to 7 days in Teflon culture dishes (Savillex,

Minnetonka MN) containing RPMI 1640 medium supplemented with 20 mM

HEPES, 20 mM L-glutamine, and 10% pooled serum as previously described18.

MDM differentiation was verified by flow cytometric analysis using appropriate cluster designation marker (data not shown) and light microscopy for typical morphologic features. Differentiated MDMs were washed and then adhered to tissue culture vessels (flasks or culture plates) containing serum-free RPMI-1640 for at least four hours. The non-adherent cells were removed by washing with

RPMI-1640 and the MDMs were incubated in RMPI-1640 containing 20mM

HEPES, 20 mM L-glutamine, and 10% human serum.

Infection of MDMs with B. anthracis spores

MDMs were seeded at a density of 1 x 106 cells/well into six-well flat- bottom tissue culture plates 17 to 19 hours before infection. The media was then replaced with 500 μl of RPMI-1640 containing 20mM HEPES, 20 mM L- glutamine and 10% human AB serum. Washed B. anthracis spores were added to each well at a multiplicity of infection (MOI) of 5 in a volume of 50 μl. The plates were centrifuged at 400 x g for 3 min and incubated at 37 °C for 30 minutes The wells were washed five times with serum-free RPMI-1640 media.

500 μl of serum-free RPMI-1640 media containing 20 mM HEPES was placed in each well and the plates were returned to the 37 ºC incubator. At each time point the media was removed from the well and the cells were lysed in 2.5 % saponin.

92 An aliquot of the cell culture media and lysate was subjected to heat treatment in a 60 ºC water bath for 30 minutes before culture. Serial dilutions of each sample were performed before plating on nutrient agar plates that were subsequently incubated at 37 º C overnight.

For microscopy experiments, MDMs were seeded as above at a density of

2.5 x 105 cells/well in four-well glass chamber slides (Nalge Nunc International,

Napierville, IL) 17 to 19 hours before infection. The media was replaced with 175

μl of RPMI-1640 containing 20mM HEPES, 20 mM L-glutamine and 10% human serum. Washed B. anthracis spores were added to each well at a multiplicity of infection (MOI) of 3 in a volume of 25 μl. The slides were incubated at 37 °C for

30 minutes and then the wells were washed five times with serum-free RPMI­

1640 media. Two hundred mls of serum-free RPMI-1640 media containing 20 mM HEPES was placed in each well and the slides were returned to the 37 ºC incubator. At each time point the media was removed from the well and the cells were fixed in situ with 4.0 % paraformaldehyde. The fixed monolayers were stained with HEMA 3 blood stain (Fisher Scientific, Pittsburgh, PA) per the manufacturer’s instructions. Infections were also performed using media containing heat inactivated human serum supplemented with factor B and D

(Fitzgerald Industries International, Inc, Concord, MA). The slides were coded to prevent investigator bias and the number of cell associated spores was counted by examining a minimum of 50 cells microscopically (1000x). Assays were

93 repeated in triplicate andstatistical significance was determined using a two-tailed

Student’s t-test with equal variance.

Spore purification

A vegetative culture of B. anthracis Sterne strain (Colorado Serum

Company, Denver, CO) was sporulated in modified G medium [0.2% yeast

extract, 0.0025% CaCl2 dihydrate, 0.05% KH2PO4, 0.00976% MgSO4 anhydrous,

0.005% MnCl2 quatrahydrate, 0.00073% ZnSO4 heptahydrate, 0.00005% FeSO4 heptahydrate, 0.2% (NH4)2SO4] until >99% sporulation was observed via

refractile bodies on light microscopy19. were washed with sterile

20 ddH2O and then purified over a Renografin 76 density gradient of 1.300g/mL .

The endospores were washed 3 times with sterile H2O to remove any remaining

protein and Renografin. The final spore preparation was resuspended in sterile

H2O. Endospore preparations were scored using serial dilutions and counting by

hemacytometer to determine total count and also plated to determine Colony

Forming Units (CFU). Endospore preparations were found to be >99% purity

with no observable vegetative cells or debris.

Spore ELISA to detect bound human immunoglobulin

Purified spores of B. anthracis Sterne were fixed in 4% paraformadehyde

for 2 hours at a concentration of 1x108 spores/ml. An aliquot of spores was

plated on nutrient agar to insure death. The fixed spores were diluted in

94 bicarbonate coating buffer to a concentration of 1x107 spores/ml. 96-well

Immulon-HB ELISA plates (Dynex Technologies, Chantilly, VA) were coated with spores by aliquoting 100 ul of the spore suspension into each well. The plate was incubated overnight at 4 ºC. The plates were then washed with PBS/0.05%

Tween-20 and examined by light inverted microscopy to insure successfully coating. The plate blocked with assay diluent (PBS/5% FBS/5% milk/0.05%

Tween-20). The complement sufficient human AB serum was prepared at dilutions from 1:50 to 1:109350 in assay diluent prior to incubation in the ELISA plate wells for 1 hour at 37 ºC. Mouse anti-human IgG conjugated to alkaline phosphatase (Sigma-Aldrich, St. Louis, MO) was prepared at 1:5000 dilution in assay diluent and incubated in the wells for 1 hour at 37 ºC. Antibody was added to wells without spores as a negative control where appropriate. Empty ELISA plate wells were tested for antibody binding independently. Following an additional three washes, 100 ul of p-nitrophenylphosphate substrate (Sigma-

Aldrich) was added to each well and the plate was incubated at 37 ºC for 30 minutes. The development reaction was stopped with 3 N NaOH and the plate was read at 405 nm. Arbitary values ranging from 7.81 to 1000 units/ml were assigned to all the dilution values (1:10 to 1:1250) from the AB serum spore antibody ELISA. The values which formed the linear portion of the curve (1:40 to 1:640) were used to construct the standard curve by spreadsheet calculation (Microsoft Excel). The linear regression of this curve was y = 1.347x -

1.2678 with a R2 value of 0.98.

95 Spore ELISA to detect bound C3 fragments

Spore coated ELISA plates were prepared as above. Complement inactivation of human serum was performed by heating serum at 56 ºC for 30 minutes. Dilutions of fresh or heat-inactivated (HI) serum were incubated in the spore coated wells for for 1 hour at 37 ºC, followed by three washes. Mouse anti­ human C3 β chain IgG (Clone no. H11) and mouse anti-human IgG (Clone no. H13) (Fitzgerald Industries International, Inc, Concord, MA) were prepared at a 1:100 dilution in assay diluent. The mouse anti-human C3a IgG recognizes native C3 α chain and the C3a split product but not bound C3b or other bound C3 cleavage products. 100 ul aliquots of these antibodies were placed in separate wells and the plate was incubated for 1 hour at 37 ºC. Goat anti-mouse IgG conjugated to horseradish peroxidase (Sigma-Aldrich) was prepared at 1:5000 dilution in assay diluent. After washing the plate, the secondary antibody was incubated in the wells for 1 hour at 37 ºC. Control wells were incubated in multiple combinations of the anti-C3 β chain and anti-C3a antibodies to determine background color development. Empty ELISA plate wells were tested for antibody binding independently (i.e. no spores incubated with serum or secondary antibodies). Following an additional three washes, 100 ul of 3,3′,5,5′­ tetramethylbenzidine (Sigma-Aldrich) was added to each well and the plate was incubated at 37 ºC for 30 minutes. The development reaction was stopped with 3

N H2SO4 and the plate was read at 405 nm.

96 C3 Western blot

Spores of Bacillus anthracis Sterne (60 μl) were incubated in phosphate buffered saline/10% human AB (fresh or HI) for 30 minutes at 37 ºC. Following incubation, the spores were washed in sterile PBS four times and resuspended in

40 ul of PBS. 60 ul of 2X SDS-PAGE sample buffer containing dye was added to each spore sample. The samples were boiled for 5 minutes, cooled on ice and centrifuged to pellet the spores. 40 ul of supernatant was loaded per well of a

10% Tris-HCl SDS-PAGE gel. Two wells were loaded with 13 and 32 ug of recombinant human native C3 as a positive control. The gel underwent electrophoresis for approximately 1 hour at 120 volts followed by transfer to a nitrocellulose membrane. The membrane was stained with Ponceau S to evaluate protein transfer followed by an overnight block in a 10% TBS/5%

FBS/5% milk solution at 4 ºC. The membrane was incubated in anti-human C3β chain antibody (1:100 dilution in a 10% TBS/5% FBS/5% milk solution) for 1 hour, followed by three washes in 10% TBS. The membrane was then incubated in goat anti-mouse IgG (1:1000 dilution) and goat anti-biotin IgG (1:2000 dilution), both conjugated to horseradish peroxidase. The membrane was developed by a chemiluminescent substrate system (Sigma-Aldrich) and autoradiography.

Fluorescence activated cell sorting (FACS) assays

For antibody detection, spores of Bacillus anthracis Sterne were incubated in 96 well sterile round bottom plates (Sigma-Aldrich) in PBS + 10% human

97 serum at 37 ºC for 30 minutes. The spores were washed three times with sterile

PBS, followed by incubation in a 1:2000 dilution of FITC conjugated mouse anti­ human IgG antibody (Jackson Immunoresearch Laboratories, Inc, West Grove,

PA) in PBS for 1 hour at 37 ºC. The spores were then fixed in 2% paraformaldehyde following three washes in PBS and submitted for FACS analysis.

For C3b detection, spores of Bacillus anthracis Sterne were incubated in

96 well sterile round bottom plates in PBS + 10% human serum at 37 ºC for 30 minutes. The spores were washed three times with sterile PBS, followed by incubation in a 1:100 dilution of mouse anti-human C3b IgG and mouse anti­ human C3a IgG + PBS for 1 hour at 37 ºC. The spores were subsequently incubated in a 1:2000 dilution of FITC conjugated anti-mouse IgG antibody for 1 hour at 37 ºC and fixed in 2% paraformaldehyde. Appropriate control spores

(spore incubated in human serum + anti-C3a antibody + secondary antibody and human serum + secondary antibody) were included in both assays.

3.3 Results

Complement proteins enhance phagocytosis of B. anthracis spores

It has been previously suggested that FcγR are mediators of the phagocytosis of B. anthracis spores via Rac1 and Cdc42 21. To evaluate the role of soluble factors present in human nonimmune serum on the phagocytosis of B. anthracis spores by macrophages, we infected MDMs with B. anthracis spores in

98 the presence of 10% fresh or HI human A/B serum or in serum-free media (SFM)

(Fig. 3.1). The macrophages were extensively washed at 30 minutes post- infection to remove any free B. anthracis spores in the media. The MDMs were lysed at each time point and the number of cell-associated bacteria was determined. Infection in the presence of a low concentration of fresh serum increased the CFUs per well at 30 minutes post-infection and the CFUs per well remained higher at 90, 150, 210, and 270 minutes post-infection as compared to

MDMs infected in the presence of HI serum (Fig. 3.1A). The CFUs per well was similar for MDMs infected in HI A/B serum or SFM. The experiments were ended at 270 minutes post-infection due to extensive MDM lysis induced by intracellular

B. anthracis and the release of intracellular bacteria into the media beginning at five to six hours post-infection (data not shown).

To further evaluate the enhancement of phagocytosis of spores by heat- labile soluble factors in serum and to account for the variability in phagocytic index between MDMs from individual donors, the ex vivo infection was repeated with six donors under serum-free conditions and four additional donors in the presence of fresh or HI nonimmune serum (Fig 3.1B). There was no difference in

SFM versus HI A/B serum at 30 minutes post-infection (P=0.554). However, fresh A/B serum increased the CFUs/well by an average of 0.7 log10 as compared to SFM (P=0.004) and HI A/B serum (P=0.011).

We next assayed for the number of cell-associated spores by microscopy.

MDMs were incubated with with B. anthracis spores in the present of 10% fresh

99 or HI nonimmune A/B serum or in SFM (Fig. 3.2). The number of intracellular spores per 50 MDMs at 30 minutes post-infection was 16 fold higher (x=822 spores/50 MDMS, n=6 donors) in the presence of 10% fresh A/B serum as compared to SFM or HI A/B serum (x=51 spores/50 MDMs (n=6), x=60 spores/50 MDMs (n=6) respectively) (P<0.001). These data confirmed our results with CFU/well measured under the same conditions.

To further identify the heat-labile component(s) in serum that enhances the phagocytosis of spores, whole guinea pig complement with or without heat inactivated serum or human C3 protein and alternative complement pathway factors B and D were incubated with spores in SFM with MDMs in microscopy experiments. The presence of guinea pig complement (3 mg/ml) supplemented with Mg++ in the absence of serum did not increase the number of phagocytosed spores per 50 MDMs (P>0.05). Since surface bound immunoglobulin can activate the classical pathway of complement activation, whole guinea pig complement was added to HI A/B serum. Purified C3 protein along with factors B and D were also added to HI A/B serum at physiologic concentrations of 0.0643 mg/ml,

0.0218 mg/ml, and 0.0005 mg/ml respectively. The addition of C3/B/D to HI serum did not restore the serum-enhanced phagocytosis of B. anthracis spores.

However, the addition of whole complement did significantly increase the phagocytic index compared to the SFM condition (P<0.05). These data suggest that a heat-stabile soluble factor in HI serum (presumed to be immunoglobulin) in addition to complement proteins of the classical pathway are involved in

100 complement-mediated opsonization and enhancement of phagocytosis of spores by macrophages.

IgG in nonimmune human serum binds to B. anthracis spores

Since B. anthracis spores incubated in nonimmune human serum containing complement exhibit a higher phagocytic index, we next determined if human immunoglobulin in the serum is bound to the surface of the spores using a newly developed spore ELISA. Nonimmune human serum contained easily detectable levels of IgG bound to B. anthracis spores as demonstrated by a significant increase in optical density (O.D.) that correlated with the concentration of serum added (Fig. 3.3B). The binding of IgG in serum to paraformaldehyde- fixed spores and unfixed spores was equivalent (data not shown). As a complementary approach to confirm IgG binding to spores, we also incubated spores in 10% human serum and assayed for bound IgG by flow cytometry.

Approximately 88% of the spores showed positive fluorescence, indicating bound human IgG, confirming the results of our ELISA spore assay (Fig. 3.6B). We next tested the sera of ten nonimmune individuals that were negative for antibodies to

B. anthracis protective antigen (PA). Nonimmune human A/B serum, which was previously shown to contain IgG that binds to B. anthracis spores, was assigned an arbitrary value of 10,000 units per ml (U/ml) and used to construct a reference curve by plotting the O.D. vs. the log10 of the dilution (Fig. 3.3C). A linear transformation was performed on the linear portion of the curve (dilutions 1:40 to

101 1:640) and the data was plotted on an O.D. vs. U/ml scale giving the assay a range of 7.81 to 250 U/ml at the dilution of serum tested (r2=0.98). A single dilution for each serum sample was tested in triplicate and assigned a value based on the dilution tested and the reference curve. Sera from all ten donors exhibited IgG spore binding activity, confirming that non-vaccinated and unexposed individuals with no detectable anti-PA antibodies have IgG that binds to B. anthracis spores (Fig. 3.3A). The mean value of the tested individuals was

4773.7 ± 1542.16 U/ml, with a maximum value of 7944.3 U/ml and a minimum value of 3032.6 U/ml.

Fixation of the complement component C3 in nonimmune serum to B. anthracis spores

Complement proteins are among the most characterized proteins of the innate immune in serum and bound C3 fragments serve as opsonins for macrophage phagocytosis22. Thus, we used the spore ELISA to assay for bound

C3 fragments on B. anthracis spores incubated in nonimmune serum. Our approach was to use two different anti-C3 mAbs; clone H13 which detects the

C3a portion of the native C3 α chain (but not C3b or other bound C3 fragments) and clone H11 which detects the C3 β chain. Cleavage of the C3a fragment away from the α chain exposes the thioester on C3b which covalently binds to the target (spore). Thus, covalent binding of C3 fragments to spores would result in recognition by antibody H11 but not antibody H13. Recognition by both

102 antibodies H11 and H13 would represent nonspecific binding of native C3 to the spores.

Incubation of B. anthracis spores in nonimmune human serum resulted in the recognition of the C3 β chain by antibody H11 but not the α chain by antibody

H13 indicating covalent binding of C3 fragments to the spores. Recognition by antibody H11 decreased as the serum was diluted to as low as 1.25% serum

(Fig. 3.4A). Binding of the C3 β chain could not be detected if the spores were incubated in 10% HI serum.

The deposition of C3 fragments on spores was confirmed by both flow cytometry and spore immunoprecipitation followed by Western blot. Spores were incubated in 10% human AB serum for 30 minutes, washed, incubated in either antibody H11 or H13, followed by a FITC-conjugated secondary antibody, fixed with paraformaldehyde and analyzed by flow cytometry. As shown in figure

3.6C, approximately 70% of the spores incubated in fresh AB serum were positive when probed with antibody H11, in contrast to the condition where spores were incubated in HI serum (data not shown). In addition, spores were not positive when probed with antibody H13 confirming that non-specific C3 binding does not occur (Fig. 3.6C). The binding of C3 fragments to B. anthracis spores was also confirmed by Western blot. Live B. anthracis spores were incubated in 10% fresh nonimmune human A/B serum for 30 minutes, washed extensively, and then boiled in sample buffer under reducing and denaturing conditions. The boiling step assists in reducing the disulfide bond between the α

103 and β chains of C3, resulting in the release of the β chain into solution. The spores were centrifuged and the proteins in the supernatant were separated by

SDS-PAGE, blotted to nitrocellulose and probed with antibody H11 by Western blotting. Recombinant native human C3 was used as a positive control. The 75 kDa β chain was observed in the positive control as well as in the supernatants from B. anthracis spores incubated in fresh nonimmune human serum but not in the supernatants from spores incubated in HI serum or in the absence of serum

(Fig. 3.5).Together the western blot assay results with our ELISAs and FACS analysis to detect C3β or C3a confirm that C3b becomes fixed to the surface of

B. anthracis spores and could therefore act as an opsonin to facilitate phagocytosis by macrophages.

3.4 Discussion

In this study, we explored complement interactions with B. anthracis spores utilizing three in vitro assays which demonstrated the deposition of the cleaved complement protein C3β but not whole C3 onto B. anthracis spores.

