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Forum: Mechanisms of Action of Cytotoxic Agents Cytolethal Distending Toxin: A Bacterial Toxin Which Disrupts the Eukaryotic Cell Cycle Vincent B. Young†,‡ and David B. Schauer*,†,§ Division of Bioengineering and Environmental Health and Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Infectious Diseases Unit, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114 Received April 17, 2000
Introduction. A variety of pathogenic bacteria elaborate toxins that play a key role in the virulence of these organisms (1). Many of these toxins have evolved to interact with elements of the host cellular machinery in a highly specific manner, resulting in the disruption of host cell function (2). For example, cholera toxin produced by the bacterium Vibrio cholerae is the prototypic A-B subunit toxin. The B subunit binds the holotoxin specifically to its GM1 receptor, and the A subunit has ADPribosyltransferase activity. Cholera toxin acts by ADP ribosylation of the R-subunit of the GTP-binding protein, Gs, stimulating adenylate cyclase activity and eventually leading to increased intestinal fluid secretion. The cytolethal distending toxin (CDT)1 family of bacterial toxins was initially described in 1988 by Johnson and Lior (3, 4). As indicated by the name, CDT causes a characteristic distension of cultured mammalian cells and eventual death. CDT homologues have been identified in some strains of Escherichia coli, Hemophilus ducreyi, Actinobacillus actinomycetemcomitans, Shigella dysenteriae, Campylobacter jejuni and other members of the genus Campylobacter, and a subset of Helicobacter species. Studies on members of the CDT family indicate that these toxins are unique in that they appear to interfere with the normal progression of the eukaryotic cell cycle. CDT Activity. (1) Cytopathic Effect. The hallmark of the cytopathic effect mediated by CDT is cellular distension. Cultured eukaryotic cells treated with CDT gradually enlarge over 48-72 h to a point at which they can occupy 10 times the surface area of a control cell. Examination of the ultrastructure of intoxicated cells reveals nuclear enlargement as well as nuclear abnormalities, including multinucleation and fragmentation (Figure 1). Additionally, abnormal accumulations of polymerized actin, resembling stress fibers, appear in CDT-treated cells (5-7). Microtubule abnormalities have * To whom correspondence should be addressed: MIT BEH/DCM, MIT Room 56-787, Cambridge, MA 02139. Phone: (617) 253-8113. Fax: (617) 258-0225. E-mail:
[email protected]. † Division of Bioengineering and Environmental Health, Massachusetts Institute of Technology. ‡ Massachusetts General Hospital. § Division of Comparative Medicine, Massachusetts Institute of Technology. 1Abbreviation: CDT, cytolethal distending toxin.
Figure 1. Cytopathic effect in HeLa cells treated with CDT from H. hepaticus. HeLa cell monolayers were treated with sterile sonicates of H. hepaticus for 72 h, then fixed and permeabilized, and stained with Texas Red-labeled phalloidin to label polymerized actin (red) and Hoechst 33342 to label DNA (blue). Compared to control cells (A), HeLa cells treated with CDT (B) exhibit marked cellular distension, the appearance of abnormal accumulations of polymerized actin, nuclear distension, and nuclear fragmentation. The bar is 25 µm long.
