Critical Reviews in Microbiology, 30:187–196, 2004Copyright c© Taylor & Francis Inc.ISSN: 1040-841X print / 1549-7828 onlineDOI: 10.1080/10408410490468795

The Adenylate Cyclase Toxins

Nidhi Ahuja, Praveen Kumar, and Rakesh BhatnagarCentre For Biotechnology, Jawaharlal Nehru University, New Delhi, India

Cyclic AMP is a ubiquitous messenger that integrates many pro-cesses of the cell. Diverse families of adenylate cyclases and phos-phodiesterases stringently regulate the intracellular concentrationof cAMP. Any alteration in the cytosolic concentration of cAMP hasa profound effect on the various processes of the cell. Disruptionof these cellular processes in vivo is often the most critical eventin the pathogenesis of infectious diseases for animals and humans.Many pathogenic bacteria secrete toxins to alter the intracellularconcentration of cAMP. These toxins either disrupt the normal reg-ulation of the host cell’s adenylate cyclases/phosphodiesterases orthey themselves catalyze the synthesis of cAMP in the host cell. Thelatter are known as the adenylate cyclase toxins. Four such toxinshave been identified: the invasive adenylate cyclase of Bordetellapertussis, the edema factor of Bacillus anthracis, ExoY of Pseu-domonas aeruginosa, and the adenylate cyclase of Yersinia pestis.These adenylate cyclase toxins enter the eukaryotic host cells andget activated by eukaryotic cofactors, like calmodulin, to triggerthe synthesis of cAMP in these cells. By accumulating cAMP inthe target cells, these toxins either modulate the cellular functionor completely deactivate the cell for further function. The immuneeffector cells appear to be the primary target of these adenylate cy-clase toxins. By accumulating cAMP in the immune effector cells,these adenylate cyclase toxins poison the immune system and thusfacilitate the survival of the bacteria in the host.

Keywords Bacterial Toxins; cAMP; Anthrax Adenylate Cyclase;Edema Factor

INTRODUCTIONCyclic AMP is a ubiquitous messenger that integrates many

cellular processes. Diverse families of adenylate cyclases(McKnight 1991) and phosphodiesterases (Beavo & Reifsnyder1990) stringently regulate the synthesis and degradation ofcAMP in the cells. These enzymes are intracellular target of manybacterial toxins. By disrupting the normal regulation of adeny-late cyclase/phosphodiesterase, these toxins alter the intracellu-lar cAMP levels in the host cells (Moss et al. 1984) and therebyhave a pronounced effect on the metabolism and function ofthe target cells. For example, cholera toxin produced by Vibriocholerae catalyses the adenosine diphosphate-ribosylation of

Address correspondence to Rakesh Bhatnagar, Centre ForBiotechnology, Jawaharlal Nehru University, New Delhi-110067, India.E-mail: rakbhat01@yahoo.com

the α subunit of Gs and thus induces intracellular accumula-tion of cyclic AMP in the target cells (Cassel & Pfeuffer 1978).The cAMP accumulation in the gut cells is the chief cause of themassive diarrhea caused by cholera toxin. On the other hand,pertussis toxin produced by Bordetella pertussis catalyzes theadenosine diphosphate ribosylation of the α subunit of Gi to in-crease the intracellular cAMP levels in the target cells (Katada &Ui 1980) and ultimately resulting in the in vivo responses such asleukocytosis, histamine sensitization, increased insulin produc-tion, and potentiation of anaphylaxis. Several acrylpeptides se-creted by Bacillus subtilis inhibit mammalian phosphodiesteraseto cause an increase in the cAMP levels in the target cells(Hosono & Suzuki 1985).

Another strategy adopted by pathogenic bacteria for increas-ing cAMP levels in the target cells is by secreting toxins thatthemselves possess the adenylate cyclase activity. These adeny-late cyclase toxins enter the target cells and get activated byeukaryotic cofactors, like calmodulin, to trigger the synthesis ofcAMP in these cells. Four such toxins have been identified: theinvasive adenylate cyclase of Bordetella pertussis, the edemafactor of Bacillus anthracis, the ExoY of Pseudomonas aerug-inosa, and the adenylate cyclase of Yersinia pestis. This reviewdescribes how these adenylate cyclase toxins incapacitate theimmune system, facilitate the establishment of infection andcontribute to microbial pathogenicity.

THE INVASIVE ADENYLATE CYCLASE FROMBORDETELLA PERTUSSIS

Bordetella are small, aerobic, gram-negative coccobacilli thatcause respiratory track infections in humans and animals. Sevendifferent species of Bordetella (Musser et al. 1986) have beenidentified: B. pertussis, B. parapertussis, B. bronchiseptica, B.avium, B. hinzii, B. holmoseii, and B. trematum. Although B.pertussis, B. parapertussis, and B. bronchiseptica have been ex-tensively studied, very little is known about the virulence of B.avium, B. Hinzii, B. holmoseii, and B. trematum.

