Some of the most potent toxins produced by plants and bacteria are members of a large family known as the AB toxins. AB toxins are generally characterized by a heterogenous complex consisting of two protein chains arranged in various monomeric or polymeric configurations. The newest class within this superfamily is the cytolethal distending toxin (Cdt). The Cdt is represented by a subfamily of toxins produced by a group of taxonomically distinct Gram negative bacteria. Members of this subfamily have a related AB-type chain or subunit configuration and properties distinctive to the AB paradigm. In this review, the unique structural and cytotoxic properties of the Cdt subfamily, target cell specificities, intoxication pathway, modes of action, and relationship to the AB toxin superfamily are compared and contrasted.
Bacteria secrete a myriad of different types of proteins that exhibit a cell damaging or “cytotoxic” activity. Examples include but are not limited to membrane-damaging proteins such as RTX (repeats in toxin) and CDC (cholesterol-dependent cytolysins), cell surface interacting proteins such as ST (heat-stable enterotoxins), and superantigens, as well as other proteins that attack cytosolic activities. One of the recurring organizational themes among secreted bacterial protein toxins that target intracellular processes in mammalian cells is a complex composed of at least two heterogeneous polypeptide chains or subunits. Each chain makes a distinct contribution to cell intoxication or toxic activity. The superfamily of cytotoxins that exhibit this structural arrangement have been generally labeled as the AB toxins [
The newest member of the AB toxin superfamily, discovered by Johnson and Lior in 1987 [
The objective of this treatise is to review current knowledge of the Cdt family and discuss how this subgroup compares and contrasts the AB toxin paradigm. Contributions from my laboratory are derived from studies of the Cdt produced by the human oral pathogen
Members of the AB toxin superfamily are presented in Table
The superfamily of AB toxins.
Class/Group | AB | ||||||||
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Phylogeny | Eukarya (eukaryotes) | Bacteria (prokaryotes) | |||||||
plant | Gram-negative | Gram-positive | |||||||
Common name | ricin | abrin subfamily | viscumin | diphtheria toxin (Dtx) | exotoxin A | botulinum toxin (Btx) | tetanus toxin (Ttx) | ||
abrin | modessin | volkensin | |||||||
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Toxic chain or subunit | N-glycoside hydrolase [type II ribosome inhibiting protein (RIP)] | NAD+-diphthamide ADP-ribosyltransferase | protease | ||||||
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Cellular target | 28S rRNA | elongation factor-2 (EF2) | cleavage of SNAREc protein SNAP-25 | cleavage of SNARE protein synaptobrevin II | |||||
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Toxic activity | inhibits protein synthesis by hydrolysis of the N-glycosidic bond of an adenine residue in the sarcin-ricin loop of the 28S rRNA | inhibits protein synthesis by ribosylation of EF2 | neurotoxicity due to prevention of release of the neurotransmitter acetylcholine | neurotoxicity due to prevention of release of the neurotransmitters glycine and | |||||
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Cell receptor | ganglioside GM3/glycoprotein/glycolipid | heparin-binding EGF-like (HB-EGF) receptor | lipoprotein receptor-related proteins LRP 1 and LRP 1B | GT1b |
Class/Group | AB2 | AB5 | AB7 | A2B7 | ? | ||||||
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Phylogeny | Bacteria (prokaryotes) | ||||||||||
Gram-negative | Gram-positive | ||||||||||
Common name | cytolethal distending toxin (Cdt) |
cholera toxin (Ctx) | Shiga toxin |
pertussis toxin (Ptx) | subtilase cytotoxin | heat labile enterotoxin (LT-I and LT-II) | enterotoxin | iota | anthrax toxin (Atx) | exfoliatin B | |
lethal factor (LF) | edema factor (EF) | ||||||||||
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multiple generaa |
