anthrax toxin
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1040-841X/01/$.50© 2001 by CRC Press LLC
Critical Reviews in Microbiology, 27(3):167–200 (2001)
Anthrax Toxin
Rakesh Bhatnagar* and Smriti Batra**
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India
* Corresponding author: Prof. Rakesh Bhatnagar, Centre for Biotechnology, Jawaharlal Nehru University,New Delhi 110067, India. Phone (91) 11 6179751: Fax (91) 6165886/ 6198234/ 6169962; Email:[email protected] and [email protected]
** Dr. Smriti Batra, Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.Phone: (91) 11 6179751; e-mail:[email protected]
ABSTRACT: Anthrax is primarily a disease of herbivores caused by Gram-positive, aerobic,spore-forming Bacillus anthracis. Humans are accidental hosts through the food of animal originand animal products. Anthrax is prevelant in most parts of the globe, and cases of anthrax havebeen reported from almost every country. Three forms of the disease have been recognized:cutaneous (through skin), gastrointestinal (through alimentary tract), and pulmonary (by inhala-tion of spores).
The major virulence factors of Bacillus anthracis are a poly-D glutamic acid capsule and athree-component protein exotoxin. The genes coding for the toxin and the enzymes responsiblefor capsule production are carried on plasmid pXO1 and pXO2, respectively. The three proteinsof the exotoxin are protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor(EF, 89 kDa). The toxins follow the A-B model with PA being the B moeity and LF/EF, thealternative A moeities. LF and EF are individually nontoxic, but in combination with PA formtwo toxins causing different pathogenic responses in animals and cultured cells. PA + LF formsthe lethal toxin and PA + EF forms the edema toxin. During the process of intoxication, PA bindsto the cell surface receptor and is cleaved at the sequence RKKR (167) by cell surface proteasessuch as furin generating a cell-bound, C-terminal 63 kDa protein (PA63). PA63 possesses abinding site to which LF or EF bind with high affinity. The complex is then internalized byreceptor-mediated endocytosis. Acidification of the vesicle leads to instertion of PA63 into theendosomal membrane and translocation of LF/EF across the bilayer into the cytosol where theyexert their toxic effects. EF has a calcium- and calmodulin-dependent adenylate cyclase activity.Recent reports indicate that LF is a protease that cleaves the amino terminus of mitogen-activatedprotein kinase kinases 1 and 2 (MAPKK1 and 2), and this cleavage inactivates MAPKK1 andthus inhibits the mitogen-activated protein kinase signal transduction pathway. We describe indetail the studies so far done on unraveling the molecular mechanisms of pathogenesis of Bacillusanthracis.
I. INTRODUCTION
Anthrax has been both a scrouge and afundamental model for infectious diseasestudies for over a century. Anthrax has oftenbeen intertwined with human history. It isbelieved to have been one of the Egyptianplagues in the time of Moses. There are de-
scriptions of anthrax involving both animalsand humans in the early literature of Hindusand Greeks. In 1752, Moret more specifi-cally characterized the disease in man, call-ing it the ‘malignant pustule.’ In 1780,Chabert described the disease in animals.The contagious nature of anthrax was notedin 1823 by Barthelemy. The first microscopic
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description of the organism was written byDelafond in 1838, and the organism wasfirst described in infected animals in 1849by Pollender. The anthrax system also holdsan eminent position in the development ofmodern germ theory and in our understand-ing of the host-parasite equation. The an-thrax bacillus was the model first used in thedevelopment of Koch’s postulates and there-fore is sometimes considered to be mankind’sfirst proven ‘germ’. Soon after Koch’s dis-covery, Pasteur generated, by growing thebacilli cultures at 42oC, a capsule-null an-thrax strain that was used as the first live,attenuated bacterial vaccine.1 In 1939, Sternereported his development of an animal vac-cine that is a spore suspension of an aviru-lent, noncapsulated live strain. The anthraxmodel has also played an important role inthe birth of cellular immunology research.Metchnikoff2 (1905), using transparent tis-sues of living animals attached to his micro-scope stage, employed the anthrax bacilli toshow that his newly discovered large bloodcells exited the circulatory system (diaped-esis), migrated toward the bacilli (chemot-axis), and ingested the virulent organisms(phagocytosis). He called these unique cellsmacrophages.
The disease anthrax is caused by Bacil-lus anthracis, a large Gram-positive bacil-lus. Synonyms for anthrax include malig-nant pustule, Siberian ulcer, malignantedema, black bane, Bradford disease,woolsorter’s disease, and ragpicker’s disease.Humans, all mammals, and several bird, rep-tile, and amphibian species are susceptibleto varying degrees. Domestic livestock andwild herbivores, for example, elephants andhippopotamuses are especially vulnerable.3
Human cases arise due to contact with theinfected animals or animal products (i.e.,wool, hide, hair, bone, and skin), by con-suming undercooked contaminated meat, orby inhalation of airborne spores. Anthrax isnearly always a fatal infection, the victim
succumbing with nonspecific, shock likesymptoms. Timely diagnosis of the diseasemay be difficult due to non-acute, ‘flu-like’symptoms in systemic anthrax, and the firstovert sign of the disease in animals is oftendeath itself.4,5
Anthrax is of continuing worldwide im-portance. It remains prevelant in many lesswell-developed parts of the world. In wildanimals, which for obvious reasons are dif-ficult to vaccinate, anthrax is a major causeof mortality and a threat to endangered spe-cies in enzootic areas.6,7 B. anthracis hasacquired some notoriety as a potential agentof biological warfare,8, 9and the Sverdlovskincident in 197910 became the stimulus for aconsiderable research thrust into numerousaspects of the disease in the 1980s. The GulfWar in 1991 heightened the awareness of thepossibility that B. anthracis could be used asa biological weapon11 and served as a re-newed stimulus to anthrax research. Also,the potential applications of anthrax toxin indelivery system for novel antigens has en-couraged interest in the study of this patho-gen.12,13,14,15,16,17
II. ANTHRAX AND BACILLUSAnthracis
A. Epidemiology
Cases of anthrax have been reported fromalmost every country. In 1958, Glassmanestimated the worldwide incidence in be-tween 20,000 to 100,000 cases. In the 1980sthe estimate decreased to approximately 2000cases annually.
Industrial cases occur primarily in Euro-pean and North American countries and areassociated with the processing of animalmaterials such as hair, wool, hides, and bones.Agricultural cases occur primarily in Asiaticand African countries and result from con-tact with diseased domestic animals or their
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products such as hair, wool, hides, bones, orcarcasses, including meat.
Several unusual epidemics have beenreported within the last 20 years. The largestepidemic in modern times occurred in Zim-babwe, where between 1979 and 1985 ap-proximately 10,000 human cases were re-ported.18 An unusual epidemic occurred inSverdlovsk, Russia, in 1979. Following anaccidental explosion in a military labora-tory, 42 human cases of inhalation anthrax(and probably more than 90 cases) occurredamong people exposed to an aerosol con-taining B. anthracis organisms.19 In the U.S.,the earliest reports of animal anthrax werefrom Louisiana in the early 1700s. Subse-quently, sporadic animal cases have beenreported from almost every state. Areas withmore regularly reported cases primarily in-clude the Great Plain States. Human anthraxwas first reported from Kentucky in 1824.Subsequently, human cases have been re-ported throughout the U.S., with the major-ity from industrialized states in the north-east.20, 21
In Europe, the major afflicted regionscontinue to be Turkey and Greece. How-ever, significant numbers of outbreaks stillregularly occur in Spain, Albania, Italy, andRomania. Central Spain suffers a quite ex-traordinary number of human cases each year— from 152 in 1990 to 50 in 1996.
In Australia sporadic outbreaks continueto occur annually in New South Wales andVictoria. In England 145 cases of humananthrax were reported between 1961 and1980, and 10 cases between 1980 to 1990.22
In Russia, in the early beginning of the cen-tury about 40 to 60,000 cases were reportedin agricultural animals and 10 to 20,000 hu-man cases annually.23 In China, anthrax isstill one of the most serious infectious dis-eases because of its wide-ranging distribu-tion.
Africa remains severely afflicted. WestAfrica is still the largest region in the world
with hyper-endemic and epidemic anthrax.The outbreak in Kruger National Park, SouthAfrica,6 and Etosha National Park, Nambia,between 1984 to 1989 killing thousands ofwild animals24 serves as a salutary reminderthat anthrax remains a potential danger inmany parts of the world.
In Philippines four outbreaks were re-corded involving 23 animals, while in Thai-land six outbreaks were recorded in animalsalongwith 148 human cases. In Bhutan thedisease is prevelant both in animals and hu-mans. In Bangladesh there were 240 out-breaks during the year 1996. In Nepal since1992 until 1997 sporadic outbreaks have beenrecorded in cattle, pigs, sheep, and horses.There were 19 outbreaks of anthrax during1996 and a total of 222 animals were af-fected.25
Animal anthrax is endemic and wide-spread in India. During 1991 to 1996 therewere 1613 outbreaks of anthrax recorded inanimals, and the mortality was 62.5% ofwhich 20% was in cattle and buffalo and80% in sheep and goats (Report on ‘Inci-dence of anthrax in India during 1991 to1996 by the Disease Surveillance Unit of theDepartment of Animal Husbandry and Dairy-ing, Ministry of Agriculture, Government ofIndia’). The disease is more prevelant incoastal areas of West Bengal, Orissa, andTamil Nadu.
B. Bacteriology
Few microbial pathogens have had asgreat an impact on the development of thescience of medical bacteriology as Bacillusanthracis. It was from studies of this etio-logic agent of anthrax, during the mid-1800sthat many of the fundamental concepts weredeveloped on which our present understand-ing of infection and immunity are based.
The anthrax bacillus is one of the largestpathogenic bacteria and ranges from 3 to 5
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µm in length and 1 to 1.5 µm in breadth. Theends of the rods are often concave and some-what swollen so that the appearance of achain of anthrax bacillus has been comparedto a jointed bamboo rod. The cells occursingly and as end-to-end pairs or shorts chainsin the body, but in culture long chains areformed. Bacillus anthracis is a Gram-posi-tive, sporulating, capsulated, nonmotile, cy-lindrical bacillus. It is not an obligate aero-bic pathogen and is capable of free living inmany environments. Like most other Bacil-lus sp. B. anthracis is a saprophyte, able tosurvive in soil, air, water, and vegetation.