B. bronchiseptica can infect a wide range of hosts includinghumans and animals like horses, dogs, cats, pigs, sheep, mice,rats, and guinea pigs. It causes a variety of respiratory disor-ders in these animals, such as, snuffles in rabbit, kennel coughin dogs, and atrophic rhinitis in pigs. B. bronchiseptica appears

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to be the evolutionary progenitor of B. pertussis and B. para-pertussis that have adapted as human pathogens (Van der Zeeet al. 1997). These three subspecies produce a common set ofvirulence factors whose expression is regulated by the bvgASlocus (Arico et al. 1991).

B. pertussis causes the highly contagious childhood disease,whooping cough (Bordet & Gengou 1906). This disease is ac-quired primarily by inhalation of infected respiratory dropletsand aerosols. The onset of the disease is gradual, with symptomsresembling those of common cold initially. In the later stages ofthe disease, a dry non-productive cough develops that is oftenassociated with the characteristic “whoop” resulting from theforced inspiration of air over a partially closed glottis (Olson1975). The intensity of the cough may be severe to cause con-vulsions and cyanosis. Leukocytosis with lymphocytosis is alsocommon during this phase of the disease.

The disease, whooping cough, is initiated when the bacte-ria adheres to and colonizes the ciliated cells in the respiratoryepithelium. The colonized bacterium secretes toxins that killthese cells (Goldman et al. 1982) and causes denuding of theepithelium. This results in decreased ciliary beating and causesprogressive accumulation of mucus and cell debris that trig-gers the cough reflex. During this initial stage of infection, thebacterium secretes an invasive adenylate cyclase that impairsthe bactericidal function of immune effector cells. It is there-fore not surprising that whooping cough is characterized by theabsence of fever, lack of adequate neutrophilic response, anda high incidence of secondary bacterial pneumonia that reflectpoor immune responses by the host (Olso 1975).

Invasive Adenylate Cyclase is an Important VirulenceFactor of B. pertussis

Studies by Weiss et al. (1984) show that adenylate cyclasetoxin is an important virulence factor of B. pertussis. Their find-ings show that Tn5 insertion mutant, deficient in adenylate cy-clase activity, is avirulent in infant mouse models of infection.Virulence of this mutant strain can be resorted by the intro-duction of the cloned adenylate cyclase gene. Cloning of theB. pertussis adenylate cyclase toxin gene was done by Glaseret al. (1988) using E. coli cya− strain (that was deficient inendogenous adenylate cyclase activity) harboring recombinantconstruct that could produce calmodulin (Glaser et al. 1988).Clones that could complement the E. coli cya defect were iden-tified on Mac-Conkey maltose plates as red colonies (indicatingmaltose degradation by enzymes induced by cAMP producedby cloned adenylate cyclase gene of B. pertussis).

Expression of the Virulence Genes in B. pertussisThe sequencing of the adenylate cyclase structural gene (cyaA)

revealed an open reading frame of 1706 codons with a poten-tial to encode a polypeptide of calculated molecular weight of177 kDa. The transcription of this gene is regulated by a sin-gle locus, called the bvg (Bordetella virulence genes) locus that

encodes members of the two-component family of signal trans-duction proteins, BvgA and BvgS. BvgS is a 135 kDa transmem-brane protein that acts as the environmental sensor (Scarlato etal. 1993). Although the actual signal to which BvgS responds invivo is not known, transcription of the bvg locus is induced at37◦C and repressed at 25◦C. The transcription is also induced inthe presence of MgSO4 or nicotinic acid. BvgS phosphorylatesBvgA, a 23 kDa transcriptional activator protein, which in turnbinds to the cis-acting sequences in the promoter region of thebvg-activated genes, facilitating their transcription. An interest-ing feature of this system is that the intracellular concentrationof BvgA correlates with the temperature, such that the bacteriacultured at the intermediate temperatures (between 25 and 37◦C)produce varying concentrations of the BvgA protein.

Another class of genes has been identified and termed as thebvg-repressed genes (Merkel & Stibitz 1995). While the detailsof this system are not completely known, Merkel et al. (1988)have hypothesized that the bvgR gene that resides immediatelydownstream of the bvgAS locus, encodes for a putative 32 kDarepressor protein that binds to the conserved sequence elementof the bvg-repressed genes. It has been further proposed thatthe binding of the phosphorylated BvgA to the bvgR promoteractivates the transcription of bvgR. Thus, at 25◦C when low con-centration of BvgA is present in the cells, the expression of thebvg-repressed genes is activated. Following the temperature up-shift to 37◦C, the intracellular concentration of BvgA increasesand the transcription of the bvg-repressed genes is downregu-lated. Using this system of temperature-regulated expression ofbacterial virulence genes, the bacterium modulates the amountof various virulence factors, including the adenylate cyclase,to optimize its interaction in different anatomical sites in thehost.

The full-length transcript of B. pertussis adenylate cyclaseproduces 200 kDa protein (Hanski & Farfel 1985). It representsthe toxic form of the protein, that is, the form capable of pene-trating the cells and catalyzing the formation of the intracellularcAMP (Glaser et al. 1988). Sequence analysis and characteri-zation of this protein reveals that the adenylate cyclase activityof the protein resides within the N-terminal 400 amino acids.The 1300 amino acids of the C-terminus of this toxin exhibitstriking similarity to the E. coli α-hemolysin. This suggests thatthis part of the protein may mediate hemolytic activity. Furtheranalysis revealed that the truncated protein carrying the first 400residues of the invasive adenylate cyclase carries full catalyticactivity. Whereas, the rest of the protein retains the hemolyticactivity. Thus, the catalytic and the hemolytic activities of the B.pertussis adenylate cyclase toxin are independent of each other(Sakamoto et al. 1992).