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Toxic chain or subunit | mammalian type I |
NAD+-diphthamide |
N-glycoside |
NAD+-diphthamide |
subtilase-like |
NAD+-diphthamide |
antigenically |
NAD+-diphthamide |
Zn2+-dependent endopeptidase F | calcium- and calmodulin (CaM)-dependent adenylyl cyclase | serine protease |
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Cellular target | nuclear DNA/phosphatidylinositol-3,4,5-triphosphate (PIP3) | guanosine nucleotide-binding proteins (G proteins) | 28S rRNA | guanosine nucleotide-binding proteins (G proteins) | cleavage of BiP/GRP78 (Hsp70 family ER chaperone) | guanosine nucleotide-binding proteins (G proteins) | actin G | mitogen-activated protein kinases (MAPKK) | cAMP-induced changes of proteins | cadherin (desmoglein I) | |
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Toxic activity | cell cycle arrest at G1 or G2/M |
alters signal |
alters signal |
alters signal |
ribosylation of actin G | removes N-terminal tail |
elevates the intracellular |
detachment | |||
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Cell receptor | terminal |
ganglioside GM1 | glycolipid Gb3 | ganglioside GD1a | N-glycolylneuraminic |
ganglioside GM1 |
ANTXR 1 (tumor endothelium marker-8 |
bToxic activities are dependent on the species-specific Cdt and target cell.
cSNARE, soluble N-ethylmaleimide-sensitive factor attachment.
The A chains or toxic components of the holotoxin are represented by enzymes including an N-glycosylase, NAD+-diphthamide ADP-ribosyltransferase, deoxyribonuclease/phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PTEN, cation-dependent metalloenzyme), protein kinase, and Zn2+ metalloprotease. The most common terminal mode of action in susceptible cells is the disruption of protein biosynthesis. However, in a few examples, the A chain acts as a potent neurotoxin or genotoxin/cyclomodulin.
The B chain is required for the binding of the holotoxin to susceptible cells which is an essential step for internalization of the A chain. There is significantly greater heterogeneity in the composition and structure of the B chain most likely due to the fact that this component of the holotoxin has evolved to recognize a broad range of target cells. These differences are evident upon a comparison of the crystal structures of several representative examples of the AB superfamily (Figure
Structure comparisons of representative AB toxins. Structures of the A and B chains are shown as ribbon backbone models generated with UCSF Chimera 1.8.1. The anthrax B chain is depicted as the assembled heptamer. Three to four A1 or A2 chains or combinations of both chains are assembled in the holotoxin. The boxed structure shows the B chain monomer. Protein data bank (PDB) files: 2AAI (ricin and viscumin), 1R4Q (Shiga toxin), 1J7N (LF), 1XFY (EF), 1TZN (PA), 2F2F (
Various A chain enzyme activities of AB toxins, other than those of the Cdt, result in the inhibition of protein synthesis, apoptosis, neurotoxicity, and alteration of cell signaling pathways (Figure
Comparison of the mode of action of the A chain of selected AB toxins. The A chain of ricin and Shiga toxin is a ribosomal inhibiting protein (N-glycoside hydrolase) that carries out a depurination by removing an adenine from the 28S rRNA. The A chain of anthrax toxin is represented by two polypeptides known as the lethal factor (LF), a Zn2+-dependent endoprotease, and edema factor (EF), a polypeptide that forms a Ca2+- and calmodulin-dependent adenylate cyclase. The A chain of cholera toxin is enzymatically cleaved to create a polypeptide (A1 chain) that ribosylates the guanosine nucleotide-binding protein Gsα. The A chain of botulinum and tetanus toxins degrades specific soluble N-ethylmaleimide-sensitive factor attachment proteins (SNARE) which inhibits vesicle fusion and delivery of neurotransmitters such as acetylcholine. Additional details are provided in the text. MAPKK: mitogen-activated protein kinase kinases; CFTR: cystic fibrosis transmembrane conductance regulator.