Capsules may be found on the bacilli insmears from an infected animal and capsuleproduction in vitro is enhanced in the pres-ence of elevated (5% or greater) CO2 andadded bicarbonate or serum.26 The capsularmaterial is a high-molecular-weight polypep-tide composed exclusively of D-glutamicacid.27,28 This is a point of particular interest,for it was the first demonstrated natural oc-currence of D-glutamic acid and of a polypep-tide composed of a single amino acid. In theabsence of the capsule, the cell wall ofB. anthracis appears layered. These layersare composed of fragments displaying ahighly patterned ultrastructure.29,30 This typeof cell surface is referred to as the surfacelayer or S-layer. It is found ubiquitously beingpresent on the surface of many archaea andeubacteria.31 The interaction between the cellwall and the S-layer proteins is very strong,although not covalent. B. anthracis simulta-neously synthesizes two S-layer proteins,named Extracellular Antigen 1 (EA1) andSurface array protein (Sap),32,33 each con-taining three S-layer homology (SLH) mo-tifs toward their amino-terminus.34 It has beenproposed that the three motifs of each pro-tein are organized as a structural domainsufficient to bind purified cell walls.35 Thecapsule, when present, is exterior to, andcompletely covers, the S-layer proteins,which form an array beneath it. Neverthe-
less, the presence of these proteins is notrequired for normal capsulation of the ba-cilli. Thus, both structures are compatible,and yet neither is required for the correctformation of the other. Therefore, Bacillusanthracis has a very complex cell wall orga-nization for a Gram-positive bacterium.
The anthrax bacillus differs from mostother aerobic pathogenic bacteria in that itforms spores that are visible as refractilebodies, either free or located centrally withinthe cell. Spores are formed most abundantlyat 32 to 35oC and only under aerobic condi-tions. Germination of the spores is usuallypolar. These spores are highly resistant toheat and many disinfecting agents and maysurvive in the environment for decades. VanNess36 hypothesized that ‘suitable soils canmaintain an organism-spore-organism cyclefor years without infecting livestock’ and‘factors such as temperature and balance ofmoisture in the soil are prerequisites foroutbreaks of the disease. These factors wouldhave little influence on the static anthraxspore.’ Many reports have confirmed thishypothesis. Under favorable conditions thespores germinate to produce vegetative bod-ies. Moisture and organic matter in soil prob-ably encourage the germination of anthraxspores. Spores can be killed in 10% formalinat 40oC in 15 min.9
The colonies of anthrax bacillus are large,rough, gray-white, and have a curled or hair-like structure, giving a ‘Medusa head’ ap-pearance. In the presence of high concentra-tions of carbon dioxide, the organisms formcapsules and colonies are smooth and mu-coid.
C. Pathogenesis
1. Pathogenicity for Lower Animals
In nature anthrax is primarily a diseaseof cattle and sheep; horses and swine are
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susceptible but are less commonly affected.Wild deer and other gregarious herbivoraare liable to occasional outbreaks. Thesmaller rodents are very sensitive to inocu-lation. Rabbits, guinea pigs, and white miceare susceptible in that order and are fatallyaffected by the subcutaneous introduction ofa very small number of virulent bacilli. Car-nivorous animals, although possessinggreater resistance than the herbivora, are nev-ertheless susceptible, as several epidemicsin zoological gardens involving leopards,lions, pumas, bears, and other animals haveshown. Certain animals possess a markednatural resistance to anthrax. Most rats arequite resistant37 especially the white rat. Themature dog is only slightly susceptible. Birds,especially pigeons, can be infected, but noteasily. Frogs are completely resistant, buttoads are very susceptible.
The route by which the bacilli enter thebody exerts an important influence in bothexperimental and natural infections. Becauseanimals usually acquire anthrax by the in-gestion of contaminated substances (feed,grass, water, or infected carcasses), theysuffer most commonly from the gastrointes-tinal form of the disease. Cutaneous anthraxcan occur, however, through the bite of blood-sucking flies. In highly susceptible animalsthe disease is acute and runs a rapid course;the case fatality in cattle and sheep is about80%. It presents all the characteristics oftypical septicemia, and local manifestationsmay be almost entirely absent. Enormousmultiplication of the bacteria takes place inthe blood and internal organs. The spleen isdeep red in color and greatly enlarged, hencethe name splenic fever. The more resistantanimal species do not develop this general-ized infection, but the bacteria remain local-ized in an abscess or carbuncle and fail tospread through the body.
When acquired via the skin, throughwounds from biting flies, the disease usuallyprogresses to death before eschars, car-
buncles, or other localized manifestationscan develop, but in less rapid cases cutane-ous or subcutaneous lesions with edematousswellings may occur.
2. Pathogenicity for Man
The Bacillus is almost always transmittedto man from lower animals rather than otherhuman beings. However, in an epidemic ofcutaneous anthrax in Gambia, there is sugges-tive evidence that human to human transmis-sion was possible.38 Human anthrax is usuallyclassified by portal of entry into the host: cuta-neous (> 95% of cases), gastrointestinal, andinhalational. In the infectious form, the spore,it enters the body and is thought to germinatewithin macrophages either at the site of inocu-lation (cutaneous or gastrointestinal) or in theregional lymph node (inhalational). The Bacil-lus then synthesizes its antiphagocytic capsuleand the lethal and edema toxins that interferewith nonspecific host defenses leading to thecharacteristic locally destructive lesion andspread by lymphatics to the systemic circula-tion and other organs.
The cutaneous form begins as a papulethat progresses over several days to a vesicleand then ulcerates. There is often edema,sometimes massive, probably due to theedema toxin that surrounds the lesions thatthen develop a characteristic black eschar(hence the name ‘anthrax’, which in Greekmeans ‘coal’). The patient may be febrilewith mild to severe systemic symptoms ofmalaise, headache, and toxicity. Oropha-ryngeal anthrax presents with severe sorethroat or an ulcer in the oropharyngeal cav-ity associated with neck swelling, fever,vomiting, and abdominal pain, which maybe similar to an acute abdomen. There maybe diarrhoea and ascites, both of which maybe hemorrhagic.39 In severe forms, generaltoxemia with shock, sepsis, and death mayoccur.
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Inhalation anthrax begins with nonspe-cific symptoms of malaise, fever, myalgia,and nonproductive cough. After a period of2 to 3 days, this is followed by sudden onsetof severe respiratory distress associated withdiaphoresis, cyanosis, and increased chestpain. There may be a widened mediastinumand pleural effusions on chest X-ray. Deathfollows in 24 to 36 h from respiratory fail-ure, sepsis, and shock.22,40
In about 5% of cases of cutaneous an-thrax, meningitis, develops as a sequel, but itcan arise from any of the forms of the dis-ease. The clinical signs of meningitis and theappearance of blood in the cerebrospinal fluidare followed rapidly by a loss of conscious-ness and death. The prognosis in this form ofanthrax is grave.
For many years there was a great confu-sion concerning the pathogenesis of anthraxinfections. It was thought that death was dueto blockage of capillaries caused by exten-sive bacteremia (log-jam theory).41 However,it was Smith and Keppie42 who identified thechemical basis of virulence of B. anthracis.They showed that sterile plasma from ex-perimentally infected guinea pigs was lethalafter being injected into other animals andopened a new era in the investigations ofanthrax.43,44
3. Diagnosis
The bacilli can be seen in the stainedsmears of the exudate. The cells form longchains giving a bamboo-like appearance withGram’s stain. The organism is differentiatedfrom other Bacillus sp. by the absence ofhemolysis, motility, growth on phenylethylalcohol blood agar, gelatin hydrolysis, andsalicin fermentation.45 It is also unusual inhaving an antiphagocytic capsule made ofD-glutamic acid. Bacterial identification isconfirmed by the production of toxin anti-gen, lysis by a specific bacteriophage, detec-
tion of capsule by fluorescent antibody, andvirulence for mice and guinea pigs.
4. Genetic Basis of Virulence
The two major virulence factors of Ba-cillus anthracis are the tripartite toxin andthe polyglutamate capsule. The capsule playsan important role in the establishment ofinfection by protecting the Bacillus againstphagocytosis and other bactericidal compo-nents of host sera. The bacilli that lose theirability to produce the capsule are avirulent.46
B. anthracis produces two protein exo-toxins: edema toxin and lethal toxin. Bothedema toxin and lethal toxin follow the gen-eral model for many protein toxins in pos-sessing a binding or B component respon-sible for binding to receptors on target cellsand an active or A component responsiblefor the toxin’s biochemical activity. How-ever, the anthrax toxins are unusual in tworespects. First, the B and A domains arelocated on separate noncovalently linkedproteins. Second, the edema and lethal tox-ins share the identical B protein, a situationunique to anthrax. The B protein is called theprotective antigen (PA: 83 kDa). This pro-tein when given together with a second pro-tein, Edema factor (EF: 89 kDa), comprisesthe edema toxin. The same B protein, PA, inconjunction with a third protein, lethal fac-tor (LF: 90 kDa) constitutes the lethal toxin.Although these three factors have come tobe equated with anthrax toxins, it is note-worthy that toxin produced in vivo differsfrom that produced and purified from cul-tures of B. anthracis in vitro in that it causesdeath more rapidly.47,48 Thus, additional fac-tors produced during the course of infectionmay enhance the lethal effects of anthraxtoxins. The three proteins of the exotoxinwere initially designated as factor I (foredema factor), factor II (for protective anti-gen), and factor III (for lethal factor).
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It was reported by Smith and Stoner in196749 that the three toxin components to-gether were more lethal and caused moreserious edemas than either PA + LF or PA+EF. Although these data were obtained withproteins presumably difficult to purify fromeach other, these results were confirmed byPezard et al.50 It is possible that lethal andedema toxins are synergistic and can coop-erate at the cellular level or modify the inter-action of bacteria with the host.
The three proteins of the toxin are en-coded by plasmids pXO1(184 kb) and thepolyglutamate capsule is encoded by pXO2(97 kb). Comparison of parental and plas-mid cured strains proved that these plasmidencode toxin51 and capsule,52, 53 respectively,and that they are required for virulence.51
Both plasmids have been physically charac-terized and mapped.54,55 The large nature ofthe virulence plasmids suggests that theyharbor other pathogenecity genes, but nonehas been cited outside those responsible forthe expression of toxin and capsule. Mutantstrains, each deficient in the production ofone toxin component, were constructed byPezard et al.50 It was found that the survivalof the strains at the inoculum site in micewas similar during the initial period of infec-tion regardless of the expression of toxins.Thus, it was inferred that pXO1-encodedvirulence factors play a minor role, if any, inlocal bacterial survival, implying that thereare as-yet-unidentified chromosomal genesrequired for survival in host tissues.
