1 September 2004

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


Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic “A-B” paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The “B” components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated “B” components form homoheptameric rings that subsequently dock with an “A” component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, “A” molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria.


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 (ι) 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).
Intoxication by C2, CDT, CST, ι, VIP, or the B. anthracis edema and lethal toxins initially involves specific, receptor-mediated binding of “B” components to a targeted cell as monomers that form homoheptamers on the cell surface or as solution-generated heptamers (schematically depicted in Fig. 1). In either scenario, the “B” oligomers are generated only after proteolysis of “B” precursor molecules. The “B” heptamer-receptor complex then acts as a docking platform that subsequently translocates an enzymatic “A” component(s) into the cytosol via acidified endosomes. Once inside the cytosol, “A” components from this binary family can inhibit normal cell functions by (i) mono-ADP-ribosylation of G-actin, which induces cytoskeletal disarray and cell death; (ii) proteolysis of mitogen-activated protein kinase kinases (MAPKK), which inhibits cell signaling; or (iii) increasing intracellular levels of cyclic AMP (cAMP) that result in edema and immunosuppression. Not only are these toxins important virulence factors representing effective vaccine targets for diseases like anthrax, but also they are useful biological tools for studying a myriad of cellular functions and delivering heterologous proteins into endosomal, as well as cytosolic, compartments. In light of heightened concerns involving B. anthracis and bioterrorism, this review provides a comprehensive glimpse at a family of related binary toxins produced by different Clostridium and Bacillus species. Important aspects of each binary toxin are highlighted, regarding biochemistry, genetics, proteolytic activation, structure, and function, as well as their applications in basic science and medicine.


The Bacillus and Clostridium genera represent ubiquitous bacilli commonly found throughout the world in soil, water, and gastrointestinal tracts of animals as well as humans. From an evolutionary perspective, the anaerobic clostridia (or their archaic relatives) probably represent some of the first bacteria on Earth, with perhaps closely related aerobic bacilli evolving thereafter during the genesis of an oxygenated atmosphere (127). Both genera grow in low-oxygen environments; however, the clostridia are better adapted for anaerobic life, with various aerotolerance levels among different species. The recent sequencing of B. anthracis (349), B. cereus (186, 347a), and C. perfringens (385) genomes, as well as the deciphering of the toxin-encoding plasmid (pXO1) from B. anthracis (315), will inevitably reveal additional similarities between these, and other, microorganisms.
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.

C. perfringens Iota Toxin

C. perfringens, also known as Bacillus aerogenes capsulatus or Clostridium welchii in older literature, was first discovered in 1892 by William Welch et al. and consists of five serotypes (A to E) classically based on the production of four lethal, dermonecrotic toxins (α, β, ε, and ι) neutralized by type-specific antiserum in mouse lethality and guinea pig dermonecrotic assays (182b, 258, 295, 457). Today, rapid genetic methods involving multiplex PCRs are more commonly used by many diagnostic laboratories for toxin typing of C. perfringens isolates (95, 96, 119, 150, 260, 412, 441, 480, 484). The binary ι toxin is produced exclusively by type E strains, implicated in sporadic diarrheic outbreaks among calves as well as lambs, and interestingly linked to a highly conserved, yet silent, enterotoxin gene localized on the same plasmid (45, 54, 178). Although C. perfringens ι toxin was initially described in 1940 by Bosworth (54), its binary nature was first elucidated in the mid-1980s by exploiting the fortuitous cross-reaction and neutralization of ι toxin with C. spiroforme antiserum (417, 421, 422). The two proteins that comprise ι toxin were designated iota a or Ia (slower moving) and iota b or Ib (faster moving), based on electrophoretic mobility in crossed immunoelectrophoresis. Ia and Ib are both nontoxic, as is the case for the individual components of all the Clostridium and Bacillus binary toxins described in this review, but when combined, they form a potent cytotoxin that is lethal to mice and dermonecrotic in guinea pigs (417, 421, 422). Later studies revealed that Ia is an ADP-ribosyltransferase (393) specific for actin (372) while Ib, which lacks any discernible enzymatic activity, binds to a cell surface protein and subsequently translocates Ia into the cytosol of a targeted cell (49, 356, 419).

C. spiroforme Toxin

Similar to the classic rod-shaped C. perfringens and enterically acting ι toxin, the distinctly coiled C. spiroforme also causes diarrheic deaths that are spontaneous or antibiotic induced in rabbits (52, 53, 70-73, 75, 183, 319, 320, 483), and perhaps in humans (25), via a binary ι-like toxin referred to as CST. Lagomorphs are most susceptible to C. spiroforme-induced diarrhea during stressful periods that include lactation, old age, weaning, and an altered diet (72). The Sa and Sb components of CST are respectively analogous to Ia and Ib of C. perfringens ι toxin, as first determined by crossed immunoelectrophoresis and neutralization studies with C. perfringens type E antiserum (52, 332, 333, 417). During the late 1970s, it was erroneously thought that C. perfringens type E represented the causative agent of various diarrheic outbreaks within rabbit colonies, since type E antiserum neutralized the in vitro cytotoxic effects of cecal contents from enterotoxemic animals (68, 70, 108, 202, 226, 319). However, C. perfringens type E was never isolated, and the real breakthrough came in 1982 when Carman and Borriello revealed a strong correlation between disease in rabbits and isolation of C. spiroforme (70), an organism not commonly associated with the normal intestinal flora (72). Overall, compared to the other bacteria and toxins portrayed in this review, C. spiroforme and particularly CST have received minimal attention and thus perhaps represent an area for future studies.

C. difficile Toxin

Closely related to the ι toxin and CST is the ι-like toxin produced by C. difficile (324, 334), consisting of CDTa and CDTb components that respectively share 80 and 82% amino acid sequence identity to C. perfringens Ia and Ib. In the United Kingdom and the United States, only 6 and 16% of all C. difficile isolates from hospitals and patients, respectively, contain both CDTa and CDTb genes (146a, 426), perhaps suggesting that this binary toxin is not essential for eliciting C. difficile-associated colitis, which is most commonly attributed to higher-molecular-weight proteins designated toxin A and toxin B (423). The protein components of CDT, CST, and ι toxin are interchangeable, thus generating biologically active chimeras that demonstrate conserved functionality among these clostridial species (166, 325, 333, 334, 417). Interestingly C. difficile, C. perfringens, and C. spiroforme are all associated with gastrointestinal diseases in humans as well as animals (52, 54, 63, 423), and the synthesis of common binary toxins with interchangeable protein components probably reveals a shared evolutionary path for these ubiquitous pathogens in a common niche.

C. botulinum C2 Toxin

C. botulinum, initially identified as Bacillus botulinus, was first described in 1895 by Emile van Ermengem following a food poisoning incident in Ellezelles, Belgium (97). Like C. perfringens, the neurotoxin types (A to G) of C. botulinum are classically determined by mouse lethality assays with toxin-specific antisera (182b, 389). However, the binary C2 enterotoxin produced by toxin types C and D is devoid of neurotoxicity but implicated in a fatal enteric disease of waterfowl, is cytopathic for many different cell types, and induces vascular permeability, necrotic-hemorrhagic lesions, and a lethal fluid accumulation in the lungs and intestinal tracts of various animals (113, 189, 191, 221, 276, 297, 298, 300, 302, 303, 305-308, 390). C2 toxin is synthesized by C. botulinum during sporulation (289) and incorporated into the spore coat (481), which is akin to the single-chain enterotoxin of C. perfringens or protective antigen from B. anthracis edema and lethal toxins (93, 130, 364, 371, 465, 466). Much of the pioneering work on characterizing the cell binding and translocation component (C2II), as well as the enzyme component (C2I), of C2 toxin was initiated by Ohishi and coworkers throughout the 1980s. Ohishi's laboratory observed, among many things, that (i) the biological effects of C2 toxin were the synergistic result of C2I plus C2II (297, 298) and (ii) C2II requires trypsin activation (designated C2IIa after proteolysis) for biological activity (299, 304, 309, 311). Simpson discovered in 1989 that C2IIa binding and subsequent entry of C2I into a targeted cell occurs by receptor-mediated endocytosis (392).
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.

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

In contrast to C. botulinum or C. perfringens, phylogenetic relatives such as B. anthracis and B. cereus are not composed of serologically distinguishable toxin types, but they do produce binary toxins respectively involved in animal or human anthrax and insecticidal effects (98, 132, 171, 230, 259). Overall, there is remarkably limited variation among B. anthracis isolates throughout the world (203), and by some accounts B. anthracis is merely a genetic variant of B. cereus and Bacillus thuringiensis, possessing plasmids that encode unique toxins and a capsule (180, 186, 214, 315, 347a, 349). The natural disease elicited by B. anthracis occurs mainly in herbivores following spore transmission via ingestion, inhalation, an open skin wound, or even biting flies, but its nefarious use as a biological weapon was first associated with espionage during World War I (439). Subsequent military events before, during, and after World War II led to more vigorous research efforts by various countries, the discovery of promising therapies as well as vaccines, and a more comprehensive understanding of anthrax pathogenesis particularly linked to the respiratory route and human disease (439).
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 ι 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.


The protein components of C. botulinum C2 toxin (302), C. difficile CDT (324), C. perfringens ι 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 ι 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 ι-toxin family, which includes CDT, CST, and ι toxin, but the signal peptide sequences are less highly conserved (40 to 61% identity); (ii) 31 to 40% identity between C2 and ι-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 ι 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).

B. anthracis PA83

The PA protein of B. anthracis edema and lethal toxins is unique and perhaps more versatile than other “B” components within the binary toxin family. For example, the PA precursor (PA83) is proteolytically activated by several routes into PA63 and these include (i) trypsin in buffer (294), (ii) an unidentified protease(s) in serum (66, 118), and (iii) ubiquitous cell surface proteases (furin or furin-like) which recognize a consensus sequence (164RKKR167) not present in the binding components from other binary toxins lacking any recognized mechanisms for cell surface activation (155, 211, 230). However, C. spiroforme Sb does have an RSAR site located 208 residues from the N terminus that perhaps reflects an evolutionary remnant of a furin cleavage site (147). Furin activation of bacterial toxins is rather common, as evidenced by the single-chain ADP-ribosyltransferases such as Corynebacterium diphtheriae diphtheria toxin and Pseudomonas aeruginosa exotoxin A, which enter cells by receptor-mediated endocytosis and subsequently inhibit protein synthesis (155, 156, 447).
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 ι toxin, and C. spiroforme CST (324, 393, 417, 418).

C. botulinum C2II

Trypsin activation of the 81-kDa C2II precursor into C2IIa (60 kDa) occurs between K181 and A182 (48), generating stable C2IIa homoheptamers in solution (32). The C2IIa complex mediates biological effects on cells, in conjunction with an ADP-ribosyltransferase (C2I), that involve the formation of ion-permeable channels in lipid membranes (373). Although C2II precursor binds to cells, it is not readily activated by cell surface proteases like PA83 and it does not dock with C2I or facilitate cytotoxicity (276, 311). X-ray crystallography of C2II has not been reported to date, but electron microscopy of C2IIa oligomers on lipid bilayers reveals annular heptameric structures with an inner diameter of 20 to 40 Å and an outer diameter of 110 to 130 Å (Fig. 2B) (32). As described below, N-terminal residues 1 to 87 of C2I are intimately involved in docking with heptameric C2II on the cell surface (37).

C. perfringens Ibp and Ia

Helen Ross and colleagues first reported in 1949 that ι toxin requires proteolytic activation (362); however, it took 35 more years before additional clues revealed that the Ib precursor (designated Ibp) was the likely target (417). Following the separation of Ia and Ibp from early-log-phase (<10-h) cultures of C. perfringens type E, as done by DEAE ion-exchange chromatography, trypsin proteolysis of fractions containing Ibp or Ia markedly (i) increases enzyme-linked immunosorbent assay (ELISA) readings for Ibp, but not Ia, versus the same untreated fractions, thus suggesting a conformational shift in Ibp that unveils cryptic epitopes recognized by Ib-specific antibodies; and (ii) increases the guinea pig dermonecrotic activity of the Ibp fraction when combined with untreated Ia. It was subsequently discovered, after cloning and sequencing of the ι-toxin gene, that proteolytic activation of Ibp into Ib occurs at A211 (323), which then facilitates Ia docking (419), formation of voltage-dependent ion-permeable channels in membranes (213), and formation of SDS-stable heptamers on cell membranes (288, 420) and in solution (49, 288). However, Ib oligomers formed in solution are seemingly less stable and do not promote cytotoxicity compared to solution-generated oligomers of PA63 or C2IIa. Additionally, Ib heptamers generated in solution versus on the cell membrane do not induce K+ release and are efficiently digested by pronase after binding to Vero cells at 37°C (288). Like C2II, which also lacks a furin cleavage site, Vero cell-bound Ibp is not activated over time (148) or with an excess of trypsin or chymotrypsin (420). To date, extensive proteolytic activation studies similar to those for C2II, Ib, and PA have not been conducted with B. cereus VIP1, C. difficile CDTb, or C. spiroforme Sb.
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).


B. anthracis PA, LF, and EF

X-ray analysis of PA (326), LF (314), and EF (103) crystal structures has provided invaluable information, as described below, and these molecules naturally represent “templates” for the components of other Clostridium and Bacillus binary toxins not crystallized to date. The PA protein contains four distinct domains that are seemingly applicable for the “B” components (C2II, CDTb, Ib, Sb, and VIP1) of other binary toxins described in this review (Fig. 2A). Various studies reveal that domains 1 (N terminus), 2, 3, and 4 (C terminus) are respectively involved in docking to an enzyme component(s), channel formation in lipid membranes, oligomerization, and binding to a specific cell surface receptor(s). For instance, domains 1 (amino acids 1 to 259) and 4 (amino acids 597 to 735) of PA83 are respectively responsible for (i) docking with EF and/or LF and (ii) binding to the cell surface via protein receptors recently identified as variants 1 and 2 of tumor endothelium marker 8, as well as human capillary morphogenesis protein 2 (60, 64, 80, 86, 92, 243, 245, 326, 361, 379, 401, 449). There is relatively little amino acid homology of PA domains 1 and 4 to their equivalents from other Clostridium and Bacillus binary toxins, which is not surprising since these regions respectively afford a unique docking site for distinct “A” components and receptor-binding specificity (33, 48, 252, 325, 418). Domain 1 contains the proteolytic cleavage site (R167) that subsequently triggers the release of a 20-kDa precursor peptide and formation of PA63 heptamers (211). The remaining segment of domain 1 (designated 1′ and consisting of residues 168 to 259) faces the channel lumen, unlike the peripherally located domain 4 (326), and is associated with two Ca2+ molecules (coordinated via residues D177 or D235 and D179, D181, E188) that evidently preserve a PA63 structure necessary for proper folding, resistance to proteolytic degradation, heptamer formation, and docking with EF or LF (138, 166a). Currently, no one has reported an association of Ca2+ with the “B” components from other binary toxins; however, concerted investigations have not been conducted evidently. Although their role is still unclear, various divalent cations (Ca2+, Co2+, and/or Zn2+) are required for Sa and Sb production in vitro by C. spiroforme (74).
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 β-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-α-helix bundle as well as β-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).
Domain 2 (encompassing amino acids 263 to 297 and 385 to 550) of LF conformationally mimics the C-terminal catalytic domain of VIP2 from B. cereus, but a critical glutamic acid necessary for ADP-ribosyltransferase activity (a property lacking in LF) is replaced by lysine. However, there is very little (15%) amino acid sequence identity between domains 2 of VIP2 and LF, perhaps suggesting an evolutionary divergence (maybe convergence?) of similar genes within this family of bacterial binary toxins. Domain 3 (amino acids 303 to 382) forms an α-helical bundle embedded between the second and third helices of domain 2, shares a hydrophobic surface with domain 4, and provides substrate specificity (314). Domain 4 (amino acids 552 to 809) contains the enzymatic active site and an HEXXH motif (686HEFGH690) common among various Zn2+ metalloproteases (210, 345), including the lambda toxin of C. perfringens (193), which may activate Ia and/or Ib of ι toxin in vitro and in vivo (148).
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.

C. botulinum C2II and C2I

In various ways, the C2 toxin is structurally similar to B. anthracis edema and lethal toxins, with a particular emphasis on the cell-binding proteins, PA and C2II (Fig. 2A). Studies reveal that the C terminus of C2II also facilitates receptor-mediated binding, since deletion of only seven C-terminal residues effectively prevents C2IIa interactions with cells (48). Antiserum specific for the C terminus (domain 4; residues 592 to 721) blocks C2IIa binding to cells as determined by Western blot analysis and cytotoxicity assays, unlike antisera toward domain 1 (residues 1 to 264) or 3 (residues 490 to 592) (48). Domain 4 antiserum neutralizes C2 cytotoxicity in vitro when preincubated with C2IIa, but not after C2IIa has bound to a cell, thus suggesting that neutralizing epitopes are sterically hindered after C2IIa-cell interactions. Deletion studies focused on the N terminus of C2II reveal that this region (residues 1 to 181), lost after proteolytic activation of the C2II precursor, may be important for proper folding of the molecule (48). Recent mutagenesis efforts with C2IIa in a conserved region encompassing amino acids 303 to 331 of domain 2, putatively involved in membrane insertion and channel formation, show that voltage gating but not chloroquine binding or translocation of C2I into the cytosol is lost following an E307K mutation (47).
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 α-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 α-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 β-sheets flanked by a pair of α-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).

C. perfringens Ib and Ia

Like PA and C2II, the importance of various domains for Ib activity has been ascertained via deletion mutagenesis and antibody studies (252, 420). Similar to PA and C2II, truncation of 10 amino acids from the C terminus (domain 4) abrogates Ib binding to Vero cells, and Ib peptides containing ≥200 C-terminal residues represent competitive inhibitors of ι cytotoxicity in vitro (252). Deletion of 27 N-terminal Ib residues within domain 1′ prevents Ia docking and intoxication, but there is little effect on Ib binding to cells as this truncated domain is an effective competitor of ι toxin (252). In this same study, three monoclonal antibodies against a common N-terminal epitope within residues 28 to 66 had no effect on Ib binding or cytotoxicity. It is possible that these immunoreagents do not occupy the Ib site necessary for Ia docking; alternatively, Ib oligomerization and/or docking of Ia may readily displace these antibodies. In contrast, two monoclonal antibodies that recognize unique Ib epitopes within C-terminal residues 632 to 655 afford protection against ι cytotoxicity via two different mechanisms. One of these antibodies prevents Ib binding to cells, as determined by flow cytometry (252) and Western blot analysis (420), while the other has no effect on Ib binding but efficiently prevents Ib oligomerization on the cell surface. Results for the latter C-terminal binding antibody further demonstrate the importance of Ib oligomerization on biological activity of ι toxin, as do studies with Ibp, a molecule that remains as a cell-bound monomer (419, 420). Similar to the polyclonal antibody studies with domain 4 of C2II (48), monoclonal antibodies that recognize the same domain of Ib do not bind or afford any in vitro protection toward cell-bound Ib and Ia (252, 420). All of the Ib monoclonal antibodies recognize Ibp or C. spiroforme Sb in an ELISA and Western blot analysis, but none cross-react with B. anthracis PA (252). Surprisingly, C2II is also recognized in an ELISA by one of the monoclonal antibodies that prevent Ib interactions with cells, but it does not neutralize C2 cytotoxicity. Such a finding is intriguing, since C2II and Ib bind unique receptors via their C terminus and share little sequence homology within this epitope (48, 135, 252). To complement the studies done with PA and enhance our understanding of ι toxin (46, 205, 206, 283, 381, 382, 399), future investigations focused on domains 2 and 3 of Ib should be done to more clearly delineate residues required for oligomerization and channel formation.
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 α-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.

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

The basic mechanism of ADP-ribosylation employed by C2, CDT, CST, ι, and VIP toxins is remarkably well conserved by diverse bacteria from many different genera. All known ADP-ribosylating toxins use NAD, a ubiquitous molecule for reduction-oxidation reactions in eukaryotic and prokaryotic cells, as a source of ADP-ribose. There are at least four bacterial groups of ADP-ribosylating toxins based on the intracellular targets: (i) elongation factor 2 (modified by C. diphtheriae diphtheria toxin and P. aeruginosa exotoxin A via an N- and C-terminal active site, respectively); (ii) heterotrimeric G-proteins (modified by B. pertussis pertussis toxin, E. coli heat-labile enterotoxin, and V. cholerae cholera toxin via an N-terminal active site); (iii) Rho and Ras GTPases (modified by C. botulinum C3 exoenzyme and P. aeruginosa exoenzyme S via a C-terminal active site); and (iv) G-actin. Members of this last group include B. cereus VIP (171), C. botulinum C2 toxin (7, 350); and the ι-toxin family represented by C. difficile CDT (334), C. perfringens ι toxin (322, 445), and C. spiroforme CST (332, 394). All actin-modifying toxins have a C-terminally located active site (Fig. 3A).
The actin-ADP-ribosylating toxins can be subdivided into two groups: (i) C2 toxin, which exclusively mono-ADP-ribosylates at R177 the isoforms of β/γ-nonmuscle, as well as γ-smooth muscle, G-actin (6, 199, 201, 310, 446) (Fig. 3B), and (ii) the ι-like toxins, which mono-ADP-ribosylate R177 of all G-actin isoforms, including α-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 ι 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 ι 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 ι as well as C2 toxins that block additional nucleation activity (30, 201, 462, 476). Additionally, the ι 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).