The A chain of all of the plant AB toxins and Shiga toxin is an N-glycosylase that removes adenine from the 28S rRNA [
Anthrax toxin contains two heterogeneous A chains, known as the edema factor (EF) and lethal factor (LF) [
The A chain of cholera toxin is activated by a cleavage step. The enzymatically active A1 fragment ribosylates the guanosine nucleotide-binding protein Gsα. This reaction maintains the G protein in a GTP-bound form that activates adenylate cyclase. As a consequence, high levels of cAMP are produced, activating the cystic fibrosis transmembrane conductance regulator (CFTR), resulting in excessive efflux of ions such as Cl− and water from the intoxicated cells [
The A or light chain of the botulinum toxin functions as a protease that degrades the SNARE (soluble N-ethylmaleimide-sensitive factor attachment) protein SNAP-25 (synaptosomal-associated protein 25) [
The Cdt is produced by a handful of facultative or microaerophilic Gram-negative bacteria that are key pathogens in diseases that involve the perturbation of a mucosal (enteritis, gastric ulcers, chancroid) or epithelial (periodontal diseases) layer (Table
the deduced amino acid sequences of all three subunits have phylogenetic relationships to eukaryotic proteins or polypeptides; the Cdt complex contains two heterogeneous A subunits—CdtA and CdtC (equivalent to the B chain in other AB toxins); the CdtB subunit (equivalent to the A chain in other AB toxins) has the potential to exhibit multiple enzymatic activities and therefore affect different cell processes or pathways; a major mode of action of the Cdt is that of a genotoxin since the toxin enters the nucleus of cells and damages the host DNA; the Cdt indirectly affects regulation of the cell cycle thereby behaving as a cyclomodulin.
The cytolethal distending toxin subfamily of AB toxins.
Sourcea | Habitat | Associated affliction or diseaseb | Cell receptor |
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Cdt-I (EPEC) | Lower intestine | Gastroenteritis | Terminal |
Cdt-II (EPEC) | Lower intestine | Gastroenteritis | |
Cdt-III (septicemia) | Blood | Septicemia | |
Cdt-IV (pVir) | Lower intestine | Gastroenteritis/urinary tract infection | |
Cdt-V (STEC) | Lower intestine | Gastroenteritis | |
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Lower intestine | Gastroenteritis | Unknown |
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Lower intestine | Typhoid fever | Unknown |
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Lower intestine | Dysentery | Unknown |
Providencia alcalifaciens | Lower intestine | Gastroenteritis | Unknown |
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Stomach/liver | Duodenal ulcers/stomach cancer/chronic hepatitis | Unknown |
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Genitalia | Chancroid | Unknown |
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Oral cavity | Localized aggressive and possibly chronic periodontitis/infectious endocarditis | GM3 (also possibly GM1 and GM2) |
bacteriophage |
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Aphid symbiont | Not applicable |
bAll diseases involving a mucosal or epithelial layer.