The genes encoding the toxin proteins,pag A (previously known as pag) (whichencodes PA), cya (which encodes EF), andlef (which encodes LF), are located non-contiguously within a 30-kb region ofpXO1.56,57,58,59,60,61 As expected, due to thepivotal role of PA in intoxication, the pag Agene is highly expressed. When B. anthracisis cultured under optimal conditions, culturesupernatants contain up to 20 mg of PA perliter, while LF and EF are present at 5 and 1
mg/l, respectively.62 Analysis of toxin geneexpression employing reporter gene fusionsuggests that the steady-state level of mRNAof pag A is four-fold higher than that of lefand 14-fold higher than that of cya.15
The cap region, which is essential forencapsulation, is located on pXO2.52,53,63,64,67
The cap region contains three open readingframes: cap B, cap C, and cap A, arranged inthat order.64 These genes are all transcribedin the same direction. The products encodedby the cap are predicted to be membrane-associated enzymes that mediate synthesisof the capsular polypeptide.64 Furthermore,the dep gene, which is associated with depo-lymerization of the capsular polymer, wasfound to be downstream of the cap regionand have the same direction of transcrip-tion.60 The cap B, cap C, cap A, and depgenes constitute an operon that is transcribedas a single mRNA.
Many bacterial pathogens have devel-oped genetic mechanisms to regulate bio-synthesis of their virulence factors.66 Selec-tive pressures will favor mechanisms thatlimit the energetically expensive synthesisof virulence factors to periods during whichthe pathogen is growing in host tissues.B. anthracis appears to be an example ofsuch a pathogen, because synthesis of thethree-component anthrax toxin and of thepoly-D-glutamate capsule requires the pres-ence of bicarbonate, a metabolite present inliving tissues. Both pXO1 and pXO2 appearto have genes encoding a bicarbonate-sens-ing mechanism, because strains cured of ei-ther plasmid still show bicarbonate-depen-dant regulation of the virulence factorencoded by the remaining plasmid.64, 67 In B.anthracis the CO2 effect is specific for tran-scription, vital for expression, and not merelydue to increased anaerobiosis.15,56,67 ThisCO2-dependant regulation is mediated by thetranscriptional activators atx A (anthrax toxinactivator) encoded by pXO1 for the toxingenes 60,68,69 and acpA, encoded by pXO2,
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for cap B 70. Green et al.52 reported that theCO2 concentration required for capsule syn-thesis by pXO2-containing strain increasesfrom 5 to 20% in the absence of pXO1. AtxA protein has sequence similarity with theacpA protein.70 Thus, the two activators canfunctionally substitute for each other in theregulation of their corresponding genes.71 Itwas shown by Uchida et al.71 that the expres-sion of genes encoding capsule synthesis isactivated not only by acpA but also atxA. Noevidence was found to suggest that acpAactivates pag expression.
B. anthracis strains carrying transposoninsertions at the atxA site are greatly de-creased for overall virulence factor expres-sion and cannot be stimulated by addition ofCO2 to the growth medium. The atxA genehas been cloned independantly by two groupsfrom pXO1, sequenced, and, when suppliedin trans, shown to rescue the CO2-nonre-sponsive phenotype.56,60,68 From the deducedsequence, the atxA protein is 476 amino acidslong with a predicted relative molecular massof 55,673.60 The atxA gene maps betweencya and pag. The atxA protein is hypoth-esized to be the trans-activating, positive-regulator, ‘DNA-interacting’ moiety of a two-component regulatory system having CO2 asits molecular environmental signal. No ana-logue for a corresponding CO2 ‘sensor’moiety of a two-component system has yetbeen identified. The atxA protein is withoutsignificant homology to other DNA binding/activator families. It may represent a newclass of transcriptional activators.72 Studiesindicate that transcription of the atxA geneitself is not affected by CO2. It is possiblethat CO2 affects translation of atxA mRNA,function of the atxA protein, and/or expres-sion of other gene products that may play arole(s) in toxin regulation.68 An atxA nullmutant does not even produce detectablelevels of PA, LF, and EF and these mutantshave been demonstrated to be avirulent inmice.68
It was shown by Hoffmaster andKoehler73 that a 300-bp gene located down-stream of pag is cotranscribed with pag Aand represses expression. This gene wasdesignated as pag R (protective antigen re-pressor). Incomplete transcription termina-tion near the 3′ end of an inverted repeatsequence downstream of pag A results inmono- and bicistronic transcripts containingpag A mRNA. There is no evidence thatattenuation at this site is regulated. Expres-sion of pag R appears to mimic pag A ex-pression.
Studies with lac Z transcriptional markerfusions to the toxin gene, along with directmeasurements of the toxin protein levels,show a coordinate regulation by atxA that isbelieved to be relevant to in vivo condi-tions.15,56,60,68,74 In the specific case of theprotective antigen gene,56 two major tran-scriptional start sites were mapped at posi-tions –58 and –26 (named ‘P1’ and ‘P2’,respectively). RNA analysis showed equiva-lent but low constitutive expression fromboth promoters under noninducing growthconditions. The presence of CO2, however,greatly increased initiation of transcriptionsolely from P1 (-58) promoter. Further sup-port for P1 as the atxA-relevant site is givenin experiments where atxA-null strains areshown to be decreased in transcription onlyfrom the P1 promoter.68 In addition of thesestudies, deletion analysis of regions upstreamof the protective antigen gene indicate thatonly 111bp 5′ to the P1 promoter are re-quired for proper activation by atx A.68 Thepresence of two promoters suggests a modelof differential regulation of pag. The paggene may be highly regulated during differ-ent stages of infection, and the relative con-tribution of the two promoters may change.B. anthracis may encounter various CO2
concentrations in various tissues. Moreover,the accumulation of acid during bacterialgrowth may cause a change in the normalCO2 concentrations in host tissues. It is pos-
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sible that steady state levels of transcriptfrom P2 remain relatively low, or that thispromoter is also induced in response to someother environmental signal.56
There are reports describing the differ-ence in the susceptibilities of warm- andcold-blooded animals to anthrax. As tem-perature is not a critical factor for germina-tion, it was hypothesized that it could beimportant for toxin production.15 The amountof PA produced increases four to sixfoldwhen cells are grown in CO2/bicarbonatemedium at 37oC when compared with growthat 28oC.15 The levels of atxA mRNA in-crease in cultures grown at 37oC relative to28oC. However, overproduction of atxA doesnot result in elevated PA synthesis whencells are grown at either temperature, furtherindicating that PA expression at 37oC cannotbe attributed directly to increased atxA ex-pression at high temperatures. Thus, addi-tional regulatory factors may be involvedand identification of such regulatory factor(s)will lead to increased understanding of themechanism by which B. anthracis sensesand responds to environmental cues that trig-ger virulence gene expression.75
III. THE TOXIN COMPONENTS
A. Protective Antigen
Protective antigen (PA), previously re-ferred to as factor II, is the most extensivelycharacterized component of anthrax toxin. Itwas the first component to be identified,76
the first to be cloned,61 and sequenced,77 andit remains the one most easily produced inlarge amounts.78,79 It is now recognized asthe central component of the anthrax toxin.PA-null Bacillus strains are greatly attenu-ated for virulence.80 The gene for PA is en-coded at the pag locus on the plasmid PXO1(formerly known as pBA1).51 A library ofpXO1 restriction fragments was constructed
in pBR322, and the resulting E. colitransformants were screened with PA-spe-cific antiserum. Two immunoreactive colo-nies were identified, and these were shownto produce biologically active PA, eventhough the bacteria produced only 5 to 10 ngof PA per millilitre of culture.61 Subsequently,the gene was subcloned and the DNA se-quence determined for a 4235-bp Hind IIIand Bam HI fragment containing pag.77 Thegene was found to contain a 2319-bp-longopen reading frame of which 2205 bp en-code an A/T-rich (69%) cysteine free, 735amino acid (82.7 kDa) secreted protein. These735 amino acids include a 29 residue exportsignal peptide. There are sequence homolo-gies between PA and the Clostridiumperfringens iota — toxin Ib (32% identity)and the Vegetative Insecticidal Protein (VIPI)from Bacillus cereus (27% identity).81,82,83
Both iota-Ib and VIP-1are binary toxins, bindto host cell receptors, are activated by pro-teolytic nicking, and are responsible for de-livering toxic enzymatic counterparts to thecytoplasm of host’s cells. These homologiesare not maintained at the C-terminal domain,the region responsible for cell receptor tro-pism.
For most bacterial toxins, for example,diphtheria toxin, tetanus toxin, neurotoxin,and C2 toxin of Clostridium botulinum, pro-teolytic cleavage is required for toxicity.84 PAhas two sites that are susceptible to cleavageby low concentrations of proteases. Thesesites define three fragments that have distinctfunctions. Treatment with 1 µg/ml chymot-rypsin produces fragments of 37 and 47 kDaby specific cleavage on the carboxyl side ofF314 in the sequence ASFFD315.78
Thermolysin cleaves in the same region, be-tween S312 and F313. The 37- and 47-kDafragments produced by chymotrypsin andthermolysin are the same size as the majordegradation products found in B. anthracisculture supernatants, as expected from thesimilarity of the major protease secreted by
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B. anthracis to thermolysin, a protease iso-lated from B. thermoproteolyticus. The frag-ments produced by cleavage near residues313 to 314 do not immediately dissociate; the‘nicked’ PA behaves like uncleaved PA dur-ing gel filtration or ion-exchange chromatog-raphy. However, the nicked PA is biologi-cally inactive.78,85 To study the role ofchymotrypsin sensitive site of PA in toxicity,residues 313 to 315 FFD were replaced withsequences FFA or AAA.86 These mutants wereas toxic as native toxin, indicating that hydro-phobic residues at this site are not essential.However, a mutated PA in which the two Pheresidues were deleted was nontoxic and PAwith a CFD sequence retained approximately20% of normal activity.
The other site in PA that is highly sen-sitive proteases is the sequence RKKR(167).Treatment with 0.1 µg/ml trypsin causescleavage at one or more of the basic resi-dues in this sequence to produce fragmentsof 20 and 63 kDa.62,78,87 The fragments re-main tightly associated in the nicked toxin,but can be separated by chromatography onmono Q resin at pH 9.0. Many differenttypes of evidence show that cleavage at thissite is a normal and obligatory step in theprocess of toxin action. Cleavage at theRKKR site was found to be biologicallyrelevant when it was discovered that PAbound to eukaryotic cells at 40C becomesnicked at the same site by a cellular pro-tease62,88,85 identified as furin. PA63 formedfrom receptor-bound PA remains bound tothe cells, where as the 20-kDa fragment isreleased to the medium.
Trypsin/ furin cleavage exposes a high-affinity binding site for LF or EF on PA63.88,89
Deletion of the cleavage site rendered PAinactive.88 The deleted PA could bind to thecell receptor, however, it was not cleavedinto fragments of 20 and 63 kDa. It did notsupport binding of radiolabeled LF to cellsand was not toxic to cells when combinedwith LF.