“B” Binding to the Cell

To reach their intracellular substrate, and like many other bacterial protein toxins, the Clostridium and Bacillus binary toxins must first bind to a targeted cell via a receptor(s) as either a preformed or cell surface-generated homoheptameric “B” complex. Recent work in discovering cell surface receptors for these toxins has been quite fruitful, especially with B. anthracis PA. The PA receptor is a ubiquitous protein (117) first identified as tumor endothelium marker (TEM) 8 (variant 2), which consists of 368 amino acids and a von Willebrand factor A domain (60) commonly found on many integrins used as ligand-binding sites (61, 473). The role played by TEM 8 in normal cells is unknown; however, its expression in tumor cells can be relatively high (414). More recent discoveries show that variant 1 of TEM 8 (245) and a ubiquitous protein expressed by the human capillary morphogenesis gene 2 (CMG2) (379) also function as PA receptors. There is 40% amino acid sequence identity between TEM 8 (variant 2) and CMG2, which also have similar molecular weights and von Willebrand factor A domains containing an embedded metal ion-dependent adhesin site important for PA and probably natural but yet unknown ligand interactions (61, 379). The high-affinity binding of PA to receptor, as represented by a 10−9 M association constant (117), is ionically (Mg2+ or Mn2+, not Ca2+) mediated via a carboxylate group provided by D683 on PA (61, 361). It has become clearer that PA can bind to various proteins possessing evolutionarily conserved, metal ion-containing domains on the cell surface (473), and perhaps further work done with the “B” components of other binary toxins will also yield exciting results that may include a common (or not so common) family of proteins exploited as cell surface receptors.
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 β-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 ι 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 ι 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 (26, 26a, 47, 50, 373), and a conserved pattern of hydrophobic and hydrophilic amino acids within C2II residues 303 to 331, a region also found in PA residues 325 to 356 of domain 2, may also play a critical role in C2IIa insertion into the membrane (47).
In contrast to C2IIa, which binds and facilitates C2I-mediated cytotoxicity in all tested vertebrate cells (31, 109, 276, 305, 428), the receptor for Ib is not as ubiquitous (419). The Ib receptor is a protein resistant to various proteases but not to pronase, as determined by flow cytometry (419) and subsequent Western blot experiments (420). Pretreatment of cells with various lectins or glycosidases does not affect Ib binding, suggesting that the receptor (or part of it) is not a carbohydrate and thus further distinguishing it from that for C2II (135). By using polarized CaCo-2 (human colon) cells, Blöcker et al. (49) discovered that the Ib receptor is localized primarily on the basolateral membrane, akin to that for PA on another human colon cell line (T84) (41). Richard et al. (356) revealed that Ib traverses CaCo-2 cells from either the apical or basolateral surface and internalizes Ia found on the distal side, even when Ia is added 3 h after Ib. In this latter study, addition of ι-toxin-neutralizing antiserum or Ib-specific monoclonal antibodies with Ia to the cell surface distal to Ib also did not affect ι cytotoxicity. Western blot experiments conducted by two different groups reveal that Ib rapidly binds to cells at 37°C and forms a large complex (>200 kDa) in less than 1 min that is evident for at least 2 h (288, 420). This is intriguing when compared to earlier work by Sakurai and Kobayashi (367) showing that Ia injected intravenously into mice 2 h after Ib administration causes death, suggesting that Ib is available for Ia docking on the cell surface over an extended period. If neutralizing ι toxin antiserum toward Ib is given only 5 min after an Ib injection, mice are not protected against Ia-induced death, and this reveals that Ib perhaps binds to cells very rapidly in vivo. Finally, in this study it was also discovered that when Ib is injected intradermally into guinea pigs and Ia is given intraperitoneally, a dermonecrotic lesion forms at the Ib injection site. Clearly, Ia is able to “find” distantly located Ib that is bound to the cell surface, and perhaps this characteristic can be exploited in future experiments involving ι toxin as a protein shuttle. Similar discoveries were also reported by Simpson (390) for C2 toxin in mice and rats. To date, receptor-binding studies for B. cereus VIP1, C. difficile CDTb, or C. spiroforme Sb are lacking in the literature.

“A” Docking to Cell-Bound “B” and Internalization

As described earlier in this review, N-terminal domains from the “A” and “B” components of each Clostridium and Bacillus binary toxin are intimately involved in docking to each other on the cell. After binding to a surface receptor, there are two major pathways for uptake of bacterial protein toxins that act intracellularly. One path involves retrograde routing through the Golgi apparatus and endoplasmic reticulum, as demonstrated by S. dysenteriae Shiga (370) and V. cholerae cholera (313) toxins. This method of cytosolic entry is inhibited by brefeldin A, a fungal macrolide that causes protein accumulation within the endoplasmic reticulum (78). The second intracellular route exploited by bacterial toxins involves translocation from acidified early endosomes into the cytosol, like that employed by single-chain C. diphtheriae diphtheria toxin (248) as well as the multiple-chain B. anthracis edema and lethal toxins (131). Subsequent transport of vesicles from early to late endosomes involves microtubules that are readily depolymerized by nocodazole, a chemically synthesized molecule that inhibits trafficking into late endosomes (32, 49, 160, 365). Since neither brefeldin A nor nocodazole influences the biological activity of C2 or ι on cells, these toxins are like the edema and lethal toxins regarding transport from early endosomes into the cytosol. However, translocation of the C2, ι, or edema and lethal toxins across the endosomal membrane is blocked by a macrolide antibiotic, bafilomycin A, which inhibits vacuolar-type ATPases responsible for acidification of the endosomal compartment (Fig. 4) (32, 49, 469). A decreased pH probably induces conformational changes and membrane insertion of the heptameric “B” component, followed by translocation of the “A” component(s) across the endosomal membrane, which is also mimicked on the cell surface by simply lowering the pH of the medium (32, 49, 50, 131, 157, 247, 267, 272, 392, 400). However, there are unique pH requirements for binary toxins such as C2 and ι, since the latter requires a lower pH (≤5.0) than the former (≤5.5) for direct cytosolic entry through the cell membrane. Currently, it is not clear if “B” heptamers of these binary toxins enter the cytosol with the “A” components or remain attached to the endosomal membrane, possibly recycling to the cell surface (311, 356).
Recent results with C2, CDT, and ι toxins reveal that the “A” components traverse the endosomal membrane via heat shock protein 90 (Hsp90) and an ill-defined mechanism (Fig. 4) (178a, 179). Hsp90 is a highly conserved ATPase that is abundantly produced by all eukaryotic cells, and in conjunction with other heat shock proteins it plays an essential housekeeping role by regulating a myriad of proteins associated with cell signaling (337). Specific inhibitors of Hsp90, such as geldanamycin or radicicol, effectively delay C2-, CDT-, or ι-induced cytotoxicity in various cell types by inhibiting, respectively, C2I, CDTa, or Ia entry into the cytosol. Interestingly, cytosolic entry of B. anthracis lethal toxin differs in that it is not affected by Hsp90 inhibitors (179), thereby suggesting distinct endosome-to-cytosol translocation mechanisms for “A” components of this binary toxin family.
It is plausible, but not definitively proven, that the enzyme components from all Clostridium and Bacillus binary toxins unfold and thread through toxin-generated channels in the membrane, perhaps entering the cytosol like that proposed for LF or the ADP-ribosyltransferase of the single-chain diphtheria toxin (120, 471). The recent work by Ratts et al. (348) shows that Hsp90 and thioredoxin reductase, found in a cytosolic complex, are both required to transport C. diphtheriae diphtheria toxin from the endosome. Additionally, geldanamycin and radicicol are both necessary for inhibiting diphtheria cytotoxicity, whereas with CDT, C2, or ι toxins, either drug alone inhibits cytotoxicity with no evident additive or synergistic effects. It is unknown whether thioredoxin reductase cleaves the disulfide bond between the “A” and “B” chains of diphtheria toxin, although this is quite possible, since it does occur with C. tetani tetanus toxin and C. botulinum neurotoxin A (209). However, disulfide bonds and reductive activation have never been described for any of the Clostridium and Bacillus binary toxins described in this review.
Inevitably, these recent discoveries with Hsp90 and its role on the entry of toxic ADP-ribosyltransferases into the cytosol, along with further investigation including other binary bacterial toxins, will likely elucidate other factors involved in the cytosolic entry of proteins from the endosome. Such studies might also lead to more efficient use of bacterial toxins as protein shuttles, in particular delving into the types of molecules transported into the cytosol of targeted cells from the endosome. Finally, results from these endeavors will also probably provide new targets for potential therapeutics against bacterial toxins that traffic through the endosome.


Protein Shuttles

Historically, the single-chain exotoxin A of P. aeruginosa and C. diphtheriae diphtheria toxin have each received much attention regarding their use as vehicles for heterologous proteins (317). As described in this section, the Clostridium and Bacillus binary toxins have also provided excellent tools for shuttling heterologous proteins, and even DNA, into cells. Again, the pioneering work on, and subsequent knowledge gleaned from, B. anthracis toxins has resulted in important advances in this field. For example, there have been many studies employing LF (amino acids 1 to 254) in which LF1-254 can be fused to either the N or C terminus of a heterologous protein (20-23, 230, 231, 261, 273, 383) and subsequently internalized by a targeted cell via heptameric PA63. However, fusions consisting of LF residues 1 to 198 or deletion of just 40 N-terminal residues from the wild-type molecule (without a fusion protein) are not cytotoxic (21), further revealing an important role played by the N terminus of LF in docking and/or translocation.
The immune system represents just one potential target for chimeras generated from B. anthracis lethal toxin. Recombinantly linked or disulfide-linked LF chimeras can induce cytotoxic T-cell responses in vivo and/or in vitro via the delivery of major histocompatibility complex class I epitopes originating from viruses (i.e., influenza virus, human immunodeficiency virus, and hepatitis C virus) or bacteria (Listeria monocytogenes) (27-29, 69, 101, 153, 154, 284, 486). An exciting variation on stimulating immunity involves the anticancer capabilities of lethal toxin (129, 216). Like previous studies with a diphtheria toxin chimera containing the C fragment of C. tetani tetanus toxin for specific targeting of neurons (128), it is also possible to direct PA toward unique surface receptors, as evidenced by fusing a c-Myc epitope consisting of 10 amino acids to the C terminus of PA, which subsequently binds to (and kills with LF) a hybridoma line expressing c-Myc antibody (233, 448).
Another novel twist on using B. anthracis lethal toxin as an antitumor compound is the exploitation of specific proteolysis by urokinase plasminogen activator, a surface protein commonly associated with malignant cells (244, 246). By replacing the furin cleavage site (RKKR) of PA83 with SGRSA, a recognition site for urokinase plasminogen activator, there is decreased toxicity and broad antitumor effects in mice when combined with the use of LF1-254 fused to the enzymatic domain of P. aeruginosa exotoxin A.
In addition to shuttling proteins into cells, DNA encoding luciferase or green fluorescent protein has been successfully transported into COS-1 cells by PA and an LF1-254 fusion containing a 16-kDa DNA-binding domain from Gal 4, a yeast transcription factor (144). In the future, this may represent an attractive alternative to the use of viral vectors, which are more common in gene therapy today (100, 194). Overall, the numerous shuttle experiments that have been done with B. anthracis lethal toxin represent “trend-setting” uses that foretell a bright future for Clostridium and Bacillus binary toxins. However, another important question that remains unanswered involves fusion constructs of any binary toxin and the maximum size of heterologous proteins transported into the cytosol. Of course size but also an ability to “unfold,” “thread,” and properly “refold” within the cytosol probably dictate the ability of any given protein to translocate into the cytosol as a biologically active form. For the most part, many aspects of getting heterologous proteins into targeted cells via any Clostridium or Bacillus binary toxin remain largely unexplored by the different laboratories working in this field.
As with any protein-based system used for targeted delivery of protein and/or DNA into cells in vivo, host-developed antibodies to the protein constituents could diminish subsequent efficacy. For instance, it has been reported that a PA-LF combination elicits higher antibody titers toward LF than does LF alone (65, 249, 328). Mahlandt et al. (249) first suggested in 1966 an additive immune response to LF with PA, and Pezard et al. (328) later reported that B. anthracis strains expressing both PA and LF (or EF) induced LF (or EF)-specific antibodies in mice, yet strains expressing LF (or EF) alone elicited very little specific antibody. Finally, vaccine studies show that spores from recombinant B. anthracis strains that express PA and LF1-254 fused to the 50-kDa C fragment of tetanus toxin result in higher-titer neutralizing antibodies toward tetanus toxin than do spores from a PA-deficient strain (65). To date, though, there have been no in vivo studies addressing specific antibodies elicited toward any Clostridium or Bacillus binary toxin when used as a shuttle system.
In light of these serological data from various groups, the potential problem of developing specific antibodies to lethal toxin-based shuttle proteins after repeated exposure might be partly circumvented, since recent evidence suggests that LF1-254 conjugates of green fluorescent protein or human immunodeficiency virus peptides can enter cells without PA and subsequently elicit a major histocompatibility complex class I-induced response from CD8+ T cells (222). The entry of LF1-254 into cells without PA clearly represents a major paradigm shift for the Clostridium and Bacillus binary toxins; however, LF can evidently enter cells through a pinocytotic route, albeit less efficiently than the PA63-facilitated translocation that may also be needed for proper trafficking of LF toward MAPKK before degradation (222, 402).
Similar to B. anthracis LF, the noncatalytic N-terminal domain (residues 1 to 225) of C. botulinum C2I (designated as C2I1-225) is also important for docking with its “B” component (C2IIa) and has been engineered as a fusion protein (31, 33) with C3 or C3-like ADP-ribosyltransferases produced by B. cereus, C. botulinum, C. limosum, and S. aureus (14, 200, 427, 474, 475). ADP-ribosyltransferases of the C3 and C3-like family are each ∼23 kDa, have 35 to 75% amino acid sequence identity among themselves but only 23% identity to “A” components from the ι-toxin family, and inactivate small GTPases (Rho A, B, and C) through mono-ADP-ribosylation of N41 (8, 11, 363, 380). The recently determined crystal structure of C. botulinum C3 exoenzyme reveals conformational similarities to the C-terminal domain of C2I containing the active site (170). Curiously, C3 and C3-like exoenzymes may enter cells via a very speculative and inefficient pinocytosis, cytolysin-mediated transport, or ill-defined secretion mechanism (247a), since neither binding nor transport components are seemingly produced by any of the host bacteria. Due to poor cell penetration, the C3 and C3-like proteins are ideal for shuttle experiments since high concentrations (>10 μg/ml) must be applied to cells for an extended period (up to 24 h) to elicit any cytotoxic effects. However, when a C2I1-225-C3 construct is added at low concentrations (100 ng/ml) with C2IIa to various primary isolated or continuously cultured cell monolayers for 3 h, nearly all are found to possess the typical “C3 morphology” within 60 min, as evidenced by cell contraction and neurite-like protrusions (33). Analysis of time-versus-concentration curves suggests that the potency of the C2I1-225-C3 fusion toxin (with C2IIa) is 300-fold higher than that of C3 toxin plus C2IIa. Upon removal of fusion toxin from the medium, cytopathic effects are reversed within hours via reorganized stress fibers and actin cytoskeleton, thus leading to normal morphology attributed to newly synthesized Rho protein (33, 35). In contrast, recovery from C2I1-225-C3 intoxication is not observed if cells are treated with inhibitors of either protein synthesis (cycloheximide or puromycin) or proteasomes (lactacystin) (35). Biochemical analysis of lysates from cells incubated with the C3 fusion toxin reveals that Rho A, B, and C, but not other Rho types or actin, are modified with ADP-ribose (33). C3 can be fused to either the N or C terminus of C2I1-225 and efficiently delivered into cells like wild-type C2I (31, 37), even if C2I1-225 is in the midst of a 75-kDa fusion protein (glutathione transferase-C2I1-225-C3).
Further investigation has shown that the minimal C2I fragment facilitating C3 transport into cells consists of residues 1 to 87, which is as effective as the larger C2I1-225 construct (37). The C2I1-87 fragment represents a minimal part of C2I needed for not only translocation but also competitive binding with C2I for C2IIa. The N terminus of C2I is important for docking with C2IIa, since deletion of just 30 amino acids from the C2I1-225-C3 fusion results in loss of cytotoxicity (with C2IIa) due to poor translocation and not inhibited binding to cell-associated C2IIa (37). Clearly, the C2 shuttle system is quite versatile for translocating heterologous proteins into cells and studying various cell functions, as further illustrated in Table 4.
In addition to the C. botulinum C2 or B. anthracis lethal toxins, a recent study with C. perfringens ι toxin reveals that it, too, acts as an effective shuttle for heterologous proteins (253). It was shown that the minimal Ia fragment necessary for efficient transport of C. botulinum C3 into Vero cells, via Ib, is the catalytically inactive Ia129-257. Linkage of C3 to the C terminus of Ia peptides of different sizes is most effective on cells, since all N-terminally linked conjugates when incubated with Ib are not cytotoxic. However, each N- or C-linked C3 conjugate of Ia is enzymatically active in vitro, as evidenced by modification of a 21-kDa protein (Rho) in bovine brain homogenates; therefore, C3 enzymatic activity is not greatly altered in any fusion construct. Additionally, most of the N-linked conjugates effectively dock to Ib on the cell surface, suggesting either that C3-Ia conjugates may be inefficiently translocated or that upon entering the cytosol, the enzyme may not adopt a biologically active conformation. These results differ from those for the lethal and C2 toxin systems, in which either the N- or C-terminal linkage of LF or C2I fragments to heterologous proteins, respectively, results in PA- or C2II-facilitated transport of biologically active proteins into cells (22, 23, 31, 37, 273). Although various in vitro studies have employed chimeric C2 and ι toxins as shuttle systems, results from in vivo studies are clearly lacking in the literature and represent the next logical step for future studies.

Vaccine Targets

Vaccines represent a historically powerful means of controlling various diseases, anthrax being one of the first. The initial vaccine study for anthrax was publicly conducted by Louis Pasteur and colleagues in the spring of 1881 at a farm near Pouilly-le-Fort, located ∼30 miles southeast of Paris. The inoculum was live, temperature-attenuated B. anthracis that subsequently afforded protection in sheep (318, 436). Following such an achievement, this vaccine was widely accepted by the veterinary community and used throughout the world for ∼50 years in cattle and sheep until the discovery of the Sterne strain, which lacks the pXO2 plasmid (capsule encoding) but not the pXO1 plasmid (toxin encoding) (134, 416). It was recognized only 20 years ago that the B. anthracis inoculum used in the Pasteur vaccine was probably cured of pXO1 plasmid after growth at an elevated temperature (271).
Today, 120 years after Pasteur's pioneering efforts, we are still pursuing B. anthracis and better methods of controlling anthrax by appropriate vaccines and therapeutics (134, 381, 399). Unlike the studies conducted during the Koch-Pasteur era, which focused primarily on disease in farm animals, current endeavors are primarily aimed at anthrax in humans from natural and, even more importantly, unnatural (i.e., biological warfare and bioterrorism) sources (134). The vaccine target of greatest interest has been the PA molecule (24, 90, 121, 134, 143, 161, 185, 187, 188, 190, 213a, 227, 237, 329, 354a, 398, 473a), ergo its name from work initially presented by Gladstone (151) and subsequently by others (59, 440, 478). Among numerous B. anthracis strains tested (n = 26), the PA molecule is evidently quite conserved at the gene and amino acid levels (342) and thus provides an appropriate target for developing broadly efficacious “second-generation” vaccines for humans (121). Antibodies toward PA protect against lethal intoxication and/or infection by B. anthracis spores, which contain PA in their coat that can be exploited by the immune system via PA- specific antibodies that enhance spore uptake, germination, and killing by macrophages (465, 466).
The current anthrax vaccine for humans in the United States, known as anthrax vaccine adsorbed (AVA) or BioThrax, was approved by the Food and Drug Administration in 1970. This vaccine is far from optimal, since it consists of formalin-treated culture filtrate from a nonproteolytic, toxin-positive/capsule-negative strain (V770-NP1-R) of B. anthracis (133, 477) in aluminum hydroxide adjuvant that is administered via six subcutaneous injections over 18 months, with an annual boost (330). Standardization of AVA has also been a problem, since the PA content is not measured in each vaccine batch and a universally accepted assay has not been established to quantitate PA specific-antibodies among vaccinees (232, 329, 354a). A slight variation of AVA, employing Sterne strain 34F2 and an aluminum phosphate adjuvant, was approved in 1979 for human use throughout the United Kingdom (134, 477). To diminish the time course and number of injections and avoid potential adverse reactions in humans, various studies aimed at developing a second-generation vaccine demonstrate that a simplified inoculum composed of purified, recombinant PA alone is efficacious when given mucosally, parenterally, or transcutaneously to animals (58, 125, 143, 254a, 398).
The veterinary vaccine is quite different from that used in humans, since it consists of B. anthracis spores (Sterne strain) that do not readily elicit PA antibodies (in guinea pigs), yet it affords better protection after one injection than that achieved after three injections of the acellular vaccine (440). It is perhaps because of this heightened immune response to spores that certain investigators are now exploring the use of PA and formaldehyde-inactivated, recombinantly detoxified spores of the Sterne strain as a potential second-generation vaccine for humans (63a).
Experimentally, various studies have been done to identify novel methods of vaccination against anthrax. For instance, parenteral vaccinations with DNA encoding PA, LF, or both have proven effective against a lethal toxin challenge in mice (343). Subcutaneous or intramuscular inoculations of mice with replication-deficient vectors, composed of Venezuelan equine encephalitis virus or adenovirus and the PA83 gene, also afford protection against a subcutaneous or intravenous challenge with B. anthracis spores or lethal toxin, respectively (227, 432a). Another report by Garmory et al. (139) also shows that mice immunized orally or intravenously with PA-expressing Salmonella enterica develop PA-specific antibodies and protection against an intraperitoneal challenge with B. anthracis spores. It is logical that antibodies against PA, particularly those targeting the C terminus, will prove efficacious, since this region binds to cell surface receptors (401, 449) and contains immunodominant epitopes that protect against intoxication as well as B. anthracis infection (126, 243, 257).
In addition to using PA, Sterne spores, or a combination of the two as vaccine targets, a dually active anthrax vaccine has been reported recently by two different groups (355, 376). These novel conjugate vaccines consist of PA plus poly-γ-d-glutamic acid (PGA) capsule that, when used in mice, effectively elicit toxin-neutralizing antibodies and complement-mediated killing of B. anthracis. Relative to PA and certainly PGA alone, conjugates containing both PA and PGA elicit higher antibody titers toward each of these antigens, with a 10- to 20-fold molar excess of decameric PGA representing an optimal immunogen (355, 376). Perhaps the concept of dually active anthrax vaccines represents a future form of prophylaxis toward not only B. anthracis, but also other pathogens that possess multiple virulence factors. It is clear that various groups are pursuing novel vaccines against anthrax, which bodes well for future prophylaxis methods that should entail less time, lower cost, and reduced reactogenicity compared with the current methods of protection.
Compared to the B. anthracis toxins, much less vaccine work has been done with other Clostridium and Bacillus binary toxins described in this review. However, various antibody studies with Ib of C. perfringens ι and C2IIa of C. botulinum C2 toxins show that their C termini, like that for PA63 (240, 243), contain toxin-neutralizing epitopes (48, 252). Studies by Sirard et al. (403) also reveal that the Sterne vaccine against anthrax can be recombinantly modified to express Ibp of ι toxin and then subsequently used to prophylactically protect mice against a lethal CST or ι toxin challenge. This brings up an interesting possibility regarding the use of this commonly accepted, spore-based vaccine as a vehicle for eliciting protective immunity against various bacteria, viruses, and toxins of veterinary and human importance (63a).
In addition to the studies with Sterne spores, there have been efforts by Ellis et al. (111) to develop veterinary vaccines against C. spiroforme. This work shows that a formalin toxoid of C. spiroforme culture supernatant containing CST elicits active immunity in weaning rabbits against a toxin challenge; however, passive immunity was not transferred by does to their young. Protection was evident after only one subcutaneous injection, but two injections elicited even better results. Unfortunately, it is unknown how effective this vaccine is toward a C. spiroforme challenge since these experiments were not done and/or not reported in this publication.