Mechanism of Secretion of the Adenylate Cyclase ToxinThe secretion of proteins by gram-negative bacteria gener-

ally requires the synthesis of these proteins with an N-terminalsignal/leader peptide. This leader peptide helps in the transloca-tion of these proteins to the periplasm. The leader peptide is then

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THE ADENYLATE CYCLASE TOXINS 189

cleaved and the protein is transferred across the outer membraneby a specific export mechanism (Hirst & Welch 1988).

The sequence analysis of the B. pertussis adenylate cyclasereveals that this protein is not synthesized along with signalsequence (Glaser et al. 1988). Moreover, Masure and Storm(1989) detected little adenylate cyclase activity in the periplasmof B. pertussis (Masure & Storm 1989). This indicated that the B.pertussis adenylate cyclase is not secreted by the general proteinsecretion mechanism. More insights into the mode of secretionof the toxin were obtained when it was discovered that threeadditional genes downstream of cyaA gene (cyaB, cyaD andcyaE) were required for the secretion of this toxin. In fact, thecyaA, B, D, and E genes are organized in a single operon andare transcribed from the cyaA promoter.

The CyaB protein (712 amino acids) shares more than 50%sequence homology with HlyB of E. coli and CyaD (440 aminoacids) exhibits striking structural similarities with HlyD of E. coli,although the overall sequence homology between CyaD andHlyD is relatively low (32%). CyaE (474 amino acids) is also es-sential for the export of CyaA. This protein may have a functionanalogous to that of TolC in the secretion of E. coli hemolysin(Wandersman & Delepelaire 1990). In contrast to HlyB/CyaBand HlyD/CyaD, TolC, and CyaE are synthesized with typicalN-terminal signal sequences.

It is interesting that the C-terminal transport signal of HlyA isabsent in the B. pertussis adenylate cyclase. However, when thisadenylate cyclase is expressed in E. coli, it is secreted from thecell by the E. coli hemolysin transport machinery (Masure et al.1990). The signal allowing this export has been demonstrated toreside at the C-terminal end of the adenylate cyclase toxin.

The adenylate cyclase and the hemolytic activities of the toxinrequire another protein, CyaC, for activation. The gene cyaCencoding this protein has been identified (Barry et al. 1991). Itbears homology to the hlyC of E. coli. This gene is transcribedin direction opposite to that of cyaA.

Mechanism of Entry of the Adenylate Cyclase Toxininto the Host Cells

No specific cell-surface receptor has been identified for B.pertussis adenylate cyclase and the exact mechanism of the toxinentry remains to be elucidated. However, the homology with theE. coli α-hemolysin system suggests that the pore-forming ac-tivity may be required for the translocation of the catalytic moi-ety into the cells. Indeed, experimental evidence suggests thatthe adenylate cyclase penetrates directly into the membranes:(a) The accumulation of the enzymes within the target cells pro-ceeds without a noticeable lag period (Friedman et al. 1987);(b) The agents that inhibit endocytosis do not affect the entryof the enzymes into the target cells (Gordon et al. 1988); (c)The adenylate cyclase activity is associated with the particulatefraction of the target cells (Farfel et al. 1987).

The penetration process of the B. pertussis adenylate cyclaseis calcium-dependent. Studies by Hewlett et al. (1991) show thatcalcium binding causes conformational changes in the toxin, cor-

responding to its transition from globular structure to an elon-gated form. Furthermore, Rogel and Hanski (1992) have sug-gested that the membrane-penetration of the adenylate cyclaseproceeds via two distinct steps (Rogel & Hanski 1992): The firststep involves insertion into the membrane and it occurs at lowcalcium concentrations. The second step occurs at high calciumconcentration and involves the unfolding of the N-terminal cat-alytic portion and its translocation through the channel createdby the insertion of the hydrophobic region of the adenylate cy-clase into the membrane. Four hydrophobic membrane-spanningregions (between the residues 500 and 700) have been identifiedin the hemolytic domain of the adenylate cyclase toxin. Thisregion may be involved in the membrane insertion, transloca-tion and in pore-formation. Another distinctive feature of thisdomain is the glycine- and the aspartate-rich repeat region thatis located between the residues 1000–1600. This region may beinvolved in the calcium binding. The last 100 residues comprisethe C-terminal secretion signal that presumably interacts withthe membrane-located CyaB protein. Several secretion signalscontained in the repeat region of this domain have also beenidentified (Sebo & Ladant 1993).

Because of its extended repeat region, the B. pertussis adeny-late cyclase toxin has been categorized into the RTX (repeat intoxin) family of bacterial toxin just like the E. coli hemolysin(Welch 1991). Both these proteins have hemolytic activity. How-ever, the main function of the hemolytic domain of the adenylatecyclase toxin is not cell lysis but to serve as a conduit for the pas-sage of the N-terminal catalytic domain (Bellalou et al. 1990).