PA63 facilitates the translocation of LFand EF across the endosomal membranesinto the cytosol.90,91,92 The importance ofPA63 in the process of translocation of cata-lytic moeities was revealed when Blausteinet al.87showed that PA63 led to the forma-tion of ion-conductive channels in artificialmembranes when exposed to acidic pH.Neither native PA nor the 20-kDa fragmentis capable of forming ion-conductive chan-nels.87 PA63 could form ion-conductive chan-nels in planar phospholipid bilayers like othertoxins such as diphtheria, tetanus, and botu-linum.93,94 Detailed electrophysiologicalanalysis of the channels shows that they arehighly cation selective, having a 20-fold pref-erence for K+ over Cl–. Quaternary ammo-nium ions bind to a single site in the lumenof the channel, thereby decreasing conduc-tance, but are driven through the channel byhigher voltages.87,93 Comparison ofpermeability to tetrahexylammonium andtetraheptylammonium ions indicates that themembrane channels are large, with a porediameter of about 12 Å.87,93 The channelsformed by diphtheria, tetanus, and botuli-num toxins are also about 12 Å, supportingthe suggestion that these channels constitutethe structure by which the catalytic subunitsof the toxins are translocated throughendosomal membranes to the cytosol.
Channel formation and membrane inter-action by PA63 also occurs in liposomes94,95
and in intact cultured Chinese Hamster Ovarycells.96 This interaction of PA63 with liposo-mal membranes occurs at a pH of approxi-mately 6.0 and increases as the pH is low-ered, at least through pH 4.7. PA63 causesthe release of K+ but not calcein from singlewall asolectin vesicles; release requires a pHgradient with the external, toxin containingsolution pH 6.0.94 Retention of calcein showsthat PA63 does not disrupt the integrity ofthe vesicle membrane, and that the K+ con-ductive channels formed have diameters ofless than or equal to 12 Å.94
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Proteolytic cleavage and removal of PA20from PA63 is not only required for binding ofEF and LF but also for the formation of PA63oligomers. The primary sequence of PA re-veals no hydrophobic regions that might bepredicted to be involved in membrane-relatedfunctions. One mechanism by which a rela-tively hydrophilic protein such as PA mightinsert stably into a membrane is to form anoligomeric structure. Small patches of hydro-phobic structure exposed in each monomercan potentially be multiplied through oligo-merization to generate a hydrophobic surfaceof significant area for interaction with thehydrophobic core of the bilayer.97 Electronmicroscopy showed that the oligomer formedin vitro is predominantly a heptamer (Figure1) with an outer diameter of 10.4 nm.98 PAinternalised by cells or exposed to low pH onthe cell surface produces oligomers that sur-vive heating in sodium dodecyl sulfate(SDS).98 The finding that nicked PA formsoligomers in solution at acidic pH, togetherwith the fact that the PA20 fragment blocksoligomerization of PA63, suggests that acidicconditions induce dissociation of PA20 fromPA63. In vivo, PA would encounter acidicconditions only after being endocytozed, butsome degree of dissociation of PA20 presum-ably must occur at the cell surface in order forPA63 to bind EF or LF from the medium.78
Low pH apparently also stabilizes the oligo-mer, because at pH 7.0 and below theheptameric ring was found to be compact andindividual subunits and the central cavity werevisible, whereas at pH 9.0 and above the ringwas diffused and individual monomers andthe central pore was difficult to discern.98
This requirement for acidic pH ensures thatPA63 does not insert into a cellular mem-brane until its arrival in an acidic compart-ment, where functional translocation can oc-cur.
Singh et al.99 reported that both monomericand oligomeric forms of PA63 bind LF in a 1:1ratio. LF binds very tightly to PA63 and oligo-
merization of PA63 is not required prior to LFbinding. The inability of chymotrypsin-nickedPA to form oligomers and its lack of toxicitywhen combined with LF suggests that the oligo-mer formation seen in cells is an essential stepin delivery of LF to the cytosol.99
If the PA63 oligomer functions in trans-location of EF and LF, its interaction withthese proteins might affect either the forma-tion of PA63 ion channels or the conduc-tance properties of the channels once formed.Finkelstein100 reported that addition of EF orLF to PA63 channels in planar lipid bilayersat acidic pH causes a reduction of conduc-tance on a time scale measured in manyseconds. These results were confirmed byZhao et al.,101 who reported that the bindingof LF to preformed PA63 channels signifi-cantly affected their ion conductance. Theblock in ion conductance caused by LF atacidic pH apparently results from the bind-ing of LF to a site on PA63 that partiallyoccludes the channel and inhibits ion trans-port. At pH 7.0 LF has only a slight inhibi-tory affect on the activity of the PA63 chan-nel, suggesting that LF weakly interacts withPA63 at neutral pH.101 This finding may berelevant to the mechanism by which LF isreleased from the channel and is able toenter the cytosol, which is near neutral pH.
The C terminal domain of PA probablybrings about the binding at the cell surface.PA proteins truncated at the carboxy termi-nus by three, five, or seven residues showprogressively greater decreases in toxicity,up to 20-fold for the deletion of seven resi-dues.102 Competition assays show that theloss in toxicity correlates with decreasedaffinity for receptor. Proteins with these smalltruncations are not significantly more sensi-tive to proteases than is native PA, indicat-ing that their structures are nearly normal. Incontrast, truncation by 12 or 14 residuesmakes the protein much more susceptible toproteases and causes a complete loss of ac-tivity.102
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PA is the essential component of theanthrax vaccine. However, effective utiliza-tion of PA is hampered due to its thermo-lability. It becomes inactive on storage at4oC within 3 to 4 weeks, while at 37oC itloses its activity after 48 h.103 Radha et al.determined certain additives that could pro-tect PA activity against thermal denaturation.The additives that have been found effectiveare magnesium sulfate (3 M), sodium citrate(1 M), and trehalose (1.5 M). The idea be-hind using these additives is that they alterthe properties of water around the proteinmolecule without having any appreciablebinding to the protein. This enhancement ofthermostability is expected to lead to thepreservation of the biological activity of theprotein at elevated temperatures.
B. Crystal Structure of ProtectiveAntigen
The crystal structure of Protective Anti-gen (PA) has been determined at 2.1 Å reso-lution.83 It is a long flat molecule and isabout 100 Å tall, approximately 50 Å wideand 30 Å deep (Figure 2). The molecule isorganized mainly into antiparallel β sheetswith only a few short helices. It has fourdomains: an amino terminal domain (do-main 1) containing two calcium ions and thecleavage site for activating the proteases; aheptamerization domain (domain 2) contain-ing a large flexible loop implicated in mem-brane insertion; a small domain of unknownfunction (domain 3); and a carboxy terminalreceptor binding domain (domain 4).
1. Domain 1
Domain 1 (residues 1 to 258) comprisesa β-sandwich with jellyroll topology, sev-eral small helices, and a pair of adjacentcalcium ions coordinated by residues in a
variant of the EF-hand motif. The first 220residues fold into 13 β-strands arranged inan antiparallel fashion in two sheets of un-equal size. Protease cleavage on the cellsurface occurs at the sequence RKKR(167)that forms an exposed loop between twostrands of the larger sheet. Residues makingup the PA20 fragment form a β-sandwichcomposed of the small sheet and 6 of the 9strands of the large sheet. That the cleavagesite does not occur between structural do-mains but within a domain explains why it ispossible to ‘nick’ PA with trypsin withoutcausing dissociation of the two resultingpolypeptide chains. Domain 1 can be viewedas having two subdomains: (1) PA20, whichforms a 4 + 6 stranded β-sandwich and (2)domain 1’ with residues 168 to 258 that foldinto a 3-stranded sheet and a random coiland form the N-terminus of the active PA63.PA20 not only disrupts hydrogen bondinginteractions in this subdomain by tearing thelarge β-sheet but also causes several hydro-phobic residues to become exposed, whichmay be involved in EF/LF binding.
2. Domain 2
Domain 2 (residues 259 to 487) (Figure3) has a β-barrel core with a modified Greekkey topology and elaborate excursions. It isorganized into a 9-stranded β-barrel and 2α−helices located at either end of the barrel.Domain 2 is the longest domain (roughly 65Å long). Domain 2 contains four large loops(1) loop 1 (residues 274 to 286) is composedentirely of charged or polar residues (2) loop2 (residues 300 to 328) contains two pheny-lalanine residues at its tip, F313 and F314,and is susceptible to proteolysis by chymot-rypsin. As with the furin/trypsin site in do-main 1, PA can be nicked with chymot-rypsin without causing dissociation into twofragments. A deletion mutant lacking thetwo Phe residues is also nontoxic and
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is defective in oligomer and channelfromation.86 It is proposed that this loop in-serts into the lipid bilayer and is involved inthe formation of porin-like β-barrel (3) loop3 (residues 339 to 352) is disordered in thecrystal form grown at pH 6.0 but in thecrystal form grown at pH 7.5 has a confor-mation in which the hydrophobic residuesW346, M350, and L352 are buried. Studiesindicate that the difference in conformationis due to pH.83 The exposure of the hydro-phobic residues at pH 6.0 may be related tothe ability of PA63 to insert into membranesat acidic pH.87,94 Modification of the resi-dues Trp (346), Leu (352) to alanine, and allthree residues (Trp 346, Met 350, and Leu352) together to alanine resulted in the lossof cytotoxic activity of PA in combinationwith LF.104 The mutant proteins were able tobind to cell surface receptor, get cleaved bytrypsin, bind LF, and oligomerize. These resi-dues might play an important role in themembrane insertion of PA and/or transloca-tion of LF/EF into the cytosol. (4) loop 4(residues 422 to 432) contains an exposedphenylalanine, Phe427, which in both crys-tal forms is in a crystal contact with domain3 of another molecule.
3. Domain 3
Domain (residues 488 to 595) has a four-stranded mixed β-sheet, two smaller sheetsand four helices; it adopts the same fold asferredoxins and resembles domain A of toxicshock syndrome toxin-1. Domain 3 is thesmallest of the domains. One helix is a partof a triangular strand-helix-loop structure thatpermits 5 residues to form a flat surfaceexposed to the solvent, constituting a‘hyrophobic patch’. In crystals of PA thispatch is occupied by Phe427 of a neighbor-ing molecule. This patch may be involved ina protein-protein interaction during host cellintoxication.
4. Domain 4
Domain 4 (residues 596 to 735) has aninitial hairpin and helix, followed by aβ-sandwich with an immunoglobulin-like fold.Domain 4 is implicated in receptor binding.
Domains 1, 2, and 3 are intimately asso-ciated, but domain 4 has limited contact withthem.
C. Lethal Factor
Lethal factor (LF), when injected alongwith PA into test animals, causes hypoten-sion, shock, and death, that closely mimicsthe symptoms seen in the disseminated formsof acute anthrax infections. It is believedthat lethal toxin is the major factor respon-sible for the overt and lethal symptoms seenin these cases. Isogenic strains of B. anthracisthat are LF deficient are 1000-fold less viru-lent than wild-type strains in the mousemodel.50 An intravenous injection of lethaltoxin causes death of the Fischer 344 rats inabout 38 minutes.105 Lethal toxin also lysesmouse peritoneal macrophages from C3Hmice90 and macrophage cell lines such asJ774A.1 and RAW264.7 in about 90 min-utes.78,106 This sensitivity of macrophages tolethal toxin provides a convenient bioassayfor researchers to study the molecular mecha-nism of action of LF.