The area of research into therapy of disease due to Clostridium and Bacillus binary toxins has historically received very little effort, but the deliberate anthrax attacks within the United States have dramatically elevated the stakes, attention, and resources now focused upon rapidly developing therapies for two major virulence factors of B. anthracis: the edema and lethal toxins (51, 79, 425). Antibiotics such as ciprofloxacin, doxycycline, and penicillin are indeed generally effective in vivo against B. anthracis (17, 38, 67, 201a, 346, 430); however, rapid diagnosis of disease as well as timely and lengthy administration of antibiotics are both critical for patients and make the difference between life and death for those suffering from inhalational forms of anthrax (122a, 192, 346). Perhaps the recent discovery of bacteriolytic phage enzymes specific for B. anthracis will afford, in the future, an additional “magic bullet” for clinicians, especially if antibiotic-resistant strains are encountered (149, 378).
To increase the survival rates among patients with inhalational anthrax, which insidiously begins as a seemingly innocuous cough and flu-like malady that can rapidly progress to shock, respiratory failure, and death, adjunct therapy directed toward edema and/or lethal toxins will probably provide a pivotal advantage (67, 344, 425). With either of these toxins, PA represents a central figure for B. anthracis survival in the host, as demonstrated by in vitro and in vivo antibody studies (42, 112, 133, 134, 213a, 238, 354a, 466) or inhibition of lethal toxin-induced cytotoxicity in vitro via a soluble receptor fragment (60). Logically, the “B” component of any bacterial binary toxin represents a prime therapeutic or vaccine target, since it is theoretically easier to neutralize a toxin before it enters a cell. Antibodies to PA, when administered promptly in vivo, do indeed provide an effective immunotherapeutic after the onset of a B. anthracis infection (213a, 238, 354a). Additionally, it was shown years ago that a recombinant PA83 lacking the furin cleavage site, and thus unable to form heptamers or dock with EF or LF, protects rats against a subsequent dose of lethal toxin via competition with wild-type PA for receptor (397). Therapeutic targeting of PA has also been advocated with a PA32 fragment (composed of domains 3 and 4) that prevents the binding of wild-type PA to cells (86).
More recently, various laboratories have developed novel means of inhibiting B. anthracis toxins involving a dominant-negative strategy via PA. Mutations of K397, D425, and/or F427 within domain 2 (4, 381, 382, 482), swapping of residues 302 to 325 (2β2-2β3 loop) from domain 2 with the C. perfringens Ib equivalent (399), and cysteine replacement of each amino acid in PA63 (285a) have generated translocation-defective molecules of PA that still oligomerize and dock with EF and/or LF. Only one dominant-negative PA molecule in a heptamer, otherwise composed of wild-type PA63, impressively protects cells in vitro against lethal toxin. These forms of PA also effectively prevent lethal toxin activity in vivo upon concomitant injection. In essence, such PA variants adsorb EF and LF out of the circulation, and future studies should ascertain whether this type of therapy represents a viable adjunct in vivo, with antibiotics, against anthrax. Additionally, the inhibition of LF docking to a PA63 heptamer and subsequent cytotoxicity has also been accomplished by biopanning a phage display library (285). By employing this technique, a dodecapeptide was discovered that proved most efficacious toward lethal toxin in vitro and in vivo when bound to a polyacrylamide backbone.
To complement the above protein- and peptide-based therapies, low-molecular-weight inhibitors of edema and lethal toxins have also been studied recently by various university, government, and company laboratories that are focusing on the inherent enzymatic activities of EF and LF (91, 280, 313a, 409, 435). Computational analyses of databases containing low-molecular-weight compounds, along with the known crystal structures of EF, LF, and PA, provide a strong starting point for drug discovery directed toward inhibiting toxin binding to cell surface receptors as well as enzymatic activity (152, 409). Ironically, it was from such a database for antitumor drugs that LF activity was first ascertained to mimic an existing compound that adversely affects the MAPKK pathway (107, 464).
Once computational analysis of databases has been done and potential inhibitors have been identified, a necessary component of discovering any toxin therapy among a myriad of candidates is the development and subsequent use of simple, accurate, and high-throughput assays like those recently demonstrated for LF involving fluor-tagged peptide substrates (91, 359, 435). As an example, a plate-based assay described by Cummings et al. (91) employs a coumarin fluor and quencher separated by 17 amino acids containing an LF cleavage site that, upon proteolysis, enables the fluor to subsequently emit a detectable signal. In many ways, this type of assay mimics the same format as that for in vitro proteolysis studies with C. botulinum neurotoxins A, B, and F (18, 375).
Recent efforts to competitively inhibit EF through a unique catalytic site involving an ATP binding region not shared with eukaryotic cyclases also has potential benefits against other pathogen-produced adenylate cyclases, like those from B. pertussis, P. aeruginosa, and Yersinia pestis (409). Soelaiman et al. (409) show that certain quinazoline compounds competitively inhibit the binding of ATP to the catalytic site of EF and also protect mouse adrenocortical cells in vitro when added before edema toxin. Unfortunately, “therapeutic-like” experiments were either not done or not reported with drug administered after toxin exposure. Additionally, it is well established that edema toxin requires Ca2+ for activity (229); however, somewhat surprising is that inhibitors of intracellular Ca2+ (i.e., dantrolene or cyclosporine A) also prevent lethal toxin activity on murine macrophages in vitro, which probably involves increased permeability of cell membranes via a Ca2+-dependent phospholipase A2-based mechanism (386). Unfortunately, these compounds are nonspecific and will likely affect many other Ca2+-dependent processes necessary for normal cell functions. Finally, the most promising drug to date against edema toxin is perhaps one already clinically approved for treating chronic hepatitis B infections via inhibition of viral DNA polymerase (384a). The cellular metabolite of adefovir dipivoxil, an acyclic nucleotide phosphonate that binds to EF with 10,000-fold-higher affinity than ATP, clearly protects various cell types in vitro from the effects of edema toxin.
For the other Clostridium and Bacillus binary toxins described in this review, specific inhibition by newly discovered compounds has not been studied because these toxins are not commonly associated with human disease and do not directly represent a bioterrorism threat. However, it is known that peptide-based inhibitors of ι toxin, composed of domain 4 from Ib, prevent the binding of wild-type Ib to cells and subsequent Ia-induced cytotoxicity (252). Similar work has not been done with the C2, CDT, CST, or VIP binary toxins. Clearly, there is much work to be done regarding therapeutics towards Clostridium and Bacillus binary toxins, and the toxins from B. anthracis represent an immediate focal point as well as global concern. Upon considering the numerous structural commonalities within this binary-toxin family, protein-based inhibitors seem like one of many largely unexplored and logical paths into the future.


As presented in this review, the binary toxins from various Bacillus and Clostridium species possess unique and versatile characteristics that are useful for (i) delivering proteins into cells, (ii) discerning the role of actin in various cellular functions, (iii) providing vaccine targets, and (iv) developing recombinantly modified therapeutics. From a historical perspective, the discovery of C. perfringens ι toxin in 1940 was the first for any Clostridium or Bacillus binary toxin (54). However, in 1956 the multicomponent structure of B. anthracis toxins was initially reported and thus represents the first binary description for any of these toxins (407, 408). Subsequently, the multicomponent nature of C. botulinum C2 toxin, C. perfringens ι toxin, C. spiroforme CST, B. cereus VIP, and C. difficile CDT was elucidated in 1980 (302), 1986 (421, 422), 1988 (332), 1992 (171), and 1997 (324), respectively. Therefore, the edema and lethal toxins of B. anthracis represent a “standard” for this binary family that is further evidenced by the number of publications written, and information determined, over time. For example, determination of the crystal structures of each B. anthracis toxin component (EF [103], LF [314], and PA [326]) and specific identification of the cell surface receptors for PA (60, 61, 245, 379) represent exciting discoveries currently lacking for the other binary toxins. Notable exceptions, however, include the crystal structures of Ia (438) and VIP2 (171).
As with any family, there are intriguing similarities but also differences among its members that become clearer over time and further study. The “B” precursors of Clostridium and Bacillus binary toxins described in this review are all proteolytically activated by serine-type proteases in solution, but the PA83 molecule from B. anthracis is also uniquely processed on a targeted cell by surface-associated furin or furin-like proteins. The distinct versatility of PA63 heptamers is also apparent with the transport of different enzymatic moieties (EF and LF) into a cell, whereas “B” heptamers from the other binary toxins transport only one type of enzyme, an ADP-ribosyltransferase specific for G-actin. In contrast to the uniform requirement for proteolytic activation of “B” components, Ia of ι toxin is the only “A” component in this binary family that is activated by proteases. The evolutionary relationships that exist among Clostridium and Bacillus binary toxins are made more evident by the generation of biologically active chimeras within the ι toxin family via interchangeable “A” and “B” molecules, which does not occur with any of the C2 or B. anthracis edema toxin and lethal toxin components.
Looking into the future, domain swapping between Clostridium and Bacillus binary toxins beyond what Singh et al. (399) initially described for PA and Ib could be an eventual reality that yields novel protein-based therapeutics targeting the B. anthracis edema and lethal toxins. Further exploration that employs gene probes and specific toxin antibodies will also probably unveil new binary toxins produced by other bacteria and perhaps those from different genera. Such discoveries could reveal additional clues to the evolutionary tracts taken by these binary toxins and of course by the bacteria that produce them. Besides the extensive pathogenesis work done with edema and lethal toxins of B. anthracis, it still remains a mystery whether the other Clostridium and Bacillus binary toxins afford any unique advantage(s) for their bacterial hosts. Again, this is a reflection of the literature not yet available for the other binary toxins.
Finally, in our opinion, it is evident that the bacterial toxins described in this review possess additional untapped promise for the future. A knowledge-based understanding of the past will hopefully spawn additional creative efforts, by various groups, involving the Clostridium and Bacillus binary toxins.
FIG. 1.
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.
FIG. 2.
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.
FIG. 3.
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 ι) that modify G-actin. Panel B modified from reference 31 with permission.
FIG. 4.
FIG. 4. Depiction of C2 intoxication via Hsp90, a process that is required for entry of C2I into the cytosol from acidified endosomes (179). A similar pathway is also used by ι toxin and CDT for intoxicating cells (178a). Translocation of C2I, CDTa, or Ia from the endosome requires an acidic pH, a process blocked by bafilomycin A (Baf) via specific inhibition of vacuolar-type ATPases located in the endosomal membrane. C2I partially unfolds during translocation across the endosomal membrane via Hsp90, a process specifically targeted by geldanamycin (GA) or radicicol (Rad), which results in trapping of C2I within the endosome.
TABLE 1. Clostridium and Bacillus binary “A-B” toxins: an overview
Toxin and componentsGene locationMol mass (kDa) (reference)Enzymatic activity (reference)
C. perfringens ι toxinPlasmid (140 kbp)  
    Ia 45 (323)ADP-ribosylation of actin (372)
    Ib 94 precursor (323)None
  81 activated (323)None
C. spiroforme CSTChromosome  
    Sa 44 (147, 333)ADP-ribosylation of actin (332)
    Sb 92 precursor (147, 333)None
  76 activated (147, 333)None
C. difficile CDTChromosome  
    CDTa 48 (324)ADP-ribosylation of actin (334)
    CDTb 99 precursor (324)None
  75 activated (324)None
C. botulinum C2 toxinChromosome  
    C2I 49 (137)ADP-ribosylation of actin (6, 310)
    C2II 81 precursor (208)None
  60 activated (48)None
B. anthracis edema and lethal toxinsPlasmid pXO1 (182 kbp)  
    EF 89 (360)Adenylate cyclase (229)
    LF 90 (62)Zn2+ metalloprotease for MAPKK (107, 453)
    PA 83 precursor (467)None
  63 activated (467)None
B. cereus VIPUnknown  
    VIP2 52aADP-ribosylation of actin (171)
    VIP1 100aNone
G. Warren, M. Koziol, M. A. Mullins, G. Nye, B. Carr, N. Desai, K. Kostichka, N. Duck, and J. J. Estruch, 1996, World Intellectual Property Organization patent application.
TABLE 2. Examples of bacterial toxins produced as preformed “A-B” structures found in solution
BacteriumToxin (reference)Structure (mol mass [kDa]/A:B ratio)
Clostridium botulinumBotulinum neurotoxins A-G (332a)Single protein (150/1:1)
Clostridium difficileToxins A and B (434a)Single protein (∼270-308/1:X)a
Clostridium novyiAlpha toxin (182a)Single protein (250/1:X)a
Clostridium sordelliiHemorrhagic and lethal toxins (252a)Single protein (∼260-300/1:X)a
Clostridium tetaniTetanus neurotoxin (332a)Single protein (150/1:1)
Corynebacterium diphtheriaeDiphtheria toxin (330a)Single protein (58/1:1)
Pseudomonas aeruginosaExotoxin A (330a)Single protein (67/1:1)
Bordetella pertussisPertussis toxin (246a)Multiple proteins (105/1:4)
Escherichia coliHeat-labile enterotoxin (181)Multiple proteins (85/1:5)
Shigella dysenteriae and other EnterobacteriaceaeShiga and Shiga-like toxins (2)Multiple proteins (∼70/1:5)
Vibrio choleraeCholera toxin (181)Multiple proteins (85/1:5)
Repetitive “B” oligopeptide domains of different lengths are evident within the C terminus of the large clostridial toxins produced by C. difficile, C. novyi, and C. sordellii.
TABLE 3. Use of C2, CST, and ι toxins as tools to determine various cellular processes involving the actin cytoskeletona
Effects (toxin)Reference(s)
Activation of neutrophils and peptide receptor dynamics (C2)16, 159, 292, 293, 468
Inhibited pseudopod formation, phagocytosis, and adhesion to laminin of murine macrophages (C2)195
Decreased actin mRNA levels by increasing G-actin concentrations in rat hepatocytes (C2)353, 354
Decreased expression of various proteins by 3T3-A31 mouse embryo cells (C2)406
Decreased esterification of cholesterol by mouse peritoneal macrophages (C2)431
Inhibited muscle contraction of guinea pig ileum but not rabbit aorta (C2)256
Inhibited histamine release from rat peritoneal mast cells (C2)55
Activation and apoptosis of human B lymphocytes (C2)114, 262-266
Increased noradrenaline release from rat PC12 adrenal pheochromocytoma cells (C2)254
Decreased catecholamine release from bovine chromaffin cells (C2)142
Decreased degranulation and serotonin release from nonadherent, rat peritoneal mast cells; increased degranulation of adherent rat 2H3-hm1 basophils (C2)339, 472
Decreased exocytosis of insulin in hamster HIT-T15 pancreatic islet cells (C2)234
Increased exocytosis of serotonin from rat 2H3-hm1 basophils (C2)340, 341
Increased steroid release from murine Y-1 adrenocortical cells (C2)89, 485
Increased intracellular Ca2+ movement in rat 2H3-hm1 leukemic basophils (C2)99
Inhibited insulin-regulated gene expression in rat L6 skeletal myoblasts (C2)437
Decreased retrograde transport from Golgi to endoplasmic reticulum in rat kidney and human cervix (HeLa) cells (C2)442, 443
Inhibited glucose transport in mouse 3T3-L1 adipocytes (C2)374
Inhibited actin expression in mouse embryocytes (3T3-A31) and rat hepatocytes (C2)44, 351, 352
Delayed human cervix (HeLa) cell cycling into mitosis (C2)34
Decreased metastasis and invasion of murine BW5147 lymphoma (C2)450
Increased endothelial permeability in various species (C2)113, 115, 377, 387, 429
Increased exocytosis but decreased endocytosis in rat melanothrophs (CST)84, 85
Decreased norepinephrine release from human SH-SY5Y neuroblastoma cells (ι)454
Decreased integrity of intercellular junctions between polarized human adenocarcinoma colon (CaCo-2) cells (ι)356
Modified from reference 31 with permission.
TABLE 4. C2-C3 fusion toxins as tools for studying the role of Rho GTPases in various cell typesa
Effects on:Reference
Aquaporin 2 translocation from rat renal cells212
Anion channel activity in bovine endothelial cells291
Cyclooxygenase-2 gene expression in rat renal cells169
Secretion of von Willebrand factor from human umbilical vein cells451
IL-1b-stimulated synthesis of IL-2 in murine EL-4 thymoma cells102
Survival of rat cerebellar neurons236
Uptake and degradation of lipoproteins by mouse J774 macrophages366
Transendothelial migration and adhesion of human monocytes424
Growth cone collapse of chicken retinal cells456
Modified from reference 31 with permission.
IL, interleukin.


B.G.S. thanks Charles Millard, the U.S. Army Medical Research Institute of Infectious Diseases, and the U.S. Army Medical Research and Materiel Command for enabling him to initiate, as well as sustain, collaborations that foster a progressive understanding of science and beyond. Much gratitude is extended to colleagues and friends at the Centre de Recherches du Service de Santé des Armées in Grenoble for their time and kind consideration afforded to B.G.S. during his sabbatical stay that included the final preparation of this paper.
Support from The Philippe Foundation is gratefully acknowledged by B.G.S. and M.R.P. for help in fostering scientific collaborations between France and the United States. M.R.P. acknowledges financial support from the Délégation Générale pour l'Armement (contract 0034056) and Institut Pasteur, which have both contributed in many ways to his laboratory and a further understanding of bacteria as well as associated toxins. K.A. and H.B. acknowledge the generous financial support afforded by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 388) that has sustained this, and other, research endeavors into bacterial toxins and their biological effects.