been observed in cells treated with CDT from C. jejuni, including perinuclear tubulin bundles and distorted spindle structures (8). Most investigators report that CDT-treated cells detach from the underlying substrate within 72-96 of the
10.1021/tx000091d CCC: $19.00 © 2000 American Chemical Society Published on Web 09/29/2000
Forum: Mechanisms of Action of Cytotoxic Agents
addition of toxin; however, it appears that the cytolethal effect of different CDTs can vary both between species and between isolates of a given species. It is not clear what the actual mechanism of death is in CDT-treated cells. CDT from H. ducreyi induces apoptosis in Jurkat T cells (9), as judged by DNA fragmentation and the loss of membrane asymmetry. Induction of apoptosis in CDTtreated epithelial cell lines has not been clearly demonstrated. We have not been able to demonstrate the appearance of DNA laddering or a sub-G1 flow cytometric peak in HeLa cells treated with CDT from Hemophilus hepaticus. It may be that differences exist between CDTs from different organisms in the ability to induce apoptosis, or perhaps more likely, cell types differ in their response to CDT. (2) Cell Cycle Arrest. Peres reported in 1997 that an E. coli strain that possessed the toxin cytotoxic necrotizing factor caused cultured cells to arrest at G2/M, resulting in cells with 4N DNA content (10). It was discovered that the cell cycle arrest was due not to cytotoxic necrotizing factor, which was carried on a plasmid called pVir, but due to a CDT that was carried on the same plasmid. Subsequently, this ability to cause cell cycle arrest has been shown to be a property of CDTs from other species as well (5, 6, 8, 11). Several groups have reported that CDT-mediated cell cycle arrest is associated with accumulation of the hyperphosphorylated, inactive form of the cyclin-dependent kinase cdk1 (Cdc2) (6, 8, 11, 12). Dephosphorylation and activation of cdk1 are essential for entry in mitosis (13). Cell cycle arrest at G2/M can be induced by DNA damage and is also associated with accumulation of phosphorylated cdk1 (13). It is not clear, however, that cell cycle arrest due to CDT is associated with preliminary induction of DNA damage (14). The actual mechanism of CDT-mediated cell cycle arrest is not clear. Although it is apparent that inactive cdk1 accumulates in CDT-treated cells, this could occur via the disruption of any of a number of different regulators of the G2/M transition (Figure 2). CDT could cause inactivation or a lack of activation of Cdc25C, a protein phosphatase that dephosphorylates and activates cdk1; CDT could cause hyperactivation of Mik1 and Wee1, kinases that phosphorylate cdk1, or CDT could even exert a direct effect on cdk1 itself. Upstream components of this regulatory cascade could also be affected such as Chk1, a kinase that indirectly inactivates Cdc25C. The complexity of the control system in place to regulate the cell cycle dictates that a careful examination of the interaction of CDT with elements of this control system will be necessary to elucidate the mechanism by which CDT causes cell cycle arrest. CDT Genetics and Biochemistry. CDT activity is encoded by three closely linked genes termed cdtA, cdtB, and cdtC. In H. duceyi, the only organisms in which transcriptional control has been studied, it appears that the three genes comprise a single transcriptional unit (15). cdt genes are predicted to encode proteins with approximate molecular masses between 20 and 30 kDa. Genetic studies have shown that all three genes are required to transfer CDT activity from CDT-expressing C. jejuni, E. coli, H. ducreyi, A. actinomycetemcomitans, and H. hepaticus to a laboratory strain of E. coli (5, 1519). The evolutionary and biologic distance that separates the bacteria that possess CDT homologues suggests that
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Figure 2. Model of control of the G2/M transition in eukaryotic cells. A prerequisite for entry into mitosis is dephosphorylation of the cyclin-dependent kinase cdk1 by Cdc25C. Dephosphorylated cdk1 in association with cyclinB (cycB) triggers entry into mitosis. In CDT-treated cells, phosphorylated cdk1 has been shown to accumulate. The phosphorylation of cdk1 is regulated by kinases (Wee1 and Mik1) as well as by the activity of the phosphatase Cdc25C. Cdc25C activity in turn is regulated by phosphorylation. N-Terminal phosphorylation of Cdc25C is required for phosphatase activity, while phophorylation of Ser216 of Cdc25C by DNA damage checkpoint kinase Chk1 results in sequestration of Cdc25C by 14-3-3 protein and a lack of cdk1 activation. Noncovalent interactions are represented by double arrows with the bimolecular species indicated by the heavy dot. P represents phosphate groups. Red lines indicate inhibition and green lines stimulation. This molecular interaction representation is depicted as outlined by Kohn (27).