LF, previously known as Factor III, is en-coded on the pXO1 plasmid.51 The gene, calledlef, has been cloned58 and sequenced,107 andfound to contain a 2427-bp open reading frameof which 99 bp encode a 33 amino acid signalpeptide and 2328 bp encode an A/T-rich (70%)cysteine-free, 776 amino acid (90.2 kDa), ma-ture secreted protein. The N-terminal domain ofLF (LF1–255) is responsible for binding to PA.LF1–255 has homology to the PA-binding domainof EF.72 Insertional mutations in LF1–255 preventbinding to PA.79 Additional support of LF1–255
being the PA binding domain is presented in
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studies of chimeric protein fusions. When ge-netically fused to heterologous marker proteins,LF1–255 has the ability to facilitate entry of thesefusions into the cytoplasm of the cells via PAdependant pathways.98,108,109,110, The ability totransfer these large heterologous proteins intocells is not dependent on overall positioning ofLF1–255. LF1–255 works equally well whether fusedto the N-terminus or to the C-terminus of the testprotein sequence.96,110 There are nine histidinesin the binding domain of LF. Modification ofthese histidines abrogates the binding of LF toPA63, implying that histidines are involved inthe binding of LF to PA63.111 LF sequenceexamination also revealed five imperfect repeatregions of 19 amino acids in length and rich incharged residues located between LF residues293-429.79 Mutagenesis of any of the first threerepeats renders LF completely unstable and in-active and mutagenesis of the fourth renders itpartially inactive. The biological function, ifany, of these repeats is unknown.72,79 Insertionsin the carboxy terminus eliminated toxicity with-out altering PA binding, suggesting that thecatalytic regions are located at the COOH termi-nus. Analysis of the amino acid sequence at thecarboxy terminus revealed that amino acids 686to 692 (HEFGHAV) contained a motif charac-teristic of metalloproteases (HEXXH, where Xis any amino acid).112 The hypothesis that LFwas a metalloprotease was supported by obser-vations that (1) protease inhibitors such as bestatinand chloromethylketones of leucine and pheny-lalanine prevented lethal toxin induced lysis ofJ774A.1 cells,112 (2) LF was found to bind ap-proximately one112,113 or more114,65 Zn atoms permolecule, (iii) substitution of alanine for poten-tial zinc-binding residues histidines 686 or 690,reduced zinc binding as well as toxicity of LF112
and (4) substitution of cysteine for glutamine687, an amino acid that forms part of the cata-lytic site, reduced LF toxicity but not zinc bind-ing,112 (5) treatment of LF with EDTA ando-phenanthroline does not remove the boundzinc atoms.114 It was suggested by Menard etal.115 that LF might act similarly to the leucotriene
A4 (LTA4) hydrolase, following the identifica-tion of several sequence similarities between thetwo proteins. Some inhibitors (bestatin,arphamenins A and B, thiorphan) of the LTA4
hydrolase and metallopeptidase activities ofLTA4 hydrolase also affect the cytotoxicity ofthe anthrax lethal factor on macrophage celllines, without interefering with the ability of thelethal factor to enter cells.115
Macrophage cell lines such as J774A.1and RAW264.7 are sensitive to anthrax le-thal toxin,78,106 while certain cell lines suchas IC-21 and all nonmacrophage cell linesand macrophages from A/J mouse strain arehighly resistant to cytolysis by lethal toxin.11
Each of these cell lines can bind PA andproteolytically modify it to PA63, but whenLF is directly introduced to the cytoplasm ofeach cell line by osmotic lysis of pinosomes,resitant cells remain unaffected.88 This sug-gests that the cell line differences in suscep-tibility lie downstream of LF entry into thecell and may reflect a differing sensitivity tothe enzymatic activity of LF. The firstchanges detected in RAW264.7 cells aftertoxin treatment are increases in permeabilityof K+ and Rb+ ions, beginning 45 min afterlethal toxin addition and a conversion ofATP to ADP and AMP. Later events includealterations in membrane permeability to Ca2+,Cr2+, Cl–, SO4
2–, amino acids and uridinebeginning at 60 min.116,5 At 75 min grosschanges in morphology are evident, and at90 min the cells begin to lyse. The eventsbeginning at 60 min require extra cellularcalcium and are blocked by osmotic stabiliz-ing agents such as 0.3 M sucrose.116 It wassuggested that increases in permeability toNa+ and water cause depletion of ATP, whichin turn leads to an influx of Ca2+ that pro-duces further damage leading to lysis. Thisview suggests that LF acts in the cytosol todamage ion pumps or to deregulate ion chan-nels. Bhatnagar et al. 117 showed that proteinsynthesis is required for expression of lethaltoxin cytotoxicity. Inhibition of protein syn-
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thesis in J774A.1 cells by inhibitors of pro-tein synthesis (cyclohexamide and puromy-cin) protects the cell against anthrax lethaltoxin cytotoxicity. This protective effect ofinhibitors is reversible after they are removed.This led to the thought that newly synthe-sized protein(s) may be implicated in thelethal factor induced cytotoxicity. Recentstudies by Bhatnagar et al.118 reveal that ac-tivation of phospholipase C and proteinkinase C are required for mediating anthraxlethal toxin cytotoxicity. There is a dramaticincrease in the levels of IP3 in the cytosol ofJ774A.1 cells when incubated in the pres-ence of PA and LF.
Evidence that the macrophages play akey role in the lethal toxin action in animalswas obtained from studies in Balb/c mice.5
Depletion of macrophages by repeated in-jection of silica renders mice resistant tolethal toxin. Sensitivity is restored by injec-tion of 108 RAW264.7 cells into the micealong with lethal toxin. The administrationof the same number of cells of the lethaltoxin-resistant mouse macrophage line IC-21 does not restore sensitivity. These resultsimplicated that macrophages play a centraland crucial role in mediating the action oflethal toxin in vivo. The production of mi-crobicidal reactive oxygen intermediates(ROI) during macrophage oxidative burst isinitiated and regulated by the reduced nico-tinamide adenine dinucleotide phosphate(NADPH) oxidase complex.
The primary species of ROI, superoxideanion (O2), is generated by the single elec-tron reduction of molecular oxygen usingthe reducing powers of NADPH. O2
– is then
further reduced to form other reactive spe-cies such as O2, H2O2 , and OH–. A highconcentration of these oxidants may consti-tute a risk for the host cells itself. ROI inexcessive amounts modify the regulatoryproteins of Ca2+ homeostasis and initiate adestructive peroxidative cascade consuminga large amount of plasma membrane lip-
ids.119 Both of these effects are disastrous tocells. Evidence from different sources hintedthat lysis of macrophage by lethal toxin wasdue to the cell’s own oxidative burst. In1986 Friedlander found that although PAreceptors were found on most cell types (andlethal toxin is fully capable of entering mostcells), cytotoxicity is apparently limited tomacrophages, a major producer of ROI.Lethal toxin challenged macrophages werefound to undergo a cascade of cellular eventsthat culminate in necrotic lysis.116 Those cel-lular pathologies resemble, at least superfi-cially, the ones seen with a high concentra-tion of reactive oxidants and include adepletion of energy stores, large calciuminfluxes through the plasma membranes, andcell bursting via colloid-osmotic lysis within1 to 2 h.116 Also, the anthrax toxin sequencedata reveal no cysteines in the entire toxincomplex, which includes three proteins to-talling 2278 amino acids.72 The lack of cys-teine residues indicates that the three toxicproteins must maintain function in an oxi-dizing milieu. Cultured macrophages treatedwith lytic concentration of lethal toxin re-lease large amounts of superoxide anion (ap-proximately 200 to 300 nmol/107 cells),120
that is, two to three times as high as that seenwith phorbol myristate acetate (PMA),a potent stimulator of oxidative burst.Cytolysis was blocked by various membrane-soluble oxidants, for example, β-mer-captoethanol, dithiothrietol, ethyl alcoholdimethylsulfoxide, etc.120 These same re-agents did not inhibit edema toxin activities,indicating that they worked at a step down-stream of toxin internalization. Mutant mu-rine macrophage lines, deficient in the abilityto produce ROI, were found to be relativelyinsensitive to the lytic effects of the toxin,whereas a line with increased oxidative burstpotential had elevated sensitivity.120
Because the symptoms of animals dyingof anthrax toxin resemble those of septicshock, measurements were made of cytokine
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induction. Sublytic concentrations of lethaltoxin-induced macrophages to express bothTNF-α and IL-1β. Macrophage productionof these cytokines was stimulated (in thepresence of PA) by small amounts of LF(10–9 to 10–5 µg/ml) resulting in TNFα levelsof 1000 to 2000 pg/ml.5 Nearly all of TNF-α was secreted from the macrophages, whileover 90% of the IL-1β remained cell associ-ated.5 During the initial stages of infection,when toxin levels are low (and ROI levels)IL-1β and other protein inflammatory me-diators are ‘stock-piled’ within the macroph-age. However, when the levels of the an-thrax lethal toxin reaches the threshold level(and thus the ROI levels) for lysis, macroph-ages may burst throughout the body, releas-ing large amounts of IL-1β and other potentstored mediators of inflammation respon-sible for dramatic ‘sudden death’ in sys-temic anthrax. Thus, ROI play a double rolein the pathogenesis of anthrax, that is, firstROI (at low levels) induce macrophagecytokine expression and then at high levelsbursting the cell and bringing about the ly-sis. Antisera of these cytokines could protectthe animals completely against anthrax le-thal toxin. This led to the convincing evi-dence that macrophages and their productscan mediate death of the host in a Gram-positive bacterial infection.
Experiments have shown that LF has toenter an intracellular acidic compartment inorder to display its toxic activity in the cytosolof macrophages.11,88,90,108 Macrolide antibiot-ics concanamycin A and bafilomycins A1, B1,C1, and D are specific and powerful inhibitorsof the vacuolar ATPase pump, which is re-sponsible for the acidification of the lumen ofendosomal and lysosomal compartments. Thesedrugs when added to the macrophage cell lineJ774A.1 inhibit the cytotoxic activity of LF.121
These inhibitors remain active long after LFaddition to macrophages, suggesting that LFenters the cytosol after having reached a lateendosomal compartment.
