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Binary Bacterial Toxins: Biochemistry, Biology, and Applications of Common Clostridium and Bacillus Proteins

Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Freiburg, Germany,¹ CNR Anaerobies, Institut Pasteur, Paris, France,² U.S. Army Medical Research Institute of Infectious Diseases, Toxinology Division, Fort Detrick, Maryland³

SUMMARY

INTRODUCTION

BACTERIA: A RICH SOURCE OF BINARY TOXINS

C. perfringens Iota Toxin

C. spiroforme Toxin

C. difficile Toxin

C. botulinum C2 Toxin

B. anthracis Edema and Lethal Toxins, and B. cereus Vegetative Insecticidal Proteins

BIOCHEMISTRY, GENETICS, AND PROTEOLYTIC ACTIVATION

B. anthracis PA83

C. botulinum C2II

C. perfringens Ibp and Ia

PROTEIN STRUCTURE AND FUNCTION

B. anthracis PA, LF, and EF

C. botulinum C2II and C2I

C. perfringens Ib and Ia

ADP-Ribosylation: a Common Enzymatic Method Used by Various Bacterial Toxins

CELL ENTRY AND INTOXICATION

''B'' Binding to the Cell

''A'' Docking to Cell-Bound ''B'' and Internalization

BACTERIAL BINARY TOXINS: VERSATILE PROTEIN SHUTTLES, VACCINE TARGETS, AND THERAPEUTICS

Protein Shuttles

Vaccine Targets

Therapeutics

CONCLUSIONS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Several proteins from gram-positive, spore-forming bacilli employ a synergistic binary mechanism for intoxicating eukaryotic cells; they include Clostridium botulinum C2 toxin, Clostridium difficile toxin (CDT), Clostridium perfringens iota () toxin, Clostridium spiroforme toxin (CST), Bacillus anthracis edema and lethal toxins, as well as the Bacillus cereus vegetative insecticidal proteins (VIP). The protein components of these related toxins do not bind cells as a preformed "A-B" complex found in solution (Table 1), thus differing from many other bacterial binary toxins that engage cells as an intact "A-B" structure composed of single- or multiple-chain proteins (Table 2). Bacillus cereus and Staphylococcus aureus also produce other multiple-chain toxins composed of proteins not associated in solution; however, these pore-forming cytolysins remain on the cell surface and are devoid of enzymatic activity, thus differing from the Clostridium and Bacillus binary toxins described in this review (149a, 341a).

View larger version (28K): [in this window] [in a new window]   FIG. 1. Basic mechanisms of cell intoxication employed by Clostridium and Bacillus binary toxins. Cell-binding "B" precursors are first activated by proteolytic cleavage in solution or on the cell surface. The furin-based, cell-associated cleavage of B. anthracis PA83 is unique, since none of the other "B" precursors are activated after binding to a cell. Activated "B" components interact with a specific cell surface receptor(s) as either preformed, ring-shaped homoheptamers or monomers that subsequently form heptamers. An enzymatic "A" component(s) docks with the cell-bound "B" heptamer, and the receptor-holotoxin complex is then taken up via receptor-mediated endocytosis into early endosomes, which become acidified by vacuolar-type ATPases. An acidic environment is essential for translocating an "A" component(s) into the cytosol, since this induces a conformational change and subsequent insertion of the "B" heptamer into an endosomal membrane to form a channel. Although not clearly defined, it is likely that an "A" component(s) is transported into the cytosol through the "B" heptamer-induced channel.

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Compared to other bacteria, Clostridium and Bacillus species have developed unique mechanisms for survival within and outside of numerous host types, as evidenced by the various diseases frequently linked to their protein toxins and spores. C. botulinum, C. difficile, C. perfringens, and C. spiroforme are associated with a plethora of animal and human diseases and intoxications such as gas gangrene, food poisoning, antibiotic-associated diarrhea, pseudomembranous colitis, and/or enterotoxemia (52, 258, 411, 423). Maladies attributed to B. anthracis or B. cereus also occur in animals and/or humans, and they respectively include three forms of anthrax (cutaneous, intestinal, and inhalational) (38, 132, 172) and food poisoning (259). As described below, the binary toxins produced by Clostridium and Bacillus species are involved in diverse diseases, and this further accentuates the differences that also exist within this toxin family.

There are intriguing physical (molecular weight and epitopes) as well as functional (cytotoxicity) variations between C2I and C2II components produced by different C. botulinum strains (301, 307), which perhaps is not surprising from an evolutionary perspective. Similar structural and functional data are unfortunately lacking for the protein components of other Clostridium and Bacillus binary toxins, with one notable exception being the highly conserved protective antigen from B. anthracis (342). Finally, based on earlier reports that C2I possesses ADP-ribosyltransferase activity specific for arginine (391), the intracellular substrate of C2 toxin was subsequently identified in 1986 as actin (7, 310) and thus represents the discovery of a new family of bacterial ADP-ribosylating proteins that target the cytoskeleton.