cdt genes may have been acquired by horizontal transfer. The degree of homology among cdt homologues is greatest for the cdtB locus. The greatest degree of homology encountered between cdt genes from bacteria that belong to different species is seen for cdtB from A. actinomycetemcomitans and H. ducreyi (96.8% amino acid identity). This is particularly striking given that cdtB homologues from two different species are generally only about 5060% identical. Sequence analysis of the cdt locus from H. ducreyi reveals several genes within 3 kb of the cdt coding region that are homologues of genes involved in transposition, raising the possibility that the gene cluster was introduced as part of a transposon (15). Adjacent to the cdt cluster from A. actinomycetemcomitans are homologues of a bacteriophage att site, an integrase, and elements of various plasmids (20). These findings, coupled with the fact that one cdt cluster in E. coli is located on a transferable plasmid (10), indicate that horizontal transfer of CDT is possible. Although all three cdt genes are required to transfer CDT activity, the biochemical nature of active CDT is not known. It is not known if all three gene products are components of CDT holotoxin, nor has the stoichiometry of subunits in the mature toxin been determined. CDT activity purified from A. actinomycetemcomitans was shown to be CdtB, the product of the cdtB gene (11). Purven et al. purified a cytotoxin from H. ducreyi using a neutralizing monoclonal antibody. Identification of this cytotoxin by determination of the N-terminal amino acid sequence revealed that it was CdtC (21). In both of these studies, the presence of other CDT subunits in the purified toxin preparation was not ruled out. Studies with neutralizing monoclonal antibodies against H. ducreyi
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and A. actinomycetemcomitans CDT indicate that CdtC specificity is associated with the highest neutralizing activity (15, 19, 21). These conflicting data indicate that further work needs to be performed to determine the exact biochemical nature of CDT. CDT and Pathogenesis. The organisms in which CDT activity has been identified represent a diverse group of pathogenic bacteria. E. coli, Shigella spp., Campylobacter spp., and the subset of Helicobacter species that possess CDT are all enteropathogenic bacteria. H. ducreyi is the causative agent of chancroid, an ulcerative condition of skin in the genital region, and A. actinomycetemcomitans is a bacterium that has been associated with juvenile peridontitis and infectious endocarditis. The presence of CDT in these pathogens implies that it may serve as a virulence factor. However, data proving a role for CDT in virulence are lacking. Although CDT was originally detected in enteropathogenic isolates of E. coli (4), two epidemiologic studies have failed to conclusively implicate CDT-producing E. coli strains as etiologic agents of gastroenteritis. A study of 546 children in Bangladesh showed a trend that did not reach statistical significance for the isolation of CDTpositive E. coli in children with diarrhea as opposed to healthy controls (22). The same group reported similar results when 187 children with diarrhea in Nigeria were screened for CDT-producing E. coli and other enteropathogenic E. coli strains (23). The latter study underscores the fact that in developing countries, there is a high incidence of carriage of pathogenic E. coli strains. Strains of A. actinomycetemcomitans vary in the amount of CDT activity that they produce. It appears that this can be due to partial deletions of the cdt cluster (20). However, the ability to produce CDT did not clearly correlate with the ability to cause peridontitis. One strain, apparently isolated from a healthy patient, did produce CDT activity, while other A. actinomycetemcomitans strains isolated from patients with peridontitis did not. Limited experimental studies have addressed the role of CDT in virulence. CDT-producing E. coli strains do not cause necrosis of rabbit skin, nor do they cause fluid secretion in the rabbit ileal loop assay (24). CDT does cause increased intestinal fluid secretion in a suckling mice assay, and this is associated with colonic tissue damage (25). However, an isogenic H. ducreyi cdtC mutant was recently shown to be as virulent as the wild type in the temperature-dependent rabbit model of chancroid (26). We are currently investigating the role of CDT in the pathogenesis of enterocolitis associated with H. hepaticus and other enterohepatic Helicobacter species. Conclusions. The CDT family of toxins has been found in a variety of pathogenic bacteria that cause disease ranging from gastroenteritis to genital ulcers to peridontitis. The exact role of CDT in the pathogenesis of any of these conditions is not known, and it is possible that CDT serves a unique role in the pathogenesis of each disease. Since evidence exists that CDT can be horizontally transferred, different pathogens may have acquired CDT and found it to be of utility in the specific pathogenic niche that each occupies, and this utility varies from pathogen to pathogen. All CDTs appear to cause cell cycle arrest at the G2/M boundary, and this suppression of cell proliferation may play a role in the pathogenesis of disease caused by CDT-
Young and Schauer
positive bacteria. Although CDT can affect many different cell types in vitro, the cell types that are targeted in vivo need to be determined to ascribe a role for CDT in virulence. There is a great deal of work that needs to be done to determine the mechanism of action of CDT. Precise biochemical characterization of the toxin itself is lacking, and this may hamper detailed analysis of the interaction of CDT with the cell cycle control machinery. However, it is possible that determination of the mechanism of CDT activity will provide important insight into the control of eukaryotic cellular proliferation and provide novel targets for immunosuppression and chemotherapy.
Note Added in Proof While this review was in press, Elwell and Dreyfus (28) reported that CdtB contains homology to type I mammalian DNases and exhibits DNase activity, further supporting the possibility that the toxin causes primary DNA damage in cells.
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