These results imply that C3b or its degradation products become fixed to B. anthracis spores via the classical pathway of complement activation. In addition, we also explored the functional significance of opsonization by C3 in relation to phagocytosis by macrophages. Most interactions between bacterial pathogens and macrophages involve either opsonins such as antibody, complement proteins, or lectins that engage their cognate receptors at the plasma membrane,

104 or cellular scavenger receptors that directly recognize conserved molecules on the surface of bacterial pathogens. Such is the case with the mannose receptor that recognizes the mannose capped lipoarabinomannan of Mycobacterium tuberculosis 23,24. Molecules associated with the surface of B. anthracis spores which interact with scavenger receptors on the macrophage in this manner have not been described. In addition, limited data exist concerning the influence that soluble factors have on the phagocytic event. Our ELISA and flow cytometry data demonstrate that sera from nonimmune human donors contain IgG that binds to

B. anthracis spores. Although the antibody from nonimmune donors binds to B. anthracis spores, it is not sufficient to directly facilitate an increase in phagocytosis of spores by MDMs as compared to infection in SFM. We have also shown by ELISA, flow cytometry, and immunoblotting that C3 fragments become fixed to the surface of B. anthracis spores and that complement proteins along with antibody are necessary to facilitate an increase in phagocytosis of spores by

MDMs. Furthermore, infecting macrophages in the presence of human C3, factor

B and or complement proteins without spore binding antibodies results in a phagocytic index that is indistinguishable to the phagocytic index in SFM or heat inactivated serum. These data implicate the classical or lectin pathways of complement activation that is likely more active in BALF fluid as compared to the alternative pathway. The nature of B. anthracis spore binding antibody in naive human serum is uncertain. One possibility is that this antibody is generated in response to natural exposure to the spores of other Bacillus spp., particularly

105 species such as Bacillus cereus or Bacillus thurigenensis, which are ubiquitous in the environment and have an exosporium. A second possibility is that these antibodies cross react with one or more antigens found in the exosporium of B. anthracis and are induced by exposure to glycoproteins or carbohydrates from unrelated bacterial species. Several examples involving cross-reactive antibodies and include antibodies generated against a polysaccharide antigen of Bacillus pumilus that cross react to antigens of group A meningococci, the presence of naturally occurring anti-phosphorylcholine IgG in human serum that has opsonophagocytic activity against Haemophilus influenzae and

Streptococcus pneumoniae and a natural antibody which mediates complement fixation to Mycobacterium leprae25-28. In addition, preimmune sera of rabbits has been shown to have cross reactive antibodies against several pathogenic and non-pathogenic bacterial organisms29.

The interactions of B. anthracis spores with tissue mononuclear phagocytes is likely most significant during an exposure by the aerosol route where macrophages are involved in facilitating germination and systemic dissemination of the organism. The interaction of B. anthracis spores with macrophages has been explored by other investigators4,30,31. Although the macrophage has been shown to be crucial in controlling the infection, macrophage depletion prior to aerosol challenge in mice has been shown to cause retention of spores in the bronchoalveolar spaces32. Thus, the nature of the environment in which the macrophage interacts with the spore likely plays a

106 role in pathogenesis. The interactions of the complement system with the spore has not been characterized previously; however, complement has been shown to be a factor in the susceptibility of mice to B. anthracis Sterne which lacks a capsule 33-35. The mechanism is most likely related to the interaction of C5 and the membrane attack complex with the vegetative organism and not the spore since spores from strains lacking the pX02 plasmid are predicted to be identical to spores from stains that carry pX02 and possess a capsule.

Determining sources of antigen responsible for the induction of B. anthracis spore-binding antibodies and the specific B. anthracis antigen(s) recognized is beyond the scope of this study; however its presence in nonimmune human serum is likely to be significant in the pathogenesis of inhalation anthrax. The current model of pathogenesis suggests that the alveolar macrophage may act as a “Trojan horse”, allowing the organism to traffic systemically with the phagocyte as an intracellular invader. This trafficking is followed by germination and escape of the vegetative organism into systemic circulation. Our data indicate that immunoglobulin and complement proteins which exist in the alveolar microenvironment of the lung can facilitate phagocytosis of B. anthracis spores by mononuclear phagocytes. Whether or not internalization of B. anthracis spores through opsonophagocytosis is protective for the host remains to be determined. It is possible that if alveolar macrophages become overwhelmed with intracellular spores and vegetative bacteria, bacteriocidal mechanisms may become impaired resulting in higher systemic

107 bacterial burdens. In this situation, interactions of macrophages with B. anthracis that involve antibodies and complement proteins represents a potential therapeutic target or point of intervention which should be explored further.

3.4 References

1. Pile J.C., Malone J.D., Eitzen E.M., & Friedlander A.M. (1998) Anthrax as a potential biological warfare agent. Arch.Intern.Med 158, 429-434.

2. Dixon T.C., Meselson M., Guillemin J., & Hanna P.C. (1999) Anthrax. N.Engl.J Med. 341, 815-826.

3. Hanna P.C. & Ireland J.A. (1999) Understanding Bacillus anthracis pathogenesis. Trends Microbiol. 7, 180-182.

4. Dixon T.C., Fadl A.A., Koehler T.M., Swanson J.A., & Hanna P.C. (2000) Early Bacillus anthracis-macrophage interactions: intracellular survival survival and escape. Cell Microbiol. 2, 453-463.

5. Guidi-Rontani C. (2002) The alveolar macrophage: the Trojan horse of Bacillus anthracis. Trends Microbiol. 10, 405-409.

6. Gordon S. (2002) Pattern recognition receptors: doubling up for the innate immune response. Cell 111, 927-930.

7. Ackerman S.K., Friend P.S., Hoidal J.R., & Douglas S.D. (1978) Production of C2 by human alveolar macrophages. Immunology 35, 369-372.

8. Cole F.S., Auerbach H.S., Goldberger G., & Colten H.R. (1985) Tissue-specific pretranslational regulation of complement production in human mononuclear phagocytes. J Immunol. 134, 2610-2616.

9. Strunk R.C., Eidlen D.M., & Mason R.J. (1988) Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways. J Clin.Invest 81, 1419-1426.

10. Ferguson J.S., Weis J.J., Martin J.L., & Schlesinger L.S. (2004) Complement protein C3 binding to Mycobacterium tuberculosis is initiated by the classical pathway in human bronchoalveolar lavage fluid. Infect.Immun. 72, 2564-2573.

108 11. Coonrod J.D. & Yoneda K. (1981) Complement and opsonins in alveolar and serum of rats with pneumonia due to . Rev.Infect.Dis. 3, 310-322.

12. Kolb W.P., Kolb L.M., Wetsel R.A., Rogers W.R., & Shaw J.O. (1981) Quantitation and stability of the fifth component of complement (C5) in bronchoalveolar lavage fluids obtained from non-human primates. Am Rev.Respir.Dis. 123, 226-231.

13. Giclas P.C., King T.E., Baker S.L., Russo J., & Henson P.M. (1987) Complement activity in normal rabbit bronchoalveolar fluid. Description of an inhibitor of C3 activation. Am Rev.Respir.Dis. 135, 403-411.

14. Watford W.T., Smithers M.B., Frank M.M., & Wright J.R. (2002) Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages. Am J Physiol Lung Cell Mol.Physiol 283, L1011-L1022.

15. Ferguson J.S., Weis J.J., Martin J.L., & Schlesinger L.S. (2004) Complement protein C3 binding to Mycobacterium tuberculosis is initiated by the classical pathway in human bronchoalveolar lavage fluid. Infect.Immun. 72, 2564-2573.

16. Gross G.N., Rehm S.R., & Pierce A.K. (1978) The effect of complement depletion on lung clearance of bacteria. J Clin.Invest 62, 373-378.

17. Schlesinger L.S., Bellinger-Kawahara C.G., Payne N.R., & Horwitz M.A. (1990) Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. J Immunol. 144, 2771-2780.

18. Gaynor C.D., McCormack F.X., Voelker D.R., McGowan S.E., & Schlesinger L.S. (1995) Pulmonary surfactant protein A mediates enhanced phagocytosis of Mycobacterium tuberculosis by a direct interaction with human macrophages. J Immunol. 155, 5343-5351.

19. Weiner M.A., Read T.D., & Hanna P.C. (2003) Identification and characterization of the gerH operon of Bacillus anthracis endospores: a differential role for purine nucleosides in germination. J Bacteriol. 185, 1462­ 1464.

20. Tamir H. & Gilvarg C. (1966) Density gradient centrifugation for the separation of sporulating forms of bacteria. J Biol.Chem. 241, 1085-1090.

21. Guidi-Rontani C. & Mock M. (2002) Macrophage interactions. Curr.Top.Microbiol.Immunol. 271, 115-141.

109 22. Sahu A. & Lambris J.D. (2001) Structure and biology of complement protein C3, a connecting link between innate and acquired immunity. Immunol.Rev. 180, 35-48.

23. Kang P.B., Azad A.K., Torrelles J.B., Kaufman T.M., Beharka A., Tibesar E., DesJardin L.E., & Schlesinger L.S. (2005) The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis. J Exp.Med 202, 987-999.

24. Schlesinger L.S., Kaufman T.M., Iyer S., Hull S.R., & Marchiando L.K. (1996) Differences in mannose receptor-mediated uptake of lipoarabinomannan from virulent and attenuated strains of Mycobacterium tuberculosis by human macrophages. J Immunol. 157, 4568-4575.

25. Goldenberg H.B., McCool T.L., & Weiser J.N. (2004) Cross-reactivity of human immunoglobulin G2 recognizing phosphorylcholine and evidence for protection against major bacterial pathogens of the human respiratory tract. J Infect.Dis. 190, 1254-1263.

26. Vann W.F., Liu T.Y., & Robbins J.B. (1976) Bacillus pumilus polysaccharide cross-reactive with meningococcal group A polysaccharide. Infect.Immun. 13, 1654-1662.

27. Filice G.A., Yeager A.S., & Remington J.S. (1980) Diagnostic significance of antibodies to Toxoplasma gondii detected after separation of immunoglobulin M from antibodies. J Clin.Microbiol 12, 336­ 342.

28. Schlesinger L.S. & Horwitz M.A. (1994) A role for natural antibody in the pathogenesis of leprosy: antibody in nonimmune serum mediates C3 fixation to the Mycobacterium leprae surface and hence phagocytosis by human mononuclear phagocytes. Infect.Immun. 62, 280-289.

29. Lathrop A.A., Huff K., & Bhunia A.K. (2006) Prevalence of antibodies reactive to pathogenic and nonpathogenic bacteria in preimmune serum of New Zealand white rabbits. J Immunoassay Immunochem. 27, 351-361.

30. Guidi-Rontani C., Levy M., Ohayon H., & Mock M. (2001) Fate of germinated Bacillus anthracis spores in primary murine macrophages. Mol.Microbiol. 42, 931-938.

31. Welkos S., Friedlander A., Weeks S., Little S., & Mendelson I. (2002) In-vitro characterisation of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody. J Med.Microbiol. 51, 821-831.

110 32. Cote C.K., Van Rooijen N., & Welkos S.L. (2006) Roles of macrophages and neutrophils in the early host response to Bacillus anthracis spores in a mouse model of infection. Infect.Immun. 74, 469-480.

33. Harvill E.T., Lee G., Grippe V.K., & Merkel T.J. (2005) Complement depletion renders C57BL/6 mice sensitive to the Bacillus anthracis Sterne strain. Infect.Immun. 73, 4420-4422.

34. Welkos S.L., Trotter R.W., Becker D.M., & Nelson G.O. (1989) Resistance to the Sterne strain of B. anthracis: phagocytic cell responses of resistant and susceptible mice. Microb.Pathog. 7, 15-35.

35. Welkos S.L., Keener T.J., & Gibbs P.H. (1986) Differences in susceptibility of inbred mice to Bacillus anthracis. Infect.Immun. 51, 795-800.

111 A.A BB. Δ56°C 50 9.0 45 Non heat-treated 8.0 P<0.05 ) 40 ) 0 0 P<0.05 1 1 7.0 000) 35 g g o o 6.0 l 30 l x100, ll ( 25 ll ( 5.0 ( e e l 20 4.0 el U/w U/w w

/ 15 3.0 F F U

10 C F 2.0

C 5 1.0 0 30 90 150 210 270 SFM A/B Serum Time post infection (min.) 30 Minutes post infection SFM A/B ( Δ56 °) A/B

Figure 3.1 – Phagocytosis of B. anthracis spores by human MDMs. (A) MDMs were infected with B. anthracis spores for 30 minutes in SFM or 10% human A/B serum (+/- Δ56°C), washed extensively, and cultured for up to 270 minutes in media. The supernatant was removed and the monolayer was washed then lysed. Lysates were plated and the CFU/well was determined as described in Materials and Methods. Data represent the average CFU/well of three experiments performed with MDMs from three donors. (B) Phagocytosis of B. anthracis spores by human MDMs was measured by determining the CFU/well in cell lysates at 30 minutes post-infection as described in Materials and Methods. Data represent the average and standard deviation for MDMs from six donors (serum-free media), and four additional donors (10% A/B serum +/- Δ56°C). Increase in CFU/well was tested for statistical significance using an ANOVA.

112 1600 P<0.001 P<0.001 SFM P<0.05 A/B Serum 1400 A/B Serum (Δ56°C) 1200

Ms 1000 D

800 *** es/50 M

r 600 o p

S 400 P>0.05 200 † † 0 Complement - + - - - + ComplementC 3 - +- -- ­- +- +­- FactC3or B ------+ -­- Factor D - - - - + ­- Factor B - - - - + ­- 30 Minutes post infection Factor D - - - - + ­- 30 minutes post infection

Figure 3.2 - Intracellular spores as detected by light microscopy per 50 human MDMs (phagocytic index). Infection in the presence of 10% human A/B serum resulted in a significantly higher phagocytic index as compared to infections in SFM or media containing heat inactivated 10% human A/B serum (Δ56°C). Supplementation of SFM with whole guinea pig complement did not increase the phagocytic index (p>0.05), nor does supplementation of heat inactivated serum with C3, Factor D and Factor B († p>0.05). Supplementation of heat inactivated serum with whole complement partially restores the phagocytic index to greater than half of the index for human A/B serum and was statistically significant when compared to the SFM (***p<0.05). Data represent the mean of four to six experiments using MDMs from six individual donors. Error bars represent standard deviation. Differences in treatment groups were tested for statistical significance using a two-tailed Student’s t-test with equal variance.

113 A. B. )

m 2.5 9000 2 405 n

( 1.5 8000 e c 1 an

7000 rb 0.5 o s 0 6000 Ab 0.5 1.5 2.5 3.5 l

m 5000 Log(10) Dilution s/ t i 4000

Un C. 3000 2.5 ) m 2000 2 05 n 4 1.5

1000 ce ( n 1 a b r 0 0.5 so

0.5 1 1.5 b A Donor Serum 0 11 .5 22 .5 Units/ml

Figure 3.3 – Detection of human IgG which binds to B. anthracis spores by ELISA. A. Sera from 10 nonimmune donors was tested for IgG that binds to B. anthracis spores by ELISA. Each dot represents the mean value (arbitrary U/ml) for each individual tested in triplicate. The horizontal bar represents the mean value. B. Serial dilutions (1:10 to 1:1280) of human A/B serum was used to construct a reference curve by plotting the O.D. vs. the log10 of the dilution. Each point represents the mean value and error bars represent the standard deviation at each dilution. The linear portion of the curve is denoted by arrows. C. A linear transformation (R2=0.98) was performed on the linear portion of the curve (dilutions 1:40 to 1:640) and the data were plotted on an O.D. vs. U/ml (log10) scale giving the assay a range of 7.81 to 250 U/ml at the dilution of serum tested. Data points represent the mean and error bars represent the standard deviation at each dilution.

114 A.A 1.6

) 1.4

nm 1.2

405 1 e (

c 0.8 n

a 0.6 b r

so 0.4 b

A 0.2 0 10.00 5.00 2.50 1.25 0.63 0.31 0.16 0.01 % Serum

C3b (AB serum) C3b (AB serum (Δ56º)) C3a (AB(AB serum)serum) B.B 2.5

) 2

05 nm 1.5 (4

ance 1 b

0.5 Absor

0 0 C3b (C3bA1B s erum) C3b (ABC3b s2er um (Δ56º)) 3 (donor serum) (donor serum(Δ56 ºC))

Figure 3.4 - Detection of C3b deposition on Bacillus anthracis Sterne spores as shown by spore C3b ELISA. A. Normal and heat inactivated AB serum was diluted from 10% down to 0.01% and incubated in 96 well ELISA plates coated with fixed Bacillus anthracis spores as described in the Materials and Methods. C3β was detected with a mouse anti-human C3β monoclonal antibody and C3a was detected with a mouse anti-human C3a monoclonal antibody. Data represent the mean and error bars the standard deviation of triplicate values at each dilution. Binding of the β chain of C3 was abrogated by heat inactivation (Δ56°C) as compared to normal serum. C3a could not be detected at any dilution tested. B. Serum samples (+/- Δ56 °C) from nonimmune individuals were tested for C3β binding to B. anthracis spores by ELISA. Dots represent the mean O.D. of triplicate values obtained at a 10% dilution of serum and the horizontal bar represents the mean. All ten donors showed binding of the β chain of C3 which was completely inhibited by heat inactivation.

115 kDa 140 100 80 75 kDa 60 50 40 1 2 3 4 5 6 7 8

Figure 3.5 - Detection of C3b deposition on Bacillus anthracis Sterne spores as shown by spore immunoprecipitation and western blot. Spore incubated in varying serum conditions for 30 minutes. The spores were boiled and pelleted and the resulting supernatant was loaded onto a 10% SDS/polyacrylamide gel. A single well was loaded with empty loading dye and the outside lanes of the gel were loaded with rainbow and biotin protein ladders. The proteins transferred to a nitrocellulose membrane were detected with a mouse monoclonal anti-C3b antibody. Spores were incubated in duplicates in PBS (3 and 4), normal AB serum (5 and 6) and heat inactivated AB serum (7 and 8). 32 and 12 μg of purified human C3b was loaded as a positive control (1 and 2) as single wells.

116 Unstained A. B. No serum 10% Hu Serum

3.6% 88.5%

Hu IgG

C. No serum 10% Hu Serum 10% Hu Se rum

0.0% 1.1% 67.1%

α -Mu IgG (FITC) C3a C3β

Figure 3.6 - Detection of spore binding antibody and C3b deposition by fluorescence assisted cytometric sorting (FACS). A. Control unstained spores incubated in PBS alone. B. Spores were incubated in 10% human AB serum followed by an anti-human mouse monoclonal antibody for 30 minutes. FACS analysis of spores shows 88.5% of gates spores are positive for human IgG binding. C. C3b deposition was probed for using an anti-C3b mouse monoclonal antibody followed by incubation with a FITC conjugated anti-mouse antibody. C3a deposition was examined using an anti-C3a mouse monoclonal antibody. Similar to the complement ELISA experiments, C3b deposition is present, however, the amount of C3a found on the spores is minimal, indicating that cleavage of the alpha chain takes place.

117 CHAPTER 4

ROLE Of SPORE ANTIBODIES IN COMPLEMENT MEDIATED PHAGOCYTOSIS OF BACILLUS ANTHRACIS SPORES IN PRIMARY MACROPHAGES

4.1 Introduction

Bacillus anthracis is a gram positive, spore forming, facultative anaerobic bacterium. Anthrax, the disease associated with infection with this organism is recognized in three forms: cutaneous, pulmonary and gastrointestinal. While the end-stage pathophysiology between the three forms is likely similar (i.e. widespread septicemia and toxemia), the early events in pathogenesis likely differ in the method in which the organism reaches systemic circulation.

Pulmonary anthrax, the form of the disease associated with the greatest morbidity and mortality, has been of particular concern in terms of bioterrorism potential. Critical to the early pathogenesis of pulmonary anthrax is the method by which the organism enters systemic circulation following deposition in the alveoli. Alveolar macrophages play a significant role in the disease by phagocytizing spores and transporting them to local lymph nodes1. Spores then germinate, escape the macrophage and disseminate. The mechanisms in which

118 the spores escape the macrophage are not clear, however, anthrolysin O, a

cholesterol-dependent phospholipase and intra-phagolysosomal secretion of

toxins have been implicated2-5.