Abrami, L., S. Liu, P. Cosson, S. H. Leppla, and F. G. van der Goot. 2003. Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process. J. Cell Biol.160:3321-3328.
Acheson, D. W. K., and G. T. Keusch. 1999. The family of Shiga toxins, p. 229-242. In J. E. Alouf and J. H. Freer (ed.), The comprehensive sourcebook of bacterial protein toxins. Academic Press, Inc., New York, N.Y.
Agrawal, A., J. Lingappa, S. H. Leppla, S. Agrawal, A. Jabbar, C. Quinn, and B. Pulendran. 2003. Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin. Nature424:329-334.
Ahuja, N., P. Kumar, S. Alam, M. Gupta, and R. Bhatnagar. 2003. Deletion mutants of protective antigen that inhibit anthrax toxin both in vitro and in vivo. Biochem. Biophys. Res. Commun.307:446-450.
Ahuja, N., P. Kumar, and R. Bhatnagar. 2001. Hydrophobic residues Phe552, Phe554, Ile562, Leu566, and Ile574 are required for oligomerization of anthrax protective antigen. Biochem. Biophys. Res. Commun.287:542-549.
Aktories, K., T. Ankenbauer, B. Schering, and K. H. Jakobs. 1986. ADP-ribosylation of platelet actin by botulinum C2 toxin. Eur. J. Biochem.161:155-162.
Aktories, K., M. Bärmann, I. Ohishi, S. Tsuyama, K. H. Jakobs, and E. Habermann. 1986. Botulinum C2 toxin ADP-ribosylates actin. Nature322:390-392.
Aktories, K., U. Braun, S. Rösener, I. Just, and A. Hall. 1989. The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3. Biochem. Biophys. Res. Commun.158:209-213.
Aktories, K., and I. Just. 1990. Botulinum C2 toxin, p. 79-95. In J. Moss and M. Vaughan (ed.), ADP-ribosylating toxins and G-proteins. American Society for Microbiology, Washington, D.C.
Aktories, K., and G. Koch. 1995. Modification of actin and of Rho proteins by clostridial ADP-ribosylating toxins, p. 491-520. In B. Iglewski, J. Moss, A. T. Tu, and M. Vaughan (ed.), Microbial toxins and virulence factors in disease. Marcel Dekker, Inc., New York, N.Y.
Aktories, K., C. Mohr, and G. Koch. 1992. Clostridium botulinum C3 ADP-ribosyltransferase. Curr. Top. Microbiol. Immunol.175:115-131.
Aktories, K., K.-H. Reuner, P. Presek, and M. Barmann. 1989. Botulinum C2 toxin treatment increases the G-actin pool in intact chicken cells: a model for the cytopathic action of actin-ADP-ribosylating toxins. Toxicon27:989-993.
Aktories, K., and A. Wegner. 1989. ADP-ribosylation of actin by clostridial toxins. J. Cell Biol.109:1385-1387.
Aktories, K., U. Weller, and G. S. Chhatwal. 1987. Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. FEBS Lett.212:109-113.
Aktories, K., M. Wille, and I. Just. 1992. Clostridial actin-ADP-ribosylating toxins. Curr. Top. Microbiol. Immunol.175:97-113.
Al-Mohanna, F. A., I. Ohishi, and M. B. Hallett. 1987. Botulinum C2 toxin potentiates activation of the neutrophil oxidase. Further evidence of a role for actin polymerization. FEBS Lett.219:40-44.
Altboum, Z., Y. Gozes, A. Barnea, A. Pass, M. White, and D. Kobiler. 2002. Postexposure prophylaxis against anthrax: evaluation of various treatment regimens in intranasally infected guinea pigs. Infect. Immun.70:6231-6241.
Anne, C., F. Cornille, C. Lenoir, and B. P. Roques. 2001. High-throughput fluorogenic assay for determination of botulinum type B neurotoxin protease activity. Anal. Biochem.291:253-261.
Arora, N. 1997. Site directed mutagenesis of histidine residues in anthrax toxin lethal factor binding domain reduces toxicity. Mol. Cell. Biochem.177:7-14.
Arora, N., K. R. Klimpel, Y. Singh, and S. H. Leppla. 1992. Fusions of anthrax toxin lethal factor to the ADP-ribosylation domain of Pseudomonas exotoxin A are potent cytotoxins which are translocated to the cytosol of mammalian cells. J. Biol. Chem.267:15542-15548.
Arora, N., and S. H. Leppla. 1993. Residues 1-254 of anthrax toxin lethal factor are sufficient to cause cellular uptake of fused polypeptides. J. Biol. Chem.268:3334-3341.
Arora, N., and S. H. Leppla. 1994. Fusions of anthrax toxin lethal factor with shiga toxin and diphtheria toxin enzymatic domains are toxic to mammalian cells. Infect. Immun.62:4955-4961.
Arora, N., L. C. Williamson, S. H. Leppla, and J. L. Halpern. 1994. Cytotoxic effects of a chimeric protein consisting of tetanus toxin light chain and anthrax toxin lethal factor in non-neuronal cells. J. Biol. Chem.269:26165-26171.
Azhar, A. M., S. Singh, P. Anand Kumar, and R. Bhatnagar. 2002. Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax. Biochem. Biophys. Res. Commun.299:345-351.
Babudieri, S., S. P. Borriello, A. Pantosti, I. Luzzi, G. P. Testore, and G. Panichi. 1986. Diarrhoea associated with toxigenic Clostridium spiroforme. J. Infect.12:278-279.
Bachmeyer, C., R. Benz, H. Barth, K. Aktories, M. Gibert, and M. Popoff. 2001. Interaction of Clostridium botulinum C2-toxin with lipid bilayer membranes and vero cells: inhibition of channel function by chloroquine and related compounds in vitro and intoxication in vivo. FASEB J.15:1658-1660.
Bachmeyer, C., F. Orlik, H. Barth, K. Aktories, and R. Benz. 2003. Mechanism of C2- toxin inhibition by fluphenazine and related compounds: investigation of their binding kinetics to the C2II-channel using the current noise analysis. J. Mol. Biol.333:527-540.
Ballard, J. D., R. J. Collier, and M. N. Starnbach. 1996. Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo. Proc. Natl. Acad. Sci. USA93:12531-12534.
Ballard, J. D., R. J. Collier, and M. N. Starnbach. 1998. Anthrax toxin as a molecular tool for stimulation of cytotoxic T lymphocytes: disulfide-linked epitopes, multiple injections, and role of CD4+ cells. Infect. Immun.66:4696-4699.
Ballard, J. D., A. M. Doling, K. Beauregard, R. J. Collier, and M. N. Starnbach. 1998. Anthrax toxin-mediated delivery in vivo and in vitro of a cytotoxic T-lymphocyte epitope from ovalbumin. Infect. Immun.66:615-619.
Ballweber, E., M. Galla, K. Aktories, S. Yeoh, A. G. Weeds, and H. G. Mannherz. 2001. Interaction of ADP-ribosylated actin with actin binding proteins. FEBS Lett.508:131-135.
Barth, H., D. Blöcker, and K. Aktories. 2002. The uptake machinery of clostridial actin ADP-ribosylating toxins—a cell delivery system for fusion proteins and polypeptide drugs. Naunyn-Schmiedeberg's Arch. Pharmacol.366:501-512.
Barth, H., D. Blöcker, J. Behlke, W. Bergsma-Schutter, A. Brisson, R. Benz, and K. Aktories. 2000. Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification. J. Biol. Chem.275:18704-18711.
Barth, H., F. Hofmann, C. Olenik, I. Just, and K. Aktories. 1998. The N-terminal part of the enzyme component (C2I) of the binary Clostridium botulinum C2 toxin interacts with the binding component C2II and functions as a carrier system for a Rho ADP-ribosylating C3-like fusion toxin. Infect Immun.66:1364-1369.
Barth, H., M. Klingler, K. Aktories, and V. Kinzel. 1999. Clostridium botulinum C2 toxin delays entry into mitosis and activation of p34cdc2 kinase and cdc25-C phosphatase in HeLa cells. Infect. Immun.67:5083-5090.
Barth, H., C. Olenik, P. Sehr, G. Schmidt, K. Aktories, and D. K. Meyer. 1999. Neosynthesis and activation of Rho by Escherichia coli cytotoxic necrotizing factor (CNF1) reverse cytopathic effects of ADP-ribosylated Rho. J. Biol. Chem.274:27407-27414.
Barth, H., J. C. Preiss, F. Hofmann, and K. Aktories. 1998. Characterization of the catalytic site of the ADP-ribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis. J. Biol. Chem.273:29506-29511.
Barth, H., R. Roebling, M. Fritz, and K. Aktories. 2002. The binary Clostridium botulinum C2 toxin as a protein delivery system: identification of the minimal protein region necessary for interaction of toxin components. J. Biol. Chem.277:5074-5081.
Bartlett, J. G., T. V. Inglesby Jr., and L. Borio. 2002. Management of anthrax. Clin. Infect. Dis.35:851-858.
Batra, S., P. Gupta, V. Chauhan, A. Singh, and R. Bhatnagar. 2001. Trp 346 and Leu 352 residues in protective antigen are required for the expression of anthrax lethal toxin activity. Biochem. Biophys. Res. Commun.281:186-192.
Beauregard, K. E., R. J. Collier, and J. A. Swanson. 2000. Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization. Cell. Microbiol.2:251-258.
Beauregard, K. E., S. Wimer-Mackin, R. J. Collier, and W. I. Lencer. 1999. Anthrax toxin entry into polarized epithelial cells. Infect. Immun.67:3026-3030.
Beedham, R. J., P. C. Turnbull, and E. D. Williamson. 2001. Passive transfer of protection against Bacillus anthracis infection in a murine model. Vaccine19:4409-4416.
Benson, E. L., P. D. Huynh, A. Finkelstein, and R. J. Collier. 1998. Identification of residues lining the anthrax protective antigen channel. Biochemistry37:3941-3948.
Bershadsky, A. D., U. Glück, O. N. Denisenko, T. V. Sklyarova, I. Spector, and A. Ben- Ze'ev. 1995. The state of actin assembly regulates actin and vinculin expression by a feedback loop. J. Cell Sci.108:1183-1193.
Billington, S. J., E. U. Wieckowski, M. R. Sarker, D. Bueschel, J. G. Songer, and B. A. McClane. 1998. Clostridium perfringens type E animal enteritis isolates with highly conserved, silent enterotoxin gene sequences. Infect. Immun.66:4531-4536.
Blaustein, R. O., T. M. Koehler, R. J. Collier, and A. Finkelstein. 1989. Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers. Proc. Natl. Acad. Sci. USA86:2209-2213.
Blöcker, D., C. Bachmeyer, R. Benz, K. Aktories, and H. Barth. 2003. Channel formation by the binding component of Clostridium botulinum C2 toxin: glutamate 307 of C2II affects channel properties in vitro and pH-dependent C2I translocation in vivo. Biochemistry42:5368-5377.
Blöcker, D., H. Barth, E. Maier, R. Benz, J. T. Barbieri, and K. Aktories. 2000. The C terminus of component C2II of Clostridium botulinum C2 toxin is essential for receptor binding. Infect. Immun.68:4566-4573.
Blöcker, D., J. Behlke, K. Aktories, and H. Barth. 2001. Cellular uptake of the binary Clostridium perfringens iota-toxin. Infect. Immun.69:2980-2987.
Blöcker, D., K. Pohlmann, G. Haug, C. Bachmeyer, R. Benz, K. Aktories, and H. Barth. 2003. Clostridium botulinum C2 toxin: low pH-induced pore formation is required for translocation of the enzyme component C2I into the cytosol of host cells. J. Biol. Chem.278:37360-37367.
Bohannon, J. 2003. Anthrax. From bioweapons backwater to main attraction. Science300:414-415.
Borriello, S., and R. Carman. 1983. Association of iota-like toxin and Clostridium spiroforme with both spontaneous and antibiotic-associated diarrhea and colitis in rabbits. J. Clin. Microbiol.17:414-418.
Borriello, S., H. A. Davies, and R. J. Carman. 1986. Cellular morphology of Clostridium spiroforme. Vet. Microbiol.11:191-195.
Bosworth, T. 1940. On a new type of toxin produced by Clostridium welchii. J. Comp. Pathol.53:245-255.
Bottinger, H., K. H. Reuner, and K. Aktories. 1987. Inhibition of histamine release from rat mast cells by botulinum C2 toxin. Int. Arch. Allergy Appl. Immunol.84:380-384.
Bourgogne, A., M. Drysdale, S. G. Hilsenbeck, S. N. Peterson, and T. M. Koehler. 2003. Global effects of virulence gene regulators in a Bacillus anthracis strain with both virulence plasmids. Infect. Immun.71:2736-2743.
Bourne, H. R., L. M. Lichtenstein, K. L. Melmon, C. S. Henney, Y. Weinstein, and G. M. Shearer. 1974. Modulation of inflammation and immunity by cyclic AMP. Science184:19-24.
Boyaka, P. N., A. Tafaro, R. Fischer, S. H. Leppla, K. Fujihashi, and J. R. McGhee. 2003. Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen. J. Immunol.170:5636-5643.
Brachman, P., S. H. Gold, S. A. Plotkin, F. R. Fekety, M. Werrin, and N. R. Ingraham. 1962. Field evaluation of a human anthrax vaccine. Am. J. Public Health52:632-645.
Bradley, K. A., J. Mogridge, M. Mourez, R. J. Collier, and J. A. Young. 2001. Identification of the cellular receptor for anthrax toxin. Nature414:225-229.
Bradley, K. A., J. Mogridge, G. J. Rainey, S. Batty, and J. A. Young. 2003. Binding of anthrax toxin to its receptor is similar to alpha integrin-ligand interactions. J. Biol. Chem.278:49342-49347.
Bragg, T. S., and D. L. Robertson. 1989. Nucleotide sequence and analysis of the lethal factor gene (lef) from Bacillus anthracis. Gene81:45-54.
Braun, M., C. Herholz, R. Straub, B. Choisat, J. Frey, J. Nicolet, and P. Kuhnert. 2000. Detection of the ADP-ribosyltransferase toxin gene (cdtA) and its activity in Clostridium difficile isolates from equidae. FEMS Microbiol. Lett.184:29-33.
Brossier, F., M. Levy, and M. Mock. 2002. Anthrax spores make an essential contribution to vaccine efficacy. Infect. Immun.70:661-664.
Brossier, F., J. C. Sirard, C. Guidi-Rontani, E. Duflot, and M. Mock. 1999. Functional analysis of the carboxy-terminal domain of Bacillus anthracis protective antigen. Infect. Immun.67:964-967.
Brossier, F., M. Weber-Levy, M. Mock, and J.-C. Sirard. 2000. Protective antigen-mediated antibody response against a heterologous protein produced in vivo by Bacillus anthracis. Infect. Immun.68:5731-5734.
Brossier, F., M. Weber-Levy, M. Mock, and J.-C. Sirard. 2000. Role of toxin functional domains in anthrax pathogenesis. Infect. Immun.68:1781-1786.
Bryskier, A. 2002. Bacillus anthracis and antibacterial agents. Clin. Microbiol. Infect.8:467-478.
Butt, M. T., R. E. Papendick, L. G. Carbone, and F. W. Quimby. 1994. A cytotoxicity assay for Clostridium spiroforme enterotoxin in cecal fluid of rabbits. Lab. Anim. Sci.44:52-54.
Cao, H., D. Agrawal, N. Kushner, N. Touzjian, M. Essex, and Y. Lu. 2002. Delivery of exogenous protein antigens to major histocompatibility complex class I pathway in cytosol. J. Infect. Dis.185:244-251.
Carman, R. J., and S. P. Borriello. 1982. Observations on an association between Clostridium spiroforme and Clostridium perfringens type E iota enterotoxaemia in rabbits. Eur. J. Chemother. Antibiot.2:143-144.
Carman, R. J., and S. P. Borriello. 1984. Infectious nature of Clostridium spiroforme-mediated rabbit enterotoxaemia. Vet. Microbiol.9:497-502.
Carman, R. J., and R. H. Evans. 1984. Experimental and spontaneous clostridial enteropathies of laboratory and free living lagomorphs. Lab. Anim. Sci.34:443-452.
Carman, R. J., S. Perelle, and M. R. Popoff. 1997. Binary toxins from Clostridium spiroforme and Clostridium perfringens, p. 359-367. In J. Rood, B. A. McClane, G. Songer, and R. Titball (ed.), The clostridia: molecular biology and pathogenesis. Academic Press, Inc., New York, N.Y.
Carman, R. J., R. L. van Tassell, and T. D. Wilkins. 1987. Production of iota toxin by Clostridium spiroforme: a requirement for divalent cations. Vet. Microbiol.15:115-120.
Carman, R. J., and T. D. Wilkins. 1991. In vitro susceptibility of rabbit strains of Clostridium spiroforme to antimicrobial agents. Vet. Microbiol.28:391-397.
Carroll, S. F., and R. J. Collier. 1984. NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD. Proc. Natl. Acad. Sci. USA81:3307-3311.
Cataldi, A., E. Labruyere, and M. Mock. 1990. Construction and characterization of a protective antigen deficient Bacillus anthracis strain. Mol. Microbiol.4:1111-1117.
Chardin, P., and F. McCormick. 1999. Brefeldin A: the advantage of being uncompetitive. Cell97:153-155.
Chaudry, G. J., M. Moayeri, S. Liu, and S. H. Leppla. 2002. Quickening the pace of anthrax research: three advances point towards possible therapies. Trends Microbiol.10:58-62.
Chauhan, V., and R. Bhatnagar. 2002. Identification of amino acid residues of anthrax protective antigen involved in binding with lethal factor. Infect. Immun.70:4477-4484.
Cheng, P. C., M. L. Dykstra, R. N. Mitchell, and S. K. Pierce. 1999. A role for lipid rafts in B cell antigen receptor signaling and antigen targeting. J. Exp. Med.190:1549-1560.
Choe, S., M. J. Bennett, G. Fujii, P. M. G. Curmi, K. A. Kantardjieff, R. J. Collier, and D. Eisenberg. 1992. The crystal structure of diphtheria toxin. Nature357:216-222.
Chopra, A. P., S. A. Boone, X. Liang, and N. S. Duesbery. 2003. Anthrax lethal factor proteolysis and inactivation of MAPK kinase. J. Biol. Chem.278:9402-9406.
Chowdhury, H. H., M. Kreft, and R. Zorec. 2002. Distinct effect of actin cytoskeleton disassembly on exo- and endo-cytic events in a membrane patch of rat melanotrophs. J. Physiol.545:879-886.
Chowdhury, H. H., M. R. Popoff, and R. Zorec. 1999. Actin cytoskeleton depolymerization with Clostridium spiroforme toxin enhances the secretory activity of rat melanotrophs. J. Physiol.521:389-395.
Cirino, N. M., D. Sblattero, D. Allen, S. R. Peterson, J. D. Marks, P. J. Jackson, A. Bradbury, and B. E. Lehnert. 1999. Disruption of anthrax toxin binding with the use of human antibodies and competitive inhibitors. Infect. Immun.67:2957-2963.
Confer, D. L., and J. W. Eaton. 1982. Phagocyte impotence caused by an invasive bacterial cyclase. Science217:948-950.
Considine, R. V., and L. L. Simpson. 1991. Cellular and molecular actions of binary toxins possessing ADP-ribosyltransferase activity. Toxicon29:913-936.
Considine, R. V., L. L. Simpson, and J. R. Sherwin. 1992. Botulinum C2 toxin and steroid production in adrenal Y-1 cells: the role of microfilaments in the toxin-induced increase in steroid release. J. Pharmacol. Exp. Ther.260:859-864.
Coulson, N. M., M. Fulop, and R. W. Titball. 1994. Bacillus anthracis protective antigen, expressed in Salmonella typhimurium SL 3261, affords protection against anthrax spore challenge. Vaccine12:1395-1401.
Cummings, R. T., S. P. Salowe, B. R. Cunningham, J. Wiltsie, Y. W. Park, L. M. Sonatore, D. Wisniewski, C. M. Douglas, J. D. Hermes, and E. M. Scolnick. 2002. A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease. Proc. Natl. Acad. Sci. USA99:6603-6606.
Cunningham, K., D. B. Lacy, J. Mogridge, and R. J. Collier. 2002. Mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen. Proc. Natl. Acad. Sci. USA99:7049-7053.
Czeczulin, J. R., P. C. Hanna, and B. A. McClane. 1993. Cloning, nucleotide sequencing, and expression of the Clostridium perfringens enterotoxin gene in Escherichia coli. Infect. Immun.61:3429-3439.
Dai, Z., J. C. Sirard, M. Mock, and T. M. Koehler. 1995. The atxA gene product activates transcription of the anthrax toxin genes and is essential for virulence. Mol. Microbiol.16:1171-1181.
Daube, G., B. China, P. Simon, K. Hvala, and J. Mainil. 1994. Typing of Clostridium perfringens by in vitro amplification of toxin genes. J. Appl. Bacteriol.77:650-655.
Daube, G., P. Simon, B. Limbourg, C. Manteca, J. Mainil, and A. Kaeckenbeeck. 1996. Hybridization of 2659 Clostridium perfringens isolates with gene probes for seven toxins (α, β, ε, ι, θ, μ, and enterotoxin) and for sialidase. Am. J. Vet. Res.57:496-501.
Devriese, P. P. 1999. On the discovery of Clostridium botulinum. J. Hist. Neurosci.8:43-50.
Dixon, T. C., A. A. Fadl, T. M. Koehler, J. A. Swanson, and P. C. Hanna. 2000. Early Bacillus anthracis-macrophage interactions: intracellular survival and escape. Cell. Microbiol.2:453-463.
Dixon, T. C., M. Meselson, J. Guillemin, and P. C. Hanna. 1999. Anthrax. N. Engl. J. Med.341:815-825.
Djouder, N., U. Prepens, K. Aktories, and A. Cavalié. 2000. Inhibition of calcium release-activated calcium current by Rac/Cdc42-inactivating clostridial cytotoxins in RBL cells. J. Biol. Chem.275:18732-18738.
Dobbelstein, M. 2003. Viruses in therapy—royal road or dead end? Virus Res.92:219-221.
Doling, A. M., J. D. Ballard, H. Shen, K. M. Krishna, R. Ahmed, R. J. Collier, and M. N. Starnbach. 1999. Cytotoxic T-lymphocyte epitopes fused to anthrax toxin induce protective antiviral immunity. Infect. Immun.67:3290-3296.
Dreikhausen, U., G. Varga, F. Hofmann, H. Barth, K. Aktories, K. Resch, and M. Szamel. 2001. Regulation by rho family GTPases of IL-1 receptor induced signaling: C3-like chimeric toxin and Clostridium difficile toxin B inhibit signaling pathways involved in IL-2 gene expression. Eur. J. Immunol.31:1610-1619.
Drum, C. L., Y. Shen, P. A. Rice, A. Bohm, and W. J. Tang. 2001. Crystallization and preliminary X-ray study of the edema factor exotoxin adenylyl cyclase domain from Bacillus anthracis in the presence of its activator, calmodulin. Acta. Crystallogr. Ser. D.57:1881-1884.