LF is a protein enzyme that is likely tofunction as a protease with cytosolic hostsubstrate. Recent studies have revealed thatLF can act as an endopeptidase.122 Severalcommercially available peptides were as-sayed as LF substrate. Cleavage productswere separated by reverse phase high-pres-sure liquid chromatography. Mass spectros-copy and peptide sequencing of the isolatedproducts indicate that LF seems to preferproline-containing substrates. Substitutionmutation within the consensus active-siteresidues completely abolished all in vitrocatalytic functions. The protease inhibitorssuch as EDTA, amino acid hydroxamates,and the novel zinc metalloprotease inhibitorZINCOV completely abolished all in vitrocatalytic functions.122
Despite the suggestive hint that LF mightbe a metalloprotease, the enzymatic substrateof LF remained unknown for several years.Then in 1998 Duesbery et al.123 and laterVitale et al.124 identified mitogen-activatedprotein kinase (MAPK) kinases 1 and 2(MAPKK 1 and 2) as substrate for LF. LFhas an activity profile similar to that ofPD09859, a compound that selectively in-hibits the mitogen-activated protein kinase(MAPK) pathway. The MAPK pathway is akey regulatory signal transduction pathwaypresent in eukaryotic cells that helps controlcell growth, embryonic development, andthe maturation of oocytes into eggs. Typi-cally in such pathways, ligand binding at thecell surface initiates a signal, which is thenrelayed to intracellular effector moleculesvia a series of phosphorylation reactions.125
One pathway that activates MAPK was out-lined years ago in frog oocytes.126 In frogoocytes, MAPK is phosphorylated and acti-vated by MAPK kinases 1 and 2. When LFwas injected into oocyte, their maturationwas prevented. Analysis of the oocyte ex-tracts indicated that LF prevented the activa-tion of MAPK.123 Analysis indicated thatLF removed residues 1-7 of MAPKK1
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(PKKKPTP). Similarly, LF was found tocleave MAPKK2 between residues 9 and10, resulting in the loss of NH2-terminal resi-dues LARR KPVLP.123 These results wereindependantly confirmed by Vitale et al.124
using a yeast two-hybrid approach. Appar-ently, the removal of this short sequence issufficient to inactivate MAPPK1 becauserecombinant MAPKK1 lacking the sameseven amino acids also lacked activity.123
Frog, mouse, and human MAPKK1 are allsubstrates of LF. The removal of the aminoterminus of the MAPKKs is accompaniedwith phosphorylation of MAPKK substratesERK1 and ERK2, the extracellular signal-regulated kinases that mediate transcriptionalactivation of specific nuclear target genes.124
It was shown by Pellizzari et al.127 thatMKK3, another dual specificity kinase thatphosphorylates and activates p38MAP ki-nase, is cleaved by LF in macrophages. Thesignalling pathways involving Mek1, Mek2,and MKK3 play a crucial role in the activa-tion of macrophages and are directly involvedin the production of cytokines, that is, in thebiosynthesis of tumor necrosis factor-α(TNF-α), interleukin (IL)-1, and IL-6.127,128
The release of TNF-α and NO induced bylipopolysaccharide/interferon γ is inhibitedby LF, and such inhibition is relevant duringthe first stages of B. anthracis infection, whena reduction of the inflammatory responsewould permit growth and diffusion of thebacterium. However, the biochemical linkbetween the MAPKK-specific proteolyticactivity of LF and macrophage cell deathremains to be established. The comparisonof the NH2- terminal sequences of MAPKK1and 2 shows each contain two proline resi-dues that are interspersed by one or twoamino acids and preceded by a series ofbasic residues. Site-directed mutagenesis atthe cleavage site indicated that both prolinesplay an important role in cleavage site rec-ognition because mutation of either Pro5 orPro7 to Ala renders MAPPKK1 resistant to
cleavage,123 while the combined mutation ofboth residues to ala prevents its cleavage.Thus, the advantage of proline residues atthe cleavage site was underscored byHammond and Hanna.122 Also in their yeasttwo-hybrid assay, Vitale et al. 124 identifieda cDNA for MAPKK2, which encoded aminoacid 31 to 400, a sequence that lies outsidethe cleavage site. Thus, additional elementswill likely play a role in LF substrate recog-nition.
Lethal toxin-induced cytotoxicity can beswitched to apopotosis under narrow condi-tions when cells are preincubated with theprotein phosphate inhibitor calyculin A,130
suggesting that apopotic and lethal-toxin-induced death mechanisms may have simi-larities and may utilize some of the samecellular components. Recent studies haveshown that the proteosome plays a role insome apopotic pathways. The proteosome isa multicatalytic protease that accounts forthe major extralysosomal degradation ofcellular proteins. It was shown by Tang andLeppla131 that proteosome inhibitors MG132,lactacystin, and ALLN were very potent ininhibiting the cytotoxicity of lethal toxin,indicating that proteosome activity is neces-sary for lethal toxin cytoxicity. The specificinhibitors efficiently protected macrophagesfrom lethal toxin but did not block cleavageof MEK1, the substrate of LF suggestingthat one of the downstream events followingthe cleavage of MEK1 or other putative sub-strates of LF is the degradation of certainprotein molecules, and that this is an essen-tial step in the cascade leading to macroph-age lysis.131
1 to 300 amino acid residues of LF havebeen viewed as the region responsible forthe high affinity binding of LF to PA. Aminoacid analysis of LF and EF reveal a commonstretch of 7 amino acids (147VYYEIGK153).It has been found that Y148A, Y149A,I151A, and K153A were deficient in theirability to lyse J774A.1 cells and their bind-
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ing ability to PA63 was drastically reduced.These four amino acids may play a crucialrole in the process of binding of LF toPA63.132
D. Edema Factor
Following infection with Bacillusanthracis, severe tissue edema occurs at thesite of bacterial challenge and in draininglymph nodes. Subcutaneous inoculation ofanthrax spores leads to prominant swellingof the skin lesion and surrounding tissues.Inhalation anthrax is associated with pulmo-nary edema. Edema is mediated by edematoxin that comprises of protective antigenand edema factor. Edema factor (EF), previ-ously referred to as factor I, is encoded at thecya locus on the pXO1 plasmid.51 Two sepa-rate groups independantly cloned thegene.57,59 The sequences of the cloned cyagenes were determined by each group.59,132
The gene was found to contain a 2400-bp-long open reading frame of which 99 bpencode a hydrophobic 33 amino acid signalsequence, and 2301 bp encode an A/T-rich(71%) cysteine-free, 767 amino acid (88.8kDa) mature secreted protein.
EF was the first component of anthraxtoxin that was shown to have an enzymaticactivity, that of adenylyl cyclase. EF is avery efficient adenylate cyclase. The cata-lytic activity is absolutely dependent on thepresence of calmodulin, and the stimulatoryactivity of calmodulin is in turn highly de-pendent on calcium.
Intradermal injection of EF, in combina-tion with PA, gives rise to experimentaledema. EF conversion of host cell ATP tocAMP is responsible for the effects of theedema toxin. Edema toxin-induced increasesin cAMP differ between cell types, but mayreach 1000-fold, representing conversion of20 to 50% of the cell’s ATP stores.91,95 Thecontribution of EF to systemic anthrax is
debated, as isogenic EF knockoutB. anthracis strains are attenuated only ten-fold in the mouse model for systemic an-thrax.50,135 The more relevant role for edematoxin is in the cutaneous form of anthrax, forwhich there is currently no good animalmodel. EF increases in cAMP may also beinvolved in early stages of the infection. Ingeneral, bacterial toxins that increase cAMPdampen innate immune responses of phago-cytes, thus contributing to establishment ofthe infection.136 Treatment of neutrophils withedema toxin inhibits both phagocytosis andoxidative burst ability of these cells, but in-creases chemotactic responses to formyl-Met-Leu-Phe (fMLP).137,138,139 Edema toxin, viaincreased cAMP levels, also affects mono-cyte cytokine profiles. Cultured monocytestreated with edema toxin are severely inhib-ited in lipopolysaccharide (LPS)-inducibletumor necrosis factor (TNF)-α expression,but secrete elevated levels of interleukin (IL)-6.140 Therefore, edema toxin has the poten-tial to disrupt the phagocytic antibacterialresponses at several key levels. Coadminis-tration of edema toxin and lethal toxin en-hances the lethality of lethal toxin.141
Sequence analysis of EF indicates thatresidues 1 to 250 at the amino terminus sharehomology with the corresponding region ofLF, both of which are responsible for bindingto PA63. Additionally, homology is foundbetween EF 265 to 570 and the catalytic re-gions of the adenylate cyclase of Bordetellapertussis, a closely related bacterial adenylatecyclase toxin that requires calmodulin as acofactor.59,95,132 The ATP binding site of EF ismaintained in residues 314 to 321, a consen-sus nucleotide binding site (GxxxxGKS/T).142
Cross-linking studies place the calmodulinbinding site at EF 613-767, while syntheticpeptide corresponding to EF 499 to 532 bindsthe cofactor calmodulin in vitro.143 Which ofthese regions (or whether both) are relevant tophysiologic calmodulin binding is not clear atpresent.
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E. Mechanism of Action
B. anthracis has two unique virulencefactors, a poly-D-glutamic acid capsule and athree-component protein exotoxin. The cap-sule enhances the virulence by making theorganism resistant to phagocytosis and mayalso protect the bacilli from lysis by cationicproteins in serum.144 The organisms that losethe ability to produce capsule are avirulent.46
Apart from this, any other role of capsule inpathogenesis has not been established. It is apoor immunogen and in the absence of toxinappears to have no value as a protectiveagent in vaccine formulation.145 The role ofthe toxin components in pathogenesis wasestablished by Smith and Keppie in 1954when they demonstrated that sterile plasmafrom experimentally infected guinea pigs waslethal after being injected into other animals.Further studies led to identification, purifi-cation, and characterization of the three toxincomponents.
The steps in the host cell intoxication aredepicted in Figure 4. Internalization of PAinto cells begins with its binding to a spe-cific cell surface receptor.62,72.78,146 Membranesurface receptors for PA are found on mostcell types.72 Escuyer and Collier 146 describeda single class of protein toxin receptor (ap-proximately 85-90 kDa) on the surface ofCHO-KI cells. Binding is specific, concen-tration dependant, saturable (Kd, 0.9 nm),and reversible at 4oC. Maximal binding re-quires the presence of Ca2+.106 Receptor isproteinaceous in nature and pretreatment ofthe cells with proteases eliminates receptorbinding.146 The number of receptors varyfrom cells to cells. CHO-KI cells contain10,000 receptors per cell, mouse macroph-ages have 30,000 receptors per cell and ratmyoblast cell line L-6 has about 50,000 re-ceptors per cell.11,72,146 Proteolytic activationby the cell-surface protease furin147 occursin a surface loop within domain 1 of PA,releasing an N-terminal 20 kDa fragment,
PA20, which plays no further role in intoxi-cation. Nicking of PA appears to be quiteslow with a half time of at least 30 min at37oC,86 as only small amounts of furin arefound on the cell surface compared with theamounts in the trans-Golgi region.148 Re-moval of PA20 requires the rupture of aβ-sheet but does not lead to disorder in do-main 1’. The new N-terminus is stabilizedthrough extensive interactions with domain2 and internally by two calcium ions main-tained by a variant of an EF-hand motif. Theloss of PA20 leads to the exposure of a largehydrophobic patch, presumably implicatedin binding EF or LF. The loss of PA20 alsoleads to heptamer formation by PA63.98 Theheptameric complex that is formed is a hol-low ring of 160 Å in diameter and 85 Å inheight in which domains 1′ and 2 face theinside of the ring.83 Most monomer-mono-mer contacts involve charged and polar resi-dues in domains 1′ and 2. The lumen has adiameter of 20 to 35 Å and is negativelycharged.