Biochemical Properties of the Adenylate Cyclase ToxinOnce inside the cell, the adenylate cyclase toxin gets acti-

vated by calmodulin to catalyze the synthesis of cAMP. Theexperimental evidence suggests that the enzyme remains asso-ciated with the membrane with its ATP-binding and calmodulinbinding sites exposed to the cytosol (Farfel et al. 1987). The ATP-binding site of the adenylate cyclase has been identified betweenresidues 54 and 70 and has the sequence 54GVATKGLGVHAK-SSDWG70. Site-directed mutagenesis of Lysine at residues 58and 65 affects the catalytic activity of this adenylate cyclasewithout affecting its binding to calmodulin (Glaser et al. 1989;Au et al. 1989). The Km of this enzyme for ATP is between0.4 to 1 mM. The calmodulin-binding site on the adenylate cy-clase has been localized around Trp242 (Glaser et al. 1989).Though calmodulin activation of this adenylate cyclase occursboth in presence and absence of calcium, in presence of 90 µMcalcium, the apparent Kd for calmodulin is ∼94 pM and in pres-ence of less than 1 nM calcium, the apparent Kd for calmodulinis ∼24 nM. Biochemical studies on this enzyme have shownthat its activity is dependent on presence of divalent cations andis not affected by the presence of α-keto acids or guanine nu-cleotides. Various amphiphiles including phosphotydylcholine,phosphotidylethanolamine and phosphotidylserine have stimu-latory effect on the catalytic activity of this enzyme (Wolff &Cook 1982).

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Role of the Invasive Adenylate Cyclasein the Pathogenesis of Whooping Cough

The mode of secretion of the adenylate cyclase toxin by theBordetella pertussis is such that the toxin has a localized site ofaction in the respiratory track. The most important contributionof the toxin to the pathogenesis is its disruption of the immuneclearance mechanisms. The immune effector cells seem to be theprimary target of the adenylate cyclase toxin. By accumulatingcAMP in these cells, the adenylate cyclase inhibits the functionof these cells and disables the host immune defense mecha-nism. Studies by Confer and Eaton (1987) show that neutrophilsand macrophages that have been exposed to the B. pertussis ex-tract containing adenylate cyclase, have impaired chemotaxis,a reduced oxidative response and a decreased killing capacity(Confer & Eaton 1987). These observations were later extendedto monocytes (Pearson et al. 1983). The treatment of the nat-ural killer cells with the B. pertussis adenylate cyclase blocksthe cytotoxicity of these cells (Hewlett et al. 1983). More recentstudies on J774A.1 cells, a monocyte-macrophage cell line, andon alveolar macrophages, have shown that the Bordetella per-tussis strains producing the invasive adenylate cyclase toxin arecapable of inducing apoptosis in these cells (Khelef et al. 1993).

B. pertussis produces two toxins, the invasive adenylate cy-clase and pertussis toxin, which are capable of causing an in-crease in the intracellular cAMP concentration in the host cells.The accumulated data suggests that both the toxins are essentialfor the virulence of the bacterium. The adenylate cyclase toxincauses a rapid increase in the intracellular cAMP levels of thetarget cells that seems to be necessary to initiate colonizationin the first few days of infection (Khelef et al. 1992). The per-tussis toxin, which is a slower acting toxin, acts in concert withthe adenylate cyclase toxin to amplify the cAMP signal there-after and thus plays an important role in the persistence of thebacteria.

EDEMA FACTOR OF BACILLUS ANTHRACISAnthrax is primarily an animal disease, transmissible to hu-

mans through contact with infected animals or contaminatedanimal products. It is caused by gram-positive, aerobic, spore-forming bacteria, called Bacillus anthracis. In the recent years,anthrax has gained notoriety as a biological warfare agent. Thelatest incidences being those of the intentional release of anthraxspores in the United States (in 2001).

The disease is initiated by the entry of the anthrax sporesinto the host body. The spores may enter the host body via asmall cut/abrasion in the skin or by insect bite to cause cuta-neous anthrax that is characterized by small pimple that laterdevelops into painless black eschars. The lesion is associatedwith substantial edema. Fever, malaise, and regional adenopa-thy occasionally occur in individuals infected with cutaneousanthrax. If treated properly, the cutaneous anthrax is rarely fatal.

Gastrointestinal anthrax occurs when anthrax spores are in-gested by eating contaminated and undercooked meat. Symp-

toms of the gastrointestinal anthrax include severe abdominalpain, hematemesis, bloody diarrhea, and pharyngeal ulcers.Edema of the neck and constriction of the airways may occurbecause of lesions in the neck. Early diagnosis is difficult andtherefore gastrointestinal anthrax results in high mortality.