Given the importance of the alveolar macrophage in the pathogenesis of

pulmonary anthrax, it is critical to understand the nature of the initial interaction of

the spore with the alveolar macrophage. Phagocytosis of most organisms

involves the recognition of conserved motifs by scavenger receptors or direct

opsonization of the spores by soluble components of the host immune system6.

Numerous soluble factors are involved in direct opsonization, however, in the alveolus, a more limited repertoire of opsonins are relevant. Among these are immunoglobulin and complement proteins. Components of the complement system are produced by macrophages and epithelial cells located within the lung7-9 and can be recovered from bronchoalveolar lavage fluid (BALF)10,10-13.

Proteolytic cleavage of C3 in BALF, however, is reduced when compared to C3 in serum12. Complement may be activated by both the classical and alternative

pathways, but the low level of factor B in BALF may favor the classical pathway13

Although immunoglobulin and the complement system are normally associated with effective clearance of bacterial organisms, the effectiveness of spore-specific antibody in the alveolus is uncertain. In other sites of inoculation, such as the dermis or the gastrointestinal tract, opsonization by antibody may be beneficial in preventing widespread germination and toxin secretion at these sites. In the alveolar air space, however, spore germination may not taken place

119 readily unless the spore gains access to an intracellular space or crosses the air- blood barrier into systemic circulation. In this circumstance, facilitating entry of a large numbers of spores into macrophages by way of the Fcγ or complement

(CR) receptors may provide a means of systemic trafficking, even if a percentage of the spores do not survive phagocytosis. Therefore, we hypothesize that spore specific antibodies induce increased phagocytosis by macrophages by means of direct opsonization and fixation of complement component C3b. In addition, we suggest that spore specific antibodies may be detrimental during aerosol exposure to B. anthracis spores due to the proposed role of the alveolar macrophage in facilitating transport of the organism to the systemic circulation.

To elucidate the effect of these antibodies, we vaccinated mice with fixed spores of B. anthracis Sterne and B. cereus. Using spore ELISAs and flow cytometry assays, we found that there is significant cross reactivity between anti-serum against B. cereus and B. anthracis spores. In addition, we found that antibodies in serum from vaccinated mice fix complement component C3b to spores in higher amounts than serum from unvaccinated mice. These anti-sera induced a higher phagocytic index when spores plus anti-sera were incubated with mouse peritoneal macrophages. The phagocytic index was significantly lowered in the presence of heat inactivated (HI) unvaccinated mouse serum, whereas heat inactivating the spore specific anti-sera only partially lowered the phagocytic index. These data support the hypothesis that phagocytosis of spores by macrophages in the presence of spore specific antibodies is both partially Fcγ

120 and dependent. In addition, we have shown that exposure

to B. cereus can generate antibodies capable of similar cross reactive opsonic

effect. The in vivo significance of these antibodies in the progression of

pulmonary anthrax will require further study. Collectively, our findings have

important implications for strategies of vaccination against B. anthracis and the

pathogenesis of pulmonary anthrax.

4.2 Materials and methods

Spore purification

Vegetative cultures of B. anthracis Sterne strain (Colorado Serum

Company) or B. cereus were sporulated in modified G medium [0.2% yeast

extract, 0.0025% CaCl2 dihydrate, 0.05% KH2PO4, 0.00976% MgSO4 anhydrous,

0.005% MnCl2 quatrahydrate, 0.00073% ZnSO4 heptahydrate, 0.00005% FeSO4 heptahydrate, 0.2% (NH4)2SO4] until >99% refractile spores were observed by

14 light microscopy . Endospores were washed with sterile ddH2O and then

purified over a Renografin 76 density gradient of 1.300g/mL15. The endospores

were washed 3 times with sterile H2O to remove any remaining protein and

Renografin and resuspended into sterile H2O. Endospore preparations were

scored using serial dilutions and counting by hemacytometer to determine total

count and also plated to determine colony forming unites (CFU). Endospore

preparations were found to be >99% pure with no observable vegetative cells or

debris.

121 Mouse vaccination

Spores of B. anthracis Sterne (BAS) and B. cereus (BC) were fixed in 4%

paraformaldehyde for 24 hours, followed by extensive washes in Ca++/Mg++ PBS.

Complete inactivation was determined by culturing spores on nutrient agar plates. Ten C57/B6 mice (Harlan Laboratories, Indianapolis, IN) were vaccinated with 1x108 spores of B. anthracis Sterne or B. cereus adsorbed in 22 μg of

aluminum hydroxide (Sigma-Aldrich, St. Louis, MO) subcutaneously. Booster

vaccinations were performed 2 weeks following initial vaccinations. PA

vaccination of C57/B6 mice were performed as described previously16.

Anti-spore ELISA

Purified spores of BAS or BC were fixed in 4% paraformadehyde for 2

hours at a concentration of 1x108 spores/ml. An aliquot of spores were plated on

nutrient agar to insure fixation and death. The fixed spores were diluted in

bicarbonate coating buffer to a concentration of 1x107 spores/ml. 96-well

Immulon-HB ELISA plates (Dynex Technologies, Chantilly, VA) were coated with

spores by aliquoting 100 µl of the spore suspension into each well. The plate was

incubated at 4 ºC overnight. The plates were then washed with PBS/0.05%

Tween-20 and blocked with assay diluent (PBS/5% FBS/5% milk/0.05% Tween­

20). The mouse serum was prepared at dilutions from 1:10 to 1:1,250 in assay

diluent prior to incubation in the ELISA plate wells for 1 hour at 37 ºC. Goat anti­

122 mouse IgG conjugated to alkaline phosphatase (Sigma-Aldrich) was prepared at a 1:5000 dilution in assay diluent. The diluted secondary antibody was incubated in the wells for 1 hour at 37 ºC. A secondary antibody alone well was used as a negative control where appropriate. Empty ELISA plate wells were tested for antibody binding independently. Following an additional three washes, 100 µl of p-nitrophenylphosphate substrate (Sigma-Aldrich) was added to each well and the plate was incubated at 37 ºC for 30 minutes. The development reaction was stopped with 3 N NaOH and the plate was read at 405 nm.

Fluorescence activated cell sorting (FACS) assay

For antibody detection, BAS spores were incubated in 96 well sterile round bottom plates (Sigma-Aldrich) in PBS + 10 % mouse serum at 37 ºC for 30 minutes. The spores were washed three times with sterile PBS, followed by incubation in a 1:2,000 dilution of FITC conjugated goat anti-mouse IgG antibody

+ PBS for 1 hour at 37 ºC. The spores were then fixed in 2 % paraformaldehyde following three washes in PBS and submitted for FACS analysis.

For C3b detection, BAS spores were incubated in 96 well sterile round bottom plates (Sigma-Aldrich) in PBS + 10% mouse serum at 37 ºC for 30 minutes. The spores were washed three times with sterile PBS, followed by incubation in a 1:100 dilution of rat anti-mouse C3b IgG (Hycult Biotechnology,

Netherlands) for 1 hour at 37ºC. The spores were subsequently incubated in a

1:2,000 dilution of FITC conjugated anti-rat IgG antibody for 1 hour at 37ºC and

123 fixed in 2% paraformaldehyde. Appropriate control spores were included in both

assays.

Mouse peritoneal macrophage isolation and culture

Culture and isolation of mouse peritoneal macrophages (MPM) were

performed as described previously17. Briefly, C57/B6 mice were euthanized by

CO2 asphyxiation and a ventral midline incision was made to expose the

peritoneum. Ten mls of RPMI 1640 media was infused into the peritoneal cavity

via a 21 gauge needle and syringe. The fluid was aspirated and deposited into a

50 ml polypropylene tube. The cells were washed twice in RPMI 1640, and then

allowed to adhere to glass chamber slides (Fischer Scientific, Kalamazoo, MI).

An aliquot of cells were stained with Wright-Giemsa or for ANBE and examined

by light microscopy for standard morphology.

Infection of mouse peritoneal macrophages with B. anthracis spores

Mouse peritoneal macrophagess were seeded at a density of 1 x 106 cells/well into six-well flat-bottom tissue culture plates 17 to 19 hours before infection. The media was then replaced with 500 μl of RPMI-1640 containing

20mM HEPES, 20 mM L-glutamine and 10% human AB serum. Washed B. anthracis spores were added to each well at a multiplicity of infection (MOI) of 10 in a volume of 50 μl. The plates were centrifuged at 400 x g for 3 min and incubated at 37 °C for 15 minutes. The wells were washed five times with serum­

124 free RPMI-1640 media. Following washes, the media was removed from the well and the cells were lysed in 2.5 % saponin. Serial dilutions of each sample were performed before plating on nutrient agar plates that were subsequently incubated at 37 º C overnight.

Mouse peritoneal macrophages were seeded at a density of 2.5 x 105 cells/well in four-well glass chamber slides (Fisher Scientific) 17 to 19 hours before infection. The media was replaced with 175 μl of RPMI-1640 containing

20mM HEPES, 20 mM L-glutamine and 10 % mouse serum. Washed B. anthracis spores were added to each well at a multiplicity of infection (MOI) of 10 in a volume of 25 μl. The slides were incubated at 37 °C for 30 minutes and then the wells were washed five times with serum-free RPMI-1640 media. Two hundred milliliters of serum-free RPMI-1640 media containing 20 mM HEPES was placed in each well and the slides were returned to the 37 °C incubator.

After 30 minutes, the media was removed from the well and the cells were fixed in situ with 4.0% paraformaldehyde. The fixed monolayers were stained with Diff­ quick blood stain (Fischer Scientific, Kalamazoo, MI) per the manufactures’ instructions. The slides were coded to prevent investigator bias and the number of intracellular spores was counted by examining a minimum of 50 cells microscopically (1000x).

125 Fcγ receptor sequestration in mouse peritoneal macrophages

The protocol for this assay was adapted from Balagopal, et al.,18 and is described briefly as follows. Eight well chamber slides were coated with poly-L­ lysine for 30 minutes followed by fixation with 2.5% glutaraldehyde for 1 hour.

The slides were washed and coated with bovine serum albumin (3 mg/ml) for 1 hour. The slides were incubated in L-glycine to quench remaining aldehyde groups. The following day, the slides were incubated in a 1:100 dilution of polyclonal rabbit anti-BSA antibody. Mouse peritoneal macrophages were plated in the slides and incubated for 30 minutes at 37 ºC to allow adherence. To test the FcγR sequestration, sheep erythrocytes were coated with anti-sheep RBC antibodies. The macrophages were incubated with coated erythrocytes for 30 minutes at a ratio of 10 erythrocytes per macrophages. The macrophages were washed and incubated in RBC lysis solution for 10 minutes to lyse extracellular erythrocytes. The macrophages were fixed in methanol and stained. Intracellular erythrocytes were enumerated by counting the erythrocytes per 50 macrophages and the percentage of macrophages exhibiting erythrophagocytosis. FcγR sequestered macrophages were infected as above in the presence of HI or normal BAS or BC antiserum. The phagocytic index was determined as described above.

126 4.3 Results

Antibodies against B. cereus react with spores of B. anthracis

Previous work from our laboratory has shown the presence of antibodies

in unvaccinated, unexposed serum donors, which binds to B. anthracis Sterne

spores. To evaluate the possibility that these antibodies are elicited against

similar species of Bacillus found in the environment, such as B. cereus or

thuringiensis, we vaccinated C57/B6 mice with paraformaldehyde fixed spores of

B. anthracis Sterne (BAS) (pX01+, pX02-) or B. cereus (BC). One week following

the second booster vaccination, the reactivity of respective sera were measured

by spore ELISA against BAS and BC. As shown by figure 4.1A and B, 1x108 spores elicited an average absorbance (405 nm) greater than 0.400 at the highest dilution for both the BC and BAS ELISAs. In addition, we measured the ability of serum from BC vaccinated mice to react with BAS spores, shown by spore ELISA in figure 4.1B, which shows an absorbance greater than 0.400 when tested in the BAS coated plates. In each of these ELISAs, the average absorbance of the unvaccinated mouse serum did not exceed 0.400, although a serum dilution dependent increase was observed in the unvaccinated mouse serum control. These data indicate that BC spores are able to elicit antibodies that bind to BAS spores, likely due to a shared epitope on the exosporium of both organisms19. We also confirmed the ability of anti-PA antibodies to bind BAS

spores (Fig 4.1C)20. These data were confirmed by flow cytometry (Fig. 4.1D).

BAS spores were incubated in PBS containing 10% serum from unvaccinated,

127 PA, BAS or BC vaccinated mice. As expected, sera from BAS vaccinated mice contained antibodies that bound nearly 100% of the spores (96.38 ± 3.75), while a similarly high percentage of spores exhibited antibody binding when incubated with BC anti-serum (83.91 ± 23.87), both of which were significantly higher than the binding observed in unvaccinated mouse serum (4.63 ± 4.61, p<0.00001 and p<0.005, respectively). The percentage of fluorescent spores after incubation in sera from PA vaccinated mice was also significantly higher than the percentage following incubation in sera from unvaccinated mice (17.04 ± 5.66, p<0.05).

Interestingly, the top absorbance values detected for anti-PA sera were similar to those of anti-BC sera (Fig. 1.4B and C); however the percentage of FITC (+) spores was less in the FACS analysis (Fig. 1.4D). These results may be due to an amplifying effect of alkaline phosphate in our ELISA and not a true quantitative measurement of PA antibody spore binding activity.

Antibodies against B. anthracis Sterne and B. cereus fix complement component C3b to the B. anthracis spores

To evaluate the ability of our generated mouse anti-sera IgG preparations to fix complement to spores, we utilized the flow cytometric assay described in

Chapter 3. The presence of C3b on the spore surface was evaluated using a rat monoclonal antibody specific for C3b, iC3b and C3c followed by a FITC conjugated rabbit anti-rat IgG antibody. Serum dilution dependent C3b binding was observed with sera from unvaccinated mice and the sera from vaccinated

128 mice (Fig. 4.3B); however, the amount of deposited C3b was greater in the presence of sera from spore vaccinated mice (69.4% and 71.5% for BC and BAS antiserum, respectively) compared to sera from unvaccinated mice (42.5%)(Fig.

4.2). In addition, anti-PA sera fixed complement to BAS spores(Fig. 4.2 and

4.3A). The average results of three assays indicated a significant difference in spores exhibiting C3b fixation comparing sera from unvaccinated mice to those vaccinated with BC spores (p<0.01) and BAS spores (p<0.01) (Fig. 4.3A). The difference in C3b deposition in the presence of sera from unvaccinated and PA vaccinated mice was not significant (Fig. 4.3A)

Antibodies against B. anthracis Sterne and B. cereus increase phagocytosis by direct FcR mediated phagocytosis and by complement opsonization.

Both FcγR mediated and complement mediated phagocytosis can play an important part in the clearance of bacterial organisms. This is particularly relevant to the dormant bacterial endospores, which do not have an active means of evading these mechanisms. To evaluate the significance of spore specific antibody opsonization and complement, we evaluated the ability of mouse peritoneal macrophages to phagocytize spores of B. anthracis in the presence of antiserum from PA, BAS and BC vaccinated mice. We first evaluated the colony forming units associated with infected macrophages (Fig. 4.4). Macrophages infected in serum-free media exhibited an average of 19.04 ± 13.95 CFU/50

129 cells. Macrophages infected in the presence of sera from BAS and BC vaccinated mice had significantly higher CFU counts (57.78 ± 21.99, p < 0.05 and 49.58 ± 16.18, p < 0.01, respectively). These data suggested that soluble components in serum from spore vaccinated mice, such as immunoglobulin and complement, enhance spore association in MPMs. Because of the inherent variability we have encountered in culture assays and due to the large numbers of non-viable spores that can exist in purified spore preparations, we turned to phagocytic index assays to investigate the role of heat labile factors, like complement, in the phagocytosis of spores. Macrophages infected in serum-free media exhibited average phagocytic indices of 2.18 ± 2.23 (standard deviation)(Fig. 4.5). In contrast, macrophages infected in the presence of unvaccinated mouse sera had average phagocytic indices of 33.75 ± 9.59 spores

(approximately 15 fold greater than indices measured in serum-free media).

These phagocytic indices were decreased significantly upon heat inactivation of the serum (3.05 ± 2.75, p<0.001), comparable to the level of those in serum-free media. The phagocytic indices for PA vaccinated mice sera also decreased significantly following heat inactivation of the sera (Fig. 4.5). When macrophages were infected in the presence of serum from BAS or BC vaccinated mice, phagocytic indices were significantly higher than those measured in serum-free media and serum from unvaccinated mice (124.08 ± 63.78 for BAS and 96.62 ±

29.21 for BC, A significant decrease in the phagocytic indices were also observed upon heat inactivation of the serum from BAS and BC vaccinated mice

130 (p<0.05 for both). However, the phagocytic indices did not decrease to the level of SFM as the indices of unvaccinated mouse sera did and a significant difference was observed between the indices of HI unvaccinated mouse serum and the indices of serum from BAS and BC vaccinated mice (p<0.05 and 0.01, respectively). These data indicate that a heat stable soluble factor not present in serum from unvaccinated mice, but present in the serum from animals vaccinated with spores, promotes phagocytosis of spores.

Sequestration of Fcy receptors partially reduces the phagocytic index of spores.

To determine the potential involvement of FcγR receptors during phagocytosis of spores in the presence of induced anti-spore antibodies, we performed a Fcγ receptor sequestration assay, which effectively removes FcγR receptors from the surface of macrophages, leaving limited numbers of receptors available to engage antibody bound particles. We tested this sequestration by incubating treated macrophages with IgG coated sheep erythrocytes. Following incubation and subsequent erythrophagocytosis, the extracellular erythrocytes were lysed and the intracellular erythrocytes were counted and expressed as the percentage of macrophage showing erythrophagocytosis or the number of erythrocytes per 50 macrophages (erythrophagocytic index). The Fcγ receptor sequestered macrophages showed a significantly decreased percentage erythrophagocytosis (Fig 4.6A.) and erythrophagocytic index (Fig 4.6B.)

131 compared to the macrophages plated on BSA alone. FcγR sequestered macrophages were then infected with spores of B. anthracis Sterne in the presence of normal and HI BAS or BC antiserum. Our data indicated a significant decrease of phagocytic indices between macrophages incubated in normal and

HI sera (Fig. 4.7). However, there was no significant difference between the phagocytic indices of macrophages and FcγR sequestered macrophages incubated in control sera from unvaccinated mice. These data indicate that deposited C3b contributed to phagocytosis despite a reduction of available Fcγ receptors at the cell surface. Finally, phagocytic indices were measured in FcγR sequestered macrophages infected in the presence of HI BC and BAS antiserum.

Our results indicated that phagocytic indices were significantly lower in macrophages depleted of FcγR (77.57 ± 19.95 versus 109.31 ± 40.25, p<0.05 for

BAS and 60.29 ± 25.36 versus 92.08 ± 30.10, p<0.001 for BC) than indices measured in normal macrophages exposed to spores in HI sera. These data indicate that Fcγ receptors mediate the association of these spores with mouse peritoneal macrophages. Consistent with our control experiments, we did not expect a complete abrogation of the phagocytic index as erythrophagocytosis could not be completely eliminated in this assay.

132 4.4 Discussion

Our study presented herein tested spore, antibody and complement interactions. Although lectins, such as surfactant protein A and D are important, particularly in the context of the alveolus, the study of the interactions with these proteins is outside the scope of this research. We have previously shown that antibody is present in sera unvaccinated and unexposed humans which binds to

Bacillus anthracis spores, but does not appear to directly facilitate opsonophagocytosis via FcγR receptors. Our data presented in Chapter 3 indicate that these pre-existing antibodies mediate the association and phagocytosis of B. anthracis spores in human MDMs by the fixation of complement C3b. The nature of this antibody is unknown, however, we have previous proposed that it may be a cross reactive antibody elicited by exposure to a species of Bacillus which has an exosporium. These species include Bacillus cereus, thuringiensis, megaterium and mycoides21,22. As many of these species are present in the environment, it is possible that exposure to the exosporium of these organisms may elicit the antibody response seen in our previous study23-25.

Although limited cross reactivity has been shown between B. anthracis polyclonal antibody and B. cereus spores26(Premanandan, data not shown), we demonstrate by both spore ELISA and flow cytometry that antibodies elicited against BC by vaccinating C57B/6 mice with paraformaldehyde fixed BC spores interact with BAS spores nearly as well as BAS specific antibody. In addition, we demonstrate the presence of these antibodies are associated with a higher

133 degree of C3b product bindings to BAS spores, showing an additional role of

spore specific antibody. We also demonstrate C3b fixation takes place in

unvaccinated mice sera and PA antisera, although it is significantly less. It is

unclear which pathway is responsible for the fixation in these serum types;

however it does not appear that anti-PA antibodies are more effective in the

fixation process than unvaccinated mouse sera.

The interaction of B. anthracis spores with alveolar macrophages is a

critical step in the pathogenesis of pulmonary anthrax. Current theories suggest

that the macrophage plays a transporting role in pathogenesis, providing a

vehicle in which the germinated organism can enter systemic circulation1,27.

Typically pathogens are phagocytized by macrophages by one of two mechanisms. First, conserved patterns integral to the organisms outer structure can be recognized by macrophage scavenger receptors. Several examples of this process in other pathogens are present in the literature. Mycobacterium tuberculosis in which lipoarabinomannan is recognized by the mannose receptor28-30. The non-opsonized capsule of Brucella abortus is thought to

interact with lectin and fibronectin receptors prior to phagocytosis31. The fimbriae

of have been shown to directly mediate binding to

CD11b/CD18, which induces cellular uptake32. The second mechanism is the

opsonization of pathogens by soluble components of the innate and acquired

immune system.

134 In our previous study (Chapter 3), we demonstrated that direct antibody binding does not enhance phagocytosis of B. anthracis spores by human MDMs in the absence of complement C3 fixation. Data presented herein indicate that polyclonal antibodies from vaccinated mice mediate association and internalization of BAS spores in mouse peritoneal macrophages by both direct and complement mediated opsonization. We demonstrated incomplete abrogation of phagocytic indices in the presence of HI BAS and BC antisera in contrast to the greater loss of phagocytic index in the presence of HI serum from unvaccinated mice. Additionally, we confirmed the involvement of Fcγ receptors in our sequestering experiments. Therefore, in our mouse model, it appears that spore specific antibodies, generated against BAS or BC, are significantly different than the antibodies detected in similar sera used in our previous study. A possible explanation for this disparity is the presence of IgM, the IgG composition, the avidity and the affinity of the antibodies involved. It is possible that since the on the spore surface are fixed, clustering of antibodies does not occur and less Fcγ receptors are engaged, preventing less effective aggregation of receptors. Alternatively, the antibody found in human serum could be a natural antibody. Natural antibodies are a spontaneously produced class of antibodies which can bind to a number of self and non-self proteins and are not considered epitope specific33. These antibodies are predominantly IgM but can be composed of IgG and IgA isotypes and have documented role in fixing complement as both a housekeeping role and in autoimmunity34-36 .

135 An interesting finding was the role of anti-PA antibodies in our model. The interaction of anti-PA antibodies with B. anthracis Sterne spore have been documented20,37. Although we show a similar interaction with spores by ELISA, the percentage of spore opsonized in PA antiserum by both antibody and complement is relatively low. In addition, the phagocytic index observed in non-

HI PA antiserum is lowered to the level of serum free media, suggesting that a majority of phagocytosis is complement mediated. These data provide an interesting possible secondary role for anti-PA antibodies generated by PA vaccination.

Our data presented herein suggest that phagocytosis of spores by macrophages in the presence of a generated spore specific antibody is both complement and FcγR dependent. These findings raise more interesting questions which are beyond the scope of our study. Determining what shared epitope is present between B. anthracis and B. cereus would be a logical next step. Previous studies have shown that BclA is the immunodominant protein in the exosporium of B. anthracis and may be responsible for the cross reactivity observed here22. In addition, the in vivo significance of these antibodies in pulmonary anthrax would be important to determine. Since the macrophage might play a role in transporting the organism to systemic circulation, overloading the cytoplasm with opsonized spores may support survival of the organism and inadvertently propagate the disease. Further studies are needed to determine the effectiveness of spore proteins as a vaccine against pulmonary anthrax.

136 4.5 References

1. Dixon T.C., Meselson M., Guillemin J., & Hanna P.C. (1999) Anthrax. N.Engl.J Med. 341, 815-826.

2. Cocklin S., Jost M., Robertson N.M., Weeks S.D., Weber H.W., Young E., Seal S., Zhang C., Mosser E., Loll P.J., Saunders A.J., Rest R.F., & Chaiken I.M. (2006) Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis. J Mol.Recognit. 19, 354-362.

3. Mosser E.M. & Rest R.F. (2006) The Bacillus anthracis cholesterol-dependent cytolysin, Anthrolysin O, kills human neutrophils, monocytes and macrophages. BMC.Microbiol 6, 56.

4. Shannon J.G., Ross C.L., Koehler T.M., & Rest R.F. (2003) Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin. Infect.Immun. 71, 3183-3189.

5. Wei Z., Schnupf P., Poussin M.A., Zenewicz L.A., Shen H., & Goldfine H. (2005) Characterization of Listeria monocytogenes expressing anthrolysin O and phosphatidylinositol-specific phospholipase C from Bacillus anthracis. Infect.Immun. 73, 6639-6646.

6. Kang P.B., Azad A.K., Torrelles J.B., Kaufman T.M., Beharka A., Tibesar E., DesJardin L.E., & Schlesinger L.S. (2005) The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis. J Exp.Med 202, 987-999.

7. Ackerman S.K., Friend P.S., Hoidal J.R., & Douglas S.D. (1978) Production of C2 by human alveolar macrophages. Immunology 35, 369-372.

8. Cole F.S., Auerbach H.S., Goldberger G., & Colten H.R. (1985) Tissue-specific pretranslational regulation of complement production in human mononuclear phagocytes. J Immunol. 134, 2610-2616.

9. Strunk R.C., Eidlen D.M., & Mason R.J. (1988) Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways. J Clin.Invest 81, 1419-1426.

10. Coonrod J.D. & Yoneda K. (1981) Complement and opsonins in alveolar secretions and serum of rats with pneumonia due to Streptococcus pneumoniae. Rev.Infect.Dis. 3, 310-322.

11. Kolb W.P., Kolb L.M., Wetsel R.A., Rogers W.R., & Shaw J.O. (1981) Quantitation and stability of the fifth component of complement (C5) in 137 bronchoalveolar lavage fluids obtained from non-human primates. Am Rev.Respir.Dis. 123, 226-231.

12. Giclas P.C., King T.E., Baker S.L., Russo J., & Henson P.M. (1987) Complement activity in normal rabbit bronchoalveolar fluid. Description of an inhibitor of C3 activation. Am Rev.Respir.Dis. 135, 403-411.

13. Watford W.T., Smithers M.B., Frank M.M., & Wright J.R. (2002) Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages. Am J Physiol Lung Cell Mol.Physiol 283, L1011-L1022.

14. Weiner M.A. & Hanna P.C. (2003) Macrophage-Mediated Germination of Bacillus anthracis Endospores Requires the gerH Operon. Infect.Immun. 71, 3954-3959.

15. Tamir H. & Gilvarg C. (1966) Density gradient centrifugation for the separation of sporulating forms of bacteria. J Biol.Chem. 241, 1085-1090.

16. Berthold I., Pombo M.L., Wagner L., & Arciniega J.L. (2005) Immunogenicity in mice of anthrax recombinant protective antigen in the presence of aluminum adjuvants. Vaccine 23, 1993-1999.

17. Kang T.J., Fenton M.J., Weiner M.A., Hibbs S., Basu S., Baillie L., & Cross A.S. (2005) Murine macrophages kill the vegetative form of Bacillus anthracis. Infect.Immun. 73, 7495-7501.

18. Balagopal A., MacFarlane A.S., Mohapatra N., Soni S., Gunn J.S., & Schlesinger L.S. (2006) Characterization of the receptor-ligand pathways important for entry and survival of in human macrophages. Infect.Immun. 74, 5114-5125.

19. Delvecchio V.G., Connolly J.P., Alefantis T.G., Walz A., Quan M.A., Patra G., Ashton J.M., Whittington J.T., Chafin R.D., Liang X., Grewal P., Khan A.S., & Mujer C.V. (2006) Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. Appl.Environ.Microbiol 72, 6355-6363.

20. Cote C.K., Rossi C.A., Kang A.S., Morrow P.R., Lee J.S., & Welkos S.L. (2005) The detection of protective antigen (PA) associated with spores of Bacillus anthracis and the effects of anti-PA antibodies on spore germination and macrophage interactions. Microb.Pathog. 38, 209-225.

21. Beaman T.C., Pankratz H.S., & Gerhardt P. (1972) Ultrastructure of the exosporium and underlying inclusions in spores of strains. J Bacteriol. 109, 1198-1209.

138 22. Steichen C., Chen P., Kearney J.F., & Turnbough C.L., Jr. (2003) Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium. J Bacteriol. 185, 1903-1910.

23. Vilain S., Luo Y., Hildreth M.B., & V.S. (2006) Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil. Appl.Environ.Microbiol 72, 4970-4977.

24. Jensen G.B., Hansen B.M., Eilenberg J., & Mahillon J. (2003) The hidden lifestyles of Bacillus cereus and relatives. Environ.Microbiol 5, 631-640.

25. Jensen G.B., Larsen P., Jacobsen B.L., Madsen B., Smidt L., & Andrup L. (2002) Bacillus thuringiensis in fecal samples from greenhouse workers after exposure to B. thuringiensis-based pesticides. Appl.Environ.Microbiol 68, 4900­ 4905.

26. Longchamp P. & Leighton T. (1999) Molecular recognition specificity of Bacillus anthracis spore antibodies. J Appl.Microbiol. 87, 246-249.

27. Dixon T.C., Fadl A.A., Koehler T.M., Swanson J.A., & Hanna P.C. (2000) Early Bacillus anthracis-macrophage interactions: intracellular survival survival and escape. Cell Microbiol. 2, 453-463.

28. Schlesinger L.S. (1993) Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors. J Immunol. 150, 2920-2930.

29. Schlesinger L.S., Kaufman T.M., Iyer S., Hull S.R., & Marchiando L.K. (1996) Differences in mannose receptor-mediated uptake of lipoarabinomannan from virulent and attenuated strains of Mycobacterium tuberculosis by human macrophages. J Immunol. 157, 4568-4575.

30. Ernst J.D. (1998) Macrophage receptors for Mycobacterium tuberculosis. Infect.Immun. 66, 1277-1281.

31. Gorvel J.P. & Moreno E. (2002) Brucella intracellular life: from invasion to intracellular replication. Vet.Microbiol 90, 281-297.

32. Hajishengallis G., Wang M., Harokopakis E., Triantafilou M., & Triantafilou K. (2006) Porphyromonas gingivalis fimbriae proactively modulate beta2 integrin adhesive activity and promote binding to and internalization by macrophages. Infect.Immun. 74, 5658-5666.

33. Binder C.J. & Silverman G.J. (2005) Natural antibodies and the of atherosclerosis. Springer Semin.Immunopathol. 26, 385-404.

139 34. Fleming S.D. (2006) Natural antibodies, and complement activation in tissue injury. Autoimmunity 39, 379-386.

35. Fleming S.D. & Tsokos G.C. (2006) Complement, natural antibodies, autoantibodies and tissue injury. Autoimmun.Rev. 5, 89-92.

36. Ochsenbein A.F. & Zinkernagel R.M. (2000) Natural antibodies and complement link innate and acquired immunity. Immunol.Today 21, 624-630.

37. Welkos S., Friedlander A., Weeks S., Little S., & Mendelson I. (2002) In-vitro characterisation of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody. J Med.Microbiol. 51, 821-831.

140 A.A. B.. 1.6 1.2 ) 1.4 ) Unvaccinated ) ) Unvaccinated 1 m 1.2 BC vaccinated m BAS vaccinated 0.8 BC vaccinated 405 n 405 n 405 nm 405 nm 1 0.8 0.6 ance ( ance ( ance ( ance ( b b b b 0.6 0.4 sor sor sor sor 0.4 b b b b A A 0.2 0.2 0 0 10 100 1000 10000 10 100 1000 10000 Dilution Dilution

C.C. DD.. 1.2 140 p < 0.0001 )

1 Unvaccinated s 120 p < 0.005 e r PA Vaccinated o 100

p 100 05 nm 0.8 4

) s 80 e ( 0.6 (+

C 60 banc 0.4 IT or F 40 s p < 0.05 % b

A 0.2 20 0 0 10 100 1000 10000 Unvaccinated PA BC BAS Dilution 10% serum

Figure 4.1 - Mouse anti-spore IgG response to vaccination. In all the following assays, IgG spore interactions were probed for using an anti-mouse IgG monoclonal antibody conjugated to alkaline phosphatase. A. Serum from unvaccinated C57/B6 mice or from mice vaccinated with 1 x 108 fixed BC spores were incubated in ELISA plates coated with BC. Serum from mice vaccinated with BC show higher absorbance values than the unvaccinated mouse serum. Data are shown from three independently run assays. B. Serum from unvaccinated C57/B6 mice or from mice vaccinated with 1 x 108 spores of BAS or BC were incubated in ELISA plates coated with BAS. Serum from mice vaccinated with BC and BAS show higher absorbance values than the unvaccinated mouse serum. Data are shown from three independently run assays. C. Serum from unvaccinated C57/BC mice or from mice vaccinated with 0.75 μg of recombinant PA was incubated in ELISA plates coated with BAS. The serum from mice vaccinated with PA show higher absorbance values than the unvaccinated mouse serum. D. Anti-spore antibody detection as shown by FACS analysis. Spores were incubated in serum for 30 minutes followed by a FITC (Alexa Fluor 488) conjugated anti-mouse secondary antibody. Shown are the mean percentage of fluorescent spores ± SD from three independent experiments (significance was determined with unpaired one-tailed Student t tests).

141 Unvaccinated PA 10.7% 25.2%

BC BAS 69.4% 71.5%

Figure 4.2 - Detection of complement protein C3b on B. anthracis Sterne spores by FACS analysis. B. anthracis Sterne spores were incubated in 10% serum from mice without vaccination or vaccinated with PA, BC spores or BAS spores followed by detection with a rat anti-mouse C3b monoclonal antibody. Detection was performed by anti-rat IgG conjugated to FITC. Representative histograms are depicted showing fluorescent spores. The open histograms represent spores incubated in mouse serum and the anti-rat IgG FITC control.

142 A.A. 100 90 p < 0.01 80 p < 0.01 70 s e r

o 60 p ) s

(+ 50 Untreated Serum C

T Heat-treated Serum I 40 F % 30 20 10 0 Unvaccinated PA BC BAS Serum Type

B.B. 90 Unvaccinated 80 PA 70 BC s

e 60 BAS por

s 50 ) + ( 40 TC

FI 30 % 20 10 0 5 2.5 1.25 0.625 % Serum

Figure 4.3 – Detection of complement protein C3b on B. anthracis Sterne spores by FACS analysis (Part 2). A. Percentage of spores with detectable C3b. Spores were incubated in 10% serum from mice without vaccination or serum from mice vaccinated with PA, BC spores or BAS spores. This incubation was followed by detection with a rat anti-mouse C3b monoclonal antibody. Detection was performed by anti-rat IgG conjugated to FITC. Figure represents the average of three assays per serum type. (p-values were calculated using a one tailed, unpaired Student t test). B. C3b deposition on BAS spores is a serum concentration dependent phenomenon. Figure shows percentage of FITC positive spores after incubation in decreasing dilutions of non-HI serum.

143 90 p<0.01 80 p<0.05

70

s 60 M P 50 100 M

/ 40 U F

C 30

20

10

0 SFM Unvaccinated PA BAS BC

Figure 4.4 – Colony forming units of B. anthracis Sterne associated with infected mouse peritoneal macrophages (MPMs). MPM monolayers in six well culture plates were infected for 15 minutes with BAS spores at an MOI of 10 with no serum (control) or in the presence of normal or HI unvaccinated, PA, BAS or BC mouse serum. After incubation, the macrophages were washed and lysed. Lysates were serially diluted, plated on nutrient agar and counted following an overnight incubation. The results are the means of three independently performed assays. p-values were calculated using a one tailed, paired Student t test.

144 250 p<0.01 p<0.05 200 † M P 150 † 50 M es/

r 100 o * p S 50 *

0 no SF Un P B B A AS C s M va po cc r e in s a t e d

Figure 4.5 - Phagocytic index of B. anthracis Sterne spores in mouse peritoneal macrophages. MPM monolayers on glass chamber slides were infected for 15 minutes with BAS spores at an MOI of 10 with no serum (control) or in the presence of normal or HI unvaccinated, PA, BAS or BC mouse serum. After incubation, the macrophages were washed, fixed and stained. The phagocytic index (spores/50 MPM) was assessed by light microscopy. The results are the means of 5 assays. * and † indicate a significant difference over the HI counterpart (p<0.001 and 0.05, respectively). p-values were calculated using a one tailed, paired Student t test.

145 A. 100 p < 0.001 90 s

i 80 os t 70 y c 60 go 50 opha

hr 40 t y r 30 E

% 20 10 0 No anti -RBC IgG BSA Anti ­-BSA

B. 180 p < 0.001 160

140 M P 120 0 M 5 100 es/ 80 cyt o r 60 yth r

E 40

20

0 No anti-RBC IgG BSA Anti ­-BSA

Figure 4.6 - Erythrophagocytosis assay in mouse peritoneal macrophages which demonstrates FcγR sequestration. MPMs were adhered to glass chamber slides coated with BSA ± rabbit polyclonal anti-BSA antibody for 30 minutes. The cells were incubated with sheep erythrocytes coated with anti-sheep RBC antibody at an MOI of 10:1 for 30 minutes. As a control the macrophages were also incubated with non-opsonized erythrocytes. Following incubation, the cells were washed, fixed and stained and the percent erythrophagocytosis (A.) and erythrocytes per 50 MPMs (B.) were assessed by light microscopy. The means ± SD from three independent assays are shown. p-values were calculated using a two tailed, unpaired Student t test.

146 300 p < 0.05 p < 0.001 250

M 200 P

50 M 150 es/ r o

p 100 S

50

0 BAS +1 +2 +3 +4 5- 6- 7- 8­- BC - - - - + + + + Δ56ºC - + - + - + - + FcγR - - + + - - + + sequestered

Figure 4.7 - Phagocytic index of B. anthracis Sterne spores in mouse peritoneal macrophages following Fc sequestration. MPMs were adhered to glass chamber slides coated with BSA ± rabbit polyclonal anti-BSA antibody for 30 minutes. MPM monolayers on glass chamber slides were infected with BAS spores at an MOI of 10 with no serum (control) or in the presence of normal or HI serum from BC or BAS vaccinated mice. Note the significant difference between the phagocytic index during normal condition or FcγR sequestration for each HI serum type (p-values were calculated using a one tailed, paired Student t test).

147 CHAPTER 5

SYNOPSIS AND FUTURE DIRECTIONS

5.1 Introduction

Our knowledge of Bacillus anthracis microbiology and anthrax pathogenesis has increased tremendously in the last decade, particularly in terms of host interactions with the organism and its exotoxins. Despite these advances, many gaps remain in our knowledge, particularly concerning the early interactions of the organism with the innate immune system.

5.2 Further characterization of expression of anthrax toxin receptors and associated proteins in mononuclear phagocytes

In Chapter 2, we examined the expression of functional anthrax toxin receptors (ATRs) in macrophage-like cell lines and primary macrophages. Our data indicates that these cells express functional ATRs as shown by increases of intracellular cyclic AMP induced by exposure to protective antigen and edema factor. In addition, we also examined the mRNA expression of the two known

148 anthrax toxin receptors, tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2) and demonstrated that primary macrophages, most notably human monocyte derived macrophages, preferentially express

CMG2 over TEM8. These data have become particularly significant considering that the process of cellular intoxication can vary depending on which ATR is engaged by toxin.

Since protein detection is ultimately the indicator of significant receptor expression, demonstration of the fully formed protein would be vital. Due to the lack of efficacious commercially available antibodies at the time in which the experiments of Chapter 2 were performed, confirmation of TEM8 or CMG2 protein receptor expression was difficult. However, at the current date, several monoclonal and polyclonal TEM8 antibodies are available as well as a few polyclonal CMG2 antibodies. Indeed several laboratories have demonstrated protein expression of TEM8 in epithelium and endothelium1,2. To our knowledge, the distribution of CMG2 has been limited to mRNA expression as shown by northern blot and RT-PCR3,4. It has become clear that each of the ATRs interact with anthrax toxins uniquely5, thus influencing the outcome of intoxication.

Therefore, confirming the protein expression of CMG2 on mononuclear phagocytes would lend ex vivo significance to published information regarding

CMG2 and TEM8 toxin interactions.

The association of low density lipoprotein receptor 6 (LRP6) with anthrax toxin receptors was a significant discovery involving the mechanism of

149 intoxication6. In this study, it was shown that inhibition of LRP6 causes decreased susceptibility of RAW 264.7 cells, a mouse macrophage-like cell line, to challenge with lethal toxin. The many differences between cell lines and primary cells were one of the reasons why we compared ATR expression in

Chapter 1. Determining the expression level of LRP6 in primary murine and human macrophages would be a significant contribution as a future direction.

5.3 Characterization of anti-spore antibodies found in naïve human serum

Chapter 3 examined the role of soluble factors in human serum such as antibody and complement in modulating the interaction between the spore and the macrophage. We demonstrated that antibody present in naïve human serum interacts with B. anthracis spores and fixes complement to the spore’s surface.

Furthermore, we determined the significance of this interaction by infecting human monocyte derived macrophages in the presence of human serum, showing that complement deposition greatly enhances the phagocytic events by the macrophage. In addition, we demonstrated that the complement fixation is likely taking place through the classical pathway, indicating the significance of these antibodies. We suspect these naturally occurring antibodies are cross reactive due to exposure of humans to other species of Bacillus in their environment. Importantly, our data implicate complement-spore-macrophage interactions as potentially significant in the pathogenesis of inhalation anthrax.

150 In Chapter 3, our data indicate that both pooled human AB serum and serum from unvaccinated, unexposed individuals contains antibody which interacts with B. anthracis spores, presumably in an epitope-dependant manner.

The epitope(s) to which this antibody is specific is unknown. The exosporial protein BclA has been identified as an immunodominant protein in the literature7,8; however, we cannot be certain that this is the peptide responsible for the generation of what we presume is a polyclonal antibody. To determine which proteins present in the exosporium react this antibody, extracted exosporial proteins could be separated by two dimensional gel electrophoresis followed by transfer to a nitrocellulose membrane. The membrane could be probed by incubation in diluted human serum previously shown to contain antibody and developed according to standard methods. The corresponding protein spots could be isolated, sequenced and identified by matrix assisted laser desorption ionisation/time of flight (MALDI-TOF) mass spectophotometry. Since many of the proteins of the exosporium have been previously identified8,9, a bioinformatics approach to determine the identity of the major epitopes could be employed.

In addition, the differences in opsonization and phagocytic enhancement observed between the human serum antibody and antibodies present in serum from vaccinated micealso indicate that further characterization is necessary.

Human MDMs incubated with spores in heat inactivated human serum showed relatively low phagocytic activity which was comparable to the activity observed in serum-free media, while mouse MPMs incubated with spores in heat

151 inactivated BAS or BC serum showed significantely higher phagocytic activity.

These data viewed as a whole indicated that the generated mouse antibody mediates FcγR phagocytosis while the antibody in human serum does not. This disparity may be due to the differences in the macrophages used or differences in the antibody itself. Performing infection studies using human MDMs or alveolar macrophages infected in the presence of mouse anti-serum or vice versa would help rule out the macrophage as the source of the discrepancy if the phagocytic indices remain unchanged. In addition, isotype identification of the antibodies in both human and mouse serum may provide valuable insight into the before mentioned disparity.

5.4 Comparison of survival of B. anthracis spores in different phagocytic pathways and further characterization of spore opsonins.

In Chapter 4, we build upon the findings in the previous chapter by generating anti-spore antibodies in mice. Specifically, we asked if antibodies generated through inactivated spore vaccination fix complement and enhance phagocytosis in a similar manner as the antibody found in human serum. In addition, we demonstrated the cross reactivity between antibodies generated against spores of Bacillus cereus with spores of Bacillus anthracis in the effort to determine if this cross reactivity may play a role in generating the anti-spore antibodies we observed in human serum.

152 A major goal of the experiments performed in Chapter 4 was to compare the amount of phagocytosis taking place in varying media conditions in a relatively short period of time (i.e. 30-60 minutes). These models were useful in demonstrating the short term influence of complement proteins and spore specific antibody in macrophage infection. It is has been shown that the intracellular trafficking and downstream events such as intracellular signaling, anti-microbial activities such as oxidative burst and cytokine modulation can be different between the phagocytic pathways mediated by FcγR vs complement receptors10-12. Such differences may have a direct impact on survival of the organism following phagocytosis which, in the event of high intracellular spore burden, may influence subsequent escape and the resulting bacteremia. In order to study survival in macrophage, multiple strategies can be utilized. One strategy is to measure the oxidative burst generation in macrophages following infection.

We would hypothesize that mouse peritoneal macrophages infected in the presence of complement free antiserum would show greater oxidative burst activity than FcγR sequestered macrophages infected in the presence of whole antiserum. A second potential experiment would involve allowing infections to proceed and culturing macrophage lysate at multiple time points after 15 minutes to 2 hours to measure intracellular bacteria. We would hypothesize that spores phagocytosed predominantely via FcγR would show a decreased CFU count compared to time zero counts, while spores phagocytosed via complement receptors would have equivalent or high CFU counts compared to time zero,

153 reflecting decreased and increased intracellular survival via FcγR and complement mediated phagoctosis, respectively. In addition, differences in the rate of intracellular germination via each trafficking pathway could be studied.

However, the usage of heat inactivated serum to investigate any potential differences in germination by these methods is problematic due to the differences in spore germination in heat inactivated versus non-heat inactivated serum (data not shown). Therefore, depletion of complement proteins by a method which leaves germinants intact would be preferable for these types of experiments.

In both Chapters 3 and 4, our data suggest an important role of complement receptors in the phagocytosis of B. anthracis spores by macrophages. However, the process of complement opsonization and phagocytosis is complex and significant functional differences, particularly in terms of signal transduction13-15, are present between the components of the complement receptor family. Our studies did not differentiate between C3b and its surface bound degradation products such as iC3b or C3dg. These degradation products can define which complement receptor is primarily engaged16-21. Further studies would be required to determine the predominant

C3b component bound to the spore’s surface. Such studies would include building upon our complement ELISAs and flow cytometry assays by using monoclonal antibodies specific to each degradation component. Extension of the time incubated in serum would to necessary to evaluate the extent of C3b degradation, however, for these experiments germination of the organism

154 becomes a confounding factor. To control for this, either a germination deficient strain or fixed spores would need to be substituted into the assays. In addition, complement receptor deficient cell lines could be infected under identical conditions to determine if a particular complement receptor dominates the high phagocytic index in non-heated inactivated serum observed in Chapter 3.

5.5 Summary

The data in this thesis focused on the macrophage, a key component of the innate immune system, and its interactions with Bacillus anthracis. We have presented many novel findings in terms of anthrax toxin receptors and complement interactions. In particular, the data of chapters 3 and 4 are the first describing a non-epitope specific opsonization of B. anthracis spores. Overall, this thesis is a significant contribution to the literature concerning anthrax pathogenesis.

5.6 References

1. Bonuccelli G., Sotgia F., Frank P.G., Williams T.M., de Almeida C.J., Tanowitz H.B., Scherer P.E., Hotchkiss K.A., Terman B.I., Rollman B., Alileche A., Brojatsch J., & Lisanti M.P. (2005) ATR/TEM8 is highly expressed in epithelial cells lining Bacillus anthracis' three sites of entry: implications for the pathogenesis of anthrax infection. Am J Physiol Cell Physiol 288, C1402-C1410.

2. Hotchkiss K.A., Basile C.M., Spring S.C., Bonuccelli G., Lisanti M.P., & Terman B.I. (2005) TEM8 expression stimulates endothelial cell adhesion and migration by regulating cell-matrix interactions on collagen. Exp.Cell Res. 305, 133-144.

3. Dowling O., Difeo A., Ramirez M.C., Tukel T., Narla G., Bonafe L., Kayserili H., Yuksel-Apak M., Paller A.S., Norton K., Teebi A.S., Grum-Tokars V., Martin G.S., 155 Davis G.E., Glucksman M.J., & Martignetti J.A. (2003) Mutations in capillary morphogenesis gene-2 result in the allelic disorders juvenile hyaline fibromatosis and infantile systemic hyalinosis. Am J Hum.Genet. 73, 957-966.

4. Banks D.J., Barnajian M., Maldonado-Arocho F.J., Sanchez A.M., & Bradley K.A. (2005) Anthrax toxin receptor 2 mediates Bacillus anthracis killing of macrophages following spore challenge. Cell Microbiol. 7, 1173-1185.

5. Rainey G.J., Wigelsworth D.J., Ryan P.L., Scobie H.M., Collier R.J., & Young J.A. (2005) Receptor-specific requirements for anthrax toxin delivery into cells. Proc.Natl.Acad.Sci U.S.A 102, 13278-13283.

6. Wei W., Lu Q., Chaudry G.J., Leppla S.H., & Cohen S.N. (2006) The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin. Cell 124, 1141-1154.

7. Swiecki M.K., Lisanby M.W., Shu F., Turnbough C.L., Jr., & Kearney J.F. (2006) Monoclonal antibodies for Bacillus anthracis spore detection and functional analyses of spore germination and outgrowth. J Immunol. 176, 6076­ 6084.

8. Steichen C., Chen P., Kearney J.F., & Turnbough C.L., Jr. (2003) Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium. J Bacteriol. 185, 1903-1910.

9. Stump M.J., Black G., Fox A., Fox K.F., Turick C.E., & Matthews M. (2005) Identification of marker proteins for Bacillus anthracis using MALDI-TOF MS and ion trap MS/MS after direct extraction or electrophoretic separation. J Sep.Sci 28, 1642-1647.

10. From the Centers for Disease Control and Prevention. (2001) Considerations for distinguishing influenza-like illness from inhalational anthrax. JAMA 286, 2537-2539.

11. Rus H., Cudrici C., & Niculescu F. (2005) The role of the complement system in innate immunity. Immunol.Res. 33, 103-112.

12. Aderem A. & Underhill D.M. (1999) Mechanisms of phagocytosis in macrophages. Annu.Rev.Immunol. 17, 593-623.

13. Shi Y., Tohyama Y., Kadono T., He J., Shahjahan Miah S.M., Hazama R., Tanaka C., Tohyama K., & Yamamura H. (2006) Protein-tyrosine kinase Syk is required for pathogen engulfment in complement-mediated phagocytosis. Blood 107, 4554-4562.

156 14. Tohyama Y. (2006) [The molecular mechanisms of complement-mediated phagocytosis--the novel function of tyrosine kinase, Syk]. Seikagaku 78, 878­ 882.

15. Tohyama Y. & Yamamura H. (2006) Complement-mediated phagocytosis-­ the role of Syk. IUBMB.Life 58, 304-308.

16. Agramonte-Hevia J., Gonzalez-Arenas A., Barrera D., & Velasco-Velazquez M. (2002) Gram-negative bacteria and phagocytic cell interaction mediated by complement receptor 3. FEMS Immunol.Med Microbiol 34, 255-266.

17. Velasco-Velazquez M.A., Barrera D., Gonzalez-Arenas A., Rosales C., & Agramonte-Hevia J. (2003) Macrophage--Mycobacterium tuberculosis interactions: role of complement receptor 3. Microb.Pathog. 35, 125-131.

18. Carroll M.C. (1998) The role of complement and complement receptors in induction and regulation of immunity. Annu.Rev.Immunol. 16, 545-568.

19. Alsenz J., Becherer J.D., Nilsson B., & Lambris J.D. (1990) Structural and functional analysis of C3 using monoclonal antibodies. Curr.Top.Microbiol Immunol. 153, 235-248.

20. Lambris J.D. & Tsokos G.C. (1986) The biology and pathophysiology of complement receptors. Anticancer Res. 6, 515-523.

21. Lambris J.D. (1988) The multifunctional role of C3, the third component of complement. Immunol.Today 9, 387-393.

157 BIBLIOGRAPHY

Abrami L., Liu S., Cosson P., Leppla S.H., & van der Goot F.G. (2003) Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin­ dependent process. J Cell Biol. 160, 321-328.

Abrami L., Reig N., & van der Goot F.G. (2005) Anthrax toxin: the long and winding road that leads to the kill. Trends Microbiol 13, 72-78.

Ackerman S.K., Friend P.S., Hoidal J.R., & Douglas S.D. (1978) Production of C2 by human alveolar macrophages. Immunology 35, 369-372.

Aderem A. & Underhill D.M. (1999) Mechanisms of phagocytosis in macrophages. Annu.Rev.Immunol. 17, 593-623.

Agramonte-Hevia J., Gonzalez-Arenas A., Barrera D., & Velasco-Velazquez M. (2002) Gram-negative bacteria and phagocytic cell interaction mediated by complement receptor 3. FEMS Immunol.Med Microbiol 34, 255-266.

Albrink W.S., Brooks S.M., Biron R.E., & Kopel M. (1960) Human inhalation anthrax. A report of three fatal cases. Am.J Pathol. 36:457-71., 457-471.

Albrink W.S. & Goodlow R.J. (1959) Experimental inhalation anthrax in the chimpanzee. Am J Pathol. 35:1055-65., 1055-1065.

Aloni-Grinstein R., Gat O., Altboum Z., Velan B., Cohen S., & Shafferman A. (2005) Oral spore vaccine based on live attenuated nontoxinogenic Bacillus anthracis expressing recombinant mutant protective antigen. Infect.Immun. 73, 4043-4053.

Alouf J.E. (2000) Cholesterol-binding cytolytic protein toxins. Int.J Med Microbiol 290, 351-356.

Alouf J.E. (2001) Pore-forming bacterial protein toxins: an overview. Curr.Top.Microbiol Immunol. 257, 1-14.

Alper T. (1979) Cellular Radiobiology., Cambridge University Press, New York.

158 Alsenz J., Becherer J.D., Nilsson B., & Lambris J.D. (1990) Structural and functional analysis of C3 using monoclonal antibodies. Curr.Top.Microbiol Immunol. 153, 235-248.

Auerbach S. & Wright G.G. (1955) Studies on immunity in anthrax. VI. Immunizing activity of protective antigen against various strains of Bacillus anthracis. J Immunol. 75, 129-133.

Balagopal A., MacFarlane A.S., Mohapatra N., Soni S., Gunn J.S., & Schlesinger L.S. (2006) Characterization of the receptor-ligand pathways important for entry and survival of Francisella tularensis in human macrophages. Infect.Immun. 74, 5114-5125.

Baldari C.T., Tonello F., Paccani S.R., & Montecucco C. (2006) Anthrax toxins: A paradigm of bacterial immune suppression. Trends Immunol. 27, 434-440.

Bales M.E., Dannenberg A.L., Brachman P.S., Kaufmann A.F., Klatsky P.C., & Ashford D.A. (2002) Epidemiologic response to anthrax outbreaks: field investigations, 1950-2001. Emerg.Infect.Dis. 8, 1163-1174.

Banchereau J., Bazan F., Blanchard D., Briere F., Galizzi J.P., van Kooten C., Liu Y.J., Rousset F., & Saeland S. (1994) The CD40 antigen and its ligand. Annu.Rev.Immunol. 12, 881-922.

Banks D.J., Barnajian M., Maldonado-Arocho F.J., Sanchez A.M., & Bradley K.A. (2005) Anthrax toxin receptor 2 mediates Bacillus anthracis killing of macrophages following spore challenge. Cell Microbiol. 7, 1173-1185.

Beaman T.C., Pankratz H.S., & Gerhardt P. (1972) Ultrastructure of the exosporium and underlying inclusions in spores of Bacillus megaterium strains. J Bacteriol. 109, 1198-1209.

Beauregard K.E., Collier R.J., & Swanson J.A. (2000) Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization. Cell Microbiol. 2, 251-258.

Beauregard K.E., Wimer-Mackin S., Collier R.J., & Lencer W.I. (1999) Anthrax toxin entry into polarized epithelial cells. Infect.Immun. 67, 3026-3030.

Bell S.E., Mavila A., Salazar R., Bayless K.J., Kanagala S., Maxwell S.A., & Davis G.E. (2001) Differential gene expression during capillary morphogenesis in 3D collagen matrices: regulated expression of genes involved in basement membrane matrix assembly, cell cycle progression, cellular differentiation and G- protein signaling. Journal of Cell Science 114, 2755-2773.

159 Belton F.C. & Strange R.E. (1954) Studies on a protective antigen produced in vitro from Bacillus anthracis: medium and methods of production. Br.J Exp.Pathol. 35, 144-152.

Berthold I., Pombo M.L., Wagner L., & Arciniega J.L. (2005) Immunogenicity in mice of anthrax recombinant protective antigen in the presence of aluminum adjuvants. Vaccine 23, 1993-1999.

Binder C.J. & Silverman G.J. (2005) Natural antibodies and the autoimmunity of atherosclerosis. Springer Semin.Immunopathol. 26, 385-404.

Bonuccelli G., Sotgia F., Frank P.G., Williams T.M., de Almeida C.J., Tanowitz H.B., Scherer P.E., Hotchkiss K.A., Terman B.I., Rollman B., Alileche A., Brojatsch J., & Lisanti M.P. (2005) Anthrax Toxin Receptor (ATR/TEM8) is Highly Expressed in Epithelial Cells Lining the Toxin's Three Sites of Entry (Lung, Skin, and Intestine). Am J Physiol Cell Physiol.

Boyaka P.N., Tafaro A., Fischer R., Leppla S.H., Fujihashi K., & McGhee J.R. (2003) Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen. J Immunol. 170, 5636-5643.

Brachman P.S. (2002) Bioterrorism: an update with a focus on anthrax. Am.J Epidemiol. 155, 981-987.

Bradley K.A., Mogridge J., Mourez M., Collier R.J., & Young J.A. (2001) Identification of the cellular receptor for anthrax toxin. Nature 414, 225-229.

Bradley K.A. & Young J.A. (2003) Anthrax toxin receptor proteins. Biochem.Pharmacol. 65, 309-314.

Brook I., Elliott T.B., Harding R.A., Bouhaouala S.S., Peacock S.J., Ledney G.D., & Knudson G.B. (2001) Susceptibility of irradiated mice to Bacillus anthracis sterne by the intratracheal route of infection. J Med.Microbiol. 50, 702-711.

Brook I., Elliott T.B., Ledney G.D., Shoemaker M.O., & Knudson G.B. (2004) Management of postirradiation infection: lessons learned from animal models. Mil.Med 169, 194-197.

Brook I., Germana A., Giraldo D.E., Camp-Hyde T.D., Bolduc D.L., Foriska M.A., Elliott T.B., Thakar J.H., Shoemaker M.O., Jackson W.E., & Ledney G.D. (2005) Clindamycin and quinolone therapy for Bacillus anthracis Sterne infection in 60Co-gamma-photon-irradiated and sham-irradiated mice. J Antimicrob.Chemother. 56, 1074-1080.

160 Brook I., Giraldo D.E., Germana A., Nicolau D.P., Jackson W.E., Elliott T.B., Thakar J.H., Shoemaker M.O., & Ledney G.D. (2005) Comparison of clarithromycin and ciprofloxacin therapy for Bacillus anthracis Sterne infection in mice with or without (60)Co gamma-photon irradiation. J Med Microbiol 54, 1157­ 1162.

Brook I. & Ledney G.D. (1992) Quinolone therapy in the management of infection after irradiation. Crit Rev.Microbiol 18, 235-246.

Brook I. & Ledney G.D. (1992) Short and long courses of ofloxacin therapy of Klebsiella pneumoniae sepsis following irradiation. Radiat.Res. 130, 61-64.

Brook I. & Ledney G.D. (1994) Effect of antimicrobial therapy on the gastrointestinal bacterial flora, infection and mortality in mice exposed to different doses of irradiation. J Antimicrob.Chemother. 33, 63-72.

Brook I. & Ledney G.D. (1994) Quinolone therapy in the prevention of endogenous and exogenous infection after irradiation. J Antimicrob.Chemother. 33, 777-784.

Brook I., Tom S.P., & Ledney G.D. (1993) Development of infection with Streptococcus bovis and Aspergillus sp. in irradiated mice after glycopeptide therapy. J Antimicrob.Chemother. 32, 705-713.

Brossier F., Levy M., & Mock M. (2002) Anthrax spores make an essential contribution to vaccine efficacy. Infect.Immun. 70, 661-664.

Candela T. & Fouet A. (2005) Bacillus anthracis CapD, belonging to the gamma­ glutamyltranspeptidase family, is required for the covalent anchoring of capsule to peptidoglycan. Mol.Microbiol 57, 717-726.

Candela T., Mock M., & Fouet A. (2005) CapE, a 47-amino-acid peptide, is necessary for Bacillus anthracis polyglutamate capsule synthesis. J Bacteriol. 187, 7765-7772.

Carroll M.C. (1998) The role of complement and complement receptors in induction and regulation of immunity. Annu.Rev.Immunol. 16, 545-568.

Carson-Walter E.B., Watkins D.N., Nanda A., Vogelstein B., Kinzler K.W., & St Croix B. (2001) Cell surface tumor endothelial markers are conserved in mice and humans. Cancer Res. 61, 6649-6655.

Chensue S.W. (2003) Exposing a killer: pathologists angle for anthrax. Am J Pathol. 163, 1699-1702.

161 Chua J., Vergne I., Master S., & Deretic V. (2004) A tale of two lipids: Mycobacterium tuberculosis phagosome maturation arrest. Curr.Opin.Microbiol 7, 71-77.

Cleret A., Quesnel-Hellmann A., Mathieu J., Vidal D., & Tournier J.N. (2006) Resident CD11c+ lung cells are impaired by anthrax toxins after spore infection. J Infect.Dis. 194, 86-94.

Cocklin S., Jost M., Robertson N.M., Weeks S.D., Weber H.W., Young E., Seal S., Zhang C., Mosser E., Loll P.J., Saunders A.J., Rest R.F., & Chaiken I.M. (2006) Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis. J Mol.Recognit. 19, 354-362.

Cole F.S., Auerbach H.S., Goldberger G., & Colten H.R. (1985) Tissue-specific pretranslational regulation of complement production in human mononuclear phagocytes. J Immunol. 134, 2610-2616.

Cominsky N.C. (1959) Histological changes in mice injected with Bacillus anthracis. Tex.Rep.Biol.Med 17, 85-93.

Coonrod J.D. & Yoneda K. (1981) Complement and opsonins in alveolar secretions and serum of rats with pneumonia due to Streptococcus pneumoniae. Rev.Infect.Dis. 3, 310-322.

Cordoba-Rodriguez R., Fang H., Lankford C.S., & Frucht D.M. (2004) Anthrax lethal toxin rapidly activates caspase-1/ICE and induces extracellular release of interleukin (IL)-1beta and IL-18. J Biol.Chem. 279, 20563-20566.

Cote C.K., Rea K.M., Norris S.L., Van Rooijen N., & Welkos S.L. (2004) The use of a model of in vivo macrophage depletion to study the role of macrophages during infection with Bacillus anthracis spores. Microb.Pathog. 37, 169-175.

Cote C.K., Rossi C.A., Kang A.S., Morrow P.R., Lee J.S., & Welkos S.L. (2005) The detection of protective antigen (PA) associated with spores of Bacillus anthracis and the effects of anti-PA antibodies on spore germination and macrophage interactions. Microb.Pathog. 38, 209-225.

Cote C.K., Van Rooijen N., & Welkos S.L. (2006) Roles of macrophages and neutrophils in the early host response to Bacillus anthracis spores in a mouse model of infection. Infect.Immun. 74, 469-480.

Crawford M.A., Aylott C.V., Bourdeau R.W., & Bokoch G.M. (2006) Bacillus anthracis toxins inhibit human neutrophil NADPH oxidase activity. J Immunol. 176, 7557-7565.

162 Cui Z. & Sloat B.R. (2006) Topical immunization onto mouse skin using a microemulsion incorporated with an anthrax protective antigen protein-encoding plasmid. Int.J Pharm. 317, 187-191. de Villiers W.J. & Smart E.J. (1999) Macrophage scavenger receptors and foam cell formation. J Leukoc.Biol. 66, 740-746.

Delvecchio V.G., Connolly J.P., Alefantis T.G., Walz A., Quan M.A., Patra G., Ashton J.M., Whittington J.T., Chafin R.D., Liang X., Grewal P., Khan A.S., & Mujer C.V. (2006) Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. Appl.Environ.Microbiol 72, 6355-6363.

Dirckx JH (1981) Virgil on anthrax. Am J Dermatopathol 3, 191-195.

Dixon T.C., Fadl A.A., Koehler T.M., Swanson J.A., & Hanna P.C. (2000) Early Bacillus anthracis-macrophage interactions: intracellular survival survival and escape. Cell Microbiol. 2, 453-463.

Dixon T.C., Meselson M., Guillemin J., & Hanna P.C. (1999) Anthrax. N.Engl.J Med. 341, 815-826.

Dowling O., Difeo A., Ramirez M.C., Tukel T., Narla G., Bonafe L., Kayserili H., Yuksel-Apak M., Paller A.S., Norton K., Teebi A.S., Grum-Tokars V., Martin G.S., Davis G.E., Glucksman M.J., & Martignetti J.A. (2003) Mutations in capillary morphogenesis gene-2 result in the allelic disorders juvenile hyaline fibromatosis and infantile systemic hyalinosis. Am J Hum.Genet. 73, 957-966.

Dragon D.C., Elkin B.T., Nishi J.S., & Ellsworth T.R. (1999) A review of anthrax in Canada and implications for research on the disease in northern bison. J Appl.Microbiol. 87, 208-213.

Dragon D.C. & Rennie R.P. (1995) The ecology of anthrax spores: tough but not invincible. Can.Vet.J 36, 295-301.

Drum C.L., Yan S.Z., Bard J., Shen Y.Q., Lu D., Soelaiman S., Grabarek Z., Bohm A., & Tang W.J. (2002) Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin. Nature 415, 396-402.

Drum C.L., Yan S.Z., Sarac R., Mabuchi Y., Beckingham K., Bohm A., Grabarek Z., & Tang W.J. (2000) An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis. J Biol.Chem. 275, 36334-36340.

163 Drysdale M., Heninger S., Hutt J., Chen Y., Lyons C.R., & Koehler T.M. (2005) Capsule synthesis by Bacillus anthracis is required for dissemination in murine inhalation anthrax. EMBO J 24, 221-227.

Drysdale M., Olson G., Koehler T.M., Lipscomb M.F., & Lyons C.R. (2007) Murine innate immune response to virulent toxigenic and non-toxigenic Bacillus anthracis strains. Infect.Immun.

Dubail I., Autret N., Beretti J.L., Kayal S., Berche P., & Charbit A. (2001) Functional assembly of two membrane-binding domains in listeriolysin O, the cytolysin of Listeria monocytogenes. Microbiology 147, 2679-2688.

Duc l.H., Hong H.A., Atkins H.S., Flick-Smith H.C., Durrani Z., Rijpkema S., Titball R.W., & Cutting S.M. (2007) Immunization against anthrax using Bacillus subtilis spores expressing the anthrax protective antigen. Vaccine 25, 346-355.

Duesbery N.S., Resau J., Webb C.P., Koochekpour S., Koo H.M., Leppla S.H., & Vande Woude G.F. (2001) Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways. Proc.Natl.Acad.Sci.U.S.A 98, 4089-4094.

Duesbery N.S. & Vande Woude G.F. (1999) Anthrax lethal factor causes proteolytic inactivation of mitogen-activated protein kinase kinase. J Appl.Microbiol. 87, 289-293.

Duesbery N.S., Webb C.P., Leppla S.H., Gordon V.M., Klimpel K.R., Copeland T.D., Ahn N.G., Oskarsson M.K., Fukasawa K., Paull K.D., & Vande Woude G.F. (1998) Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 280, 734-737.

Duong S., Chiaraviglio L., & Kirby J.E. (2006) Histopathology in a murine model of anthrax. Int.J Exp.Pathol. 87, 131-137.

Duverger A., Jackson R.J., van Ginkel F.W., Fischer R., Tafaro A., Leppla S.H., Fujihashi K., Kiyono H., McGhee J.R., & Boyaka P.N. (2006) Bacillus anthracis edema toxin acts as an adjuvant for mucosal immune responses to nasally administered vaccine antigens. J Immunol. 176, 1776-1783.

Elliott T.B., Brook I., Harding R.A., Bouhaouala S.S., Shoemaker M.O., & Knudson G.B. (2002) Antimicrobial therapy for Bacillus anthracis-induced polymicrobial infection in (60)Co gamma-irradiated mice. Antimicrob.Agents Chemother. 46, 3463-3471.

Enkhtuya J., Kawamoto K., Kobayashi Y., Uchida I., Rana N., & Makino S. (2006) Significant passive protective effect against anthrax by antibody to

164 Bacillus anthracis inactivated spores that lack two virulence plasmids. Microbiology 152, 3103-3110.

Ernst J.D. (1998) Macrophage receptors for Mycobacterium tuberculosis. Infect.Immun. 66, 1277-1281.

Erwin J.L., DaSilva L.M., Bavari S., Little S.F., Friedlander A.M., & Chanh T.C. (2001) Macrophage-derived cell lines do not express proinflammatory cytokines after exposure to Bacillus anthracis lethal toxin. Infect.Immun. 69, 1175-1177.

Ezzell J.W. & Welkos S.L. (1999) The capsule of Bacillus anthracis, a review. J Appl.Microbiol. 87, 250.

Fellows P.F., Linscott M.K., Ivins B.E., Pitt M.L., Rossi C.A., Gibbs P.H., & Friedlander A.M. (2001) Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine 19, 3241-3247.

Ferguson J.S., Weis J.J., Martin J.L., & Schlesinger L.S. (2004) Complement protein C3 binding to Mycobacterium tuberculosis is initiated by the classical pathway in human bronchoalveolar lavage fluid. Infect.Immun. 72, 2564-2573.

Filice G.A., Yeager A.S., & Remington J.S. (1980) Diagnostic significance of immunoglobulin M antibodies to Toxoplasma gondii detected after separation of immunoglobulin M from immunoglobulin G antibodies. J Clin.Microbiol 12, 336­ 342.

Firoved A.M., Miller G.F., Moayeri M., Kakkar R., Shen Y., Wiggins J.F., McNally E.M., Tang W.J., & Leppla S.H. (2005) Bacillus anthracis edema toxin causes extensive tissue lesions and rapid lethality in mice. Am J Pathol. 167, 1309-1320.

Fisher N. & Hanna P. (2005) Characterization of Bacillus anthracis germinant receptors in vitro. J Bacteriol. 187, 8055-8062.

Fleming S.D. (2006) Natural antibodies, autoantibodies and complement activation in tissue injury. Autoimmunity 39, 379-386.

Fleming S.D. & Tsokos G.C. (2006) Complement, natural antibodies, autoantibodies and tissue injury. Autoimmun.Rev. 5, 89-92.

Flick-Smith H.C., Eyles J.E., Hebdon R., Waters E.L., Beedham R.J., Stagg T.J., Miller J., Alpar H.O., Baillie L.W., & Williamson E.D. (2002) Mucosal or parenteral administration of microsphere-associated Bacillus anthracis protective antigen protects against anthrax infection in mice. Infect.Immun. 70, 2022-2028.

165 Flick-Smith H.C., Walker N.J., Gibson P., Bullifent H., Hayward S., Miller J., Titball R.W., & Williamson E.D. (2002) A recombinant carboxy-terminal domain of the protective antigen of Bacillus anthracis protects mice against anthrax infection. Infect.Immun. 70, 1653-1656.

Flick-Smith H.C., Waters E.L., Walker N.J., Miller J., Stagg A.J., Green M., & Williamson E.D. (2005) Mouse model characterisation for anthrax vaccine development: comparison of one inbred and one outbred mouse strain. Microb.Pathog. 38, 33-40.

Fritz D.L., Jaax N.K., Lawrence W.B., Davis K.J., Pitt M.L., Ezzell J.W., & Friedlander A.M. (1995) Pathology of experimental inhalation anthrax in the rhesus monkey. Lab Invest 73, 691-702.

From the Centers for Disease Control and Prevention. (2001) Considerations for distinguishing influenza-like illness from inhalational anthrax. JAMA 286, 2537­ 2539.

Galloway D.R. & Baillie L. (2004) DNA vaccines against anthrax. Expert.Opin.Biol.Ther. 4, 1661-1667.

Garmory H.S., Titball R.W., Griffin K.F., Hahn U., Bohm R., & Beyer W. (2003) Salmonella enterica serovar typhimurium expressing a chromosomally integrated copy of the Bacillus anthracis protective antigen gene protects mice against an anthrax spore challenge. Infect.Immun. 71, 3831-3836.

Gaur R., Gupta P.K., Banerjea A.C., & Singh Y. (2002) Effect of nasal immunization with protective antigen of Bacillus anthracis on protective immune response against anthrax toxin. Vaccine 20, 2836-2839.

Gaynor C.D., McCormack F.X., Voelker D.R., McGowan S.E., & Schlesinger L.S. (1995) Pulmonary surfactant protein A mediates enhanced phagocytosis of Mycobacterium tuberculosis by a direct interaction with human macrophages. J Immunol. 155, 5343-5351.

Giclas P.C., King T.E., Baker S.L., Russo J., & Henson P.M. (1987) Complement activity in normal rabbit bronchoalveolar fluid. Description of an inhibitor of C3 activation. Am Rev.Respir.Dis. 135, 403-411.

Gleiser C.A., Berdjis C.C., Hartman H.A., & Gochenour W.S. (1963) Pathology of experimental respiratory anthrax in macaca mullata. Br.J Exp.Pathol. 44:416-26., 416-426.

Gleiser C.A., Gochenour W.S., Jr., & Ward M.K. (1968) Pulmonary lesions in dogs and pigs exposed to a cloud of anthrax spores. J Comp Pathol. 78, 445­ 448. 166 Goldenberg H.B., McCool T.L., & Weiser J.N. (2004) Cross-reactivity of human immunoglobulin G2 recognizing phosphorylcholine and evidence for protection against major bacterial pathogens of the human respiratory tract. J Infect.Dis. 190, 1254-1263.

Goodman J.W. & D.E.Nitecki (1966) Immunochemical studies on the poly- gamma-D-glutamyl capsule of Bacillus anthracis. I. Characterization of the polypeptide and of the specificity of its reaction with rabbit antisera. Biochemistry 5, 657-663.

Gordon S. (2002) Pattern recognition receptors: doubling up for the innate immune response. Cell 111, 927-930.

Gordon S. & Taylor P.R. (2005) Monocyte and macrophage heterogeneity. Nat.Rev.Immunol. 5, 953-964.

Gordon V.M., Leppla S.H., & Hewlett E.L. (1988) Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin. Infect.Immun. 56, 1066­ 1069.

Gorvel J.P. & Moreno E. (2002) Brucella intracellular life: from invasion to intracellular replication. Vet.Microbiol 90, 281-297.

Gross G.N., Rehm S.R., & Pierce A.K. (1978) The effect of complement depletion on lung clearance of bacteria. J Clin.Invest 62, 373-378.

Gu M., Hine P.M., James J.W., Giri L., & Nabors G.S. (2007) Increased potency of BioThrax((R)) anthrax vaccine with the addition of the C-class CpG oligonucleotide adjuvant CPG 10109. Vaccine 25, 526-534.

Guidi-Rontani C. (2002) The alveolar macrophage: the Trojan horse of Bacillus anthracis. Trends Microbiol. 10, 405-409.

Guidi-Rontani C., Levy M., Ohayon H., & Mock M. (2001) Fate of germinated Bacillus anthracis spores in primary murine macrophages. Mol.Microbiol. 42, 931-938.

Guidi-Rontani C. & Mock M. (2002) Macrophage interactions. Curr.Top.Microbiol.Immunol. 271, 115-141.

Guidi-Rontani C., Weber-Levy M., Labruyere E., & Mock M. (1999) Germination of Bacillus anthracis spores within alveolar macrophages. Mol.Microbiol. 31, 9­ 17.

167 Hahn B.L., Sharma S., & Sohnle P.G. (2005) Analysis of epidermal entry in experimental cutaneous Bacillus anthracis infections in mice. J Lab Clin.Med 146, 95-102.

Hahn U.K., Alex M., Czerny C.P., Bohm R., & Beyer W. (2004) Protection of mice against challenge with Bacillus anthracis STI spores after DNA vaccination. Int.J Med Microbiol 294, 35-44.

Hahn U.K., Boehm R., & Beyer W. (2006) DNA vaccination against anthrax in mice-combination of anti-spore and anti-toxin components. Vaccine 24, 4569­ 4571.

Hajishengallis G., Wang M., Harokopakis E., Triantafilou M., & Triantafilou K. (2006) Porphyromonas gingivalis fimbriae proactively modulate beta2 integrin adhesive activity and promote binding to and internalization by macrophages. Infect.Immun. 74, 5658-5666.

Hanks S., Adams S., Douglas J., Arbour L., Atherton D.J., Balci S., Bode H., Campbell M.E., Feingold M., Keser G., Kleijer W., Mancini G., McGrath J.A., Muntoni F., Nanda A., Teare M.D., Warman M., Pope F.M., Superti-Furga A., Futreal P.A., & Rahman N. (2003) Mutations in the gene encoding capillary morphogenesis protein 2 cause juvenile hyaline fibromatosis and infantile systemic hyalinosis. Am J Hum.Genet. 73, 791-800.

Hanna P. (1999) Lethal toxin actions and their consequences. J Appl.Microbiol. 87, 285-287.

Hanna P.C., Acosta D., & Collier R.J. (1993) On the role of macrophages in anthrax. Proc.Natl.Acad.Sci.U.S.A 90, 10198-10201.

Hanna P.C. & Ireland J.A. (1999) Understanding Bacillus anthracis pathogenesis. Trends Microbiol. 7, 180-182.

Harrop R. & Carroll M.W. (2006) Viral vectors for cancer immunotherapy. Front Biosci. 11, 804-817.

Harrop R., John J., & Carroll M.W. (2006) Recombinant viral vectors: cancer vaccines. Adv.Drug Deliv.Rev. 58, 931-947.

Harvill E.T., Lee G., Grippe V.K., & Merkel T.J. (2005) Complement depletion renders C57BL/6 mice sensitive to the Bacillus anthracis Sterne strain. Infect.Immun. 73, 4420-4422.

Heffernan B.J., Thomason B., Herring-Palmer A., Shaughnessy L., McDonald R., Fisher N., Huffnagle G.B., & Hanna P. (2006) Bacillus anthracis phospholipases

168 C facilitate macrophage-associated growth and contribute to virulence in a murine model of inhalation anthrax. Infect.Immun. 74, 3756-3764.

Heninger S., Drysdale M., Lovchik J., Hutt J., Lipscomb M.F., Koehler T.M., & Lyons C.R. (2006) Toxin-deficient mutants of Bacillus anthracis are lethal in a murine model for pulmonary anthrax. Infect.Immun. 74, 6067-6074.

Hestvik A.L., Hmama Z., & Av-Gay Y. (2005) Mycobacterial manipulation of the host cell. FEMS Microbiol Rev. 29, 1041-1050.

Hoover D.L., Friedlander A.M., Rogers L.C., Yoon I.K., Warren R.L., & Cross A.S. (1994) Anthrax edema toxin differentially regulates lipopolysaccharide- induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP. Infect.Immun. 62, 4432-4439.

Hotchkiss K.A., Basile C.M., Spring S.C., Bonuccelli G., Lisanti M.P., & Terman B.I. (2005) TEM8 expression stimulates endothelial cell adhesion and migration by regulating cell-matrix interactions on collagen. Exp.Cell Res. 305, 133-144.

Huber M., Vor Dem E.U., Grunow R., & Bessler W.G. (2005) Generation of mouse polyclonal and human monoclonal antibodies against Bacillus anthracis toxin. Drugs Exp.Clin.Res. 31, 35-43.

Iacono-Connors L.C., Schmaljohn C.S., & Dalrymple J.M. (1990) Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants. Infect.Immun. 58, 366-372.

Iacono-Connors L.C., Welkos S.L., Ivins B.E., & Dalrymple J.M. (1991) Protection against anthrax with recombinant virus-expressed protective antigen in experimental animals. Infect.Immun. 59, 1961-1965.

Ivins B.E., Pitt M.L., Fellows P.F., Farchaus J.W., Benner G.E., Waag D.M., Little S.F., Anderson G.W., Jr., Gibbs P.H., & Friedlander A.M. (1998) Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques. Vaccine 16, 1141-1148.

Ivins B.E., Welkos S.L., Little S.F., Crumrine M.H., & Nelson G.O. (1992) Immunization against anthrax with Bacillus anthracis protective antigen combined with adjuvants. Infect.Immun. 60, 662-668.

Jensen G.B., Hansen B.M., Eilenberg J., & Mahillon J. (2003) The hidden lifestyles of Bacillus cereus and relatives. Environ.Microbiol 5, 631-640.

Jensen G.B., Larsen P., Jacobsen B.L., Madsen B., Smidt L., & Andrup L. (2002) Bacillus thuringiensis in fecal samples from greenhouse workers after exposure to B. thuringiensis-based pesticides. Appl.Environ.Microbiol 68, 4900-4905. 169 Jones W.I., Jr., Klein F., Lincoln R.E., Walker J.S., Mahlandt B.G., & Dobbs J.P. (1967) Antibiotic treatment of anthrax infection in mice. J Bacteriol. 94, 609-614.

Joyce J., Cook J., Chabot D., Hepler R., Shoop W., Xu Q., Stambaugh T., Aste- Amezaga M., Wang S., Indrawati L., Bruner M., Friedlander A., Keller P., & Caulfield M. (2006) Immunogenicity and protective efficacy of Bacillus anthracis poly-gamma-D-glutamic acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy. J Biol.Chem. 281, 4831-4843.

Kalns J., Morris J., Eggers J., & Kiel J. (2002) Delayed treatment with doxycycline has limited effect on anthrax infection in BLK57/B6 mice. Biochem.Biophys.Res.Commun. 297, 506-509.

Kang P.B., Azad A.K., Torrelles J.B., Kaufman T.M., Beharka A., Tibesar E., DesJardin L.E., & Schlesinger L.S. (2005) The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis. J Exp.Med 202, 987-999.

Kang T.J., Fenton M.J., Weiner M.A., Hibbs S., Basu S., Baillie L., & Cross A.S. (2005) Murine macrophages kill the vegetative form of Bacillus anthracis. Infect.Immun. 73, 7495-7501.

Kassam A., Der S.D., & Mogridge J. (2005) Differentiation of human monocytic cell lines confers susceptibility to Bacillus anthracis lethal toxin. Cell Microbiol 7, 281-292.

Kayal S. & Charbit A. (2006) Listeriolysin O: a key protein of Listeria monocytogenes with multiple functions. FEMS Microbiol Rev. 30, 514-529.

Kenney R.T., Yu J., Guebre-Xabier M., Frech S.A., Lambert A., Heller B.A., Ellingsworth L.R., Eyles J.E., Williamson E.D., & Glenn G.M. (2004) Induction of protective immunity against lethal anthrax challenge with a patch. J Infect.Dis. 190, 774-782.

Kenney R.T., Yu J., Guebre-Xabier M., Frech S.A., Lambert A., Heller B.A., Ellingsworth L.R., Eyles J.E., Williamson E.D., & Glenn G.M. (2004) Induction of protective immunity against lethal anthrax challenge with a patch. J Infect.Dis. 190, 774-782.

Keppie J., Smith H., & Harris-Smith P.W. (1955) The chemical basis of the virulence of Bacillus anthracis. III. The role of the terminal bacteraemia in death of guinea-pigs from anthrax. Br.J Exp.Pathol. 36, 315-322.

Keynan A.H.H. (1965) In: Spores III In L. L. Campbell and H. 0. Halvorson [ed.], edn, p. 174-179 American Society for Microbiology, Ann Arbor, Michigan.

170 Kim J.G., Keshava C., Murphy A.A., Pitas R.E., & Parthasarathy S. (1997) Fresh mouse peritoneal macrophages have low scavenger receptor activity. J Lipid Res. 38, 2207-2215.

Kim S.O., Jing Q., Hoebe K., Beutler B., Duesbery N.S., & Han J. (2003) Sensitizing anthrax lethal toxin-resistant macrophages to lethal toxin-induced killing by tumor necrosis factor-alpha. J Biol.Chem. 278, 7413-7421.

Kirby J.E. (2004) Anthrax lethal toxin induces human endothelial cell apoptosis. Infect.Immun. 72, 430-439.

Klein F., Dearmon I.A., Jr., Lincoln R.E., Mahlandt B.G., & Fernelius A.L. (1962) Immunological studies of anthrox. II. Levels of immunity against Bacillus anthracis obtained with protective antigen and live vaccine. J Immunol. 88, 15­ 19.

Klimpel K.R., Molloy S.S., Thomas G., & Leppla S.H. (1992) Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc.Natl.Acad.Sci.U.S.A 89, 10277­ 10281.

Klinman D.M. (2006) CpG oligonucleotides accelerate and boost the immune response elicited by AVA, the licensed anthrax vaccine. Expert.Rev.Vaccines. 5, 365-369.

Klinman D.M., Xie H., Little S.F., Currie D., & Ivins B.E. (2004) CpG oligonucleotides improve the protective immune response induced by the anthrax vaccination of rhesus macaques. Vaccine 22, 2881-2886.

Kobiler D., Gozes Y., Rosenberg H., Marcus D., Reuveny S., & Altboum Z. (2002) Efficiency of protection of guinea pigs against infection with Bacillus anthracis spores by passive immunization. Infect.Immun. 70, 544-560.

Koch R. (1876) Die Aetiologie der Milzbrand-Krankheit, begründet auf die Entwicklungsgeschichte des Bacillus anthracis. Beiträge zur Biologie der Pflanzen 2, 277-310.

Koehler T.M. (2002) Bacillus anthracis genetics and virulence gene regulation. Curr.Top.Microbiol.Immunol. 271, 143-164.

Kolb W.P., Kolb L.M., Wetsel R.A., Rogers W.R., & Shaw J.O. (1981) Quantitation and stability of the fifth component of complement (C5) in bronchoalveolar lavage fluids obtained from non-human primates. Am Rev.Respir.Dis. 123, 226-231.

171 Koya V., Moayeri M., Leppla S.H., & Daniell H. (2005) Plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge. Infect.Immun. 73, 8266-8274.

Kozel T.R., Murphy W.J., Brandt S., Blazar B.R., Lovchik J.A., Thorkildson P., Percival A., & Lyons C.R. (2004) mAbs to Bacillus anthracis capsular antigen for immunoprotection in anthrax and detection of antigenemia. Proc.Natl.Acad.Sci U.S.A 101, 5042-5047.

Kozel T.R., Thorkildson P., Brandt S., Welch W.H., Lovchik J.A., AuCoin D.P., Vilai J., & Lyons C.R. (2007) Protective and immunochemical activities of monoclonal antibodies reactive with the Bacillus anthracis polypeptide capsule. Infect.Immun. 75, 152-163.

Kramer M.J. & Roth I.L. (1970) Electron Microscopy of the Phagocytosis of Capsulated Bacillus anthracis. Infect.Immun. 2, 216-221.

Kubler-Kielb J., Liu T.Y., Mocca C., Majadly F., Robbins J.B., & Schneerson R. (2006) Additional conjugation methods and immunogenicity of Bacillus anthracis poly-gamma-D-glutamic acid-protein conjugates. Infect.Immun. 74, 4744-4749.

Kumar P., Ahuja N., & Bhatnagar R. (2002) Anthrax edema toxin requires influx of calcium for inducing cyclic AMP toxicity in target cells. Infect.Immun. 70, 4997­ 5007.

Lambris J.D. (1988) The multifunctional role of C3, the third component of complement. Immunol.Today 9, 387-393.

Lambris J.D. & Tsokos G.C. (1986) The biology and pathophysiology of complement receptors. Anticancer Res. 6, 515-523.

Lathrop A.A., Huff K., & Bhunia A.K. (2006) Prevalence of antibodies reactive to pathogenic and nonpathogenic bacteria in preimmune serum of New Zealand white rabbits. J Immunoassay Immunochem. 27, 351-361.

Lee J.S., Hadjipanayis A.G., & Welkos S.L. (2003) Venezuelan equine encephalitis virus-vectored vaccines protect mice against anthrax spore challenge. Infect.Immun. 71, 1491-1496.

Leppla S.H. (1982) Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc.Natl.Acad.Sci.U.S.A 79, 3162-3166.

Leppla S.H. (1984) Bacillus anthracis calmodulin-dependent adenylate cyclase: chemical and enzymatic properties and interactions with eucaryotic cells. Adv.Cyclic.Nucleotide.Protein Phosphorylation.Res. 17, 189-198. 172 Leppla S.H. (1988) Production and purification of anthrax toxin. Methods Enzymol. 165, 103-116.

Leppla S.H. (1995) Anthrax Toxins. In: Bacterial Toxins and Virulence Factors in Disease (ed. J. Moss, B. Iglewski, M. Vaughan, & A. T. Tu), p. 543-572 Dekker, New York.

Lety M.A., Frehel C., Dubail I., Beretti J.L., Kayal S., Berche P., & Charbit A. (2001) Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes. Mol.Microbiol 39, 1124-1139.

Li Z.N., Mueller S.N., Ye L., Bu Z., Yang C., Ahmed R., & Steinhauer D.A. (2005) Chimeric influenza virus hemagglutinin proteins containing large domains of the Bacillus anthracis protective antigen: protein characterization, incorporation into infectious influenza viruses, and antigenicity. J Virol 79, 10003-10012.

Little S.F. & Ivins B.E. (1999) Molecular pathogenesis of Bacillus anthracis infection. Microbes.Infect. 1, 131-139.

Liu S. & Leppla S.H. (2003) Cell surface tumor endothelium marker 8 cytoplasmic tail-independent anthrax toxin binding, proteolytic processing, oligomer formation, and internalization. J Biol.Chem. 278, 5227-5234.

Longchamp P. & Leighton T. (1999) Molecular recognition specificity of Bacillus anthracis spore antibodies. J Appl.Microbiol. 87, 246-249.

Lyons C.R., Lovchik J., Hutt J., Lipscomb M.F., Wang E., Heninger S., Berliba L., & Garrison K. (2004) Murine model of pulmonary anthrax: kinetics of dissemination, histopathology, and mouse strain susceptibility. Infect.Immun. 72, 4801-4809.

Makino S., Uchida I., Terakado N., Sasakawa C., & Yoshikawa M. (1989) Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis. J Bacteriol. 171, 722-730.

Masure H.R., Shattuck R.L., & Storm D.R. (1987) Mechanisms of bacterial pathogenicity that involve production of calmodulin-sensitive adenylate cyclases. Microbiol.Rev. 51, 60-65.

Matyas G.R., Friedlander A.M., Glenn G.M., Little S., Yu J., & Alving C.R. (2004) Needle-free skin patch vaccination method for anthrax. Infect.Immun. 72, 1181­ 1183.

173 McConnell M.J., Hanna P.C., & Imperiale M.J. (2006) Cytokine response and survival of mice immunized with an adenovirus expressing Bacillus anthracis protective antigen domain 4. Infect.Immun. 74, 1009-1015.

Metcalfe N. (2004) The history of woolsorters' disease: a Yorkshire beginning with an international future? Occup.Med (Lond) 54, 489-493.

Mikesell P., Ivins B.E., Ristroph J.D., & Dreier T.M. (1983) Evidence for plasmid- mediated toxin production in Bacillus anthracis. Infect.Immun. 39, 371-376.

Moayeri M., Haines D., Young H.A., & Leppla S.H. (2003) Bacillus anthracis lethal toxin induces TNF-alpha-independent hypoxia-mediated toxicity in mice. J Clin.Invest 112, 670-682.

Mock M. & Ullmann A. (1993) Calmodulin-activated bacterial adenylate cyclases as virulence factors. Trends Microbiol. 1, 187-192.

Mogridge J., Cunningham K., & Collier R.J. (2002) Stoichiometry of anthrax toxin complexes. Biochemistry 41, 1079-1082.

Morens D.M. (2003) Characterizing a "new" disease: epizootic and epidemic anthrax, 1769-1780. Am J Public Health 93, 886-893.

Mosser E.M. & Rest R.F. (2006) The Bacillus anthracis cholesterol-dependent cytolysin, Anthrolysin O, kills human neutrophils, monocytes and macrophages. BMC.Microbiol 6, 56.

Nanda A., Carson-Walter E.B., Seaman S., Barber T.D., Stampfl J., Singh S., Vogelstein B., Kinzler K.W., & St Croix B. (2004) TEM8 interacts with the cleaved C5 domain of collagen alpha 3(VI). Cancer Res. 64, 817-820.

Navacharoen N., Sirisanthana T., Navacharoen W., & Ruckphaopunt K. (1985) Oropharyngeal anthrax. J Laryngol.Otol. 99, 1293-1295.

O'Brien J., Friedlander A., Dreier T., Ezzell J., & Leppla S. (1985) Effects of anthrax toxin components on human neutrophils. Infect.Immun. 47, 306-310.

Ochsenbein A.F. & Zinkernagel R.M. (2000) Natural antibodies and complement link innate and acquired immunity. Immunol.Today 21, 624-630.

Ogasawara Y., Amexis G., Yamaguchi H., Kajigaya S., Leppla S.H., & Young N.S. (2006) Recombinant viral-like particles of parvovirus B19 as antigen carriers of anthrax protective antigen. In Vivo 20, 319-324.

Okinaka R., Cloud K., Hampton O., Hoffmaster A., Hill K., Keim P., Koehler T., Lamke G., Kumano S., Manter D., Martinez Y., Ricke D., Svensson R., & 174 Jackson P. (1999) Sequence, assembly and analysis of pX01 and pX02. J Appl.Microbiol. 87, 261-262.

Okinaka R.T., Cloud K., Hampton O., Hoffmaster A.R., Hill K.K., Keim P., Koehler T.M., Lamke G., Kumano S., Mahillon J., Manter D., Martinez Y., Ricke D., Svensson R., & Jackson P.J. (1999) Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J Bacteriol. 181, 6509-6515.

Oncu S., Oncu S., & Sakarya S. (2003) Anthrax--an overview. Med Sci Monit. 9, RA276-RA283.

Palecanda A. & Kobzik L. (2001) Receptors for unopsonized particles: the role of alveolar macrophage scavenger receptors. Curr.Mol.Med 1, 589-595.

Pandey J. & Warburton D. (2004) Knock-on effect of anthrax lethal toxin on macrophages potentiates cytotoxicity to endothelial cells. Microbes.Infect. 6, 835­ 843.

Park J.M., Greten F.R., Li Z.W., & Karin M. (2002) Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science 297, 2048-2051.

Park J.M., Ng V.H., Maeda S., Rest R.F., & Karin M. (2004) Anthrolysin O and other gram-positive cytolysins are toll-like receptor 4 agonists. J Exp.Med 200, 1647-1655.

Passalacqua K.D., Bergman N.H., Herring-Palmer A., & Hanna P. (2006) The superoxide dismutases of Bacillus anthracis do not cooperatively protect against endogenous superoxide stress. J Bacteriol. 188, 3837-3848.

Peiser L. & Gordon S. (2001) The function of scavenger receptors expressed by macrophages and their role in the regulation of inflammation. Microbes.Infect. 3, 149-159.

Pellizzari R., Guidi-Rontani C., Vitale G., Mock M., & Montecucco C. (1999) Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNgamma-induced release of NO and TNFalpha. FEBS Lett. 462, 199-204.