cEPEC: enteropathogenic
The
Deduced amino acid sequence comparisons indicated that the
In spite of the evidence for genomic plasticity, there is no obvious selective pressure on the
The
Biologically active Cdt is a trimer having the AB2 configuration (Figure
Organization of the CdtA, CdtB, and CdtC subunits in the
The “B chain” of the Cdt is a heterodimer composed of the products of the
It was predicted from the crystal structure of the
We found that CdtC binds poorly to CHO cells and does not enhance the binding of CdtA [
In contrast to the glycoprotein data, the individual
The G-protein-coupled receptor, TMEM181, has been identified as a third potential Cdt cell receptor [
The identity of the Cdt receptor(s) has been a challenging problem. Based on the structural analyses and
The biologically active or toxic component of the Cdt, comparable to the A chain of the other classes of AB toxins, is also an enzyme. Based on a position-specific iterated (PSI) BLAST analysis [
CdtB-induced inhibitory pathways. (a) Schematic representation of stages in the cell cycle showing the locations of the G1 and G2 DNA damage checkpoints. (b) Cell cycle arrest at G2/M can be measured by flow cytometry or cell sorting. Populations that are arrested at G2/M accumulate cells that have a 4
During normal growth, the protein kinases Myt1 and Wee1 phosphorylate the effector kinase Cdc2/Cdk1 at positions Thr14 and Tyr15, respectively (Figure
The pathways for cell cycle arrest in response to the Cdt appear to be cell-type specific and have been presented in various reviews [
Human foreskin fibroblasts exposed to the
ATM induced Chk2 can also send the cells on an apoptotic pathway due to accumulation of phosphorylated p53 [
Activation of the ATM pathway in response to the
There are common features of the AB toxins for assembly of the holotoxin complex and intoxication of target cells. Synthesis and assembly of both the A and B chains or subunits are required for these toxins to be effective against susceptible cells. The B chains, in either monomeric or multimeric forms are essential for the delivery of the A chain into the cell. Since these toxins act on internal pathways or processes, unlike pore-forming or membrane-damaging toxins, it is essential that the A chains are transported across the plasma membrane. Endocytosis appears to be a common mechanism for internalization of the A chain. Anthrax toxin can be considered a minor exception to the general mode of AB toxin assembly since active holotoxins are heterogeneous. Holotoxins can consist of the B chain polymer together with various combinations of the A chains (see Figure
Comparison of representative examples of several of the most common classes of AB toxin demonstrates the similarities and differences among the cell intoxication mechanisms (Figure
Comparison of cell intoxication pathways of representative AB toxins. Based on the available information, the toxins shown may or may not bind to lipid rafts and enter the cell by either a clathrin-dependent or independent receptor-mediated endocytosis. Anthrax, botulinum, and tetanus toxins are not translocated to the Golgi apparatus and endoplasmic reticulum (ER) but enter the cytosol from early endosomes. Additional details are provided in the text. Unique features of Cdt intoxication are (i) endocytosis of CdtB and CdtC rather than the intact holotoxin, (ii) dynamin II participation in early endosome formation, (iii) translocation of CdtB by a non-ER-associated degradation (ERAD) pathway, and (iv) translocation of CdtB across the nuclear membrane via a nuclear localization signal (NLS).
There are many similarities between the mechanism of cell intoxication by ricin and Shiga toxin. This is expected since both contain an A chain comprised of N-glycosidase. The Shiga toxin-receptor Gb3 resides in the outer leaflet of the plasma membrane [
Anthrax is an A2B7 class toxin with each of the LF and EF A chains having distinct modes of action [
Botulinum toxin is made as a single inactive polypeptide of approximately 150 kDa that is internalized by receptor-mediated endocytosis. However, relatively little is known about this process in comparison to the other members of the AB toxin family. The polypeptide appears to bind to a receptor in the early endosome (Figure
Studies of the
As discussed in Section
Experiments performed primarily with the
CdtB enters the nucleus of the cell via the ER. In a fluorescent tagging approach combined with the isolation of nuclei from intoxicated CHO cells, we found that the
The ability of the Cdts to exhibit a broad cell tropism is one of the intriguing properties of this subclass of Ab toxins. The species-specific Cdts, as a group, usually cause cell distention, cell cycle arrest in either the G1/S or G2/M transition phase, and/or apoptosis in a variety of mammalian cell types [
Human epithelial-like cell lines such as HeLa [
We have shown that KB cells, thought to have originated from epidermoid carcinomas of the larynx, are sensitive to the
The results of studies examining the ability of the Cdt to inhibit the proliferation of fibroblasts are mixed. In one report, Chinese hamster lung (Don) fibroblasts were reported to be sensitive to the