Inhalation of aerosolized spores causes inhalation anthrax.This form shows a biphasic clinical pattern with a benign initialphase followed by an acute second phase that is almost alwaysfatal. The initial phase begins as a nonspecific illness consistingof malaise, fatigue, myalgia, mild fever, non-productive cough,and occasionally a sensation of precordial oppression. This ill-ness may resemble as the mild upper respiratory track infectionsuch as cold or the flu. After two to four days, the patient mayshow signs of improvement. However, then there is a suddenonset of severe respiratory distress with dyspnea, cyanosis, res-piratory stridor, and profound diaphoresis. In several cases, sub-cutaneous edema of the chest and neck has been described. Thepulse, respiratory rate, and temperature become elevated. Deathoccurs in most persons infected with inhalation anthrax within24 hours after the onset of the acute phase.

The spores that enter the host body through different routesof infection are efficiently phagocytosed by the macrophages(Shafa et al. 1966). Inside these macrophages the spores germi-nate to form vegetative bacteria (Guidi-Rontani et al. 1999a).A germination operon ger X found on the pXO1 plasmid of B.anthracis has been implicated in the germination process (Guidi-Rontani et al. 1999b). The deletion of this operon affects the ger-mination in the macrophages and reduces the virulence of thebacterium. The bacterium proliferates within the macrophages,expresses the toxin genes, kills the macrophages and escapes intothe bloodstream to establish systemic infection. The physiolog-ical body temperature and CO2 levels favors the transcriptionalactivation of the capsule and toxin genes (Sirad et al. 1994).

The capsule of B. anthracis is a polymer of poly-D-glutamicacid (Zwartouw & Smith 1956). It is an important virulencefactor that enables the bacteria to evade the host-immune systemand provoke septicemia. The plasmid pXO2 encodes the genes,capA, capB, and capC that are required for the synthesis of the B.anthracis capsule (Makino et al. 1989). Another gene of the caplocus, dep, is required for the depolymerization of the capsulepolymer and controlling the size of the capsule (Uchida et al.1993).

Anthrax Toxin Complex: An Important VirulenceDeterminant of B. anthracis

The other important virulence factor of B. anthracis is thethree-component exotoxin called the anthrax toxin complex(Leppla 1991). This toxin complex, comprising of anthrax pro-tective antigen (PA), anthrax lethal factor (LF), and anthraxedema factor (EF), is primarily responsible for most of the symp-toms of the disease (Smith et al. 1955, 1956). The three structuralgenes, pagA, lef, and cya, encoding PA, LF, and EF, respectively,are all located on pXO1 (Robertson et al. 1990; Robertson &Leppla 1986; Robertson et al. 1988; Vodkin & Leppla 1983).

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THE ADENYLATE CYCLASE TOXINS 191

The three toxin genes along with their regulatory elements, re-solvase, transposase and the gerX operon map within the 44.8 kbpregion that is flanked by inverted IS1627 elements, and is knownas “the pathogenecity island” (Robertson et al. 1990). Recom-binant gene libraries have been prepared by digestion of pXO1with BamH1 and ligation to pBR322, and later by partial MboIdigestion and ligation to pUC8. Screening was later done to se-lect clones containing the EF gene. Mock et al. (1988) cloned EFgene by a different approach. Their approach involved direct ge-netic selection for the complementation of adenylate cyclase ac-tivity in cyaA− mutant of E. coli. The clones were later selectedon the basis of their ability to ferment maltose. The gene for EFhas been sequenced. Sequence analysis of the gene reveals thatit codes for a 800-residue protein with a typical amino-terminalBacillus signal sequence of 33 residues that is removed uponsecretion (Robertson et al. 1988; Escuyer et al. 1988).

The expression of the three toxin genes is coordinately regu-lated by bicarbonate and temperature at the transcription level.Two groups using different approaches independently isolateda pXO1-encoded transcriptional activator, AtxA that is requiredfor the toxin genes expression in vivo and in vitro (Koehler et al.1994; Uchida et al. 1993). The Sterne strain derivative, devoidof AtxA, is avirulent in the mouse model for anthrax. AtxAalso regulates the transcription of genes responsible for capsuleproduction (Uchida 1997). Another gene regulator, pagR wasrecently identified. It was shown to repress expression of thepag operon and the other pXO1-encoded genes (Hoffmaster &Koehler 1999). Further studies are required to identify additionalregulatory factors and to understand the mechanism by whichthey influence the expression of the target genes in B. anthracis.

Mechanism of Entry of the Edema Toxininto the Target Cells

Individually, all the three proteins are non-toxic. However,the combination of PA and LF, called the lethal toxin, causesdeath in experimental animals (Smith & Keppie 1954). Whereas,combination of PA and EF, known as the edema toxin, inducesan increase in intracellular cAMP levels of the susceptible cells(Leppla 1982) and elicits skin edema after subcutaneous injec-tion (Stanley & Smith 2001).