Drum, C. L., S. Z. Yan, J. Bard, Y. Q. Shen, D. Lu, S. Soelaiman, Z. Grabarek, A. Bohm, and W. J. Tang. 2002. Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin. Nature415:396-402.
Drum, C. L., S. Z. Yan, R. Sarac, Y. Mabuchi, K. Beckingham, A. Bohm, Z. Grabarek, and W. J. Tang. 2000. An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis. J. Biol. Chem.275:36334-36340.
Duesbery, N. S., and G. F. Vande Woude. 1999. Anthrax lethal factor causes proteolytic inactivation of mitogen-activated protein kinase kinase. J. Appl. Microbiol.87:289-293.
Duesbery, N. S., C. P. Webb, S. H. Leppla, V. M. Gordon, K. R. Klimpel, T. D. Copeland, N. G. Ahn, M. K. Oskarsson, K. Fukasawa, K. D. Paull, and G. F. Vande Woude. 1998. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science280:734-737.
Eaton, P., and D. S. Fernie. 1980. Enterotoxaemia involving Clostridium perfringens iota toxin in a hysterectomy-derived rabbit colony. Lab. Anim.14:347-351.
Eckhardt, M., H. Barth, D. Blöcker, and K. Aktories. 2000. Binding of Clostridium botulinum C2 toxin to asparagine-linked complex and hybrid carbohydrates. J. Biol. Chem.275:2328-2334.
Egelman, E. H. 2001. Actin allostery again? Nat. Struct. Biol.8:735-736.
Ellis, T. M., A. R. Gregory, and G. D. Logue. 1991. Evaluation of a toxoid for protection of rabbits against enterotoxaemia experimentally induced by trypsin-activated supernatant of Clostridium spiroforme. Vet. Microbiol.28:93-102.
Enserink, M. 2002. Anthrax. “Borrowed immunity” may save future victims. Science295:777.
Ermert, L., H. Brückner, D. Walmrath, F. Grimminger, K. Aktories, N. Suttorp, H.-R. Duncker, and W. Seeger. 1995. Role of endothelial cytoskeleton in high-permeability edema due to botulinum C2 toxin in perfused rabbit lungs. Am. J. Physiol.268:L753-L761.
Ermert, L., H.-R. Duncker, H. Bruckner, F. Grimminger, T. Hansen, R. Rossig, K. Aktories, and W. Seeger. 1997. Ultrastructural changes of lung capillary endothelium in response to botulinum C2 toxin. J. Appl. Physiol.82:382-388.
Ermert, L., R. Rossig, T. Hansen, H. Schutte, K. Aktories, and W. Seeger. 1996. Differential role of actin in lung endothelial and epithelial barrier properties in perfused rabbit lungs. Eur. Respir. J.9:93-99.
Erwin, J. L., L. M. DaSilva, S. Bavari, S. F. Little, A. M. Friedlander, and T. C. Chanh. 2001. Macrophage-derived cell lines do not express proinflammatory cytokines after exposure to Bacillus anthracis lethal toxin. Infect. Immun.69:1175-1177.
Escuyer, V., and R. J. Collier. 1991. Anthrax protective antigen interacts with a specific receptor on the surface of CHO-K1 cells. Infect. Immun.59:3381-3386.
Ezzell, J. W., and T. G. Abshire. 1992. Serum protease cleavage of Bacillus anthracis protective antigen. J. Gen. Microbiol.138:543-549.
Fach, P., and M. R. Popoff. 1997. Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide agglutination test. Appl. Environ. Microbiol.63:4232-4236.
Falnes, P. O., S. Choe, I. H. Madshus, B. A. Wilson, and S. Olsnes. 1994. Inhibition of membrane translocation of diphtheria toxin A-fragment by internal disulfide bridges. J. Biol. Chem.269:8402-8407.
Fellows, P. F., M. K. Linscott, B. E. Ivins, M. L. Pitt, C. A. Rossi, P. H. Gibbs, and A. M. Friedlander. 2001. Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine19:3241-3247.
Fernie, D. S., J. M. Knights, R. O. Thomson, and R. J. Carman. 1984. Rabbit enterotoxaemia: purification and preliminary characterization of a toxin produced by Clostridium spiroforme. FEMS Microbiol. Lett.21:207-211.
Fine, A. M., J. B. Wong, H. S. Fraser, G. R. Fleisher, and K. D. Mandl. 2004. Is it influenza or anthrax? A decision analytic approach to the treatment of patients with influenza-like illnesses. Ann. Emerg. Med.43:318-328.
Finkelstein, A. 1994. The channel formed in planar lipid bilayers by the protective antigen component of anthrax toxin. Toxicology87:29-41.
Fivaz, M., L. Abrami, Y. Tsitrin, and F. G. van der Goot. 2001. Not as simple as just punching a hole. Toxicon39:1637-1645.
Flick-Smith, H. C., J. E. Eyles, R. Hebdon, E. L. Waters, R. J. Beedham, T. J. Stagg, J. Miller, H. O. Alpar, L. W. Baillie, and E. D. Williamson. 2002. Mucosal or parenteral administration of microsphere-associated Bacillus anthracis protective antigen protects against anthrax infection in mice. Infect. Immun.70:2022-2028.
Flick-Smith, H. C., N. J. Walker, P. Gibson, H. Bullifent, S. Hayward, J. Miller, R. W. Titball, and E. D. Williamson. 2002. A recombinant carboxy-terminal domain of the protective antigen of Bacillus anthracis protects mice against anthrax infection. Infect. Immun.70:1653-1656.
Fox, G. E., E. Stackabrandt, R. B. Hespell, J. Gibson, J. Mariloff, T. A. Dyer, R. S. Wolfe, W. E. Balch, R. S. Tanner, L. J. Magrum, et al. 1980. The phylogeny of prokaryotes. Science209:457-463.
Francis, J. W., R. H. Brown Jr., D. Figueiredo, M. P. Remington, O. Castillo, M. A. Schwarzschild, P. S. Fishman, J. R. Murphy, and J. C. vanderSpek. 2000. Enhancement of diphtheria toxin potency by replacement of the receptor binding domain with tetanus toxin C-fragment: a potential vector for delivering heterologous proteins to neurons. J. Neurochem.74:2528-2536.
Frankel, A. E., B. L. Powell, N. S. Duesbery, G. F. Vande Woude, and S. H. Leppla. 2002. Anthrax fusion protein therapy of cancer. Curr. Protein Pept. Sci.3:399-407.
Frieben, W. R., and C. L. Duncan. 1973. Homology between enterotoxin protein and spore structural protein in Clostridium perfringens type A. Eur. J. Biochem.39:393-401.
Friedlander, A. M. 1986. Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J. Biol. Chem.261:7123-7126.
Friedlander, A. M. 2000. Anthrax: clinical features, pathogenesis, and potential biological warfare threat. Curr. Clin. Top. Infect. Dis.20:335-349.
Friedlander, A. M., P. R. Pittman, and G. W. Parker. 1999. Anthrax vaccine. Evidence for safety and efficacy against inhalational anthrax. JAMA282:2104-2106.
Friedlander, A. M., S. L. Welkos, and B. E. Ivins. 2002. Anthrax vaccines. Curr. Top. Microbiol. Immunol.271:33-60.
Fritz, G., P. Schroeder, and K. Aktories. 1995. Isolation and characterization of a Clostridium botulinum C2 toxin-resistant cell line: evidence for possible involvement of the cellular C2II receptor in growth regulation. Infect. Immun.63:2334-2340.
Fry, D. C., S. A. Kuby, and A. S. Mildvan. 1986. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins. Proc. Natl. Acad. Sci. USA83:907-911.
Fujii, N., T. Kubota, S. Shirakawa, K. Kimura, I. Ohishi, K. Moriishi, E. Isogai, and H. Isogai. 1996. Characterization of component-I gene of botulinum C2 toxin and PCR detection of its gene in clostridial species. Biochem. Biophys. Res. Commun.220:353-359.
Fukao, T. 2004. Immune system paralysis by anthrax lethal toxin: the roles of innate and adaptive immunity. Lancet Infect. Dis.4:166-170.
Gao-Sheridan, S., S. Zhang, and R. J. Collier. 2003. Exchange characteristics of calcium ions bound to anthrax protective antigen. Biochem. Biophys. Res. Commun.300:61-64.
Garmory, H. S., R. W. Titball, K. F. Griffin, U. Hahn, R. Bohm, and W. Beyer. 2003. Salmonella enterica serovar typhimurium expressing a chromosomally integrated copy of the Bacillus anthracis protective antigen gene protects mice against an anthrax spore challenge. Infect. Immun.71:3831-3836.
Garred, O., E. Dubinina, A. Polessakaya, S. Olsnes, J. Kozlov, and K. Sandvig. 1997. Role of the disulfide bond in Shiga toxin A-chain for toxin entry into cells. J. Biol. Chem.272:11414-11419.
Garred, O., B. van Deurs, and K. Sandvig. 1995. Furin-induced cleavage and activation of Shiga toxin. J. Biol. Chem.270:10817-10821.
Gasman, S., S. Chasserot-Golaz, M. Popoff, D. Aunis, and M.-F. Bader. 1999. Involvement of Rho GTPases in calcium-regulated exocytosis from adrenal chromaffin cells. J. Cell Sci.112:4763-4771.
Gaur, R., P. K. Gupta, A. C. Banerjea, and Y. Singh. 2002. Effect of nasal immunization with protective antigen of Bacillus anthracis on protective immune response against anthrax toxin. Vaccine20:2836-2839.
Gaur, R., P. K. Gupta, A. Goyal, W. Wels, and Y. Singh. 2002. Delivery of nucleic acid into mammalian cells by anthrax toxin. Biochem. Biophys. Res. Commun.297:1121-1127.
Geipel, U., I. Just, and K. Aktories. 1990. Inhibition of cytochalasin D-stimulated G-actin ATPase by ADP-ribosylation with Clostridium perfringens iota toxin. Biochem. J.266:335-339.
Geipel, U., I. Just, B. Schering, D. Haas, and K. Aktories. 1989. ADP-ribosylation of actin causes increase in the rate of ATP exchange and inhibition of ATP hydrolysis. Eur. J. Biochem.179:229-232.
Geric, B., S. Johnson, D. D. Gerding, M. Grabnar, and M. Rupnik. 2003. Frequency of binary toxin genes among Clostridium difficile strains that do not produce large clostridial toxins. J. Clin. Microbiol.41:5227-5232.
Gibert, M., S. Perelle, G. Daube, and M. R. Popoff. 1997. Clostridium spiroforme toxin genes are related to C. perfringens iota toxin genes but have a different genomic localization. Syst. Appl. Microbiol.20:337-347.
Gibert, M., L. Petit, S. Raffestin, A. Okabe, and M. R. Popoff. 2000. Clostridium perfringens iota-toxin requires activation of both binding and enzymatic components for cytopathic activity. Infect. Immun.68:3848-3853.
Gilligan, P. H. 2002. Therapeutic challenges posed by bacterial bioterrorism threats. Curr. Opin. Microbiol.5:489-495.
Gilmore, M. S., M. C. Callegan, and B. D. Jett. 1999. Enterococcus faecalis cytolysin and Bacillus cereus bi- and tri-component haemolysins, p. 419-434. In J. E. Alouf and J. H. Freer (ed.), The comprehensive sourcebook of bacterial protein toxins. Academic Press, Inc., New York, N.Y.
Gkiourtzidis, K., J. Frey, E. Bourtzi-Hatzopoulou, N. Iliadis, and K. Sarris. 2001. PCR detection and prevalence of alpha-, beta-, beta 2-, epsilon-, iota- and enterotoxin genes in Clostridium perfringens isolated from lambs with clostridial dysentery. Vet. Microbiol.82:39-43.
Gladstone, G. P. 1946. Immunity to anthrax. Protective antigen present in cell-free culture filtrates. Br. J. Exp. Pathol.27:349-418.
Glick, M., D. D. Robinson, G. H. Grant, and W. G. Richards. 2002. Identification of ligand binding sites on proteins using a multi-scale approach. J. Am. Chem. Soc.124:2337-2344.
Gold, J. A., Y. Hoshino, S. Hoshino, M. B. Jones, A. Nolan, and M. D. Weiden. 2004. Exogenous gamma and alpha/beta interferon rescues human macrophages from cell death induced by Bacillus anthracis. Infect. Immun.72:1291-1297.
Goletz, T. J., K. R. Klimpel, N. Arora, S. H. Leppla, J. M. Keith, and J. A. Berzofsky. 1997. Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein. Proc. Natl. Acad. Sci. USA94:12059-12064.
Goletz, T. J., K. R. Klimpel, S. H. Leppla, J. M. Keith, and J. A. Berzofsky. 1997. Delivery of antigens to the MHC class I pathway using bacterial toxins. Hum. Immunol.54:129-136.
Gordon, V. M., K. R. Klimpel, N. Arora, M. A. Henderson, and S. H. Leppla. 1995. Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases. Infect. Immun.63:82-87.
Gordon, V. M., and S. H. Leppla. 1994. Proteolytic activation of bacterial toxins: role of bacterial and host cell proteases. Infect. Immun.62:333-340.
Gordon, V. M., S. H. Leppla, and E. L. Hewlett. 1988. Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin. Infect. Immun.56:1066-1069.
Goyard, S., C. Orlando, J. M. Sabatier, E. Labruyere, J. d'Alayer, G. Fontan, J. van Rietschoten, M. Mock, A. Danchin, A. Ullmann, and A. Monneron. 1989. Identification of a common domain in calmodulin-activated eukaryotic and bacterial adenylate cyclases. Biochemistry28:1964-1967.
Granum, P. E., and S. Brynestad. 1999. Bacterial toxin as food poisons, p. 669-681. In J. E. Alouf and J. H. Freer (ed.), The comprehensive sourcebook of bacterial protein toxins. Academic Press, Inc., New York, N.Y.
Grimminger, F., U. Sibelius, K. Aktories, I. Just, and W. Seeger. 1991. Suppression of cytoskeletal rearrangement in activated human neutrophils by botulinum C2 toxin. Impact on cellular signal transduction. J. Biol. Chem.266:19276-19282.
Gruenberg, J., and K. E. Howell. 1989. Membrane traffic in endocytosis: insights from cell-free assays. Annu. Rev. Cell Biol.5:453-481.
Gu, M. L., S. H. Leppla, and D. M. Klinman. 1999. Protection against anthrax toxin by vaccination with a DNA plasmid encoding anthrax protective antigen. Vaccine17:340-344.
Guidi-Rontani, C. 2002. The alveolar macrophage: the Trojan horse of Bacillus anthracis. Trends. Microbiol.10:405-409.
Guidi-Rontani, C., M. Levy, H. Ohayon, and M. Mock. 2001. Fate of germinated Bacillus anthracis spores in primary murine macrophages. Mol. Microbiol.42:931-938.
Guidi-Rontani, C., M. Weber-Levy, E. Labruyere, and M. Mock. 1999. Germination of Bacillus anthracis spores within alveolar macrophages. Mol. Microbiol.31:9-17.
Guidi-Rontani, C., M. Weber-Levy, M. Mock, and V. Cabiaux. 2000. Translocation of Bacillus anthracis lethal and oedema factors across endosome membranes. Cell Microbiol.2:259-264.
Guignot, J., M. Mock, and A. Fouet. 1997. AtxA activates the transcription of genes harbored by both Bacillus anthracis virulence plasmids. FEMS Microbiol. Lett.147:203-207.
Gülke, I., G. Pfeifer, J. Liese, M. Fritz, F. Hofmann, K. Aktories, and H. Barth. 2001. Characterization of the enzymatic component of the ADP-ribosyltransferase toxin CDTa from Clostridium difficile. Infect. Immun.69:6004-6011.
Gupta, P. K., H. Chandra, R. Gaur, R. K. Kurupati, S. Chowdhury, V. Tandon, Y. Singh, and K. Maithal. 2003. Conformational fluctuations in anthrax protective antigen: a possible role of calcium in the folding pathway of the protein. FEBS Lett.554:505-510.
Gupta, P. K., R. K. Kurupati, H. Chandra, R. Gaur, V. Tandon, Y. Singh, and K. Maithal. 2003. Acid induced unfolding of anthrax protective antigen. Biochem. Biophys. Res. Commun.311:229-232.
Gupta, P., A. Singh, V. Chauhan, and R. Bhatnagar. 2001. Involvement of residues 147VYYEIGK153 in binding of lethal factor to protective antigen of Bacillus anthracis. Biochem. Biophys. Res. Commun.280:158-163.
Hahn, A., H. Barth, M. Kress, P. R. Mertens, and M. Goppelt-Strübe. 2002. Role of Rac and Cdc42 in lysophosphatidic acid-mediated cyclooxygenase-2 gene expression. Biochem. J.362:33-40.
Hale, M. L., J. C. Marvaud, M. R. Popoff, and B. G. Stiles. 2004. Detergent-resistant membrane microdomains facilitate Ib oligomer formation and biological activity of Clostridium perfringens iota-toxin. Infect. Immun.72:2186-2193.
Han, S., A. S. Arvai, S. B. Clancy, and J. A. Tainer. 2001. Crystal structure and novel recognition motif of Rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis. J. Mol. Biol.305:95-107.
Han, S., J. A. Craig, C. D. Putnam, N. B. Carozzi, and J. A. Tainer. 1999. Evolution and mechanism from structures of an ADP-ribosylating toxin and NAD complex. Nat. Struct. Biol.6:932-936.
Hanna, P. 1998. Anthrax pathogenesis and host response. Curr. Top. Microbiol. Immunol.225:13-35.
Hanna, P. 1999. Lethal toxin actions and their consequences. J. Appl. Microbiol.87:285-287.
Hanna, P. C., D. Acosta, and R. J. Collier. 1993. On the role of macrophages in anthrax. Proc. Natl. Acad. Sci. USA90:10198-10201.
Hanna, P. C., S. Kochi, and R. J. Collier. 1992. Biochemical and physiological changes induced by anthrax lethal toxin in J774 macrophage-like cells. Mol. Biol. Cell3:1269-1277.
Hanna, P. C., B. A. Kruskal, R. A. Ezekowitz, B. R. Bloom, and R. J. Collier. 1994. Role of macrophage oxidative burst in the action of anthrax lethal toxin. Mol. Med.1:7-18.
Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol.141:929-942.
Hart, B., and P. Hooper. 1967. Enterotoxaemia of calves due to Clostridium welchii type E. Aust. Vet. J.43:360-363.
Haug, G., K. Aktories, and H. Barth. 2003. The host cell chaperone Hsp90 is necessary for cytotoxic action of the binary iota-like toxins. Infect. Immun.72:3066-3068.
Haug, G., J. Leemhuis, D. Tiemann, D. K. Meyer, K. Aktories, and H. Barth. 2003. The host cell chaperone Hsp90 is essential for translocation of the binary Clostridium botulinum C2 toxin into the cytosol. J. Biol. Chem.278:32266-32274.
Helgason, E., O. A. Okstad, D. A. Caugant, H. A. Johansen, A. Fouet, M. Mock, I. Hegna, and A.-B. Kolsto. 2000. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—one species on the basis of genetic evidence. Appl. Environ. Microbiol.66:2627-2630.
Hirst, T. R. 1999. Cholera toxin and Escherichia coli heat-labile enterotoxin, p. 104-129. In J. E. Alouf and J. H. Freer (ed.), The comprehensive sourcebook of bacterial protein Toxins. Academic Press, Inc., New York, N.Y.
Hoffmaster, A. R., and T. M. Koehler. 1997. The anthrax toxin activator gene atxA is associated with CO2-enhanced non-toxin gene expression in Bacillus anthracis. Infect. Immun.65:3091-3099.
Hofmann, F., A. Herrmann, E. Habermann, and C. von Eichel-Streiber. 1995. Sequencing and analysis of the gene encoding the α-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile. Mol. Gen. Gene.247:670-679.
L. V. Holdeman, E. P. Cato, and W. E. C. Moore (ed.) 1977. VPI anaerobe laboratory manual, p. 131-133. Southern Printing Co., Blacksburg, Va.
Holmes, H. T., R. J. Sonn, and N. M. Patton. 1988. Isolation of Clostridium spiroforme from rabbits. Lab. Anim. Sci.38:167-168.
Hoover, D. L., A. M. Friedlander, L. C. Rogers, I. K. Yoon, R. L. Warren, and A. S. Cross. 1994. Anthrax edema toxin differentially regulates lipopolysaccharide induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP. Infect. Immun.62:4432-4439.
Iacono-Connors, L. C., S. L. Welkos, B. E. Ivins, and J. M. Dalrymple. 1991. Protection against anthrax with recombinant virus-expressed protective antigen in experimental animals. Infect. Immun.59:1961-1965.
Ivanova, N., A. Sorokin, I. Anderson, N. Galleron, B. Candelon, V. Kapatral, A. Bhattacharyya, G. Reznik, N. Mikhailova, A. Lapidus, L. Chu, M. Mazur, E. Goltsman, N. Larsen, M. D'Souza, T. Walunas, Y. Grechkin, G. Pusch, R. Haselkorn, M. Fonstein, S. D. Ehrlich, R. Overbeek, and N. Kyrpides. 2003. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature423:87-91.
Ivins, B. E., M. L. Pitt, P. F. Fellows, J. W. Farchaus, G. E. Benner, D. M. Waag, S. F. Little, G. W. Anderson, P. H. Gibbs, and A. M. Friedlander. 1998. Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques. Vaccine16:1141-1148.
Ivins, B. E., S. L. Welkos, G. B. Knudson, and S. F. Little. 1990. Immunization against anthrax with aromatic compound-dependent (Aro) mutants of Bacillus anthracis and with recombinant strains of Bacillus subtilis that produce anthrax protective antigen. Infect. Immun.58:303-308.
Iwasaki, M., I. Ohishi, and G. Sakaguchi. 1980. Evidence that botulinum C2 toxin has two dissimilar components. Infect. Immun.29:390-394.
Jendrek, S., S. F. Little, S. Hem, G. Mitra, and S. Giardina. 2003. Evaluation of the compatibility of a second generation recombinant anthrax vaccine with aluminum- containing adjuvants. Vaccine21:3011-3018.
Jensen, W. I., and R. M. Duncan. 1980. The susceptibility of the mallard duck (Anas platyrhynchos) to Clostridium botulinum C2 toxin. Jpn. J. Med. Sci. Biol.33:81-86.
Jernigan, J. A., D. S. Stephens, D. A. Ashford, C. Omenaca, M. S. Topiel, M. Galbraith, M. Tapper, T. L. Fisk, S. Zaki, T. Popovic, R. F. Meyer, C. P. Quinn, S. A. Harper, S. K. Fridkin, J. J. Sejvar, C. W. Shepard, M. McConnell, J. Guarner, W. J. Shieh, J. M. Malecki, J. L. Gerberding, J. M. Hughes, and B. A. Perkins. 2001. Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg. Infect. Dis.7:933-944.
Jin, F., O. Matsushita, S. Katayama, S. Jin, C. Matsushita, J. Minami, and A. Okabe. 1996. Purification, characterization, and primary structure of Clostridium perfringens lambda-toxin, a thermolysin-like metalloprotease. Infect. Immun.64:230-237.