The binary toxins produced by B. anthracis were the first multicomponent bacterial toxins ever described in the literature (408), and they consists of three synergistically acting proteins (413) now known as edema factor (EF), lethal factor (LF), and protective antigen (PA) (407). From a historical perspective, this discovery is quite fitting, since Robert Koch and Louis Pasteur initially used B. anthracis to prove profound scientific concepts involved in disease (Koch's Postulates in 1876) and immunology (Pasteur's vaccine studies in 1881). The PA molecule combines with EF and/or LF on the cell surface to respectively form edema and/or lethal toxins, which represent virulence factors that work synergistically toward bacterial survival and dissemination (77, 172, 204, 230, 279, 296, 321, 327, 335, 336). Unlike the protein components of CDT, CST, and toxin, those of C2 as well as the edema and lethal toxins do not form biologically active chimeras with other binary toxins (325, 418).

Relative to any other Clostridium or Bacillus binary toxin described in this review, much less information is available for the newly (1990s) discovered B. cereus VIP, composed of VIP1, a cell-binding component, and VIP2, an ADP-ribosyltransferase that targets actin (171). In addition to its insect-killing properties on Northern and Western corn rootworms via VIP, B. cereus is involved in human food poisoning (158a) and is considered a nonlethal intestinal symbiont of various soil-dwelling insects such as roaches, sow bugs, and termites that, when foraging, inadvertently ingest Bacillus as well as Clostridium spores (251). Future studies with specific antibodies and gene probes for the various components of binary toxins described in this review will probably yield new toxins produced by different species of Bacillus, Clostridium, and perhaps other bacterial genera.

BIOCHEMISTRY, GENETICS, AND PROTEOLYTIC ACTIVATION

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The protein components of C. botulinum C2 toxin (302), C. difficile CDT (324), C. perfringens toxin (422), C. spiroforme CST (333), B. anthracis edema and lethal toxins (408, 413), and B. cereus VIP (171) are produced as separate "A" and "B" molecules not associated in solution. Table 1 lists the gene locations, molecular weights, and enzymatic activities of these bacterial binary toxins. The isoelectric points of mature "A" components range from 5.2 (C. perfringens Ia) to a distinctly high 9.3 (C. difficile CDTa), while the "B" component range encompasses pH 4.5 (C. difficile CDTb) to 6.2 (C. botulinum C2II) (331). The cell-binding components are enzymatically inert (as ascertained by existing assays) and produced by the bacterium as precursor molecules, with isoelectric point shifts of less than 0.8 pH unit following activation by various serine-type proteases such as chymotrypsin, furin, or trypsin derived from the bacterium, host, or exogenous addition in vitro (122, 211, 325, 417). The resultant loss of an N-terminal peptide (20 kDa) from a "B" precursor induces conformational changes that facilitate homoheptamerization, either in solution or on the cell surface, and subsequent docking with an "A" component(s). To further understand the various mechanisms for proteolytic activation of bacterial toxins, we recommend the comprehensive review by Gordon and Leppla (156).

Like other multicomponent toxins, the enzymatic and binding proteins of all Clostridium and Bacillus binary toxins are encoded by distinct genes that have a 27 to 31% G+C content (331). As an example, the genes for B. anthracis EF, LF, and PA are located on a large (182-kbp) plasmid called pXO1 (271, 312, 315) and span a 23-kbp region (230). Among the clostridia, "A"- and "B"-component genes are transcribed in the same orientation from a common operon consisting of an "A" gene located 40 to 50 nucleotides upstream of the "B" gene, with a known exception being the open reading frames for C2 toxin, which are separated by 247 nucleotides (137, 147, 208, 323, 331). There is also another significant difference at the genetic level since C. botulinum C2 toxin, C. difficile CDT, and C. spiroforme CST are chromosome encoded, in contrast to the plasmid-localized C. perfringens toxin. Each "A" and "B" component of the Clostridium and Bacillus binary toxins, but not those from C2, is respectively synthesized with a signal peptide consisting of 29 to 49 and 39 to 47 residues (331). These findings are consistent with proteins secreted during logarithmic growth; however, the C2 toxin is evident from sporulating C. botulinum only after sporangium lysis (289, 331). It was recently shown that an extracellular chaperone protein, PrsA, is important for efficient folding and secretion of PA83 from Bacillus species via a Sec-dependent route (477), but it is unknown if similar chaperones play any role with binary toxin components produced by the other bacilli.