Human CD4+ and CD8+ T lymphocytes as well as monocytes undergo cell cycle arrest, without cell distention, in the G2 phase when treated with extracts containing the
Macrophages may be potential
The AB toxins can be associated with either noninfectious disease or infectious disease. The noninfectious disease types are the toxins produced by plants. These toxins can promote disease when the toxin itself or cellular material containing the toxin is inhaled or ingested by the host. AB toxins produced by bacteria are of the infectious disease type. Diseases associated with these toxins become established when the host becomes infected with the microorganism or spores (anthrax, botulinum, and tetanus toxins) produced by the microorganism. In most cases, the bacterial AB toxins are produced by one to several species within a single genus of the bacterium and represent the primary “virulence factor” in the disease. For example, the botulinum toxin is produced by
In site of the genetic diversity of the various Cdts, there is a commonality of function based on a connection between the Cdt-producing strains of bacteria and diseases that compromise tissue layers comprised of epithelial or epithelial-related cells. Due to the complexity of mucosal related diseases, cause, and effect, relationships between the toxin and clinical symptoms of disease have had to rely on indirect associations. Often the relationship is based solely on the isolation of Cdt-producing strains from a diseased subject as in a case study reporting the isolation of
Attempting to establish a major role for the Cdt in the virulence of
Other studies have examined the role of the
Clearly, the variety of cell types and infectious diseases associated with the Cdt subfamily of AB toxins has added layers of complexity in attempts to unequivocally demonstrate a cause and effect relationship between the toxin and disease at the clinical level. Further complications can be attributed to the presence of multiple virulence factors, including endotoxin, in Cdt+ strains. Application of standard epidemiological, serological, and genetic approaches including: (i) identification of Cdt+ strains or evidence of
Similar conflicting results were obtained when systemic and neutralizing
In spite of the conflicting data from epidemiological studies, complexities of polymicrobial diseases like LAP and chronic periodontitis, and the limitations of animal models to adequately represent human periodontal disease, significant progress has been made towards identifying an
In an elegant study, Ohara and coworkers [
My laboratory examined the effect of recombinant
We extended the analysis of
The morphological changes observed in
The Cdt is an intriguing and novel member of the superfamily of AB toxins and continues to pose significant challenges in studies of the biology of cytotoxins and their role in disease almost 30 years after the discovery of the first member of this subgroup. One Cdt enigma is that, unlike the other AB toxins, it seems to fail to live up to the tremendous potential it has to be a prominent virulence component in bacterial species associated with major diseases. For example,
Another Cdt puzzle is the molecular basis for the extensive heterogeneity. The different Cdts exhibit significant deduced amino acid sequence diversity, a broad host cell range exemplified by heterogeneity in cell attachment mechanisms and variability in the end-point of intoxication. Some Cdts appear to bind specifically structured glycan residues on glycoproteins or glycolipids, certain gangliosides, and/or some types of transmembrane G-protein-coupled receptor proteins. Also, intoxication by some Cdts leads to cell cycle arrest at either the G1 or G2/M checkpoint or cell death via an apoptotic pathway. Exhaustive phylogenetic analyses and comparisons within each of the three subunits across the species-specific Cdts have been performed to attempt to understand this heterogeneity [
Therefore, the extensive body of work that exists for the Cdt has to be extended in order to solve the mysteries of what is arguably the most fascinating AB toxin. As pointed out by a number of other groups working in this field, the obvious current challenges are to decipher the details of the Cdt intoxication process, determine whether there are truly multiple specific receptors or a defined interplay between components in highly specialized regions of the cell membrane, and identify cause and effect relationships to confirm Cdt-mediated pathogenicity. Although the results of current studies are leading to a consensus of opinion that environmental factors dictate different outcomes for Cdt behavior, there may turn out to be more commonality among the species-specific Cdts than expected.
The author declares that there is no potential conflict of interests with respect to the authorship and/or publication of this paper.
While attempts were made to be as thorough as possible within the constraints of the review format, a sincere apology is made if some studies were inadvertently omitted. The research performed in the laboratory was supported by the National Institutes of Health Grants DE012593 and DE017679 from the National Institute of Dental and Craniofacial Research.