Jooss, K., and N. Chirmule. 2003. Immunity to adenovirus and adeno-associated viral vectors: implications for gene therapy. Gene Ther.10:955-963.
Jun, C. D., M. K. Han, U. H. Kim, and H. T. Chung. 1996. Nitric oxide induces ADP-ribosylation of actin in murine macrophages: association with the inhibition of pseudopodia formation, phagocytic activity, and adherence on a laminin substratum. Cell Immunol.174:25-34.
Jung, M., I. Just, J. van Damme, J. Vandekerckhove, and K. Aktories. 1993. NAD- binding site of the C3-like ADP-ribosyltransferase from Clostridium limosum. J. Biol. Chem.268:23215-23218.
Jupin, C., S. Anderson, C. Damais, J. E. Alouf, and M. Parant. 1988. Toxic shock syndrome toxin 1 as an inducer of human tumor necrosis factors and gamma interferon. J. Exp. Med.167:752-761.
Just, I., U. Geipel, A. Wegner, and K. Aktories. 1990. De-ADP-ribosylation actin by Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin. Eur. J. Biochem.192:723-727.
Just, I., E. S. Hennessey, D. R. Drummond, K. Aktories, and J. C. Sparrow. 1993. ADP-ribosylation of Drosophila indirect-flight-muscle actin and arthrin by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. Biochem. J.291:409-412.
Just, I., C. Mohr, G. Schallehn, L. Menard, J. R. Didsbury, J. Vandekerckhove, J. van Damme, and K. Aktories. 1992. Purification and characterization of an ADP-ribosyltransferase produced by Clostridium limosum. J. Biol. Chem.267:10274-10280.
Just, I., M. Wille, C. Chaponnier, and K. Aktories. 1993. Gelsolin-actin complex is target for ADP-ribosylation by Clostridium botulinum C2 toxin in intact human neutrophils. Eur. J. Pharmacol. Mol. Pharmacol.246:293-297.
Karginov, V. A., T. M. Robinson, J. Riemenschneider, B. Golding, M. Kennedy, J. Shiloach, and K. Alibek. 2004. Treatment of anthrax infection with combination of ciprofloxacin and antibodies to protective antigen of Bacillus anthracis. FEMS Immunol. Med. Microbiol.40:71-74.
Katz, L., J. T. Lamont, J. S. Trier, E. B. Sonnenblick, S. W. Rothman, S. A. Broitman, and S. Rieth. 1978. Experimental clindamycin associated colitis in rabbits. Evidence for toxin-mediated mucosal damage. Gastroenterology74:246-252.
Keim, P., and K. L. Smith. 2002. Bacillus anthracis evolution and epidemiology. Curr. Top. Microbiol. Immunol.271:21-32.
Keppie, J., P. W. Harris-Smith, and H. Smith. 1963. The chemical basis of the virulence of Bacillus anthracis. IX. Its aggressins and their mode of action. Br. J. Exp. Pathol.44:446-453.
Khanna, H., A. P. Chopra, N. Arora, A. Chaudhry, and Y. Singh. 2001. Role of residues constituting the 2β1 strand of domain II in the biological activity of anthrax protective antigen. FEMS Microbiol. Lett.199:27-31.
Khanna, H., P. K. Gupta, A. Singh, R. Chandra, and Y. Singh. 2001. Participation of residue F552 in domain III of the protective antigen in the biological activity of anthrax lethal toxin. Biol. Chem.382:941-946.
Kim, S. O., Q. Jing, K. Hoebe, B. Beutler, N. S. Duesbery, and J. Han. 2003. Sensitizing anthrax lethal toxin-resistant macrophages to lethal toxin-induced killing by tumor necrosis factor-alpha. J. Biol. Chem.278:7413-7421.
Kimura, K., T. Kubota, I. Ohishi, E. Isogai, H. Isogai, and N. Fujii. 1998. The gene for component-II of botulinum C2 toxin. Vet. Microbiol.62:27-34.
Kirby, J. E. 2004. Anthrax lethal toxin induces human endothelial cell apoptosis. Infect. Immun.72:430-439.
Kistner, A., and E. Habermann. 1992. Reductive cleavage of tetanus toxin and botulinum neurotoxin A by the thioredoxin system from brain. Evidence for two redox isomers of tetanus toxin. Naunyn Schmiedebergs Arch. Pharmacol.345:227-234.
Klimpel, K. R., N. Arora, and S. H. Leppla. 1994. Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity. Mol. Microbiol.13:1093-1100.
Klimpel, K. R., S. S. Molloy, G. Thomas, and S. H. Leppla. 1992. Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc. Natl. Acad. Sci. USA89:10277-10281.
Klussmann, E., G. Tamma, D. Lorenz, B. Wiesner, K. Maric, F. Hofmann, K. Aktories, G. Valenti, and W. Rosenthal. 2001. An inhibitory role of Rho in the vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells. J. Biol. Chem.276:20451-20457.
Knapp, O., R. Benz, M. Gibert, J. C. Marvaud, and M. R. Popoff. 2002. Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. J. Biol. Chem.277:6143-6152.
Kobiler, D., Y. Gozes, H. Rosenberg, D. Marcus, S. Reuveny, and Z. Altboum. 2002. Efficiency of protection of guinea pigs against infection with Bacillus anthracis spores by passive immunization. Infect. Immun.70:544-550.
Koehler T. M. 2002. Bacillus anthracis genetics and virulence gene regulation. Curr. Top. Microbiol. Immunol.271:143-164.
Koehler, T. M., and R. J. Collier. 1991. Anthrax toxin protective antigen: low-pH- induced hydrophobicity and channel formation in liposomes. Mol. Microbiol.5:1501-1506.
Koo, H.-M., M. VanBrocklin, M. J. McWilliams, S. H. Leppla, N. S. Duesbery, and G. F. Vande Woude. 2002. Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase. Proc. Natl. Acad. Sci. USA99:3052-3057.
Krakauer, T. 1995. Inhibition of toxic shock syndrome toxin-1-induced cytokine production and T cell activation by interleukin-10, interleukin-4, and dexamethasone. J. Infect. Dis.172:988-992.
Krakauer, T., and B. G. Stiles. 2003. Staphylococcal enterotoxins, toxic shock syndrome toxin-1, and streptococcal pyrogenic exotoxins: some basic biology of bacterial superantigens. Recent Res. Dev. Infect. Immun.1:1-27.
Kumar, P., N. Ahuja, and R. Bhatnagar. 2001. Purification of anthrax edema factor from Escherichia coli and identification of residues required for binding to anthrax protective antigen. Infect. Immun.69:6532-6536.
Kumar, P., N. Ahuja, and R. Bhatnagar. 2002. Anthrax edema toxin requires influx of calcium for inducing cyclic AMP toxicity in target cells. Infect. Immun.70:4997-5007.
Kurazono, H., M. Hosokawa, H. Matsuda, and G. Sakaguchi. 1987. Fluid accumulation in the ligated intestinal loop and histopathological changes of the intestinal mucosa caused by Clostridium botulinum C2 toxin in the pheasant and chicken. Res. Vet. Sci.42:349-353.
Kushner, N., D. Zhang, N. Touzjian, M. Essex, J. Lieberman, and Y. Lu. 2003. A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen. Proc. Natl. Acad. Sci. USA100:6652-6657.
Labruyere E., M. Mock, D. Ladant, S. Michelson, A. M. Gilles, B. Laoide, and O. Barzu. 1990. Characterization of ATP and calmodulin-binding properties of a truncated form of Bacillus anthracis adenylate cyclase. Biochemistry29:4922-4928.
Lacy, D. B., M. Mourez, A. Fouassier, and R. J. Collier. 2002. Mapping the anthrax protective antigen binding site on the lethal and edema factors. J. Biol. Chem.277:3006-3010.
Lafont, F., G. Tran Van Nhieu, K. Hanada, P. Sansonetti, and F. G. van der Goot. 2002. Initial steps of Shigella infection depend on the cholesterol/sphingolipid raft-mediated CD44-IpaB interaction. EMBO J.21:4449-4457.
Lamont, J. T., E. B. Sonnenblick, and S. Rothman. 1979. Role of clostridial toxin in the pathogenesis of clindamycin colitis in rabbits. Gastroenterology76:356-361.
Lee, J. S., A. G. Hadjipanayis, and S. L. Welkos. 2003. Venezuelan equine encephalitis virus-vectored vaccines protect mice against anthrax spore challenge. Infect. Immun.71:1491-1496.
Lencer, W. I., C. Constable, S. Moe, P. A. Rufo, A. Wolf, M. G. Jobling, S. P. Ruston, J. L. Madara, R. K. Holmes, and T. R. Hirst. 1997. Proteolytic activation of cholera toxin and Escherichia coli labile toxin by entry into host epithelial cells: signal transduction by a protease-resistant toxin variant. J. Biol. Chem.272:15562-15568.
Leppla, S. H. 1982. Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations in eukaryotic cells. Proc. Natl. Acad. Sci. USA79:3162-3166.
Leppla, S. H. 1999. The bifactorial Bacillus anthracis lethal and oedema toxins, p. 243-263. In J. E. Alouf and J. H. Freer (ed.), The comprehensive sourcebook of bacterial protein toxins. Academic Press, Inc., New York, N.Y.
Leppla, S. H., N. Arora, and M. Varughese. 1999. Anthrax toxin fusion proteins for intracellular delivery of macromolecules. J. Appl. Microbiol.87:284.
Leppla, S. H., J. B. Robbins, R. Schneerson, and J. Shiloach. 2002. Development of an improved vaccine for anthrax. J. Clin. Investig.110:141-144.
Levens, D. L. 2003. Reconstructing MYC. Genes Dev.17:1071-1077.
Li, G., E. Rungger-Brändle, I. Just, J.-C. Jonas, K. Aktories, and C. B. Wollheim. 1994. Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets. Mol. Biol. Cell5:1199-1213.
Li, M., F. Dyda, I. Benhar, I. Pastan, and D. R. Davies. 1996. Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation. Proc. Natl. Acad. Sci. USA93:6902-6906.
Linseman, D. A., T. Laessig, M. K. Meintzer, M. McClure, H. Barth, K. Aktories, and K. A. Heidenreich. 2001. An essential role for Rac/Cdc42 GTPases in cerebellar granule neuron survival. J. Biol. Chem.276:39123-39131.
Little, S. F., and B. E. Ivins. 1999. Molecular pathogenesis of Bacillus anthracis infection. Microbes Infect.1:131-139.
Little, S. F., B. E. Ivins, P. F. Fellows, and A. M. Friedlander. 1997. Passive protection by polyclonal antibodies against Bacillus anthracis infection in guinea pigs. Infect. Immun.65:5171-5175.
Little, S. F., S. H. Leppla, J. W. Burnett, and A. M. Friedlander. 1994. Structure-function analysis of Bacillus anthracis edema factor by using monoclonal antibodies. Biochem. Biophys. Res. Commun.199:676-682.
Little, S. F., S. H. Leppla, and E. Cora. 1988. Production and characterization of monoclonal antibodies to the protective antigen component of Bacillus anthracis toxin. Infect. Immun.56:1807-1813.
Little, S. F., S. H. Leppla, and A. M. Friedlander. 1990. Production and characterization of monoclonal antibodies against the lethal factor component of Bacillus anthracis lethal toxin. Infect. Immun.58:1606-1613.
Little, S. F, and J. R. Lowe. 1991. Location of receptor-binding region of protective antigen from Bacillus anthracis. Biochem. Biophys. Res. Commun.180:531-537.
Little, S. F., J. M. Novak, J. R. Lowe, S. H. Leppla, Y. Singh, K. R. Klimpel, B. C. Lidgerding, and A. M. Friedlander. 1996. Characterization of lethal factor binding and cell receptor binding domains of protective antigen of Bacillus anthracis using monoclonal antibodies. Microbiology142:707-715.
Liu, S., H. Aaronson, D. J. Mitola, S. H. Leppla, and T. H. Bugge. 2003. Potent antitumor activity of a urokinase-activated engineered anthrax toxin. Proc. Natl. Acad. Sci. USA100:657-662.
Liu, S., and S. H. Leppla. 2003. Cell surface tumor endothelium marker 8 cytoplasmic tail-independent anthrax toxin binding, proteolytic processing, oligomer formation, and internalization. J. Biol. Chem.278:5227-5234.
Liu, S., R. L. Schubert, T. H. Bugge, and S. H. Leppla. 2003. Anthrax toxin: structures, functions and tumour targeting. Exp. Opin. Biol. Ther.3:843-853.
Locht, C., and R. Antoine. 1999. Bordetella pertussis protein toxins, p. 130-146. In J. E. Alouf and J. H. Freer (ed.), The comprehensive sourcebook of bacterial protein toxins. Academic Press, Inc., New York, N.Y.
Lord, J. M., D. C. Smith, and L. M. Roberts. 1999. Toxin entry: how bacterial proteins get into mammalian cells. Cell. Microbiol.1:85-91.
Madden, J. C., N. Ruiz, and M. Caparon. 2001. Cytolysin-mediated translocation (CMT): a functional equivalent of type III secretion in gram-positive bacteria. Cell104:143-152.
Madshus, I. H., H. Stenmark, K. Sandvig, and S. Olsnes. 1991. Entry of diphtheria toxin-protein A chimeras into cells. J. Biol. Chem.266:17446-17453.
Mahlandt, B. G., F. Klein, R. E. Lincoln, B. W. Haines, W. I. Jones, Jr., and R. H. Friedman. 1966. Evaluation of the immunogenicity of three components of anthrax toxin. J. Immunol.96:727-733.
Majoul, I., D. Ferrari, and H. D. Soling. 1997. Reduction of protein disulfide bonds in an oxidizing environment—the disulfide bridge of cholera toxin A-subunit is reduced in the endoplasmic reticulum. FEBS Lett.401:104-108.
Margulis, L., J. Z. Jorgensen, S. Dolan, R. Kolchinsky, F. A. Rainey, and S.-C. Lo. 1998. The Arthromitus stage of Bacillus cereus: intestinal symbionts of animals. Proc. Natl. Acad. Sci. USA95:1236-1241.
Martinez, R. D., and T. D. Wilkins. 1992. Comparison of Clostridium sordellii toxin HT and LT with toxins A and B of Clostridium difficile. J. Med. Microbiol.36:30-36.
Marvaud, J.-C., T. Smith, M. L. Hale, M. R. Popoff, L. A. Smith, and B. G. Stiles. 2001. Clostridium perfringens iota-toxin: mapping of receptor binding and Ia docking domains on Ib. Infect. Immun.69:2435-2441.
Marvaud, J.-C., B. G. Stiles, A. Chenal, D. Gillet, M. Gibert, L. A. Smith, and M. R. Popoff. 2002. Clostridium perfringens iota toxin. Mapping of the Ia domain involved in docking with Ib and cellular internalization. J. Biol. Chem.277:43659-43666.
Matter, K., F. Dreyer, and K. Aktories. 1989. Actin involvement in exocytosis from PC12 cells: studies on the influence of botulinum C2 toxin on stimulated noradrenaline release. J. Neurochem.52:370-376.
Matyas, G. R., A. M. Friedlander, G. M. Glenn, S. Little, J. Yu, and C. R. Alving. 2004. Needle-free skin patch vaccination method for anthrax. Infect. Immun.72:1181-1183.
Mauss, S., C. Chaponnier, I. Just, K. Aktories, and G. Gabbiani. 1990. ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. Eur. J. Biochem.194:237-241.
Mauss, S., G. Koch, V. A. Kreye, and K. Aktories. 1989. Inhibition of the contraction of the isolated longitudinal muscle of the guinea-pig ileum by botulinum C2 toxin: evidence for a role of G/F-actin transition in smooth muscle contraction. Naunyn Schmiedeberg's Arch. Pharmacol.340:345-351.
Maynard, J. A., C. B. Maassen, S. H. Leppla, K. Brasky, J. L. Patterson, B. L. Iverson, and G. Georgiou. 2002. Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity. Nat. Biotechnol.20:597-601.
McDonel, J. L. 1986. Toxins of Clostridium perfringens types A, B, C, D and E, p. 477-517. In F. Dorner and J. Drews (ed.), Pharmacology of bacterial toxins. Pergamon Press, Inc., New York, N.Y.
McKillip, J. L. 2000. Prevalence and expression of enterotoxins in Bacillus cereus and other Bacillus spp., a literature review. Antonie Leeuwenhoek77:393-399.
Meer, R. R., and J. G. Songer. 1997. Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens. Am. J. Vet. Res.58:702-705.
Mehra, V., H. Khanna, R. Chandra, and Y. Singh. 2001. Anthrax-toxin-mediated delivery of a 19 kDa antigen of Mycobacterium tuberculosis into the cytosol of mammalian cells. Biotechnol. Appl. Biochem.33:71-74.
Melamed, I., G. P. Downey, K. Aktories, and C. M. Roifman. 1991. Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes. J. Immunol. Methods147:1139-1146.
Melamed, I., R. A. Franklin, and E. W. Gelfand. 1995. Microfilament assembly is required for anti-IgM dependent MAPK and p90rsk activation in human B lymphocytes. Biochem. Biophys. Res. Commun.209:1102-1110.
Melamed, I., and E. W. Gelfand. 1999. Microfilament assembly is involved in B-cell apoptosis. Cell Immunol.194:136-142.
Melamed, I., L. Stein, and C. M. Roifman. 1994. Epstein-Barr virus induces actin polymerization in human B cells. J. Immunol.153:1998-2003.
Melamed, I., C. E. Turner, K. Aktories, D. R. Kaplan, and E. W. Gelfand. 1995. Nerve growth factor triggers microfilament assembly and paxillin phosphorylation in human B lymphocytes. J. Exp. Med.181:1071-1079.
Menard, A., K. D. Altendorf, D. D. Breves, M. Mock, and C. Montecucco. 1996. The vacuolar ATPase proton pump is required for the cytotoxicity of Bacillus anthracis lethal toxin. FEBS Lett.386:161-164.
Menard, A., J. E. Papini, M. Mock, and C. Montecucco. 1996. The cytotoxic activity of Bacillus anthracis lethal factor is inhibited by leukotriene A4 hydrolase and metallopeptidase inhibitors. Biochem. J.320:687-691.
Miethke, T., C. Wahl, K. Heeg, B. Echtenacher, P. H. Krammer, and H. Wagner. 1992. T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor. J. Exp. Med.175:91-98.
Mignot, T., M. Mock, and A. Fouet. 2003. A plasmid-encoded regulator couples the synthesis of toxins and surface structures in Bacillus anthracis. Mol. Microbiol.47:917-927.
Mikesell, P., B. E. Ivins, J. D. Ristroph, and T. M. Dreier. 1983. Evidence for plasmid-mediated toxin production in Bacillus anthracis. Infect. Immun.39:371-376.
Miller, C. J., J. L. Elliott, and R. J. Collier. 1999. Anthrax protective antigen: prepore-to-pore conversion. Biochemistry38:10432-10441.
Milne, J. C., S. R. Blanke, P. C. Hanna, and R. J. Collier. 1995. Protective antigen- binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino- or carboxy-terminus. Mol. Microbiol.15:661-666.
Milne, J. C., and R. J. Collier. 1993. pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen. Mol. Microbiol.10:647-653.
Milne, J. C., D. Furlong, P. C. Hanna, J. S. Wall, and R. J. Collier. 1994. Anthrax protective antigen forms oligomers during intoxication of mammalian cells. J. Biol. Chem.269:20607-20612.
Miyake, M., and I. Ohishi. 1987. Response of tissue-cultured cynomolgus monkey kidney cells to botulinum C2 toxin. Microb. Pathog.3:279-286.
Miyata, S., J. Minami, E. Tamai, O. Matsushita, S. Shimamota, and A. Okabe. 2002. Clostridium perfringens epsilon-toxin forms a heptameric pore within the detergent-insoluble microdomains of Madin-Darby canine kidney cells and rat synaptosomes. J. Biol. Chem.277:39463-39468.
Moayeri, M., D. Haines, H. A. Young, and S. H. Leppla. 2003. Bacillus anthracis lethal toxin induces TNF-α-independent hypoxia-mediated toxicity in mice. J. Clin. Investig.112:670-682.
Mock, M., and T. Mignot. 2003. Anthrax toxins and the host: a story of intimacy. Cell. Microbiol.5:15-23.
Mock, M., and B. P. Roques. 2002. Progress in rapid screening of Bacillus anthracis lethal factor activity. Proc. Natl. Acad. Sci. USA99:6527-6529.
Mogridge, J., K. Cunningham, and R. J. Collier. 2002. Stoichiometry of anthrax toxin complexes. Biochemistry41:1079-1082.
Mogridge, J., K. Cunningham, D. B. Lacy, M. Mourez, and R. J. Collier. 2002. The lethal and edema factors of anthrax toxin bind only to oligomeric forms of the protective antigen. Proc. Natl. Acad. Sci. USA99:7045-7048.
Mogridge, J., M. Mourez, and R. J. Collier. 2001. Involvement of domain 3 in oligomerization by the protective antigen moiety of anthrax toxin. J. Bacteriol.183:2111-2116.
Moriya, O., M. Matsui, M. Osorio, H. Miyazawa, C. M. Rice, S. M. Feinstone, S. H. Leppla, J. M. Keith, and T. Akatsuka. 2002. Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells treated with an anthrax toxin fusion protein. Vaccine20:789-796.
Mourez, M., R. S. Kane, J. Mogridge, S. Metallo, P. Deschatelets, B. R. Sellman, G. M. Whitesides, and R. J. Collier. 2001. Designing a polyvalent inhibitor of anthrax toxin. Nat. Biotechnol.19:958-961.
Mourez, M., M. Yan, D. B. Lacy, L. Dillon, L. Bentsen, A. Marpoe, C. Maurin, E. Hotze, D. Wigelsworth, R.-A. Pimental, J. D. Ballard, R. J. Collier, and R. K. Tweten. 2003. Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis. Proc. Natl. Acad. Sci. USA100:13803-13808.
Munier, H., A. Bouhss, A. M. Gilles, E. Krin, P. Glaser, A. Danchin, and O. Barzu. 1993. Structural flexibility of the calmodulin-binding locus in Bordetella pertussis adenylate cyclase. Reconstitution of catalytically active species from fragments or inactive forms of the enzyme. Eur. J. Biochem.217:581-586.
Nagahama, M., A. Kihara, T. Miyawaki, M. Mukai, Y. Sakaguchi, S. Ochi, and J. Sakurai. 2000. Characterization of the enzymatic component of Clostridium perfringens iota-toxin. J. Bacteriol.182:2096-2103.
Nagahama, M., K. Nagayasu, K. Kobayashi, and J. Sakurai. 2002. Binding component of Clostridium perfringens iota-toxin induces endocytosis in Vero cells. Infect. Immun.70:1909-1914.
Nakamura, S., T. Serikawa, K. Yamakawa, S. Nishida, S. Kozaki, and G. Sakaguchi. 1978. Sporulation and C2 toxin production by Clostridium botulinum type C strains producing no C1 toxin. Microbiol. Immunol.22:591-596.