Production of B. anthracis toxin is controlled by the positive regulatory gene atxA, also located on pXO1, via protein binding 110 bp upstream of the ATG start codon, and its importance in pathogenesis is further evidenced by the observation that less virulent strains lack the atxA gene (94, 214). The 56-kDa protein encoded by atxA is unique, with little sequence homology to other known transcriptional activators (94). In addition to the genes for edema and lethal toxins, atxA regulates the expression of others on pXO1 (182), the pXO2 plasmid (93 kbp) that encodes the capsule (165), and the chromosome (56, 270). Bicarbonate, carbon dioxide, and temperature also represent environmental factors that regulate the synthesis of B. anthracis edema and lethal toxins, as well as capsule (214, 230, 270). Gene products or environmental factors that may affect the production of other bacterial binary toxins described in this review are currently unknown, although divalent cations seemingly play a role in CST synthesis via an undefined mechanism (74).

Comparisons of amino acid sequences among the Clostridium and Bacillus binary-toxin components reveal similar evolutionary paths, since they share (i) 80 to 85% identity within the -toxin family, which includes CDT, CST, and toxin, but the signal peptide sequences are less highly conserved (40 to 61% identity); (ii) 31 to 40% identity between C2 and -family toxins; (iii) 26 to 30% identity between PA and clostridial "B" components; and (iv) 29 to 31% identity between VIP and equivalent clostridial toxin components, which overall suggests that these toxin genes were derived from a common ancestor. Clearly, the "B" components are structurally conserved between Clostridium and Bacillus binary toxins (Fig. 2A), and over time these proteins have adapted to transporting unique "A" components into cells. For example, there are striking differences between "A" components such as B. anthracis EF (an adenylcyclase) and LF (a metalloprotease), which are quite distinct structurally and enzymatically from the ADP-ribosyltransferases within the C2 or toxin families. Although unproven, it is very plausible that the binary toxin genes originated from an ancestral form of clostridia and were horizontally transferred between Bacillus and Clostridium species via plasmids capable of inserting into the bacterial chromosome, as perhaps evidenced by the CDT, CST, and C2 toxin genes. However, with one known exception found in a C. spiroforme strain, insertion sequences do not commonly flank these toxin genes. This omission may indicate that genetic rearrangements and/or deletions occurred after successful transfer (331).

View larger version (176K): [in this window] [in a new window]   FIG. 2. The cell-binding "B" proteins of Clostridium and Bacillus binary toxins are activated by serine-type proteases, share varying sequence homology, and form heptameric ring-like structures. (A) Proteolytic cleavage sites, domain functions, and amino acid lengths of PA83, C2II, and Ibp precursor molecules are shown on the left. Below C2II and Ibp are the percent identities (and in parentheses are the percent homologies) for the amino acid sequences from each domain relative to PA83. Percent sequence identities of the "B" precursors (top), activated "B" proteins (middle), and N-terminal peptides released after proteolysis of "B" precursors (bottom) are shown on the right. Sequences were found in either the DNA Data Bank of Japan (DDBJ) with accession number D88982 (C2II) or GenBank with accession numbers M22589 (PA), I40862 (Ib), X97969 (Sb), and L76081 (CDTb). Modified from reference 31 with permission. (B) C2IIa heptamers on lipid bilayers as detected by electron microscopy. Modified from reference 32 with permission.

Activation of PA83 on the cell surface is quite robust, since proteolysis occurs at 4°C or on chemically fixed cells (230). Studies with a furin-resistant variant of PA83 reveal that this molecule is not readily internalized into cells, suggesting that surface-associated PA83 is not "wasted" (i.e., internalized prior to proteolytic activation, oligomerization, and transport of EF and/or LF into the cytosol) (40). Additionally, the 20-kDa peptide generated after proteolysis of the PA83 precursor slows PA63 clearance from the cell surface (40), perhaps by preventing heptamer formation, localization into lipid rafts (1), and ultimately endocytosis, which further optimizes EF and LF docking opportunities. The 20-kDa peptide also does not form membrane channels like PA63 (215). To our knowledge, similar work with the 20-kDa precursor peptides from other binary toxins described in this review has not been reported in the literature. Following proteolysis, whether in solution or on the cell surface, PA63 readily assembles into sodium dodecyl sulfate (SDS)-resistant, hydrophobic homoheptamers that form pH-dependent (pH 7), ion-permeable channels in membranes obstructed by known channel blockers (39, 46, 245, 272, 274, 275, 294, 326). X-ray crystallography of the PA83 monomer reveals four distinct domains, and the PA63 heptamer, as first demonstrated by electron microscopy in 1994 by Milne et al. (275), forms a ringed structure 160 Å in diameter and 85 Å in length, with a central lumen diameter of 35 Å (326).

Another unique aspect of PA versus the cell-binding components of other binary toxins is that the PA63 heptamer provides a cell surface docking site for two different proteins, EF and LF (281, 282). Both EF and LF possess a common, N-terminal heptapeptide (VYYEIGK) that is integral for competitive docking interactions with PA63 (168, 219, 224, 229). This sequence does not appear in the enzymatic components of other binary toxins, further supporting previous experimental data showing that PA does not bind or internalize heterologous "A" molecules (325). In fact, the only known complementation of heterologous components that exists within the binary-toxin family occurs among the enterically acting C. difficile CDT, C. perfringens toxin, and C. spiroforme CST (324, 393, 417, 418).

Other proteases such as pepsin, proteinase K, subtilisin, and thermolysin activate Ibp more efficiently in solution than trypsin does, and as more recently discovered, Ia is also proteolytically activated by these enzymes, with a resultant loss of 9 to 13 amino acids from the N terminus (148). It is still uncertain whether proteolysis of Ia increases (i) efficiency of docking to cell-bound Ib, (ii) efficiency of translocation into cells, and/or (iii) ADP-ribosyltransferase activity. Proteolytic activation of Ia is unique among the "A" components from binary toxins; however, upon further examination, they too may possess similar proteolysis patterns.

It is noteworthy that among another family of "A-B" toxins composed of heterologous proteins that form holotoxins in solution, like Escherichia coli heat-labile, Shigella dysenteriae Shiga, and Vibrio cholerae cholera enterotoxins, the enzymatic "A" components are also processed by serine-type proteases. However, following proteolysis, these "A" components form A₁ and A₂ subunits linked by a disulfide bond subsequently reduced by protein disulfide isomerase located in the endoplasmic reticulum (2, 140, 141, 181, 228, 250).

PROTEIN STRUCTURE AND FUNCTION

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Within domain 1', residues R₁₇₈, K₁₉₇, R₂₀₀, F₂₀₂, L₂₀₃, P₂₀₅, I₂₀₇, I₂₁₀, and K₂₁₄ have been identified by two different groups as being critical for the docking of EF and LF (80, 92). The maximum number of EF or LF molecules interacting with a PA63 heptamer has been controversial, since estimates range from seven (400) via nondenaturing gel electrophoresis to a more convincing three (92, 281, 282) when determined by multiple methods that include gel filtration chromatography, multiangle laser light scattering, and analysis of oligomer-deficient variants of PA63. Similar stoichiometric analysis of A-B interactions has not been done with the other binary toxins. Additionally, it is still not known if all enzyme molecules docked with a PA63 heptamer are efficiently translocated into the cytosol. It is possible that translocation of an EF or LF molecule induces conformational changes in the PA63 heptamer that either facilitate, or perhaps even inhibit, the subsequent passage of other "A" molecules.

The importance of domain 4, and particularly residues 671 to 721, in PA binding to cells was first described by Little et al. on the basis of epitope mapping with monoclonal antibodies (240, 242, 243). Subsequent mutagenesis efforts by other groups show that Y₆₈₁, N₆₈₂, D₆₈₃, and P₆₈₆ represent key residues critical for PA binding to its receptor (61, 361, 449). Further evidence that an exposed loop (residues 703 to 722) is important for PA binding to cells was obtained by Brossier et al. (64) via deletions of 9 or 16 amino acids from this region. Other studies of domain 4 reveal that truncations of only 5 to 12 amino acids from the far C terminus of PA prevent binding to cells (401, 449), suggesting an important role in direct binding and/or conformational integrity of PA (230). Similar investigations with C2II and Ib also unveil a pattern of exquisite sensitivity regarding deletions within the C terminus and subsequent effects on biological activity (48, 252), thus demonstrating a conserved structural trait among "B" components from this binary-toxin family.

A comparison of amino acid sequences between PA, C2II, and Ib reveals 27 to 38% identity; localized primarily within central domains 2 (amino acids 259 to 487) and 3 (amino acids 488 and 596) (Fig. 2A), that respectively participate in channel formation/enzyme translocation and oligomerization of PA63 monomers (43, 46, 47, 205, 206, 208, 213, 283, 324, 326, 373, 381, 382). As previously described, PA83 is readily converted into PA63 and homoheptamers by serine-type proteases in solution or on cell membranes (66, 118, 245, 272, 275, 294, 326). After proteolysis at pH <7, domain 2 undergoes a conformational shift that stabilizes a PA63 heptamer which binds to cells and docks with the enzyme but is translocation defective (272). However, if PA63 heptamers are proteolytically generated and maintained at pH >8, they are less stable (as evidenced by their SDS solubility at room temperature) but represent a biologically active "prepore" that binds to cells, docks with EF/LF, and subsequently translocates EF/LF into the cytosol following endosomal acidification (272). Domain 2 contains a "Greek key" motif (residues 262 to 368) that unfolds to form a ß-hairpin amphipathic loop (residues 302 to 325) which inserts into the membrane (357), thus promoting an acid-driven prepore-to-pore conversion (43, 167, 290). Although PA63 has little sequence homology to the alpha-hemolysin of Staphylococcus aureus, there are striking similarities in how these heptameric, pore-forming proteins produced by quite different bacteria insert into membranes (410). Further investigations of domain 2 identify PA residues D₄₂₅ and F₄₂₇, which are conserved in C2II, CDTb, Ib, and Sb (147, 208, 323, 324), as critical for channel formation as well as translocation (381, 382). Another group has shown that alanine mutations of residues W₃₄₆, M₃₅₀, and L₃₅₂ within domain 2 result in a PA63 heptamer unable to facilitate LF-induced cytotoxicity, perhaps because of dysfunctional membrane insertion and/or enzyme translocation (39). Analysis of PA63 crystals produced at pH 6 and 7.5 shows that residues 342 to 355 become exposed at the lower pH (326), thus promoting a more hydrophobic state and oligomerization (39, 215, 275). It is also within domains 2 and 3 that alanine mutations of Q₂₇₇ (buried) and F₅₅₄ (surface exposed) respectively increase the thermostability of wild-type PA, which might be useful in improving vaccine stability (396). Additional mutagenesis studies of a surface-exposed, hydrophobic "patch" in domain 3 reveal that alanine replacement of highly conserved residues F₅₅₂, F₅₅₄, I₅₆₂, L₅₆₆, or I₅₇₄, also evident in the "B" components of other Clostridium and Bacillus binary toxins, equivocally results in an oligomer-defective molecule of PA63 (5, 206, 283). Except for a recent study by Blöcker et al. with C. botulinum C2II (47), there has been very little structure-function analysis within domains 2 and 3 of "B" components from the other binary toxins.

The crystal structure of LF, a Zn²⁺ metalloprotease specific for the N terminus of a conserved family of eukaryotic proteins (MAPKK) involved in cell signal transduction (106, 107, 210, 452, 453), was recently reported by Pannifer et al. (314). Upon entering a cell via PA, where macrophages have been considered the primary target for lethal toxin (131, 174) but recent work suggests that other cell types are adversely affected too (3, 208a, 278), LF binds to various MAPKK on the latter's C-terminal regulatory region and cleaves within a proline-rich, N-terminal site that subsequently inhibits MAPKK interaction with the substrate and enzymatic activity (83, 107, 452). Additionally, cleavage of MAPKK may also result in modification by ubiquitin and rapid degradation by proteasomes (433). However, the role that truncated MAPKK play in toxicity is still uncertain, as evidenced by their cleavage in macrophages resistant to lethal toxin-induced cell death (321, 459). Perhaps susceptibility to lethal toxin is dictated by a kinesin-like motor protein (Kif1C), since polymorphic forms of Kif1C evident in resistant, but not susceptible, murine macrophages result in protection via an unknown mechanism not involving toxin entry or processing (459). A PA-LF combination increases the permeability of macrophage membranes, depletes intracellular ATP levels, and ultimately causes cell lysis, but these effects are all readily prevented by reducing agents, amines, bestatin, monensin, or inhibitors of metallopeptidases and possibly caspases (131, 175, 176, 207, 208a, 268, 316, 335, 336, 433). In addition to the in vitro effects described above, mice devoid of macrophages are resistant to lethal-toxin-induced mortality (174).

Akin to a host's adverse reaction to endotoxin or bacterial superantigens (i.e., S. aureus enterotoxins), proinflammatory cytokines may also play a role in B. anthracis edema toxin and lethal-toxin activity, but this concept still remains controversial (116, 152a, 173, 174, 184, 197, 207, 217, 218, 269, 278, 321, 335, 336). An apparent augmentation of cytokine-induced damage involves lethal toxin and indirect repression of select nuclear hormone receptors for estrogen, glucocorticoid, and progesterone that normally provide a protective anti-inflammatory response for a host (460). In addition to cytokine-elicited damage by the B. anthracis toxins, a cytokine-independent hypoxia induced by lethal toxin causes terminal necrosis of the liver and metaphyseal bone marrow (278, 344). Overall, lethal toxin seemingly represents a defense mechanism employed by the bacterium to weaken the host immune system by eliminating or impairing the immunological responsiveness of major cell types (i.e., macrophages and dendritic cells) naturally involved in pathogen clearance (137a). However, as recently shown by Salles et al. (369), a low percentage of macrophages can adapt to and resist high concentrations of lethal toxin when initially exposed to a lower yet not uniformly lethal dose of toxin in vitro. It will be interesting to see if this phenomenon is also apparent in vivo and perhaps is linked to quorum sensing. Finally, B. anthracis spores use macrophages as a germination site (162, 163, 465, 466), and perhaps lethal toxin, as well as other less well defined virulence factors, may facilitate the survival and dissemination of B. anthracis throughout the host from this mobile, normally pathogen-hostile environment (97a, 162a, 321). Since the bacterium is not directly transmitted from an infected individual to another possible host, eventual killing of the host and subsequent deposition of spores into the soil logically appear to be important mechanisms for B. anthracis survival and dissemination.

Like PA, LF also contains four distinct domains. The N-terminal domain 1 (amino acids 1 to 267) is important for docking to PA (345), blocks PA63-induced channels (487), and shares a multi--helix bundle as well as ß-sheet structure with domain 4, perhaps reflecting domain duplication (Fig. 3A). Although structural similarities exist between domains 1 and 4, there is very little sequence homology and there are no functional commonalities. As mentioned above, it is within domain 1 that LF and EF both contain a common VYYEIGK sequence important for docking to PA63 heptamers (168, 219). Monoclonal antibodies against LF prevent docking with cell-bound heptamers of PA63 and cross-react with EF, presumably via a shared epitope within domain 1 (241). In addition to the conserved VYYEIGK sequence, residues H₃₅, H₄₂, D₁₈₇, and F₁₉₀ play an important role in the proper LF conformation necessary for docking to PA63 heptamers (19, 395).

View larger version (51K): [in this window] [in a new window]   FIG. 3. (A) Enzymatic "A" proteins of Clostridium and Bacillus binary toxins with known catalytic sites and docking domains for "B" heptamers. (B) ADP-ribosylation of G-actin at R177 by C2I, according to the B. cereus VIP2 model as proposed by Han et al. (171). In the left-hand panel, the hydrophobic cleft of the C2I catalytic domain is depicted with bound NAD. Amino acids E387 to E389 stabilize an intermediate state before nucleophilic attack on G-actin R177, thus yielding mono-ADP-ribosylated G-actin and nicotinamide (right-hand panel). The same mechanism is utilized by other ADP-ribosylating toxins (CDT, CST, and ) that modify G-actin. Panel B modified from reference 31 with permission.

The EF molecule, a Ca²⁺/calmodulin-dependent adenylate cyclase (229) that shares structural homology (24% sequence identity) and epitopes with the Bordetella pertussis, but not mammalian, adenylate cyclases (158), has also been crystallized with and without calmodulin (103, 104). The N terminus of EF (or LF) docks with PA via an aforementioned VYYEIGK sequence (219, 223, 239), while residues 291 to 776 are sufficient for catalysis (Fig. 3A) (105). Residues 342 to 358 contain a motif (GXXXXGKT) commonly found in other ATP-binding proteins (136), including the B. pertussis adenylate cyclase that has 88% homology with EF in this region of 17 amino acids (223). Intriguingly, C. difficile CDTb, C. perfringens Ib, and C. spiroforme Sb also have a similar ATP-binding motif within the N terminus that appears dysfunctional and unnecessary for the biological activity of these common "B" components (147). The increased levels of intracellular cAMP induced by EF, which can be 1,000-fold higher than basal levels (230, 434), are nonlethal and transient, since the intracellular half-life of EF is 2 h (229).

As eloquently described over 20 years ago, the activity of EF is intimately dependent on calmodulin (229), a conserved Ca²⁺-sensing protein (16.5 kDa) that is found in all eukaryotic cells and binds to numerous proteins directly or indirectly involved in the cytoskeleton, ion flow, transcription, and vesicular trafficking (104). Calmodulin interactions with EF are dependent on Ca²⁺ levels, which, when too high, inhibit binding to EF and catalysis. Second, hydrophobic and hydrophilic residues within EF regions 501 to 540 (K₅₂₅ in particular) and 616 to 798 induce a conformational shift in EF, after calmodulin binding, which exposes the catalytic core and H₃₅₁ found in three globular domains encompassed by residues 294 to 622 (104, 223, 286, 384). Calmodulin interactions with EF also stabilize EF residues 579 to 591, which may also be important in ATP binding and catalysis. Once EF enters a cell, it triggers Ca²⁺ influx and subsequently elevates intracellular cAMP levels, as demonstrated in various cell types that include leukocytes (220). Increased cAMP levels in leukocytes can profoundly decrease the host immune response (57, 87) by inhibiting lymphocyte proliferation (405), phagocytosis (296), oxidative burst (479), and proinflammatory cytokine release (184); thus, EF seems to be another clever bacterial tool that enables B. anthracis to survive and flourish during an infection (220). The effects of EF are no doubt acting in concert with those of LF, the latter affecting MAPKK signaling pathways important in macrophage activation, nitric oxide production, and cytokine release (172, 279, 321, 335, 336). Additionally, the synergy between EF and LF can increase melanogenesis, thus generating perhaps the dark eschar commonly associated with cutaneous anthrax (216). The many complementary and conspiring roles that edema and lethal toxins play in B. anthracis survival via host impairment will become even more evident in the future, with time and well-crafted experiments.

For the C2I molecule, residues 1 to 87 mediate binding to C2IIa heptamers and translocation across the cytoplasmic membrane (Fig. 3A) (37). Alignment of C2I with VIP2 from B. cereus reveals that amino acids 1 to 225 of C2I correspond to the N domain of VIP2 (residues 60 to 275) containing -helices 1 to 4 (residues 1 to 87 in C2I and 60 to 133 in VIP2). Residues 12 to 29 of C2I are akin to the first -helical structure encompassing residues 71 to 85 in VIP2, while the other N-terminal helices are also exposed on the protein surface (171). Further analysis of VIP2 crystals shows two structurally homologous domains possessing similar folding patterns, a result most probably generated by gene duplication. X-ray crystallography of other bacterial ADP-ribosyltransferases such as B. pertussis pertussis toxin (415), C. diphtheriae diphtheria toxin (82), E. coli heat-labile enterotoxin (404), P. aeruginosa exotoxin A (235), and VIP2 (171) reveals that within the C terminus there are (i) two antiparallel ß-sheets flanked by a pair of -helices and (ii) a highly conserved catalytic domain containing an ₃₈₇EXE₃₈₉ motif found in prokaryotic as well as eukaryotic ADP-ribosyltransferases (444). However, the sequence homology of these toxins within the C terminus is low. Further studies of the EXE motif of C. botulinum C2I show that an E₃₈₇Q mutation prevents ADP-ribosyltransferase, but not NAD-glycohydrolase, activity while the same alteration of E₃₈₉ inhibits both (36). These results are similar to those derived from mutagenesis of P. aeruginosa exoenzyme S, an ADP-ribosyltransferase specific for Ras GTPases (347, 358).

Although no one has published the crystal structure of Ib, that of Ia has been recently reported by Tsuge et al. (438). Analysis of Ia reveals two domains that have conformational, but little sequence, similarity (Fig. 3A). The catalytic C domains of Ia (residues 211 to 413) and VIP2 (residues 266 to 461) (171) are also quite homologous, with 40% sequence identity and a similar distribution of surface charges. However, one obvious difference between Ia and VIP2 is the spatial orientation of the first glutamic acid found within the conserved catalytic motif, EXE. Like C2I (36), the initial glutamic acid within the ₃₇₈EXE₃₈₀ motif of Ia is important for ADP-ribosyltransferase, but not NAD-glycohydrolase, activity (287). Further analysis of Ia by site-directed mutagenesis or mass spectrometry of cyanogen bromide/trypsin-generated peptides reveals that C-terminal residues R₂₉₅ and E₃₈₀, which are conserved among various ADP-ribosyltransferases (76, 196, 368, 432), are also important for Ia catalysis (287, 323, 444) (Fig. 3A). An additional motif, ₃₃₈STS₃₄₀, found in Ia is also commonly located near the active site of many other ADP-ribosyltransferases. Although the ADP-ribosyltransferases of C. botulinum (C2I) or C. difficile (CDTa) have not been crystallized to date, structure-function studies show that the same amino acids are also necessary for enzymatic activity (36, 166). Extensive mutagenesis studies of Ia that focus on the NAD-binding cavity reveal that Y₂₄₆ and N₂₅₅ are important for ADP-ribosyltransferase, but not NAD-glycohydrolase, activity, unlike Y₂₅₁ involvement in both (287). All ADP-ribosyltransferases within the binary toxin family (C2I, CDTa, Ia, Sa, and VIP2) target globular (G)-actin, which is a common and remarkably conserved protein found throughout nature and plays a pivotal role in the cytoskeleton and intracellular trafficking of all eukaryotic cells (84, 85, 110, 351, 470).

In contrast to the C-terminal similarities, the N-terminal domains of Ia (residues 1 to 210) and VIP2 (60 to 265) have only 20% sequence identity, dissimilar surface charges, and different conformations, as further evidenced by Ia possessing an additional -helix (residues 61 to 66). Relative to "A" components of other binary toxins, the Ib docking region on Ia is more centrally located within the N-terminal domain (residues 129 to 257) (253) than C2I residues 1 to 87, needed for binding to C2II (37); LF residues 1 to 254, needed for interactions with PA (21); or CDTa residues 1 to 240, needed for docking to CDTb (166) (Fig. 3A). Overall, these data probably reflect evolutionary variation among the "A" and "B" proteins comprising these related Clostridium and Bacillus binary toxins.

The actin-ADP-ribosylating toxins can be subdivided into two groups: (i) C2 toxin, which exclusively mono-ADP-ribosylates at R₁₇₇ the isoforms of ß/-nonmuscle, as well as -smooth muscle, G-actin (6, 199, 201, 310, 446) (Fig. 3B), and (ii) the -like toxins, which mono-ADP-ribosylate R₁₇₇ of all G-actin isoforms, including -actin of skeletal muscle (255, 445). The "A" components of CDTa, Ia, and Sa have an LKDKE sequence between N-terminal residues 10 to 19 that is commonly associated with the binding of actin (331, 338). The C2I molecule has a different actin-binding sequence and location, ₄₄LKTKE₄₈, which may also explain its unique substrate specificity. The enzymatic activity of toxin is inhibited by EDTA chelation of divalent cations associated with actin, but low temperature (0°C) decreases activity only 50% compared with that at 37°C (198). It has also been shown that enzymatic components of C2 and toxins, in the presence of nicotinamide excess, remove the ADP-ribosyl moiety from modified actin; however, C2I does not displace ADP-ribose from Ia-modified actin of skeletal muscle (198). Filamentous (F)-actin does not represent a substrate target. However, ADP-ribosylation of G-actin inhibits monomer assembly into F-actin strands (6, 7), which leads to decreasing F-actin, but increasing G-actin, concentrations within a cell (12, 461, 463). An actin-gelsolin complex, in which gelsolin facilitates actin nucleation and subsequent polymerization, is also modified by as well as C2 toxins that block additional nucleation activity (30, 201, 462, 476). Additionally, the and C2 toxins ADP-ribosylate G-actin complexed with ATPase, which results in an increased exchange, but decreased hydrolysis, of ATP (145, 146). From the perspective of bacterial survival, disruption of a eukaryotic cytoskeleton and reduction of ATP hydrolysis can prevent phagocytosis (16), intracellular trafficking, and ultimately induce cell death with subsequent release of valuable nutrients. From a scientist's perspective, toxins that modify actin have become invaluable tools for studying the cytoskeleton and numerous cell processes such as endothelium permeability, exocytosis and endocytosis, leukocyte activation, migration, etc. (Table 3). Previous reviews of this topic are acknowledged and recommended for further reading (9, 10, 13, 15, 88, 300).

View this table: [in this window] [in a new window]   TABLE 3. Use of C2, CST, and toxins as tools to determine various cellular processes involving the actin cytoskeletona

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To further understand the binding and oligomerization properties of "B" components on cell surfaces, recent studies have delved into the potential role played by lipid rafts. Lipid rafts are cholesterol-rich, detergent-insoluble (at 4°C) "structures" or "microdomains" located on the outer cell membrane that inadvertently serve as dispersed attachment, entry, and sometimes exit sites cleverly pirated by various bacteria, viruses, and toxins (124, 177, 225, 277, 388). As recently reported, lipid rafts and a clathrin-dependent process promote the oligomerization as well as internalization of PA; however, the PA receptor is not initially raft associated (1). The assembly of PA63 into lipid rafts is cholesterol dependent and interrupted by ß-methylcyclodextrin, a compound that depletes cell membranes of cholesterol (1). Via an ill-defined process mimicking ligand-dependent clustering of B-cell receptors into rafts (81), activated PA63 "forces" receptor localization into lipid rafts that subsequently generates PA63 heptamers and entry of EF and LF into the cytosol by acidified endosomes and cation-selective channels (46, 123, 157, 274). It is likely, but not definitively proven, that EF and LF travel into the cytosol through the lumen of a PA63-induced channel; however, translocated EF probably remains associated with the endosomal membrane (164). PA83, which does not form heptamers or ion channels, does not bind EF or LF, and does not facilitate any recognized toxicity, also shares these same attributes with other precursor molecules such as C2II and Ibp (46, 213, 288, 373, 420). Although little work has been done with lipid rafts and the other Clostridium or Bacillus binary toxins, recent results do suggest that C. perfringens Ib, but not Ibp, localizes into these membrane microdomains on Vero cells that are sensitive to toxin (169a).

In addition to PA, receptor-binding studies have also been done rather extensively with C2II. The C2II precursor and proteolytically activated C2IIa bind to cells (304); however, only C2IIa has hemagglutinating properties with human as well as animal erythrocytes, which is a process competitively inhibited by various sugars such as N-acetylgalactosamine, N-acetylglucosamine, L-fucose, galactose, and mannose (428). This study also shows that trypsin or pronase pretreatment of human erythrocytes prevents C2IIa-induced hemagglutination, thus suggesting that the receptor for C2II/C2IIa is a glycoprotein. Further revelations regarding the C2II receptor were provided by Fritz et al. (135) via chemical mutagenesis of CHO cells (designated RK14) that subsequently do not bind C2IIa because they lack the N-acetylglucosaminyltransferase I activity necessary for forming asparagine-linked carbohydrates (109). The altered gene contains a premature stop codon for W₉₆. From these experiments, it can be concluded that the receptor for C2IIa contains a complex or hybrid carbohydrate structure. In contrast, the RK14 cells are still susceptible to toxin, and this finding further demonstrates that C2IIa and Ib recognize unique receptors. C2IIa, like PA63 and Ib, also forms voltage-dependent channels in lipid membranes (