PA plays a central role during intoxication by anthrax toxin. Itis the receptor-binding moiety that facilitates the delivery of theother two components, LF and EF, into the cell. During intoxi-cation, protective antigen binds to its receptors on the surface ofsusceptible cells. The cleavage of the receptor-bound PA by thecell surface proteases, such as furin, results in the release of a 20kDa fragment from the N-terminal of the protein (Klimpel et al.1992). The 63 kDa fragment of PA (PA63) oligomerizes to formring-shaped heptamer (Milne et al. 1994; Ahuja et al. 2001). LFor EF bind competitively to the site exposed on release of 20 kDafragment of PA (Kumar et al. 2001; Gupta et al. 2001). This entirecomplex undergoes receptor-mediated endocytosis. The acidi-fication of the endosome causes major conformational changesin the PA molecule, leading to the insertion of the heptamer

into the endosomal membrane (Batra et al. 2001; Blaustein etal. 1989; Milne et al. 1993). LF and EF are translocated acrossthe endosomal membrane to the cytosol through these pores(Guidi-Rontani 2000). After reaching the cell cytosol, LF andEF, exert their toxic effects. LF is a metalloprotease that cleavesseveral isoforms of MAP kinase kinases within mammaliancells (Duesbery et al. 1998; Pellizzari et al. 1999; Vitale 1998).Whereas, EF is a calcium/calmodulin-dependent adenylate cy-clase that causes an increase in the intracellular cAMP levels ofthe host cells (Leppla 1982).

Structure-Function Relationship of the Edema ToxinX-ray crystallographic structure of EF was solved both with

and without the bound calmodulin (Drum et al. 2002). This pro-tein has four domains. The N-terminal 300 amino acids of LFand EF have extensive homology and are considered to be the do-mains that bind to PA63. Both the proteins have high proportionof highly charged residues at the N-terminus (residues 1–28 ofEF and homologous residues 11–38 of LF). Recent studies fromauthors laboratory show that residues 136VYYEIGK142 of EF(that are identical to the residues 147VYYEIGK153 in LF) areinvolved in the binding of EF to PA63 (Kumar et al. 2001; Guptaet al. 2001). Alanine-substitution of residues Tyr137, Tyr138,Ile140, and Lys142, makes EF defective in binding to PA andincapable of inducing cAMP toxicity in CHO cells.

The catalytic domain of EF begins soon after the bindingdomain. The catalytic domain lies entirely within the stretch262–767. This portion of the protein may be expressed as a stableand active truncated protein. The catalytic activity of EF requiresassociation of eukaryotic calcium binding protein, calmodulin.The dependence on calmodulin is absolute. No activity can bedetected in its absence. Calcium-calmodulin complex is a muchpotent activator of EF than calmodulin alone. Thus in the absenceand presence of 50 µM calcium, the concentrations of calmod-ulin needed to activate EF are 5 µM and 2 nM, respectively(Leppla 1984). Calmodulin binding site extends over a large sur-face area and four discrete regions of EF are involved: 510–540,615–634, 647–672, and 695–721. The binding of calmodulinto EF induces a conformational change in EF such that a high-affinity catalytic site is formed to which ATP subsequently binds(Drum et al. 2002).

Residues 314–321, matching the GxxxxGKS consensus, havebeen identified as the ATP binding site by site-directed mutage-nesis. Substitution of Lys320 with Asn causes more than 10-foldloss in the catalytic activity of EF (Robertson et al. 1988). Thecrystal structure of EF reveals that 3′-deoxy ATP and a singlemetal ion are well positioned for catalysis with His351 as thecatalytic base (Drum et al. 2002).

After gaining access to the cell cytoplasm, EF gets activatedby calmodulin, to catalyze the synthesis of cAMP in the hostcells. Studies on the enzymatic activity of EF demonstratedthat EF has a high catalytic activity with a Vmax of 1.2 mmolcAMP/min/mg protein. The adenylate cyclase activity of EF isvery sensitive to concentrations of calcium, showing optimal

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activity at 0.2 mM and inhibition at higher concentrations ofcalcium (Leppla 1984).

To gain more insight into the requirement of extracellularcalcium for causing edema toxin-mediated cAMP toxicity, westudied the accumulation of cAMP in the target cells in the pres-ence or absence of extracellular calcium (Kumar et al. 2002).Although, the response generated by edema toxin in differentcells, varied in intensity and in the time of initiation, the de-pendence on extracellular calcium was a common feature. Itwas observed that after the translocation of edema factor intothe cell cytosol, increased influx of calcium is triggered in thehost cell that plays a critical role in determining the ensuingcAMP response (Kumar et al. 2002). The absence of calcium orthe presence of calcium channel antagonists in the extracellularmedium prevented the cAMP accumulation by edema toxin inthe host cells.

Contribution of the Edema Toxinto the Pathogenesis of Anthrax

As reflected in its name, edema toxin induces local edemawhen injected in the skin of experimental animals. Subcutaneousinoculation of anthrax spores also lead to prominent swelling ofthe skin lesions and the surrounding tissues. Inhalation anthrax isalso associated with pulmonary edema and profound mediastinalwidening resulting from the edema of lymph nodes. These patho-logical symptoms are primarily caused by anthrax edema toxin.Studies by Hoover et al. (1994) suggests that the edema toxindifferentially regulates lipopolysaccharide-induced productionof TNF-α and IL-6 by increasing the intracellular cAMP levelsin monocytes. The disruption of the cytokine network may im-pair cellular antimicrobial responses and may also contribute toclinical signs and symptoms of anthrax.

The action of anthrax edema toxin on neutrophils has beenwell studied. By increasing the intracellular cAMP concentra-tions in neutrophils, anthrax edema toxin inhibits phagocytosisand blocks both particulate as well as phorbol myristate acetate-induced chemiluminescence (O’Brien et al. 1985). The edematoxin also inhibits the priming of the neutrophils by lipopolysac-charide. The studies by Alexeyev et al. (1994) confirm that theneutrophil function is impaired in the cutaneous form of anthrax.

Among the immune effector cells, the lymphocytes are mostsensitive to edema toxin-induced increase in cAMP concentra-tions (Kumar et al. 2002). The anthrax edema toxin-inducedmassive elevation in the cAMP levels of lymphocytes can leadto alteration of critical immunoregulatory genes (Bordor et al.2001), apoptosis (Colic et al. 2001), decrease in T-cell prolifer-ation (Skalhegg et al. 1992), and decrease in immune response(Bourne et al. 1974). In contrast to the response generated inthe lymphocytes, the edema toxin generates poor and delayedcAMP response in macrophages and neutrophils (Kumar et al.2002).

However, even a slight increase in cAMP levels of thesephagocytic cells is enough to cause inhibition of phagocytosis(Razin et al. 1978), superoxide production (Takei et al. 1998),

and bacterial killing by these cells. Thus, by accumulating cAMP,edema toxin may be disrupting bactericidal functions of immuneeffector cells and disabling the host defense mechanism, therebyfacilitating replication and survival of the invading bacterium.

EXOY: THE ADENYLATE CYCLASEOF PSEUDOMONAS AERUGINOSA

Pseudomonas aeruginosa is a gram-negative, aerobic rod, be-longing to the bacterial family Pseudomonadaceae. The Pseu-domonas are common inhabitants of soil and water. They occurregularly on the surfaces of plants and occasionally on the sur-faces of animals. In fact, these bacteria are common pathogen ofplants and animals. Three Pseudomonas species are known toinfect humans: (a) Pseudomonas mallei that causes glanders dis-ease in horses that is transmissible to humans; (b) Pseudomonaspseudomallei causes melioidosis, a highly fatal tropical diseaseof humans and other mammals; and (c) Pseudomonas aerugi-nosa is an opportunistic pathogen that causes urinary tract in-fections, respiratory system infections, dermatitis, soft tissueinfections, bacteremia, and a variety of systemic infections, par-ticularly in patients with severe burns, and in cancer and AIDSpatients who are immunosuppressed (Bodey et al. 1983).

Pseudomonas aeruginosa is notorious for its resistance to an-tibiotics and is, therefore, a particularly dangerous and dreadedpathogen. The bacterium is naturally resistant to many antibi-otics due to the permeability barrier afforded by its outer mem-brane LPS. Secondly, this bacterium colonizes the surfaces inform of a biofilm that makes the cells impervious to therapeu-tic concentrations antibiotics. Moreover, Pseudomonas main-tains antibiotic resistance plasmids. Thus, only a few antibioticsare effective against Pseudomonas, including fluoroquinolones,gentamicin and imipenem, and even these antibiotics are noteffective against all strains.

Most Pseudomonas infections are both invasive and toxino-genic. The pathogenesis of Pseudomonas infections is multi-factorial and is attributed to a large number of virulence deter-minants possessed by the bacterium. These include proteases,alginate, phospholipases, and toxins. The ultimate Pseudomonasinfection may be seen as composed of three distinct stages:(1) bacterial attachment and colonization; (2) local invasion;and (3) disseminated systemic disease. Particular bacterial de-terminants of virulence mediate each of these stages and are ulti-mately responsible for the characteristic syndromes that accom-pany the disease. One particular virulence determinant is ExoSwhose expression is correlated with the spread of the bacteriumfrom epithelial colonization sites to the bloodstream (Frank1997).

The expressions of all the genes of the ExoS regulon are co-ordinately controlled at the transcriptional level by ExsA (Yahr& Frank 1994). ExsA regulates its own synthesis and a seriesof operons encoding proteins that are involved in the intoxica-tion of eukaryotic cells by a process that is referred to as theType III secretion. The Type III secretion system comprises of

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three classes of gene products that include components of thesecretory apparatus, proteins that mediate the translocation ofthe effectors into the host cell cytoplasm and the effector pro-teins that disrupt the normal cellular processes.

The Type III-mediated effector translocation is postulatedto inhibit the phagocytic response to infection, allow bacterialreplication and promote epithelial injury. Four effector proteinsof the ExoS regulon have been identified (Frank 1997; Finck-Barbancon et al. 1997): ExoS that is an ADP-ribosyl transferase,ExoU that causes acute cytotoxicity, ExoT whose function is notknown and ExoY that is an adenylate cyclase.