Nassi, S., R. J. Collier, and A. Finkelstein. 2002. PA63 channel of anthrax toxin: an extended β-barrel. Biochemistry41:1445-1450.
Nilius, B., T. Voets, J. Prenen, H. Barth, K. Aktories, K. Kaibuchi, G. Droogmans, and J. Eggermont. 1999. Role of Rho and Rho kinase in the activation of volume- regulated anion channels in bovine endothelial cells. J. Physiol.516:67-74.
Norgauer, J., I. Just, K. Aktories, and L. A. Sklar. 1989. Influence of botulinum C2 toxin on F-actin and N-formyl peptide receptor dynamics in human neutrophils. J. Cell Biol.109:1133-1140.
Norgauer, J., E. Kownatzki, R. Seifert, and K. Aktories. 1988. Botulinum C2 toxin ADP-ribosylates actin and enhances O2 production and secretion but inhibits migration of activated human neutrophils. J. Clin. Investig.82:1376-1382.
Novak, J. M., M. P. Stein, S. F. Little, S. H. Leppla, and A. M. Friedlander. 1992. Functional characterization of protease-treated Bacillus anthracis protective antigen. J. Biol. Chem.267:17186-17193.
Oakley, C., and G. Warrack. 1953. Routine typing of Clostridium welchii. J. Hyg. (Cambridge)51:102-107.
O'Brien, J., A. Friedlander, T. Dreier, J. Ezzell, and S. Leppla. 1985. Effects of anthrax toxin components on human neutrophils. Infect. Immun.47:306-310.
Ohishi, I. 1983. Lethal and vascular permeability activities of botulinum C2 toxin induced by separate injections of the two toxin components. Infect. Immun.40:336-339.
Ohishi, I. 1983. Response of mouse intestinal loop to botulinum C2 toxin: enterotoxic activity induced by cooperation of nonlinked protein components. Infect. Immun.40:691-695.
Ohishi, I. 1987. Activation of botulinum C2 toxin by trypsin. Infect. Immun.55:1461-1465.
Ohishi, I. 2000. Structure and function of actin-adenosine-diphosphate-ribosylating toxins, p. 253-273. In K. Aktories and I. Just. (ed.), Bacterial protein toxins. Springer Verlag KG, Berlin, Germany.
Ohishi, I., and Y. Hama. 1992. Purification and characterization of heterologous component IIs of botulinum C2 toxin. Microbiol. Immunol.36:221-229.
Ohishi, I., M. Iwasaki, and G. Sakaguchi. 1980. Purification and characterization of two components of botulinum C2 toxin. Infect. Immun.30:668-673.
Ohishi, I., M. Iwasaki, and G. Sakaguchi. 1980. Vascular permeability activity of botulinum C2 toxin elicited by cooperation of two dissimilar protein components. Infect. Immun.31:890-895.
Ohishi, I., and M. Miyake. 1985. Binding of the two components of C2 toxin to epithelial cells and brush borders of mouse intestine. Infect. Immun.48:769-775.
Ohishi, I., M. Miyake, H. Ogura, and S. Nakamura. 1984. Cytopathic effect of botulinum C2 toxin on tissue-culture cells. FEMS Microbiol. Lett.23:281-284.
Ohishi, I., and Y. Odagiri. 1984. Histopathological effect of botulinum C2 toxin on mouse intestines. Infect. Immun.43:54-58.
Ohishi, I., and Y. Okada. 1986. Heterogeneities of two components of C2 toxin produced by Clostridium botulinum types C and D. J. Gen. Microbiol.132:125-131.
Ohishi, I., and G. Sakaguchi. 1980. Oral toxicities of Clostridium botulinum type C and D toxins of different molecular sizes. Infect. Immun.28:303-309.
Ohishi, I., and G. Sakaguchi. 1982. Production of C2 toxin by Clostridium botulinum types C and D as determined by its vascular permeability activity. Infect. Immun.35:1-4.
Ohishi, I., and S. Tsuyama. 1986. ADP-ribosylation of nonmuscle actin with component I of C2 toxin. Biochem. Biophys. Res. Commun.136:802-806.
Ohishi, I., and A. Yanagimoto. 1992. Visualizations of binding and internalization of two nonlinked protein components of botulinum C2 toxin in tissue culture cells. Infect. Immun.60:4648-4655.
Okinaka, R. T., K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson. 1999. Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J. Bacteriol.181:6509-6515.
Orlandi, P. A., P. K. Curran, and P. H. Fishman. 1993. Brefeldin A blocks the response of cultured cells to cholera toxin. Implications for intracellular trafficking in toxin action. J. Biol. Chem.268:12010-12016.
Panchal, R. G., A. R. Hermone, T. L. Nguyen, T. Y. Wong, R. Schwarzenbacher, J. Schmidt, D. Lane, C. McGrath, B. E. Turk, J. Burnett, M. J. Aman, S. Little, E. A. Sausville, D. W. Zaharevitz, L. C. Cantley, R. C. Liddington, R. Gusssio, and S. Bavari. 2004. Identification of small molecule inhibitors of anthrax lethal factor. Nat. Struct. Mol. Biol.11:67-72.
Pannifer, A. D., T. Y. Wong, R. Schwarzenbacher, M. Renatus, C. Petosa, J. Bienkowska, D. B. Lacy, R. J. Collier, S. Park, S. H. Leppla, P. Hanna, and R. C. Liddington. 2001. Crystal structure of the anthrax lethal factor. Nature414:229-233.
Pannucci, J., R. T. Okinaka, R. Sabin, and C. R. Kuske. 2002. Bacillus anthracis pXO1 plasmid sequence conservation among closely related bacterial species. J. Bacteriol.184:134-141.
Park, J. M., F. R. Greten, A.-W. Li, and M. Karin. 2002. Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science297:2048-2051.
Pastan, I., V. Chaudhary, and D. J. FitzGerald. 1992. Recombinant toxins as novel therapeutic agents. Annu. Rev. Biochem.61:331-354.
Pasteur, L. 1881. De l'attenuation des virus et de leur retour a la virulence. C. R. Acad. Sci. Agric. Bulg.92:429-435.
Patton, N. M., H. T. Holmes, R. J. Riggs, and P. R. Cheeke. 1978. Enterotoxemia in rabbits. Lab. Anim. Sci.28:536-540.
Peeters, J. E., R. Geeroms, R. J. Carman, and T. D. Wilkins. 1986. Significance of Clostridium spiroforme in the enteritis-complex of commercial rabbits. Vet. Microbiol.12:25-31.
Pellizzari, R., C. Guidi-Rontani, G. Vitale, M. Mock, and C. Montecucco. 1999. Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNgamma-induced release of NO and TNFalpha. FEBS Lett.462:199-204.
Perelle, S., M. Domenighini, and M. R. Popoff. 1996. Evidence that Arg-295, Glu-378, and Glu-380 are active-site residues of the ADP-ribosyltransferase activity of iota toxin. FEBS Lett.395:191-194.
Perelle, S., M. Gibert, P. Boquet, and M. R. Popoff. 1993. Characterization of Clostridium perfringens iota-toxin genes and expression in Escherichia coli. Infect. Immun.61:5147-5156.
Perelle, S., M. Gibert, P. Bourlioux, G. Corthier, and M. R. Popoff. 1997. Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196. Infect. Immun.65:1402-1407.
Perelle, S., S. Scalzo, S. Kochi, M. Mock, and M. R. Popoff. 1997. Immunological and functional comparison between Clostridium perfringens iota toxin, C. spiroforme toxin, and anthrax toxins. FEMS Microbiol. Lett.146:117-121.
Petosa, C., R. J. Collier, K. R. Klimpel, S. H. Leppla, and R. C. Liddington. 1997. Crystal structure of the anthrax toxin protective antigen. Nature385:833-838.
Pezard, C., P. Berche, and M. Mock. 1991. Contribution of individual toxin components to virulence of Bacillus anthracis. Infect. Immun.59:3472-3477.
Pezard, C., M. Weber, J. C. Sirard, P. Berche, and M. Mock. 1995. Protective immunity induced by Bacillus anthracis toxin-deficient strains. Infect. Immun.63:1369-1372.
Pitt, M. L., S. F. Little, B. E. Ivins, P. Fellows, J. Barth, J. Hewetson, P. Gibbs, M. Dertzbaugh, and A. M. Friedlander. 2001. In vitro correlate of immunity in a rabbit model of inhalational anthrax. Vaccine19:4768-4773.
Pittman, P. R., G. Kim-Ahn, D. Y. Pifat, K. Coonan, P. Gibbs, S. Little, J. G. Pace- Templeton, R. Myers, G. W. Parker, and A. M. Friedlander. 2002. Anthrax vaccine: immunogenicity and safety of a dose-reduction, route-change comparison study in humans. Vaccine20:1412-1420.
Pizza, M., V. Masignani, and R. Rappuoli. 1999. Molecular, functional and evolutionary aspects of ADP-ribosylating toxins, p. 45-72. In J. E. Alouf and J. H. Freer (ed.), The comprehensive sourcebook of bacterial protein toxins. Academic Press, Inc., New York, N.Y.
Popoff, M. R. 2000. Molecular biology of actin-ADP-ribosylating toxins, p. 275-306. In K. Aktories and I. Just (ed.), Handbook of experimental pharmacology, vol. 145. Bacterial protein toxins. Springer-Verlag KG, Berlin, Germany.
Popoff, M. R., and P. Boquet. 1988. Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin. Biochem. Biophys. Res. Commun.152:1361-1368.
Popoff, M. R., and J. C. Marvaud. 1999. Structural and genomic features of clostridial neurotoxins, p. 174-201. In J. E. Alouf and J. H. Freer (ed.), The comprehensive sourcebook of bacterial protein toxins. Academic Press, Inc., New York, N.Y.
Popoff, M. R., F. W. Milward, B. Bancillon, and P. Boquet. 1989. Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells. Infect. Immun.57:2462-2469.
Popoff, M. R., E. J. Rubin, D. M. Gill, and P. Boquet. 1988. Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain. Infect. Immun.56:2299-2306.
Popov, S. G., R. Villasmil, J. Bernardi, E. Grene, J. Cardwell, T. Popova, A. Wu, D. Alibek, C. Bailey, and K. Alibek. 2002. Effect of Bacillus anthracis lethal toxin on human peripheral blood mononuclear cells. FEBS Lett.527:211-215.
Popov, S. G., R. Villasmil, J. Bernardi, E. Grene, J. Cardwell, A. Wu, D. Alibek, C. Bailey, and K. Alibek. 2002. Lethal toxin of Bacillus anthracis causes apoptosis of macrophages. Biochem. Biophys. Res. Commun.293:349-355.
Pratt, W. B., and D. O. Toft. 2003. Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp. Biol. Med.228:111-133.
Prekeris, R., M. W. Mayhew, J. B. Cooper, and D. M. Terrian. 1996. Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the regulation of synaptic function. J. Cell Biol.132:77-90.
Prepens, U., H. Barth, J. Wilting, and K. Aktories. 1998. Influence of Clostridium botulinum C2 toxin on FcεRI-mediated secretion and tyrosine phosphorylation in RBL cells. Naunyn-Schmiedeberg's Arch. Pharmacol.357:323-330.
Prepens, U., I. Just, F. Hofmann, and K. Aktories. 1997. ADP-ribosylating and glucosylating toxins as tools to study secretion in RBL cells. Adv. Exp. Med. Biol.419:349-353.
Prepens, U., I. Just, C. von Eichel-Streiber, and K. Aktories. 1996. Inhibition of FcεRI-mediated activation of rat basophilic leukemia cells by Clostridium difficile toxin B (monoglucosyltransferase). J. Biol. Chem.271:7324-7329.
Prevost, G. 1999. The bi-component staphylococcal leucocidins and γ-haemolysins (toxins), p. 402-418. In J. E. Alouf and J. H. Freer (ed.), The comprehensive sourcebook of bacterial protein toxins. Academic Press, Inc., New York, N.Y.
Price, L. B., M. Hugh-Jones, P. J. Jackson, and P. Keim. 1999. Genetic diversity in the protective antigen gene of Bacillus anthracis. J. Bacteriol.181:2358-2362.
Price, B. M., A. L. Liner, S. Park, S. H. Leppla, A. Mateczun, and D. R. Galloway. 2001. Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein. Infect. Immun.69:4509-4515.
Prince, A. S. 2003. The host response to anthrax lethal toxin: unexpected observations. J. Clin. Investig.112:656-658.
Quinn, C. P., Y. Singh, K. R. Klimpel, and S. H. Leppla. 1991. Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis. J. Biol. Chem.266:20124-20130.
Quintiliani, R., Jr., and R. Quintiliani. 2003. Inhalational anthrax and bioterrorism. Curr. Opin. Pulm. Med.9:221-226.
Radke, J., K. J. Pederson, and J. T. Barbieri. 1999. Pseudomonas aeruginosa exoenzyme S is a biglutamic acid ADP ribosyltransferase. Infect. Immun.67:1508-1510.
Rasko, D. A., J. Ravel, O. A. Okstad, E. Helgason, R. Z. Cer, L. Jiang, K. A. Shores, D. E. Fouts, N. J. Tourasse, S. V. Angiuoli, J. Kolonay, W. C. Nelson, A. B. Kolsto, C. M. Fraser, and T. D. Read. 2004. The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1. Nucleic Acids Res.32:977-988.
Ratts, R., H. Zeng, E. A. Berg, C. Blue, M. E. McComb, C. E. Costello, J. C. vanderSpeck, and J. R. Murphy. 2003. The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex. J. Cell Biol.160:1139-1150.
Read, T. D., S. N. Peterson, N. Tourasse, L. W. Baillie, I. T. Paulsen, K. E. Nelson, H. Tettelin, D. E. Fouts, J. A. Eisen, S. R. Gill, et. al. 2003. The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature423:81-86.
Reuner, K. H., P. Presek, C. B. Boschek, and K. Aktories. 1987. Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells. Eur. J. Cell Biol.43:134-140.
Reuner, K. H., K. Schlegel, I. Just, K. Aktories, and N. Katz. 1991. Autoregulatory control of actin synthesis in cultured rat hepatocytes. FEBS Lett.286:100-104.
Reuner, K. H., A. van der Does, P. Dunker, I. Just, K. Aktories, and N. Katz. 1996. Microinjection of ADP-ribosylated actin inhibits actin synthesis in hepatocyte-hepatoma hybrid cells. Biochem. J.319:843-849.
Reuner, K. H., M. Wiederhold, P. Dunker, I. Just, R. M. Bohle, M. Kroger, and N. Katz. 1995. Autoregulation of actin synthesis in hepatocytes by transcriptional and posttranscriptional mechanisms. Eur. J. Biochem.230:32-37.
Reuner, K. H., M. Wiederhold, K. Schlegel, I. Just, and N. Katz. 1995. Autoregulation of actin synthesis by physiological alterations of the G-actin level in hepatocytes. Eur. J. Clin. Chem. Clin. Biochem.33:569-574.
Reuveny, S., M. D. White, Y. Y. Adar, Y. Kafri, Z. Altboum, Y. Gozes, D. Kobiler, A. Shafferman, and B. Velan. 2001. Search for correlates of protective immunity conferred by anthrax vaccine. Infect. Immun.69:2888-2893.
Rhie, G.-E., M. H. Roehrl, M. Mourez, R. J. Collier, J. J. Mekalanos, and J. Y. Wang. 2003. A dually active anthrax vaccine that confers protection against both bacilli and toxins. Proc. Natl. Acad. Sci. USA100:10925-10930.
Richard, J. F., G. Mainguy, M. Gibert, J. C. Marvaud, B. G. Stiles, and M. R. Popoff. 2002. Transcytosis of iota-toxin across polarized CaCo-2 cells. Mol. Microbiol.43:907-917.
Richardson, J. S. 1977. β-sheet topology and the relatedness of proteins. Nature268:495-500.
Riese, M. J., and J. T. Barbieri. 2002. Membrane localization contributes to the in vivo ADP-ribosylation of Ras by Pseudomonas aeruginosa ExoS. Infect. Immun.70:2230-2232.
Rivera, V. R., G. A. Merrill, J. A. White, and M. A. Poli. 2003. An enzymatic electrochemiluminescence assay for the lethal factor of anthrax. Anal. Biochem.321:125-130.
Robertson, D. L., M. T. Tippetts, and S. H. Leppla. 1988. Nucleotide sequence of the Bacillus anthracis edema factor gene (cya): a calmodulin-dependent adenylate cyclase. Gene73:363-371.
Rosovitz, M. J., P. Schuck, M. Varughese, A. P. Chopra, V. Mehra, Y. Singh, L. M. McGinnis, and S. H. Leppla. 2003. Alanine scanning mutations in domain 4 of anthrax toxin protective antigen reveal residues important for binding to the cellular receptor and to a neutralizing monoclonal antibody. J. Biol. Chem.278:30936-30944.
Ross, H. E., M. E. Warren, and J. M. Barnes. 1949. Clostridium welchii iota toxin: its activation by trypsin. J. Gen. Microbiol.3:148-152.
Rubin, E. J., D. M. Gill, P. Boquet, and M. R. Popoff. 1988. Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum. Mol. Cell Biol.8:418-426.
Ryu, S., and R. G. Labbe. 1989. Coat and enterotoxin-related proteins in Clostridium perfringens spores. J. Gen. Microbiol.135:3109-3118.
Sakai, T., S. Yamashina, and S. Ohnishi. 1991. Microtubule-disrupting drugs blocked delivery of endocytosed transferrin to the cytocenter, but did not affect return of transferrin to plasma membrane. J. Biochem. (Tokyo)109:528-533.
Sakr, S., R. J. Eddy, H. Barth, F. Wang, S. Greenberg, F. R. Maxfield, and I. Tabas. 2001. The uptake and degradation of matrix-bound lipoproteins by macrophages require an intact actin cytoskeleton, Rho family GTPases, and myosin ATPase activity. J. Biol. Chem.276:37649-37658.
Sakurai, J., and K. Kobayashi. 1995. Lethal and dermonecrotic activities of Clostridium perfringens iota toxin: biological activities induced by cooperation of two nonlinked components. Microbiol. Immunol.39:249-253.
Sakurai, J., M. Nagahama, J. Hisatsune, N. Katunuma, and H. Tsuge. 2003. Clostridium perfringens iota-toxin, ADP-ribosyltransferase: structure and mechanism of action. Adv. Enzyme Regul.43:361-377.
Salles, I. I., A. E. Tucker, D. E. Voth, and J. D. Ballard. 2003. Toxin-induced resistance in Bacillus anthracis lethal toxin-treated macrophages. Proc. Natl. Acad. Sci. USA100:12426-12431.
Sandvig, K., and B. Van Deurs. 1996. Endocytosis, intracellular transport, and cytotoxic action of shiga toxin and ricin. Physiol. Rev.76:949-966.
Sarker, M. R., U. Singh, and B. A. McClane. 2000. An update on Clostridium perfringens enterotoxin. J. Nat. Toxins9:251-266.
Schering, B., M. Barmann, G. S. Chhatwal, U. Geipel, and K. Aktories. 1988. ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin. Eur. J. Biochem.171:225-229.
Schmid, A., R. Benz, I. Just, and K. Aktories. 1994. Interaction of Clostridium botulinum C2 toxin with lipid bilayer membranes: formation of cation-selective channels and inhibition of channel function by chloroquine and peptides. J. Biol. Chem.269:16706-16711.
Schmid, G., A. Schürmann, C. Huppertz, F. Hofmann, K. Aktories, and H.-G. Joost. 1998. Inhibition of insulin-stimulated glucose transport in 3T3-L1 cells by Clostridium difficile toxin B, Clostridium sordellii lethal toxin, and Clostridium botulinum C2 toxin. Naunyn-Schmiedeberg's Arch. Pharmacol.357:385-392.
Schmidt, J. J., and R. G. Stafford. 2003. Fluorigenic substrates for the protease activities of botulinum neurotoxins, serotypes A, B, and F. Appl. Environ. Microbiol.69:297-303.
Schneerson, R., J. Kubler-Kielb, T. Y. Liu, Z. D. Dai, S. H. Leppla, A. Yergey, P. Backlund, J. Shiloach, F. Majadly, and J. B. Robbins. 2003. Poly(γ-d-glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of Bacillus anthracis: a potential addition to the anthrax vaccine. Proc. Natl. Acad. Sci. USA100:8945-8950.
Schnittler, H.-J., S. W. Schneider, H. Raifer, F. Luo, P. Dieterich, I. Just, and K. Aktories. 2001. Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress. Pflügers Arch.-Eur. J. Physiol.442:675-687.
Schuch, R., D. Nelson, and V. A. Fischetti. 2002. A bacteriolytic agent that detects and kills Bacillus anthracis. Nature418:884-888.
Scobie, H. M., G. J. Rainey, K. A. Bradley, and J. A. Young. 2003. Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc. Natl. Acad. Sci. USA100:5170-5174.
Sekine, A., M. Fujiwara, and S. Narumiya. 1989. Asparagine residue in the ρ gene product is the modification site for botulinum ADP-ribosyltransferase. J. Biol. Chem.264:8602-8605.
Sellman, B. R., M. Mourez, and R. J. Collier. 2001. Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax. Science292:695-697.
Sellman, B. R., S. Nassi, and R. J. Collier. 2001. Point mutations in anthrax protective antigen that block translocation. J. Biol. Chem.276:8371-8376.
Sharma, M., H. Khanna, N. Arora, and Y. Singh. 2000. Anthrax toxin-mediated delivery of cholera toxin-A subunit into the cytosol of mammalian cells. Biotechnol. Appl. Biochem.32:69-72.
Shen, Y., Y. S. Lee, S. Soelaiman, P. Bergson, D. Lu, A. Chen, K. Beckingham, Z. Grabarek, M. Mrksich, and W. J. Tang. 2002. Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins. EMBO J.21:6721-6732.
Shen, Y., N. L. Zhukovskaya, M. I. Zimmer, S. Soelaiman, P. Bergson, C. R. Wang, C. S. Gibbs, and W. J. Tang. 2004. Selective inhibition of anthrax edema factor by adefovir, a drug for chronic hepatitis B virus infection. Proc. Natl. Acad. Sci. USA101:3242-3247.
Shimizu, T., K. Ohtani, H. Hirakawa, K. Ohshima, A. Yamashita, T. Shiba, N. Ogasawara, M. Hattori, S. Kuhara, and H. Hayashi. 2002. Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc. Natl. Acad. Sci. USA99:996-1001.
Shin, S., G. H. Hur, Y. B. Kim, K. J. Park, Y. M. Park, and W. S. Lee. 2000. Intracellular calcium antagonist protects cultured peritoneal macrophages against anthrax lethal toxin-induced cytotoxicity. Cell Biol. Toxicol.16:137-144.
Sibelius, U., K. Hattar, A. Schenkel, T. Noll, E. Csernok, W. L. Gross, W. J. Mayet, H. M. Piper, W. Seeger, and F. Grimminger. 1998. Wegener's granulomatosis: anti-proteinase 3 antibodies are potent inductors of human endothelial cell signaling and leakage response. J. Exp. Med.187:497-503.
Simons, K., and R. Ehehalt. 2002. Cholesterol, lipid rafts, and disease. J. Clin. Investig.110:597-603.