Pellizzari R., Guidi-Rontani C., Vitale G., Mock M., & Montecucco C. (2000) Lethal factor of Bacillus anthracis cleaves the N-terminus of MAPKKs: analysis of the intracellular consequences in macrophages. Int.J Med.Microbiol. 290, 421­ 427.

Perkins S.D., Flick-Smith H.C., Garmory H.S., Essex-Lopresti A.E., Stevenson F.K., & Phillpotts R.J. (2005) Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax. Genet.Vaccines.Ther. 3, 3. 175 Petosa C., Collier R.J., Klimpel K.R., Leppla S.H., & Liddington R.C. (1997) Crystal structure of the anthrax toxin protective antigen. Nature 385, 833-838.

Pezard C., Berche P., & Mock M. (1991) Contribution of individual toxin components to virulence of Bacillus anthracis. Infect.Immun. 59, 3472-3477.

Pezard C., Duflot E., & Mock M. (1993) Construction of Bacillus anthracis mutant strains producing a single toxin component. J Gen.Microbiol. 139 ( Pt 10), 2459­ 2463.

Phipps A.J., Premanandan C., Barnewall R.E., & Lairmore M.D. (2004) Rabbit and nonhuman primate models of toxin-targeting human anthrax vaccines. Microbiol.Mol.Biol.Rev. 68, 617-629.

Pickering A.K. & Merkel T.J. (2004) Macrophages release tumor necrosis factor alpha and interleukin-12 in response to intracellular Bacillus anthracis spores. Infect.Immun. 72, 3069-3072.

Pickering A.K., Osorio M., Lee G.M., Grippe V.K., Bray M., & Merkel T.J. (2004) Cytokine response to infection with Bacillus anthracis spores. Infect.Immun. 72, 6382-6389.

Pile J.C., Malone J.D., Eitzen E.M., & Friedlander A.M. (1998) Anthrax as a potential biological warfare agent. Arch.Intern.Med 158, 429-434.

Popov S.G., Popova T.G., Grene E., Klotz F., Cardwell J., Bradburne C., Jama Y., Maland M., Wells J., Nalca A., Voss T., Bailey C., & Alibek K. (2004) Systemic cytokine response in murine anthrax. Cell Microbiol 6, 225-233.

Portnoy D.A., Auerbuch V., & Glomski I.J. (2002) The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell- mediated immunity. J Cell Biol. 158, 409-414.

Premanandan C., Lairmore M.D., Fernandez S., & Phipps A.J. (2006) Quantitative measurement of anthrax toxin receptor messenger RNA in primary mononuclear phagocytes. Microb.Pathog. 41, 193-198.

Price B.M., Liner A.L., Park S., Leppla S.H., Mateczun A., & Galloway D.R. (2001) Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein. Infect.Immun. 69, 4509-4515.

Quesnel-Hellmann A., Cleret A., Vidal D.R., & Tournier J.N. (2006) Evidence for adjuvanticity of anthrax edema toxin. Vaccine 24, 699-702.

176 Rainey G.J., Wigelsworth D.J., Ryan P.L., Scobie H.M., Collier R.J., & Young J.A. (2005) Receptor-specific requirements for anthrax toxin delivery into cells. Proc.Natl.Acad.Sci U.S.A 102, 13278-13283.

Rhie G.E., Roehrl M.H., Mourez M., Collier R.J., Mekalanos J.J., & Wang J.Y. (2003) A dually active anthrax vaccine that confers protection against both bacilli and toxins. Proc.Natl.Acad.Sci U.S.A 100, 10925-10930.

Ribot W.J., Panchal R.G., Brittingham K.C., Ruthel G., Kenny T.A., Lane D., Curry B., Hoover T.A., Friedlander A.M., & Bavari S. (2006) Anthrax lethal toxin impairs innate immune functions of alveolar macrophages and facilitates Bacillus anthracis survival. Infect.Immun. 74, 5029-5034.

Riemenschneider J., Garrison A., Geisbert J., Jahrling P., Hevey M., Negley D., Schmaljohn A., Lee J., Hart M.K., Vanderzanden L., Custer D., Bray M., Ruff A., Ivins B., Bassett A., Rossi C., & Schmaljohn C. (2003) Comparison of individual and combination DNA vaccines for B. anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus. Vaccine 21, 4071-4080.

Rmali K.A., Al Rawi M.A., Parr C., Puntis M.C., & Jiang W.G. (2004) Upregulation of tumour endothelial marker-8 by interleukin-1beta and its impact in IL-1beta induced angiogenesis. Int.J Mol.Med 14, 75-80.

Rmali K.A., Puntis M.C., & Jiang W.G. (2005) Prognostic values of tumor endothelial markers in patients with colorectal cancer. World J Gastroenterol. 11, 1283-1286.

Rus H., Cudrici C., & Niculescu F. (2005) The role of the complement system in innate immunity. Immunol.Res. 33, 103-112.

Ruthel G., Ribot W.J., Bavari S., & Hoover T.A. (2004) Time-lapse confocal imaging of development of Bacillus anthracis in macrophages. J Infect.Dis. 189, 1313-1316.

Sahu A. & Lambris J.D. (2001) Structure and biology of complement protein C3, a connecting link between innate and acquired immunity. Immunol.Rev. 180, 35­ 48.

Sastry K.S., Tuteja U., & Batra H.V. (2003) Generation and characterization of monoclonal antibodies to protective antigen of Bacillus anthracis. Indian J Exp.Biol. 41, 123-128.

Schlesinger L.S. (1993) Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors. J Immunol. 150, 2920-2930.

177 Schlesinger L.S., Bellinger-Kawahara C.G., Payne N.R., & Horwitz M.A. (1990) Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. J Immunol. 144, 2771­ 2780.

Schlesinger L.S. & Horwitz M.A. (1994) A role for natural antibody in the pathogenesis of leprosy: antibody in nonimmune serum mediates C3 fixation to the Mycobacterium leprae surface and hence phagocytosis by human mononuclear phagocytes. Infect.Immun. 62, 280-289.

Schlesinger L.S., Kaufman T.M., Iyer S., Hull S.R., & Marchiando L.K. (1996) Differences in mannose receptor-mediated uptake of lipoarabinomannan from virulent and attenuated strains of Mycobacterium tuberculosis by human macrophages. J Immunol. 157, 4568-4575.

Schneerson R., Kubler-Kielb J., Liu T.Y., Dai Z.D., Leppla S.H., Yergey A., Backlund P., Shiloach J., Majadly F., & Robbins J.B. (2003) Poly(gamma-D­ glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of Bacillus anthracis: a potential addition to the anthrax vaccine. Proc.Natl.Acad.Sci U.S.A 100, 8945-8950.

Scobie H.M., Rainey G.J., Bradley K.A., & Young J.A. (2003) Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc.Natl.Acad.Sci.U.S.A 100, 5170-5174.

Scorpio A., Blank T.E., Day W.A., & Chabot D.J. (2006) Anthrax vaccines: Pasteur to the present. Cell Mol.Life Sci 63, 2237-2248.

Scorpio A., Chabot D.J., Day W.A., O'brien D.K., Vietri N.J., Itoh Y., Mohamadzadeh M., & Friedlander A.M. (2007) Poly-{gamma}-Glutamate Capsule-Degrading Enzyme Treatment Enhances Phagocytosis and Killing of Encapsulated Bacillus anthracis. Antimicrob.Agents Chemother. 51, 215-222.

Seto K. & Takizawa T. (1969) Dose-response relationship between inoculum dose of anthrax spores and survival time of mice. Natl.Inst.Anim Health Q.(Tokyo) 9, 129-135.

Shannon J.G., Ross C.L., Koehler T.M., & Rest R.F. (2003) Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin. Infect.Immun. 71, 3183-3189.

Shi Y., Tohyama Y., Kadono T., He J., Shahjahan Miah S.M., Hazama R., Tanaka C., Tohyama K., & Yamamura H. (2006) Protein-tyrosine kinase Syk is required for pathogen engulfment in complement-mediated phagocytosis. Blood 107, 4554-4562.

178 Sirisanthana T. & Brown A.E. (2002) Anthrax of the gastrointestinal tract. Emerg.Infect.Dis. 8, 649-651.

Sirisanthana T., Navachareon N., Tharavichitkul P., Sirisanthana V., & Brown A.E. (1984) Outbreak of oral-oropharyngeal anthrax: an unusual manifestation of human infection with Bacillus anthracis. Am.J Trop.Med.Hyg. 33, 144-150.

Sirisanthana T., Nelson K.E., Ezzell J.W., & Abshire T.G. (1988) Serological studies of patients with cutaneous and oral-oropharyngeal anthrax from northern Thailand. Am.J Trop.Med.Hyg. 39, 575-581.

Sloat B.R. & Cui Z. (2006) Nasal immunization with anthrax protective antigen protein adjuvanted with polyriboinosinic-polyribocytidylic acid induced strong mucosal and systemic immunities. Pharm.Res. 23, 1217-1226.

Sloat B.R. & Cui Z. (2006) Strong mucosal and systemic immunities induced by nasal immunization with anthrax protective antigen protein incorporated in liposome-protamine-DNA particles. Pharm.Res. 23, 262-269.

Smith K.A. (2005) Wanted, an Anthrax vaccine: Dead or Alive? Med Immunol. 4, 5.

Spencer R.C. (2003) Bacillus anthracis. J Clin.Pathol. 56, 182-187.

Steele A.D., Warfel J.M., & D'Agnillo F. (2005) Anthrax lethal toxin enhances cytokine-induced VCAM-1 expression on human endothelial cells. Biochem.Biophys.Res.Commun. 337, 1249-1256.

Steichen C., Chen P., Kearney J.F., & Turnbough C.L., Jr. (2003) Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium. J Bacteriol. 185, 1903-1910.

Sternbach G. (2003) The history of anthrax. J Emerg.Med 24, 463-467.

Sterne M., Nichol J., & Lambrechts M.C. (1942) The effect of large scale active immunization against anthrax. J.S.African Vet.Med.Assoc. 13, 53-63.

Sterne M. (1939) The use of anthrax vaccines prepared from avirulent (uncapsulated) variants of Bacillus anthracis. Onderstepoort J.Vet.Sci.An.Ind. 13, 307-312.

Sterne M. (1988) Anthrax vaccines. J Am.Vet.Med.Assoc. 192, 141.

Stokes M.G., Titball R.W., Neeson B.N., Galen J.E., Walker N.J., Stagg A.J., Jenner D.C., Thwaite J.E., Nataro J.P., Baillie L.W., & Atkins H.S. (2006) Oral administration of a Salmonella-based vaccine expressing Bacillus anthracis 179 protective antigen confers protection against aerosolized B. anthracis. Infect.Immun.

Strunk R.C., Eidlen D.M., & Mason R.J. (1988) Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways. J Clin.Invest 81, 1419-1426.

Stump M.J., Black G., Fox A., Fox K.F., Turick C.E., & Matthews M. (2005) Identification of marker proteins for Bacillus anthracis using MALDI-TOF MS and ion trap MS/MS after direct extraction or electrophoretic separation. J Sep.Sci 28, 1642-1647.

Swiecki M.K., Lisanby M.W., Shu F., Turnbough C.L., Jr., & Kearney J.F. (2006) Monoclonal antibodies for Bacillus anthracis spore detection and functional analyses of spore germination and outgrowth. J Immunol. 176, 6076-6084.

Tamir H. & Gilvarg C. (1966) Density gradient centrifugation for the separation of sporulating forms of bacteria. J Biol.Chem. 241, 1085-1090.

Tigertt W.D. (1980) Anthrax. William Smith Greenfield, M.D., F.R.C.P., Professor Superintendent, the Brown Animal Sanatory Institution (1878-81). Concerning the priority due to him for the production of the first vaccine against anthrax. J Hyg.(Lond) 85, 415-420.

Tippetts M.T. & Robertson D.L. (1988) Molecular cloning and expression of the Bacillus anthracis edema factor toxin gene: a calmodulin-dependent adenylate cyclase. J Bacteriol. 170, 2263-2266.

Tizard I. (1999) Grease, anthraxgate, and kennel cough: a revisionist history of early veterinary vaccines. Adv.Vet.Med 41, 7-24.

Tohyama Y. (2006) [The molecular mechanisms of complement-mediated phagocytosis--the novel function of tyrosine kinase, Syk]. Seikagaku 78, 878­ 882.

Tohyama Y. & Yamamura H. (2006) Complement-mediated phagocytosis--the role of Syk. IUBMB.Life 58, 304-308.

Tucker A.E., Salles I.I., Voth D.E., Ortiz-Leduc W., Wang H., Dozmorov I., Centola M., & Ballard J.D. (2003) Decreased glycogen synthase kinase 3-beta levels and related physiological changes in Bacillus anthracis lethal toxin-treated macrophages. Cell Microbiol 5, 523-532.

Turell M.J. & Knudson G.B. (1987) Mechanical transmission of Bacillus anthracis by stable flies (Stomoxys calcitrans) and mosquitoes (Aedes aegypti and Aedes taeniorhynchus). Infect.Immun. 55, 1859-1861. 180 Turnbull P.C. (1991) Anthrax vaccines: past, present and future. Vaccine 9, 533­ 539.

Ulmer T.S., Soelaiman S., Li S., Klee C.B., Tang W.J., & Bax A. (2003) Calcium- dependence of the interaction between calmodulin and anthrax edema factor. J Biol.Chem.

Van Ness G.B. (1971) Ecology of anthrax. Science 172, 1303-1307.

Van Oers M.M. (2006) Vaccines for viral and parasitic diseases produced with baculovirus vectors. Adv.Virus Res. 68, 193-253.

Vann W.F., Liu T.Y., & Robbins J.B. (1976) Bacillus pumilus polysaccharide cross-reactive with meningococcal group A polysaccharide. Infect.Immun. 13, 1654-1662.

Vasconcelos D., Barnewall R., Babin M., Hunt R., Estep J., Nielsen C., Carnes R., & Carney J. (2003) Pathology of inhalation anthrax in cynomolgus monkeys (Macaca fascicularis). Lab Invest 83, 1201-1209.

Velasco-Velazquez M.A., Barrera D., Gonzalez-Arenas A., Rosales C., & Agramonte-Hevia J. (2003) Macrophage--Mycobacterium tuberculosis interactions: role of complement receptor 3. Microb.Pathog. 35, 125-131.

Vergne I., Chua J., Singh S.B., & Deretic V. (2004) Cell biology of Mycobacterium tuberculosis phagosome. Annu.Rev.Cell Dev.Biol. 20, 367-394.

Vieira O.V., Botelho R.J., & Grinstein S. (2002) Phagosome maturation: aging gracefully. Biochem.J 366, 689-704.

Vilain S., Luo Y., Hildreth M.B., & Brozel V.S. (2006) Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil. Appl.Environ.Microbiol 72, 4970-4977.

Wang J.Y. & Roehrl M.H. (2005) Anthrax vaccine design: strategies to achieve comprehensive protection against spore, bacillus, and toxin. Med Immunol. 4, 4.

Warfel J.M., Steele A.D., & D'Agnillo F. (2005) Anthrax lethal toxin induces endothelial barrier dysfunction. Am J Pathol. 166, 1871-1881.

Watford W.T., Smithers M.B., Frank M.M., & Wright J.R. (2002) Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages. Am J Physiol Lung Cell Mol.Physiol 283, L1011-L1022.

181 Wei W., Lu Q., Chaudry G.J., Leppla S.H., & Cohen S.N. (2006) The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin. Cell 124, 1141-1154.

Wei Z., Schnupf P., Poussin M.A., Zenewicz L.A., Shen H., & Goldfine H. (2005) Characterization of Listeria monocytogenes expressing anthrolysin O and phosphatidylinositol-specific phospholipase C from Bacillus anthracis. Infect.Immun. 73, 6639-6646.

Weiner M.A. & Hanna P.C. (2003) Macrophage-Mediated Germination of Bacillus anthracis Endospores Requires the gerH Operon. Infect.Immun. 71, 3954-3959.

Weiner M.A., Read T.D., & Hanna P.C. (2003) Identification and characterization of the gerH operon of Bacillus anthracis endospores: a differential role for purine nucleosides in germination. J Bacteriol. 185, 1462-1464.

Welkos, S, D.Becker, A.Friedlander, and R.Trotter. Pathogenesis and host resistance to Bacillus anthracis: a mouse model. J.R.H.Pinkerton. Proceedings of the International Workshop on Anthrax [68], 49-52. 1990. Salisbury, England, Salisbury Medical Society.

Welkos S., Friedlander A., Weeks S., Little S., & Mendelson I. (2002) In-vitro characterisation of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody. J Med.Microbiol. 51, 821-831.

Welkos S.L. & Friedlander A.M. (1988) Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice. Microb.Pathog. 5, 127-139.

Welkos S.L. & Friedlander A.M. (1988) Pathogenesis and genetic control of resistance to the Sterne strain of Bacillus anthracis. Microb.Pathog. 4, 53-69.

Welkos S.L., Keener T.J., & Gibbs P.H. (1986) Differences in susceptibility of inbred mice to Bacillus anthracis. Infect.Immun. 51, 795-800.

Welkos S.L., Trotter R.W., Becker D.M., & Nelson G.O. (1989) Resistance to the Sterne strain of B. anthracis: phagocytic cell responses of resistant and susceptible mice. Microb.Pathog. 7, 15-35.

Werner E., Kowalczyk A.P., & Faundez V. (2006) Anthrax toxin receptor 1/tumor endothelium marker 8 mediates cell spreading by coupling extracellular ligands to the actin cytoskeleton. J Biol.Chem. 281, 23227-23236.

Williamson E.D., Hodgson I., Walker N.J., Topping A.W., Duchars M.G., Mott J.M., Estep J., Lebutt C., Flick-Smith H.C., Jones H.E., Li H., & Quinn C.P.

182 (2005) Immunogenicity of recombinant protective antigen and efficacy against aerosol challenge with anthrax. Infect.Immun. 73, 5978-5987.

Wright G.G., Green T.W., & Kanode R.G., Jr. (1954) Studies on immunity in anthrax. V. Immunizing activity of alum-precipitated protective antigen. J Immunol. 73, 387-391.

Xie H., Gursel I., Ivins B.E., Singh M., O'Hagan D.T., Ulmer J.B., & Klinman D.M. (2005) CpG oligodeoxynucleotides adsorbed onto polylactide-co-glycolide microparticles improve the immunogenicity and protective activity of the licensed anthrax vaccine. Infect.Immun. 73, 828-833.

Xing Z., Santosuosso M., McCormick S., Yang T.C., Millar J., Hitt M., Wan Y., Bramson J., & Vordermeier H.M. (2005) Recent advances in the development of adenovirus- and poxvirus-vectored tuberculosis vaccines. Curr.Gene Ther. 5, 485-492.

Zaucha G.M., Pitt L.M., Estep J., Ivins B.E., & Friedlander A.M. (1998) The pathology of experimental anthrax in rabbits exposed by inhalation and subcutaneous inoculation. Arch.Pathol.Lab Med 122, 982-992.

Zeng M., Xu Q., Hesek E.D., & Pichichero M.E. (2006) N-fragment of edema factor as a candidate antigen for immunization against anthrax. Vaccine 24, 662­ 670.

Zhao P., Liang X., Kalbfleisch J., Koo H.M., & Cao B. (2003) Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model. Hum.Antibodies 12, 129-135.

183