Biochemical Properties of ExoYExoY is a 42-kDa protein that is encoded by 1,134 bp long

gene (Yahr et al. 1998). Residues 41–107 and 209–221 of ExoYshare homology with sequences of EF of Bacillus anthracis andthe invasive adenylate cyclase of Bordetella pertussis. The firstconserved region (extending from 41–107) contains ATP/GTP-binding site that binds to the α-phosphate of the bound nu-cleotide and the second conserved region (extending from209–221) that bind to the β- and γ -phosphate of the bound nu-cleotide, shares identity with a range of nucleotide-binding pro-teins including pyruvate kinase and phosphofructokinase (Glaseret al. 1991). However, ExoY does not contain any sequence thatmay have homology to the calmodulin-binding domains of theadenylate cyclases of Bordetella pertussis or of Bacillus an-thracis. Another distinguishing feature of ExoY is that unlikethe other two adenylate cyclases, this protein contains five cys-teines (Yahr et al. 1998).

ExoY is a heat-labile protein. It requires a eukaryotic cy-tosolic protein, distinct from calmodulin, for the stimulation ofits adenylate cyclase activity (Yahr et al. 1998). However thisprotein has not been identified as yet. The stimulation of ExoYadenylate cyclase activity by a eukaryotic cofactor may repre-sent a regulatory strategy to prevent the generation of cAMPwithin the bacterium before intoxication of the host cells. Sitedirected mutagenesis has revealed that the conserved residuesLys81, Lys88, Asp212, and Asp214 of ExoY are crucial for theadenylate cyclase activity of ExoY.

The infection of the eukaryotic cells with Pseudomonas aerug-inosa strains, producing catalytically active ExoY, results inthe elevation of the intracellular cAMP and cell morphologychanges. However the cell morphology changes or cAMP ac-cumulation were not observed when the Type III translocationapparatus was compromised by mutation, or when the catalyt-ically inactive form of ExoY was provided in trans. Direct ad-dition of ExoY to the cells also does not cause increase in thecAMP levels. This data provides convincing proof that the ExoYis delivered into the cytosol of eukaryotic cells via the type IIItranslocation mechanism. This mechanism of entry into the cellslimits ExoY intoxication to site of colonization. Although muchneeds to be learned about the role of ExoY in pathogenesis, itseems likely to be the one of considerable significance.

ADENYLATE CYCLASE OF YERSINIA PESTISThe genus Yersinia is composed of 11 species, three of which

are pathogenic in humans. The three pathogens, Y. pestis,Y. enterocolitica, and Y. pseudotuberculosis, cause a broad spec-trum of disease ranging from pneumonic plague to acute gas-troenteritis. Yersinia pestis, which is a gram-negative, nonmotile,nonsporulating, non-lactose fermenting, pleomorphic bacillus,causes the disease plague. Plague bacilli are aerobic andfacultative anaerobic.

Yersinia pestis is primarily a rodent pathogen, with humansbeing an accidental host when bitten by an infected rat flea. Theflea draws viable bacteria into its intestinal tract. The bacteriamultiply in the flea and block the flea’s proventriculus. Y. pestisis regurgitated when the flea gets its next blood meal, and theinfection is thus transferred to a new host. While growing inthe flea, Y. pestis loses its capsular layer. So, in the human hostmost bacteria are phagocytosed and killed by the polymorphonu-clear leukocytes. However, few bacilli are taken up by the tis-sue macrophages. These bacilli survive in the macrophages andresynthesize their virulence factors. They kill the macrophagesand are released into the extracellular environment. Y. pestis soonestablishes a systemic infection. Liver, spleen and lungs becomeinfected and the patient develops a severe bacterial pneumonia.During the coughing fits, the patient exhales viable organismsinto the air. As the epidemic spreads, it eventually shifts into apredominantly pneumonic form that is difficult to control andhas 100% mortality.

The important virulence factors of the bacteria are: the an-tiphagocytic capsule, the plasminogen activator (that degradesthe compliment components C3b and C5a), the invasin andYadA proteins (that facilitate the attachment of the bacteriato the eukaryotic host cells) and the Yop proteins (that disturbthe dynamics of cytoskeleton and block the production of pro-inflammatory cytokines, thereby facilitating the survival of theinvading Yersinia). Three forms of adenylate cyclase have beenidentified in Y. pestis: membrane-bound, cytoplasmic, and theextracellular. The extracellular adenylate cyclase has been puri-fied and its physiochemical properties studied (Shevchenko et al.1987). This 30 kDa protein has an optimal activity at pH 7.0and 7.2 and between 37–40◦C temperature.

Not much data is available on the role of this adenylate cy-clase. However, it has been shown that this protein suppressesthe oxidative metabolism of peritoneal leukocytes in white mice(Mishankin et al. 1989). It lowers the level of chemilumines-cence to 50–70% and has an appreciable cytotoxic effect on theperitoneal macrophages (Mishankin 1992). These results sug-gest that this adenylate cyclase may be an important virulencefactor that may contribute to development of plague infection.

To summarize, the adenylate cyclase toxins represent an im-portant group of bacterial toxins. They are potent virulence fac-tors. Much work still needs to be done to fully understand theirrole in pathogenesis of infections. These toxins provide an inter-esting model system to study how cAMP regulates basic cellularand metabolic processes. The adenylate cyclase toxins may serve

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as powerful probes in cell biology and membrane biochemistry.They may also serve as a tool by which cellular processes canbe modulated.

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