Simpson, L. L. 1981. The origin, structure, and pharmacological activity of botulinum toxin. Pharmacol. Rev.33:155-187.
Simpson, L. L. 1982. A comparison of the pharmacological properties of Clostridium botulinum type C1 and C2 toxins. J. Pharmacol. Exp. Ther.223:695-701.
Simpson, L. L. 1984. Molecular basis for the pharmacological actions of Clostridium botulinum type C2 toxin. J. Pharmacol. Exp. Ther.230:665-669.
Simpson, L. L. 1989. The binary toxin produced by Clostridium botulinum enters cells by receptor-mediated endocytosis to exert its pharmacologic effects. J. Pharmacol. Exp. Ther.251:1223-1228.
Simpson, L. L., B. G. Stiles, H. H. Zepeda, and T. D. Wilkins. 1987. Molecular basis for the pathological actions of Clostridium perfringens iota toxin. Infect. Immun.55:118-122.
Simpson, L. L., B. G. Stiles, H. Zepeda, and T. D. Wilkins. 1989. Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases. Infect. Immun.57:255-261.
Singh, A., V. Chauhan, A. Sodhi, and R. Bhatnagar. 2002. Asp 187 and Phe 190 residues in lethal factor are required for the expression of anthrax lethal toxin activity. FEMS Microbiol. Lett.212:183-186.
Singh, S., N. Ahuja, V. Chauhan, E. Rajasekaran, S. Mohsin Waheed, R. Bhat, and R. Bhatnagar. 2002. Gln277 and Phe554 residues are involved in thermal inactivation of protective antigen of Bacillus anthracis. Biochem. Biophys. Res. Commun.296:1058-1062.
Singh, Y., V. K. Chaudhary, and S. H. Leppla. 1989. A deleted variant of Bacillus anthracis protective antigen is non-toxic and blocks anthrax toxin action in vivo. J. Biol. Chem.264:19103-19107.
Singh, Y., B. E. Ivins, and S. H. Leppla. 1998. Study of immunization against anthrax with the purified recombinant protective antigen of Bacillus anthracis. Infect. Immun.66:3447-3448.
Singh, Y., H. Khanna, A. P. Chopra, and V. Mehra. 2001. A dominant negative mutant of Bacillus anthracis protective antigen inhibits toxin action in vivo. J. Biol. Chem.276:22090-22094.
Singh, Y., K. R. Klimpel, S. Goel, P. K. Swain, and S. H. Leppla. 1999. Oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells. Infect. Immun.67:1853-1859.
Singh, Y., K. R. Klimpel, C. P. Quinn, V. K. Chaudhary, and S. H. Leppla. 1991. The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity. J. Biol. Chem.266:15493-15497.
Singh, Y., S. H. Leppla, R. Bhatnagar, and A. M. Friedlander. 1989. Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells. J. Biol. Chem.264:11099-11102.
Sirard, J.-C., M. Weber, E. Duflot, M. R. Popoff, and M. Mock. 1997. A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins. Infect. Immun.65:2029-2033.
Sixma, T. K., S. E. Pronk, K. H. Kalk, E. S. Wartna, B. A. M. van Zanten, B. Witholt, and W. G. Hol. 1991. Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli.Nature351:371-377.
Skalhegg, B. S., B. F. Landmark, S. O. Doskeland, V. Hansson, T. Lea, and T. Jahnsen. 1992. Cyclic AMP-dependent protein kinase type I mediates the inhibitory effects of 3′,5′-cyclic adenosine monophosphate on cell replication in human T lymphocytes. J. Biol. Chem.267:15707-15714.
Sklyarova, T., V. Kostyukovski, V. Sharov, V. Prisyazhnoy, and O. Denisenko. 1995. Alterations in protein synthesis induced by C2 toxin in 3T3 cells. FEBS Lett.363:273-276.
Smith, H. 2002. Discovery of the anthrax toxin: the beginning of studies of virulence determinants regulated in vivo. Int. J. Med. Microbiol.291:411-417.
Smith, H., A. E. Williams, J. H. Pearce, J. Keppie, P. W. Harris-Smith, R. B. Fitsgeorge, and K. Witt. 1956. The chemical basis of the virulence of Bacillus anthracis. VII. Two components of the anthrax toxin: their relationship to known immunizing aggressins. Br. J. Exp. Pathol.37:263-271.
Soelaiman, S., B. Q. Wei, P. Bergson, Y. S. Lee, Y. Shen, M. Mrksich, B. K. Shoichet, and W. J. Tang. 2003. Structure-based inhibitor discovery against adenylyl cyclase toxins from pathogenic bacteria that cause anthrax and whooping cough. J. Biol. Chem.278:25990-25997.
Song, L., M. R. Hobaugh, C. Shustak, S. Cheley, H. Bayley, and J. E. Gouaux. 1996. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science274:1859-1866.
Songer, J. G. 1996. Clostridial enteric diseases of domestic animals. Clin. Microbiol. Rev.9:216-234.
Songer, J. G., and R. M. Meer. 1996. Genotyping of Clostridium perfringens by polymerase chain reaction is a useful adjunct to diagnosis of clostridial enteric disease in animals. Anaerobe2:197-203.
Stanley, J. L., and H. Smith. 1961. Purification of factor I and recognition of a third factor of the anthrax toxin. J. Gen. Microbiol.26:49-66.
St. Croix, B., C. Rago, V. Velculescu, G. Traverso, K. E. Romans, E. Montgomery, A. Lal, G. J. Riggins, C. Lengauer, B. Vogelstein, and K. W. Kinzler. 2000. Genes expressed in human tumor endothelium. Science289:1197-1202.
Stein, P. E., A. Boodhoo, G. D. Armstrong, S. A. Cockle, M. H. Klein, and R. J. Read 1994. The crystal structure of pertussis toxin. Structure2:45-57.
Sterne, M. 1939. The use of anthrax vaccines prepared from avirulent (unencapsulated) variants of Bacillus anthracis. Oderstepoort J. Vet. Sci. Anim. Ind.13:307-312.
Stiles, B. G. 1987. Purification and characterization of Clostridium perfringens iota toxin. Ph.D. thesis, Virginia Polytechnic Institute and State University, Blacksburg.
Stiles, B. G., D. Blöcker, M. L. Hale, M. A. Guetthoff, and H. Barth. 2002. Clostridium botulinum C2 toxin: binding studies with fluorescence-activated cytometry. Toxicon40:1135-1140.
Stiles, B. G., M. L. Hale, J.-C. Marvaud, and M. R. Popoff. 2000. Clostridium perfringens iota toxin: binding studies and characterization of cell surface receptor by fluorescence-activated cytometry. Infect. Immun.68:3475-3484.
Stiles, B. G., M. L. Hale, J.-C. Marvaud, and M. R. Popoff. 2002. Clostridium perfringens iota toxin: characterization of the cell-associated iota b complex. Biochem. J.367:801-809.
Stiles, B. G., and T. D. Wilkins. 1986. Clostridium perfringens iota toxin: synergism between two proteins. Toxicon24:767-773.
Stiles, B. G., and T. D. Wilkins. 1986. Purification and characterization of Clostridium perfringens iota toxin: dependence on two nonlinked proteins for biological activity. Infect. Immun.54:683-688.
Stoddart, B., and M. H. Wilcox. 2002. Clostridium difficile.Curr. Opin. Infect. Dis.15:513-518.
Strey, A., A. Janning, H. Barth, and V. Gerke. 2002. Endothelial Rho signaling is required for monocyte transendothelial migration. FEBS Lett.517:261-266.
Stubbs, M. T. 2002. Anthrax X-rayed: new opportunities for biodefence. Trends Pharmacol. Sci.23:539-541.
Stubbs, S., M. Rupnik, M. Gibert, J. Brazier, B. Duerden, and M. Popoff. 2000. Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile. FEMS Microbiol. Lett.186:307-312.
Sugai, M., T. Enomoto, K. Hashimoto, K. Matsumoto, Y. Matsuo, H. Ohgai, Y.-M. Hong, S. Inoue, K. Yoshikawa, and H. Suginaka. 1990. A novel epidermal cell differentiation inhibitor (EDIN): purification and characterization from Staphylococcus aureus. Biochem. Biophys. Res. Commun.173:92-98.
Sugii, S., and S. Kozaki. 1990. Hemagglutinating and binding properties of botulinum C2 toxin. Biochim. Biophys. Acta1034:176-179.
Suttorp, N., M. Polley, J. Seybold, H. Schnittler, W. Seeger, F. Grimminger, and K. Aktories. 1991. Adenosine diphosphate-ribosylation of G-actin by botulinum C2 toxin increases endothelial permeability in vitro. J. Clin. Investig.87:1575-1584.
Swartz, M. N. 2001. Recognition and management of anthrax—an update. N. Engl. J. Med.345:1621-1626.
Tabas, I., X. Zha, N. Beatini, J. N. Myers, and F. R. Maxfield. 1994. The actin cytoskeleton is important for the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages. J. Biol. Chem.269:22547-22556.
Takada, T., K. Iida, and J. Moss. 1995. Conservation of a common motif in enzymes catalyzing ADP-ribose transfer. J. Biol. Chem.270:541-544.
Tan, Y., N. R. Hackett, J. L. Boyer, and R. G. Crystal. 2003. Protective immunity evoked against anthrax lethal toxin after a single intramuscular administration of an adenovirus-based vaccine encoding humanized protective antigen. Hum. Gene Ther.14:1673-1682.
Tang, G., and S. H. Leppla. 1999. Proteasome activity is required for anthrax lethal toxin to kill macrophages. Infect. Immun.67:3055-3060.
Tang, W. J., J. Krupinski, and A. G. Gilman. 1991. Expression and characterization of calmodulin-activated (type I) adenylylcyclase. J. Biol. Chem.266:8595-8603.
Thelestam, M., E. Chaves-Olarte, M. Moos, and C. von Eichel-Streiber. 1999. Clostridial toxins acting on the cytoskeleton, p. 147-173. In J. E. Alouf and J. H. Freer (ed.), The comprehensive sourcebook of bacterial protein toxins. Academic Press, Inc., New York, N.Y.
Tonello, F., M. Seveso, O. Marin, M. Mock, and C. Montecucco. 2002. Screening inhibitors of anthrax lethal factor. Nature418:386.
Trachtman, P. 2002. Hero for our time. Smithsonian2002(Jan.):34-41.
Tsakiridis, T., A. Bergmann, R. Somwar, C. Taha, K. Aktories, T. F. Cruz, A. Klip, and G. P. Downey. 1998. Actin filaments facilitate insulin activation of the Src and collagen homologous/mitogen-activated protein kinase pathway leading to DNA synthesis and c-fos expression. J. Biol. Chem.273:28322-28331.
Tsuge, H., M. Nagahama, H. Nishimura, J. Hisatsune, Y. Sakaguchi, Y. Itogawa, N. Katunuma, and J. Sakurai. 2003. Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin. J. Mol. Biol.325:471-483.
Turnbull, P. C. 2002. Introduction: anthrax history, disease and ecology. Curr. Top. Microbiol. Immunol.271:1-19.
Turnbull, P. C., M. G. Broster, J. A. Carman, R. J. Manchee, and J. Melling. 1986. Development of antibodies to protective antigen and lethal factor components of anthrax toxin in humans and guinea pigs and their relevance to protective immunity. Infect. Immun.52:356-363.
Uzal, F. A., J. J. Plumb, L. L. Blackall, and W. R. Kelly. 1997. PCR detection of Clostridium perfringens producing different toxins in faeces of goats. Lett. Appl. Microbiol.25:339-344.
Valderrama, F., J. M. Duran, T. Babia, H. Barth, J. Renau-Piqueras, and G. Egea. 2001. Actin microfilaments facilitate the retrograde transport from the Golgi complex to the endoplasmic reticulum in mammalian cells. Traffic2:717-726.
Valderrama, F., A. Luna, T. Babia, J. A. Martinez-Menarguez, J. Ballesta, H. Barth, C. Chaponnier, J. Renau-Piqueras, and G. Egea. 2000. The Golgi-associated COPI-coated buds and vesicles contain beta/gamma-actin. Proc. Natl. Acad. Sci. USA97:1560-1565.
van Damme, J., M. Jung, F. Hofmann, I. Just, J. Vandekerckhove, and K. Aktories. 1996. Analysis of the catalytic site of the actin ADP-ribosylating Clostridium perfringens iota toxin. FEBS Lett.380:291-295.
Vandekerckhove, J., B. Schering, M. Bärmann, and K. Aktories. 1987. Clostridium perfringens iota toxin ADP-ribosylates skeletal muscle actin in Arg-177. FEBS Lett.225:48-52.
Vandekerckhove, J., B. Schering, M. Bärmann, and K. Aktories. 1988. Botulinum C2 toxin ADP-ribosylates cytoplasmic β/γ-actin in arginine 177. J. Biol. Chem.263:696-700.
Van Ness, B. G., J. B. Howard, and J. W. Bodley. 1980. ADP-ribosylation of elongation factor 2 by diphtheria toxin. J. Biol. Chem.255:10710-10716.
Varughese, M., A. Chi, A. V. Teixeira, P. J. Nicholls, J. M. Keith, and S. H. Leppla. 1998. Internalization of a Bacillus anthracis protective antigen-c-Myc fusion protein mediated by cell surface anti-c-Myc antibodies. Mol. Med.4:87-95.
Varughese, M., A. V. Teixeira, S. Liu, and S. H. Leppla. 1999. Identification of a receptor-binding region within domain 4 of the protective antigen component of anthrax toxin. Infect. Immun.67:1860-1865.
Verschueren, H., I. Van der Taelen, J. Dewit, J. De Braekeleer, P. De Baetelier, K. Aktories, and I. Just. 1995. Effects of Clostridium botulinum C2 toxin and cytochalasin D on in vitro invasiveness, motility and F-actin content of a murine T-lymphoma cell line. Eur. J. Cell Biol.66:335-341.
Vischer, U. M., H. Barth, and C. B. Wollheim. 2000. Regulated von Willebrand factor secretion is associated with agonist-specific patterns of cytoskeletal remodeling in cultured endothelial cells. Arterioscler. Thromb. Vasc. Biol.20:883-891.
Vitale, G., L. Bernardi, G. Napolitani, M. Mock, and C. Montecucco. 2000. Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor. Biochem. J.352:739-745.
Vitale, G., R. Pellizzari, C. Recchi, G. Napolitani, M. Mock, and C. Montecucco. 1998. Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. Biochem. Biophys. Res. Commun.248:706-711.
Viviani, B., C. L. Galli, and M. Marinovich. 1996. Is actin polymerization relevant to neurosecretion? A study on neuroblastoma cells. Biochem. Biophys. Res. Commun.223:712-717.
Reference deleted.
Wahl, S., H. Barth, T. Ciossek, K. Aktories, and B. K. Mueller. 2000. Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase. J. Cell Biol.149:263-270.
Walker, P., I. Batty, and J. Egerton. 1979. The typing of C. perfringens and the veterinary background. Papua New Guinea Med. J.22:50-56.
Reference deleted.
Watters, J. W., K. Dewar, J. Lehoczky, V. Boyartchuk, and W. F. Dietrich. 2001. Kif1C, a kinesin-like motor protein, mediates mouse macrophage resistance to anthrax lethal factor. Curr. Biol.11:1503-1511.
Webster, J. I., L. H. Tonelli, M. Moayeri, S. S. Simons, S. H. Leppla, and E. M. Sternberg. 2003. Anthrax lethal factor represses glucocorticoid and progesterone receptor activity. Proc. Natl. Aad. Sci. USA100:5706-5711.
Wegner, A., and K. Aktories. 1988. ADP-ribosylated actin caps the barbed ends of actin filaments. J. Biol. Chem.263:13739-13742.
Wegner, A., K. Aktories, A. Ditsch, I. Just, B. Schoepper, N. Selve, and M. Wille. 1994. Actin-gelsolin interaction. Adv. Exp. Med. Biol.358:97-104.
Weigt, C., I. Just, A. Wegner, and K. Aktories. 1989. Nonmuscle actin ADP-ribosylated by botulinum C2 toxin caps actin filaments. FEBS Lett.246:181-184.
Weinstein, J. N., T. G. Myers, P. M. O'Connor, S. H. Friend, A. J. Fornace, Jr., K. W. Kohn, T. Fojo, S. E. Bates, L. V. Rubinstein, N. L. Anderson, et al. 1997. An information-intensive approach to the molecular pharmacology of cancer. Science275:343-349.
Welkos, S., A. Friedlander, S. Weeks, S. Little, and I. Mendelson. 2002. In vitro characterization of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody. J. Med. Microbiol.51:821-831.
Welkos, S., S. Little, A. Friedlander, D. Fritz, and P. Fellows. 2001. The role of antibodies to Bacillus anthracis and anthrax toxin components in inhibiting the early stages of infection by anthrax spores. Microbiology147:1677-1685.
Welkos, S. L., J. R. Lowe, F. Eden-McCutchan, M. Vodkin, S. H. Leppla, and J. J. Schmidt. 1988. Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis. Gene69:287-300.
Wenzel-Seifert, K., H. Lentzen, K. Aktories, and R. Seifert. 1997. Complex regulation of human neutrophil activation by actin filaments: dihydrocytochalasin B and botulinum C2 toxin uncover the existence of multiple cation entry pathways. J. Leukoc. Biol.61:703-711.
Werner, G., H. Hagenmaier, H. Drautz, A. Baumgartner, and H. Zahner. 1984. Metabolic products of microorganisms. 224. Bafilomycins, a new group of macrolide antibiotics. Production, isolation, chemical structure and biological activity. J. Antibiot.37:110-117.
Wertman, K. F., and D. G. Drubin. 1992. Actin constitution: guaranteeing the right to assemble. Science258:759-760.
Wesche, J., J. L. Elliott, P. O. Falnes, S. Olsnes, and R. J. Collier. 1998. Characterization of membrane translocation by anthrax protective antigen. Biochemistry37:15737-15746.
Wex, C. B., G. Koch, and K. Aktories. 1997. Effects of Clostridium botulinum C2 toxin-induced depolymerisation of actin on degranulation of suspended and attached mast cells. Naunyn-Schmiedeberg's Arch. Pharmacol.355:319-327.
Whittaker, C. A., and R. O. Hynes. 2002. Distribution and evolution of von Willebrand/integrin A domains: widely dispersed domains with roles in cell adhesion and elsewhere. Mol. Biol. Cell13:3369-3387.
Wild, M. A., H. Xin, T. Maruyama, M. J. Nolan, P. M. Calveley, J. D. Malone, M. R. Wallace, and K. S. Bowdish. 2003. Human antibodies from immunized donors are protective against anthrax toxin in vivo. Nat. Biotechnol.21:1305-1306.
Wilde, C., and K. Aktories. 2001. The Rho-ADP-ribosylating C3 exoenzyme from Clostridium botulinum and related C3-like transferases. Toxicon39:1647-1660.
Wilde, C., G. S. Chhatwal, G. Schmalzing, K. Aktories, and I. Just. 2001. A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3. J. Biol. Chem.276:9537-9542.
Wille, M., I. Just, A. Wegner, and K. Aktories. 1992. ADP-ribosylation of the gelsolin-actin complex by clostridial toxins. J. Biol. Chem.267:50-55.
Williams R. C., M. L. Rees, M. F. Jacobs, Z. Pragai, J. E. Thwaite, L. W. Baillie, P. T. Emmerson, and C. R. Harwood. 2003. Production of Bacillus anthracis protective antigen is dependent on the extracellular chaperone, PrsA. J. Biol. Chem.278:18056-18062.
Wright, G. G., T. W. Green, and R. G. Kanode, Jr. 1954. Immunizing activity of alum-precipitated protective antigen. J. Immunol.73:387-391.
Wright, G. G., and G. L. Mandell. 1986. Anthrax toxin blocks priming of neutrophils by lipopolysaccharide and by muramyl dipeptide. J. Exp. Med.164:1700-1709.
Yamagishi, T., K. Sugitani, K. Tanishima, and S. Nakamura. 1997. Polymerase chain reaction test for differentiation of five toxin types of Clostridium perfringens. Microbiol. Immunol.41:295-299.
Yamakawa, K., S. Nishida, and S. Nakamura. 1983. C2 toxicity in extract of Clostridium botulinum type C spores. Infect. Immun.41:858-860.
Yan, M., and R. J. Collier. 2003. Characterization of dominant-negative forms of anthrax protective antigen. Mol. Med.9:46-51.
Yonushonis, W. P., M. J. Roy, R. J. Carman, and R. E. Sims. 1987. Diagnosis of spontaneous Clostridium spiroforme iota enterotoxemia in a barrier rabbit breeding colony. Lab. Anim. Sci.37:69-71.
Yoo, H. S., S. U. Lee, K. Y. Park, and Y. H. Park. 1997. Molecular typing and epidemiological survey of prevalence of Clostridium perfringens types by multiplex PCR. J. Clin. Microbiol.35:228-232.
Zepeda, H., R. V. Considine, H. L. Smith, J. R. Sherwin, I. Ohishi, and L. L. Simpson. 1988. Actions of the Clostridium botulinum binary toxin on the structure and function of Y-1 adrenal cells. J. Pharmacol. Exp. Ther.246:1183-1189.
Zhang, Y., Y. Kida, K. Kuwano, Y. Misumi, Y. Ikehara, and S. Arai. 2001. Role of furin in delivery of a CTL epitope of an anthrax toxin-fusion protein. Microbiol. Immunol.45:119-125.
Zhao, J., J. C. Milne, and R. J. Collier. 1995. Effect of anthrax toxin's lethal factor on ion channels formed by the protective antigen. J. Biol. Chem.270:18626-18630.

Information & Contributors


Published In

cover image Microbiology and Molecular Biology Reviews
Microbiology and Molecular Biology Reviews
Volume 68Number 31 September 2004
Pages: 373 - 402
PubMed: 15353562


Published online: 1 September 2004


Request permissions for this article.



Holger Barth [email protected]
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Freiburg, Germany
Klaus Aktories
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Freiburg, Germany
Michel R. Popoff
CNR Anaerobies, Institut Pasteur, Paris, France
Bradley G. Stiles [email protected]
U.S. Army Medical Research Institute of Infectious Diseases, Toxinology Division, Fort Detrick, Maryland

Metrics & Citations



  • For recently published articles, the TOTAL download count will appear as zero until a new month starts.
  • There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.
  • Citation counts come from the Crossref Cited by service.


If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. For an editable text file, please select Medlars format which will download as a .txt file. Simply select your manager software from the list below and click Download.

View Options

Figures and Media






Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy