BW-ch14.pdf

Staphylococcal Enterotoxin B and Related Toxins

Chapter 14

STAPHYLOCOCCAL ENTEROTOXIN B

AND RELATED TOXINS

RobeRt G. UlRich, P h D*; catheRine l. Wilhelmsen, DVm, P h D, cbsP † ; a n d teResa KRaKaUeR, P h D ‡

INTRODUCTION

DESCRIPTION OF THE AGENT

PATHOGENESIS

CLINICAL DISEASE

Fever

Respiratory Symptoms

Headache

Nausea and Vomiting

Other Signs and Symptoms

DETECTION AND DIAGNOSIS

MEDICAL MANAGEMENT

IMMUNOTHERAPY

VACCINES

SUMMARY

* Microbiologist, Department of Immunology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

21702

† Lieutenant Colonel, Veterinary Corps, US Army (Ret); Biosafety Officer, Office of Safety, Radiation Protection, and Environmental Health, US Army

Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702; formerly, Chief, Division of Toxinology, US Army

Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

‡ Microbiologist, Department of Immunology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

21702

311

Medical Aspects of Biological Warfare

INTRODUCTION

the gram-positive bacteria Streptococcus pyogenes

and Staphylococcus aureus extensively colonize the hu-

man population and are frequent opportunistic patho-

gens. these bacteria secrete a variety of enzymatic and

nonenzymatic virulence factors that are responsible for

many disease symptoms. among these factors, staphy-

lococcal enterotoxins (ses), toxic shock syndrome toxin

(tsst-1), and streptococcal pyrogenic exotoxins of S

pyogenes share a common three-dimensional protein

fold characteristic of the bacterial products called

“superantigens” because of their profound effects

upon the immune system. most strains of S aureus and

S pyogenes examined harbor genes for superantigens

and are likely to produce at least one of these products.

the staphylococcal enterotoxins are most frequently

associated with food poisoning, yet not all superanti-

gens are enterotoxins, and more severe physiological

consequences, such as a life-threatening toxic shock

syndrome, may result from exposure to any of the

superantigens through a nonenteric route. high dose,

microgram-level exposures to staphylococcal entero-

toxin b (seb) will result in fatalities, and inhalation

exposure to nanogram or lower levels may be severely

incapacitating. 1 in addition, the severe perturbation of

the immune system caused by superantigen exposure

may lower the infectious or lethal dose of replicating

agents such as influenza virus. 2

seb is a prototype enterotoxin and potential bio-

logical threat agent produced by many isolates of

S aureus . During the 1960s, seb was studied exten-

sively as a biological incapacitant in the Us offensive

program. Us scientists had completed studies that

clearly demonstrated the effectiveness of seb as a

biological weapon before the ban on offensive toxin

weapons announced by President nixon in Febru-

ary 1970 (3 months after replicating agent weapons

were banned). seb was exceptionally suitable as a

biological agent because its effect was produced with

much less material than was necessary with synthetic

chemicals, and it presumably had an exceptional

“safety ratio” (calculated by dividing the effective

dose for incapacitation by the dose producing lethal-

ity). however, the safety ratio is misleading because

the coadministration of seb or related toxins with

replicating pathogens may profoundly lower the

lethal dose. available countermeasures and diag-

nostics have focused on seb because of its historical

significance in past biowarfare efforts; however, seb

represents many (perhaps hundreds) of related bio-

logically active superantigens that are readily isolated

and manipulated by recombinant Dna techniques.

all of these superantigens are presumed to have a

similar mode of biological action, but very little data

are available for confirmation.

DESCRIPTION OF THE AGENT

an examination of genes encoding superantigens

of S aureus and S pyogenes indicates a common origin

or perhaps an exchange of genetic elements between

bacterial species. the great diversity of superantigens

and the highly mobile nature of their genetic ele-

ments also suggest an accelerated rate of evolution.

staphylococcal and streptococcal strains that colonize

domestic animals are potential genetic reservoirs for

new toxin genes, 3 and the transfer of these sequences

may contribute to hybrid polypeptides. however, the

many similarities among severe diseases caused by

S aureus and S pyogenes superantigens 4 imply a com-

mon mechanism of pathology. amino acid sequence

comparisons indicate that superantigens can be loosely

compiled into three major subgroups and numerous

sequence variations 5 ; whereas genetic analysis shows

that they are all likely derived from common ancestral

genes. Despite significant sequence divergence, with

similarities as low as 14%, overall protein folds are

similar among staphylococcal and streptococcal supe-

rantigens. the toxin genes have evolved by strong se-

lective pressures to maintain receptor-binding surfaces

by preserving three-dimensional protein structure. the

contact surfaces with human leukocyte antigen DR

(hla-DR) receptors involve variations of conserved

structural elements, 6,7 which include a ubiquitous hy-

drophobic surface loop, a polar-binding pocket present

in most superantigens, and one or more zinc-binding

sites found in some toxins. comparison of antibody

recognition among superantigens 8 suggests that anti-

genic variation is maximized while three-dimensional

structures, and hence receptor-binding surfaces, are

conserved. From a practical standpoint, this observa-

tion indicates that a large panel of antibody probes will

be required for proper identification of samples.

molecular details of the biological actions of bacte-

rial superantigens are well established. superantigens

target cells mediating innate and adaptive immunity,

resulting in an intense activation and subsequent

pathology associated with aberrant host immune

responses. class ii molecules of the major histocom-

patibility complex (mhc) are the primary receptors,

and the mhc-bound superantigen in turn stimulates

t cells. most superantigens share a common mode

for binding class ii mhc molecules, with additional

stabilizing interactions that are unique to each one. 9

312

Staphylococcal Enterotoxin B and Related Toxins

a second, zinc-dependent molecular binding mode

for some superantigens increases t-cell signaling and

may impart greater toxicities in some cases. in normal

t-cell responses to peptide antigens, the cD4 mol-

ecule stabilizes interactions between t-cell antigen

receptors and class ii mhc molecules on antigen-

presenting cells (Figure 14-1). superantigens also

cross-link t-cell antigen receptors and class ii mhc

molecules, mimicking the cD4 molecule, 10 and hence

stimulate large numbers of t cells. in addition, each

superantigen preferentially stimulates t cells bearing

distinct subsets of antigen receptors, predominantly

dictated by the specific Vβ chain. an intense and

rapid release of cytokines such as interferon-γ, inter-

leukin-6 and tumor necrosis factor-α is responsible

for the systemic effects of the toxins. 11 in addition to

direct t-cell activation, the gastrointestinal illness

especially prominent after ingestion of staphylococ-

cal enterotoxins is also associated with histamine and

leukotriene release from mast cells. 12 Furthermore,

the cD44 molecule reportedly provides protection

from liver damage in mice caused by seb exposure

through a mechanism linked to activation-induced

apoptosis of immune cells. 13

individuals within the human population may re-

spond differently to superantigen exposure as a result

of mhc polymorphisms, age, and many physiological

factors. each toxin exhibits varying affinities toward

the hla-DR, DQ, and DP isotypes and distinct alleles

of class ii mhc molecules, observed by differences

in t-cell responses in vitro. in addition, primates, in-

cluding humans, are most sensitive to superantigens

compared to other mammals. 14 lethal or incapacitating

doses of toxin may be lowered by coexposure to endo-

toxin from gram-negative bacteria 11 or hepatotoxins, 15

or by infection with replicating agents. 2

Rodents and other domestic animals infected with

strains that produce tsst-1 and se 16,17 are potential

environmental reservoirs. both ovine- and-bovine spe-

cific staphylococcal toxins, which are associated with

mastitis, are almost identical to tsst-1 in amino acid

sequence. 18 toxigenic strains are frequent or universal

in both clinical and nonclinical isolates of S aureus and

S pyogenes , and these strains contribute significantly to

several diseases. approximately 50% of nonmenstrual

toxic shock syndrome (tss) cases are linked to tsst-1,

while the remaining cases are attributable to se, with

seb predominating. 19 Kawasaki’s syndrome and some

forms of arthritis are loosely associated with organisms

producing streptococcal pyrogenic exotoxins (sPes),

sea, and tsst-1. 20 in addition, streptococcal pneumo-

nia with accompanying tss-like symptoms is caused

by sPe-producing bacteria. 21

most of the streptococcal superantigens are encoded

by mobile genetic elements. sPe-a, sPe-c, sea, and

see are all phage-borne, while seD is plasmid-en-

coded. a chromosomal cluster of se and se-like genes

is present in strains of S aureus. 22 because little evi-

dence of genetic drift exists, it has been hypothesized

that the majority of staphylococcal and streptococcal

tss-like bacterial isolates have each descended from

single clones. 23 Production of many ses is dependent

on the phase of cell-growth cycle, environmental ph,

and glucose concentration. transcriptional control of

tsst-1, seb, sec, and seD is mediated through the

accessory gene regulator (agr) locus, 24 whereas sea

expression appears to be independent of agr. strains

that are agr-negative are generally low toxin producers.

antigen-presenting cell

peptide

T cell antigen

receptor

HLA-DR

T lymphocyte

SEB

HLA-DR

SEB

TCR

TCR-[HLA-DR]

TCR-[HLA-DR]-SEB

Fig. 14-1. molecular model of receptor binding. staphylococ-

cal enterotoxins and other bacterial superantigens target the

multireceptor communication between t cells and antigen-

presenting cells that is fundamental to initiating pathogen-

specific immune clearance. the superantigen inserts itself

between the antigen receptor of t cells and the class ii major

histocompatibility complex molecule displaying peptides

from potential pathogens. toxin exposure results in hy-

peractivation of the immune system, and the pathology is

mediated by tumor necrosis factor-α, interferon-γ, and other

cytokines.

hla-DR: human leukocyte antigen DR

seb: staphylococcal enterotoxin b

tcR: t cell receptor

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Medical Aspects of Biological Warfare

however, there are also considerable differences in

production levels among agr-positive isolates. in ad-

dition, a feedback-mediated regulatory mechanism for

increasing expression of seb and tsst-1 and suppress-

ing all other exotoxins has been demonstrated. 25

at the cellular level, the interaction of superantigens

with receptors on antigen-presenting cells and t cells

leads to intracellular signaling. 26 high concentrations

of seb elicit phosphatidyl inositol production and

activation of protein kinase c and protein tyrosine ki-

nase pathways, 26–28 similar to mitogenic activation of t

cells. ses also activate transcription factors nF-κb and

aP-1, resulting in the expression of proinflammatory

cytokines, chemokines, and adhesion molecules. both

interleukin-1 and tumor necrosis factor-α can directly

activate the transcription factor nF-κb in many cell

types, including epithelial cells and endothelial cells,

perpetuating the inflammatory response. another

mediator, interferon-γ, produced by activated t cells

and natural killer cells, synergizes with tumor ne-

crosis factor-α and interleukin-1 to enhance immune

reactions and promote tissue injury. the substances

induced directly by seb and other superantigens—

chemokines, interleukin-8, monocyte chemoattractant

protein-1, macrophage inflammatory protein-1α, and

macrophage inflammatory protein-1β—can selectively

chemoattract and activate leukocytes. thus, cellular

activation by seb and other superantigens leads to

severe inflammation, hypotension, and shock. addi-

tional mediators contributing to seb-induced shock

include prostanoids, leukotrienes, and tissue factor

from monocytes; superoxide and proteolytic enzymes

from neutrophils; tissue factor; and chemokines from

endothelial cells. activation of coagulation via tissue

factor leads to disseminated intravascular coagulation,

tissue injury, and multiorgan failure. se-induced tss

thus presents a spectrum and progression of clinical

symptoms, including fever, tachycardia, hypotension,

multiorgan failure, disseminated intravascular coagu-

lation, and shock.

Given the complex pathophysiology of toxic shock,

the understanding of the cellular receptors and signal-

ing pathways used by staphylococcal superantigens,

and the biological mediators they induce, has provided

insights to selecting appropriate therapeutic targets.

Potential targets to prevent the toxic effects of ses

include ( a ) blocking the interaction of ses with the

mhc, tcRs, 26 or other costimulatory molecules 29–32 ;

( b ) inhibition of signal transduction pathways used

by ses 26 ; ( c ) inhibition of cytokine and chemokine

production 33,34 ; and ( d ) inhibition of the downstream

signaling pathways used by proinflammatory cyto-

kines and chemokines.

most therapeutic strategies in animal models of

seb-induced shock have targeted proinflammatory

mediators. therapeutic regimens include corticoste-

roids and inhibitors of cytokines, caspases, or phos-

phodiesterases. although several clinical trials of

treatment of sepsis with high-dose corticosteroids were

unsuccessful, a multicenter clinical trial using lower

doses of corticosteroids for longer periods reduced

the mortality rate of septic shock. 35 a newer interven-

tion targeting the coagulation pathway by activated

protein c improved the survival of septic patients

with high aPache (acute Physiology and chronic

health evaluation, a system for classifying patients

in the intensive care unit) score. 36 because coagulation

and endothelial dysfunction are important facets of

seb-induced shock, activated protein c may also be

useful in treating tss.

limited therapeutics for treating superantigen-

induced toxic shock are currently available. intrave-

nous immune globulin was effective as a treatment

in humans after the onset of tss. antibody-based

therapy targeting direct neutralization of seb or other

superantigens represents another form of therapeu-

tics, most suitable during the early stages of exposure

before cell activation and the release of proinflamma-

tory cytokines. because some neutralizing antibodies

cross-react among different superantigens, 8 a relatively

small mixture of antibodies might be effective in treat-

ing exposures to a greater variety of superantigens.