PA is capable of forming ion-conductivechannels in artificial bilayers and cellularmembranes.87,96,149 In both systems pro-teolytic activation of full-length PA and theexposure of the PA63 to acidic pH, presum-ably in an acidic intracellular compartment,are required for membrane insertion andchannel formation. Thus, inhibitors of inter-nalization and endosome acidification blockconversion of cell-associated PA63 to highmolecular mass species,91,90,98 and acidifica-tion of the medium induces oligomer forma-tion either in the presence or absence ofthese inhibitors. Therefore, the conditionsfor PA63 oligomerization correlate with thoserequired for channel formation and translo-cation of EF and LF across cellular mem-branes, suggesting that the oligomer mayplay a role in both activities.
After furin processing, the two A moi-eties compete for the same high-affinity bind-ing site on PA63. An analysis of binding
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curves and binding ratios of LF to PA63 ondenaturing gels suggests that each PA63monomer binds one LF or EF.99 Because ofthe unique requirement that receptor-boundPA be cleaved before it can bind LF/EF, therate of PA internalization relative to pro-teolytic activation is a critical determinant ofthe efficiency of LF/EF delivery to thecystosol. It was reported by Singh et al.99
that approximately half the PA entered cellsin 30 to 40 min. Trypsin nicking had noeffect on the rate of internalization, suggest-ing that the receptor does not distinguishbetween various forms of PA. This rate ofinternalization is very slow (3% per min)compared with that for receptors for manyprotein ligands (25% per min) but is similarto the rate at which diphtheria toxin is inter-nalized.150 Because LF and EF bind cellspreincubated with PA63 and block ion-con-ductance of preformed PA63 membranechannels,101 membrane-bound heptamer islikely to be oriented with domain 1′ exposedto the extracellular environment, availableto bind EF and LF, and with domain 4 andthe bottom of domain 2 next to the mem-brane.83 The complex of PA63-LF or PA63-EF is endocytozed to an acidic membrane-bound compartment, the endosome byreceptor-mediated endocytosis. It is not clearwhich event oligomerization or LF/EF bind-ing occurs first.113 The endosomes undergoacidification and this acidification, of theendosome, a normal cellular phenomenon,is a prerequisite for anthrax toxin transloca-tion through the endosomal membrane intothe cystosol.
Because there are no extensive hydro-phobic areas on the surface of the heptamerat neutral pH, it is likely that a significantconformational change occurs after mem-brane insertion. Based on proteolysis andmutagenesis experiments.85,86 Petosa et al.83
proposed that a large chymotrypsin-sensi-tive loop in domain 2 (residues 302 to 325)inserts into the membrane. This loop is com-
posed of alternating hydrophobic residuesand is likely to form an amphipathic β-hair-pin that would span the lipid bilayers. Oneβ-hairpin is contributed by each of theprotomers forming a 14-stranded β-barrelformed from seven β-hairpins. It was re-ported by Werche et al.151 that translocationof protein into cells by the anthrax toxinpathway requires at least partial unfolding ofthe protein.
It is in the cytosol of the cell where thecatalytic moieties act. EF brings about a dra-matic increase in cAMP concentration rising1000-fold to reach 2000 µmol per milligramof cell protein.91,95 This represents conver-sion of 20 to 50% of the ATP present. In-creases in cAMP concentration have manyeffects on cellular metabolism, all of whichare believed to be mediated through cAMP-dependent protein kinase. Treatment of hu-man polymorphonuclear neutrophils (PMNs)with edema toxin inhibits phagocytosis ofopsonized vegetative B. anthracis,137 an ef-fect that is reversible after toxin removal.Edema toxin stimulates the chemotactic re-sponse of these cells to N-formyl-Met-Leu-Phe.138 Edema toxin blocks priming of PMNsby muramyl dipeptide or lipopolysaccharide,decreasing the release of superoxide anioncaused by later stimulation with the chemo-tactic peptide.139
Lethal factor brings about lysis of mousemacrophages, and it has proven to be a use-ful system to study changes occurring in thecell after toxin treatment.90 Hanna et al.116
showed that there is an increase in levels ofcytokines such as 1L-1β and TNF α, whichcauses death. Also, there is an increase in thepermeability of Na+, K+, and Rb+, ions, hy-drolysis of ATP, which further leads to aninflux of Ca2+, causing damage to the cell.106
Unlike other bacterial toxins such as diph-theria toxin, cholera toxin, etc., where in-hibitor of protein synthesis have lethal effectin susceptible cells,152 protein synthesis inhi-bition is not the cause of cell death rather
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protein synthesis is required for anthrax toxinaction.117 There is a dramatic increase in thelevels of IP3 in toxin treated J774A.1 mac-rophages, which indicates the role of phos-pholipase C in process of cytotoxicity.118
However, until recently, the enzymatic ac-tivity of LF and the substrate on which LFacts was not known. Recently, it has beenshown that LF acts by inhibiting the MAPkinase signal transduction pathway.123 It pro-teolytically cleaves the amino terminus ofMAPKK1 and 2 and this removal of aminoterminus inactivates MAPKK1. Thus, LFprevents the association of MAPKK1 withits substrate and further inhibits the MAPKsignal transduction pathway.123
IV. ANTHRAX TOXIN PRODUCTIONAND PURIFICATION
Culture supernatants of B. anthracis havebeen the major source for purification ofanthrax toxin proteins. Toxin is usually pro-duced from Sterne-type strains, such as theprotease-deficient strain V770-NP1-R, al-though a rifampicin resistant mutant, SRI-1,is reported to secrete 50 to 75% more toxin.62
Anaerobic growth promotes PA production,and media compositions therefore were op-timized for anaerobic conditions.153 Underthe best conditions found, B. anthracis Sternestrains (pXO1+, pXO2–) produce culture su-pernatants containing PA, LF, and EF atapproximately 20, 5, and 1 µg per milliliter,respectively. In the defined medium used,the three toxin proteins constitute more thanhalf of the total extracellular protein.62
The defined ‘R medium’ proposed in1983154 was derived from a previously usedmedium155 by replacement of the casaminoacid mixture with a mixture of purified aminoacids. Stanley et al.156 described the purifica-tion of different components of anthrax toxinfrom the toxic plasma of guinea-pigs dyingfrom anthrax by precipitation, fractionation
on dimethyl amino ethyl (DEAE) cellulosecolumn, followed by ultracentrifugation.However, the purified components were of-ten cross-contaminated with each other.44,156
Harris-Smith et al.157 described conditionsfor the elaboration of toxin in vitro by add-ing 30% (v/v) of serum in the medium. Theysuggested that the serum components playan important part in toxin production. Thorneet al.158 described the isolation of toxin com-ponents from fritted glass filters from cul-tures grown in casamino acids medium with-out serum. Leppla62 described large-scalepurification of anthrax toxin componentsfrom 50-l fermentor culture supernatantsusing sequential chromatography on hy-droxyapatite and DEAE sepharose. Compo-nents eluted in the order PA> LF>EF andeach was evident as a peak of UV-absorbingmaterial. The yields from this purificationprocedure were 400 mg PA, 75 mg LF, and20 mg EF.62 The toxin components werealso purified by ion-exchange and hydro-phobic interaction chromatography159 orwith monoclonal antibody adsorbent col-umns.160
To avoid the problem of cross-contami-nation, different methodologies were tried.B. anthracis in which one or more of thetoxin components were inactivated135 wereused to produce subsets of the toxin compo-nents. Ivins and Welkos161 expressed the PAgene in B. subtilis. The yields from this sys-tem were equivalent to that of PA purifiedfrom B. anthracis Sterne strain. A shuttleplasmid, pYS5, derived from pUB110 andpVC8f(+)T was able to replicate in bothBacillus species and E. coli.88 Production oftoxin from pYS5 plasmid in B. anthracisand B. subtilis requires a rich medium andvigorous aeration. A rich medium contain-ing tryptone and yeast extract (FA medium)gave yields of PA exceeding 50 µg/ml cul-ture supernatant.88 Another approach to im-proving production of toxin proteins fromB. anthracis and B. subtilis was the use of
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protease-deficient strains. Two strains ofB. subtilis, DB104, lacking two proteases162
and WB600, lacking six proteases,163 wereconstructed. Proteolysis in the culture super-natants was reduced in these strains.
B. anthracis appears to produce less ex-tracellular protease than B. subtilis. Morethan half the protease activity in the superna-tant is due to a single metalloprotease, onethat is probably very similar to the well char-acterized B. cereus metalloprotease.164 StrainBH441 contains transposon Tn917 insertedin the protease gene. Recombinant proteinssecreted from this host appear to suffer lessdegradation, and this is particularly evidentfor proteins containing alterations expectedto destabilize their native conformations.Farchaus et al.165 described purification ofPA from a recombinant avirulent strain ofB. anthracis that was PA positive, kanamy-cin resistant, LF negative, EF negative, andcapsule negative Sterne-1 (pPA102). Cul-tures grown in fermentor using high tryptoneand high yeast medium could yield 20 to 30mg of secreted PA per liter.165 PA has alsobeen expressed and purified from baculovirusvector and vaccinia virus.166 PA has beenpurified from E. coli from our laboratory.167
It was expressed as a fusion protein inE. coli. The recombinant PA was shown tobe biologically and functionally active.
It has been difficult to produce milli-gram amounts of recombinant LF proteins.159
Klimpel et al.112 expressed LF taking advan-tage of efficient expression of PA from pYS5type plasmids. LF was expressed as fusionprotein along with 167 amino acids of PAjoined by a sequence that can be cleaved byFactor Xa protease. The fusion proteins werepurified from protease-deficient strain BH441of B. anthracis and then cleaved by FactorXa protease to yield the desired LF native ormutant proteins.112 Attempts to express andpurify LF from B. subtilis and E. coli werenot very successful.72,78 In other approaches,fusion proteins of LF 1-254 connected to
Pseudomonas exotoxin A (PE) or otherpolypeptides have been expressed using glu-tathione-S-transferase (GST). This systemallowed affinity purification of GST-LF1-254-PE fusions.110 Full-length LF 1-776could not be purified from E. coli because itundergoes extensive proteolytic degradation.In our laboratory LF has been expressed asa fusion protein with six histidine residues inE. coli. Recombinant LF was purified byaffinity chromatography, gel filtration, andfinally ion-exchange chromatography. Theyield of purified LF was 1.5 mg/l.168 Re-cently, Park and Leppla169 have optimizedthe expression and purification of B.anthracis lethal factor leading to yields of 20to 30 mg/L. They constructed a new vectorfor expression of LF based on the vectorpYS5, which is capable of producing PA atconcentrations of 50 mg per liter of culturesupernatant when grown under optimum con-ditions in modified FA medium.88 In the newvector, pSJ115, the structural gene for LF(lef) was fused with PA signal sequence andexpressed under the control of the PA pro-moter. During secretion the PA signal peptideis cleaved to release the 90-kDa LF protein.LF was purified from the culture mediumusing three chromatographic steps (Phenyl-Sepharose, Q-Sepharose and hydroxyapatite).
In our laboratory edema factor gene hasbeen expressed under the transcriptional regu-lation of T5 promoter (unpublished data).EF was purified to homogeneity by a twostep procedure involving metal-chelate af-finity chromatography and cation-exchangechromatography. One liter of culture yielded2.5 mg of biologically active EF.
V. IMMUNITY AND VACCINATIONAGAINST ANTHRAX
The optimal method of preventing lethalinfections such as anthrax is prophylaxis throughvaccination; however, ‘specific prophylaxis of
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anthrax still remains one of the most urgentproblems in modern immunology.’170
Anthrax was the first disease for whichdefinite immunity was produced by the useof bacteria. It was Chauveau in 1879 whofirst noticed that sheep were more resistantto anthrax after having survived a previousinoculum of blood heated at 55°C from ani-mals that died due to anthrax. Both Greenfieldin England and Pasteur in France developedvaccines from heat-attenuated cultures ofB. anthracis. Pasteur’s vaccine schedule in-volved two inoculations 2 weeks apart. Thefirst dose consisted of B. anthracis cells fromcultures that had been incubated at 42 to43°C for 15 to 20 days (Pasteur I vaccine)and was pathogenic only for mice and youngguinea pigs. The second dose consisted ofcells from cultures incubated at 42 to 43°Cfor only 10 to 2 days and were rather lessattenuated (Pasteur II vaccine). The Pasteurduplex vaccine became widely used for cattleand sheep in Europe and South Americaover the next 50 years. In the 1930s,Pasteurian vaccine was replaced by singlevaccines consisting spores suspended in 50%glycerol. The strains were attenuated to suchan extent as to be nonvirulent for rabbits, butvirulent for guinea pigs, and the intricatemanipulations needed to meet this require-ment rendered these vaccines impractical inthe long run. The current hypothesis to ex-plain Pasteur’s mechanism of attenuation ofB. anthracis is that by growing virulent cellsat high temperatures, he induced the loss ofpXO1 toxin plasmid, resulting in an increasedproportion of pXO1–, pXO2+ cells in thecultures. Studies have clearly demonstratedthat pure cultures of pXO1–, pXO2+
B. anthracis cells are not effective live vac-cines.161 It is believed that efficacy ofPasteur’s vaccine can be attributed to thepresence of small numbers of pXO1+, pXO2+
bacteria, and that subclinical infection bythese bacteria would have introduced a pro-tective immune response.
Pasteurian vaccine was later replaced byCarbozoo. This vaccine consisted of sporesfrom virulent strains suspended in 10% sa-ponin. The vaccine proved to be quite effec-tive; however, the high saponin content re-sulted in adverse effects.3 Then came theSterne’s live spore vaccine that becameworld’s most potent vaccine against an-thrax.171,172 The strain used was a rough aviru-lent dissociant derived from subculture of anisolate (Strain 34F2) from a case of bovineanthrax on 50% horse serum nutrient agarwith incubation under a 30% CO2 atmo-sphere for 24 h.173 His final formulation con-sisted of 600,000 to 1,200,000 spores per mlsuspended in 0.5% saponin in 50% glycer-ine saline. The formulation of livestock vac-cine used in most countries of the worldtoday remains essentially as specified bySterne and still uses his strain 34F2.
The elucidation of the nature of the three-component anthrax toxin and its relevanceto pathogenesis and immunity174 led to thedevelopment in the 1950s of nonliving orchemical vaccines for human use. At present,three vaccines are commercially available:the Russian, the UK, and the USA vaccines.The Russian vaccine is produced by the TblisiResearch Institute of Vaccines and Serumsand consists of a live spore vaccine preparedfrom a derivative (STI) of the Sterne strainadministered in the shoulder by scarifica-tion. Its efficacy is unknown, but it is knownto have a high number of side effects andcontraindications to its use. The human an-thrax vaccine for use in the USA (AnthraxVaccine Adsorbed or AVA) is produced bythe Michigan Biological Products Institute(formerly, the Michigan Department of Pub-lic Health) and consists primarily of protec-tive antigen produced in fermentor culturesof a toxinogenic, nonencapsulated strain ofBacillus anthracis, V770-NP1-R153,175 andadsorbed onto aluminium hydroxide. The UKvaccine is an alum-precipitated culture fil-trate of the Sterne strain 34F2. Immunization
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can induce local pain, edema, and erythema,and frequent boosters are required.20 Theydo not provide protection against all naturalstrains of B. anthracis.161 As a result thecurrently used vaccines are regarded unsat-isfactory, and there is a need to develop anew vaccine that is safe, nonreactogenic,efficacious against all forms of the disease,and that requires a minimum number of in-oculations to achieve maximum long-lastingimmunity.
The protective antigen component of theanthrax toxin has proven to be the essentialcomponent in vaccines for inducing protec-tive immunity. It was Gladstone76 who rec-ognized the immunizing properties of thisprotein and showed that it extended time todeath for guinea-pigs and provided protec-tion in rabbits by a spore challenge. Studiesshow that live spores vaccines administeredto guinea-pigs in a single dose conferredsignificantly better protection than the hu-man vaccines, although they elicited signifi-cantly lower anti-PA and anti-LF titers at thetime of challenge with virulent Bacillusanthracis.176 Therefore, substantial anti-PAand anti-LF titres may not indicate solid pro-tective immunity against anthrax infection.
An interesting inverse relationship hasbeen demonstrated in certain laboratory spe-cies between susceptibility to infection andsusceptibility to anthrax toxin; guinea-pigsare susceptible to infection but relativelyresistant to the effect of toxins, while rats arerelatively resistant to lethal infection butkilled by much smaller doses of toxin.5 There-fore, there appears to be separate mecha-nisms involved in host defense against sporechallenge and lethal toxicity. This may ex-plain the lack of correlation between PAneutralization titers and the outcome of chal-lenge. The greater effectiveness of the livevaccines compared with that of the humanvaccines may be the result of the involve-ment of antigens other than PA, LF, and EFin protective immunity. Alternatively, the
dynamic relationship between the toxin com-ponents may be all important105 and perhaps,for maximal effect, the toxin must be pre-sented to the immune system at a certainrate, concentration, and order. It is possiblethat live vaccines come closer than chemicalvaccines to achieving this.
The role that antibodies play in vaccine-induced resistance to or protection againstanthrax infection has not been fully defined.Neutralization of anthrax intoxication andprotection against infection have been re-ported after the passive administration of apolyclonal antiserum prepared by infectionwith either Sterne spore or in vitro-producedantigens,43,177,178,179 thus demonstrating therole that the toxin plays in pathogenesis andthe protective capability of antibodies. Mono-clonal antibodies that block the binding ofPA to cells or of LF or EF to PA63 protectagainst intoxication.180,181,182,183 Active im-munity studies using PA combined withaluminium salt adjuvants, which are potentstimulators of the humoral response, furthersupport a role of antibodies in immunity.145,184
The differing levels of protection from alethal anthrax challenge afforded to miceimmunized with spores of the mutant strainsdeficient in production of one or two of thetoxin components, not only confirm the roleof PA as the major protective immunogen inthe humoral response but also indicate a sig-nificant contribution of LF and EF toimmunoprotection.185 It was also observedthat PA-deficient strains were also able toprovide some protection, thereby suggestingthat immune mechanisms other than the hu-moral response may be involved in immu-nity to anthrax.185
As PA administered on its own inducespoor protection, studies have been performedon the effect of combining adjuvants withPA. To date, only those adjuvants composedof whole bacterial cells, and those that arehighly potent have afforded protection equalto or exceeding that of the live spore vac-
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cines.186,187 Studies involving the combina-tion of native PA with Ribi Tri-Mix adju-vant (MPL-TDM-CWS: Monophosphoryllipid A (MPL), a derivative of diphosphoryllipid A from Salmonella minnesota R595that had been detoxified by removal of thephosphate group at the reducing end of themolecule; trehalose dimycolate (TDM), thepurified cord factor from Mycobacteriumphlei; and deproteinized cell wall skeleton(CWS) from either M. phlei or the BCGstrain of M. bovis) have previously indicatedthat a high degree of protection is affordedagainst an aerosolized challenge with vari-ous strains of B. anthracis.188 The high levelof protection generated may be due to partlythe fact that MPL is a potent stimulator ofcell-mediated immunity. MPL is a stimula-tor of interleukin-1 and interferon produc-tion and B cell proliferation. TDM activatesmacrophages and may act as an antigen ‘an-chor’ or carrier in antigen-adjuvant emul-sions, aiding in bringing the immunogen toantigen-presenting cells.189 The developmentof adjuvants with potential for human use,together with high-resolution antigen purifi-cation methods will increase the possibilityof a new generation vaccine against anthraxthat is both safe and highly efficacious.
Recent efforts at developing an improvedhuman vaccine are multifaceted. Recombi-nant DNA methodology is being used to cre-ate possible live vaccine strains ofB. anthracis,134,145 B. subtilis,161,190 Salmonellatyphimurium,191 Lactobacillus,192 Baculovirus,and Vaccinia virus166 that produce PA but notLF or EF. Transposon Tn916 mutagenesishas been used to generate Aro– mutants ofB. anthracis that are effective as live vaccinesin laboratory animals.145,190
Research examining the possibility ofinducing protection against anthrax by im-munizing with a DNA vaccine encoding PAhas also been done.193 An important advan-tage of DNA vaccines over conventionalprotein antigen is that the anti-PA response
is characterized by the production of neu-tralizing antibodies as well as the activationof antigen-specific Th1 and Th2 cytokine-secreting cells. Also, a combination sched-ule comprising priming with plasmid DNAand boosting with free protein appeared tohave some advantages in terms of achievinga rapidly enhanced secondary immune re-sponse in vivo.194
Thus, continued research is essential toelucidate the mechanism(s) of immunity toanthrax and to design effective means ofdiagnosis, treatment, and protection againstthe disease.
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