Vaccines of seb and sea with altered critical residues

involved in binding class ii mhc molecules were also

used successfully to vaccinate mice and monkeys

against seb-induced disease. 37,38

PATHOGENESIS

Rhesus macaques ( Macaca mulatta ) have been used

extensively as a model for lethal disease caused by

inhaled seb. Rabbits, endotoxin-primed mice, and ad-

ditional animal models have been developed. because

seb and related toxins primarily affect primates, the

following unpublished rhesus monkey data are highly

relevant for understanding potential human pathol-

ogy. Young and mature adult male and female rhesus

monkeys developed signs of seb intoxication 39 after

being exposed to a lethal dose of aerosolized seb for

10 minutes in a modified henderson head-only aerosol

exposure chamber. 40 these animals demonstrated no

detectable anti-seb antibody before exposure. after

inhalation exposure, microscopic lymphoproliferation

of t-cell–dependent areas of the lymphoid system,

consistent with the potent stimulatory effect of seb

314

Staphylococcal Enterotoxin B and Related Toxins

on the rhesus monkey immune system, was appar-

ent. immunohistochemical analysis, using anti-cD3

antibody, of the large lymphocytes present in the pul-

monary vasculature of the monkeys identified these

lymphocytes as t cells. 41

Generally, the seb-intoxicated rhesus monkeys de-

veloped gastrointestinal distress within 24 hours post-

exposure. clinical signs were mastication, anorexia,

emesis, and diarrhea. after mild, brief, self-limiting

gastrointestinal signs, the monkeys had a variable

period of up to 40 hours of clinical improvement. at

approximately 48 hours postexposure, the monkeys

generally had an abrupt onset of rapidly progressive

lethargy, dyspnea, and facial pallor, culminating in

death or euthanasia within 4 hours of onset.

at necropsy, most of the monkeys had similar gross

pulmonary lesions. the lungs were diffusely heavy

and wet, with multifocal petechial hemorrhages and

areas of atelectasis. clear serous-to-white frothy fluid

often drained freely from the laryngeal orifice. the

small and large intestines frequently had petechial

hemorrhages and mucosal erosions. typically, the

monkeys had mildly swollen lymph nodes, with moist

and bulging cut surfaces.

most of the monkeys also had similar microscopic

pulmonary lesions. the most obvious lesion was

marked multifocal to coalescing interstitial pulmonary

edema involving multiple lung lobes. Peribronchovas-

cular connective tissue spaces were distended by pale,

homogeneous, eosinophilic, proteinaceous material

(edema), variably accompanied by entrapped, beaded

fibrillar strands (fibrin), extravasated erythrocytes,

neutrophils, macrophages, and small and large lym-

phocytes. Perivascular lymphatics were generally

distended by similar eosinophilic material and inflam-

matory cells. most of the monkeys had intravascular

circulating and marginated neutrophils, monocytes,

mononuclear phagocytes, and lymphocytes, including

large lymphocytes with prominent nucleoli (lympho-

blasts), some in mitosis (Figure 14-2). extravascular

extension of these cell types was interpreted as exo-

cytosis/chemotaxis.

loss of airway epithelium was inconsistent. some

monkeys had multifocal, asymmetric denudation of

bronchial epithelium, with near total loss of bronchiolar

epithelium. Former bronchioles were recognized only

by their smooth muscle walls. scant bronchial intralumi-

nal exudate consisted of mucoid material, neutrophils,

macrophages, and sloughed necrotic cells.

a common finding was multifocal alveolar flood-

ing and acute purulent alveolitis. alveolar septa

were distended by congested alveolar capillaries.

alveolar spaces were filled with pale, homogeneous,

eosinophilic material (edema), with deeper embedded

eosinophilic beaded fibrillar strands (fibrin), or with

condensed, curvilinear, eosinophilic deposits hugging

the alveolar septal contours (hyaline membranes). a

variably severe cellular infiltrate of neutrophils, eosino-

phils, small lymphocytes, large lymphocytes (lympho-

blasts), erythrocytes, and alveolar macrophages filled

alveolar spaces. Replicate pulmonary microsections

stained with phosphotungstic-acid–hematoxylin

demonstrated alveolar fibrin deposition. Replicate

microsections stained with Giemsa revealed scarce

sparsely granulated connective-tissue mast cells.

in the upper respiratory tract, the tracheal and

bronchial lamina propria was thickened by clear

space or pale, homogeneous, eosinophilic material

(edema), neutrophils, small and large lymphocytes,

and (possibly preexisting) plasma cells. the edema

and cellular infiltrate extended transtracheally into the

Fig. 14-2. lung of a rhesus monkey that died from inhaled

staphylococcal enterotoxin b. ( a ) marked perivascular

interstitial edema and focal loss of bronchial epithelium

can be seen (hematoxylin-eosin stain, original magnifica-

tion x 10). ( b ) the intravascular mononuclear cells include

lymphocytes, lymphoblasts, monocytes, and mononuclear

phagocytes (hematoxylin-eosin stain, original magnifica-

tion x 50).

315

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