No abstract

Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). The genes encoding this toxin in C. jejuni 81-176 were cloned and sequenced. The nucleotide sequence of the genes revealed that there are three genes, cdtA, cdtB, and cdtC, encoding proteins with predicted sizes of 30,116, 28,989, and 21,157 Da, respectively. All three proteins were found to be related to the Escherichia coli CDT proteins, yet the amino acid sequences have diverged significantly. All three genes were required for toxic activity in a HeLa cell assay. HeLa cell assays of a variety of C. jejuni and C. coli strains suggested that most C. jejuni strains produce significantly higher CDT titers than do C. coli strains. Southern hybridization experiments demonstrated that the cdtB gene is present on a 6.0-kb ClaI fragment in all but one of the C. jejuni strains tested; the cdtB gene was on a 6.9-kb ClaI fragment in one strain. The C. jejuni 81-176 cdtB probe hybridized weakly to DNAs from C. coli strains. The C. jejuni 81-176 cdtB probe did not hybridize to DNAs from representative C. fetus, C. lari, C. "upsaliensis," and C. hyointestinalis strains, although the HeLa cell assay indicated that these strains make CDT. PCR experiments indicated the probable presence of cdtB sequences in all of these Campylobacter species. Campylobacter jejuni is established as one of the most frequent bacterial causes of diarrheal disease in humans throughout the world (6, 20, 33). The disease can be variable, but patients typically have watery diarrhea that often contains mucus and may progress to bloody diarrhea (5, 6). Whether specific disease symptoms correlate with specific virulence factors or pathogenic traits is not known. C. jejuni has been reported to produce toxins, although definitive reports of these toxins have not appeared, and it is not clear how many different toxins C. jejuni actually produces (11, 16, 18, 21, 23, 28). Consequently, the role of any toxin in pathogenesis has not been established. Cytolethal distending toxin (CDT) production by C. jejuni was first described by Johnson and Lior (16) in 1988. CDT activity in culture supernatants caused several cultured cell lines, including HeLa and Vero cells, to become slowly distended over a 2-to 4-day period, after which the cells disintegrated. The activity was heat sensitive, trypsin sensitive, and nondialyzable (16). Johnson and Lior screened over 500 C. jejuni isolates obtained from many different countries and found that approximately 41% produced CDT. The CDT-positive isolates included representatives of all four C. jejuni biotypes and numerous serogroups. They also tested C. coli, C. lari, and C. fetus strains and showed that about 42% of these strains produced CDT (16). These Campylobacter species, as well as C. hyointestinalis and C. "upsaliensis," have all been shown to cause diarrheal disease in humans, although they apparently cause the disease much less frequently in the United States than C. jejuni does (8, 10, 26, 32, 33, 37). Johnson and Lior (13-15) also report...

Campylobacter jejuni produces a toxin, called cytolethal distending toxin (CDT), which causes direct DNA damage leading to invocation of DNA damage checkpoint pathways. The affected cells arrest in G 1 or G 2 and eventually die. CDT consists of three protein subunits, CdtA, CdtB, and CdtC, with CdtB recently identified as a nuclease. However, little is known about the functions of CdtA or CdtC. In this work, enzyme-linked immunosorbent assay-based experiments were used to show, for the first time, that both CdtA and CdtC bound with specificity to the surface of HeLa cells, whereas CdtB did not. Varying the order of the addition of subunits for reconstitution of the holotoxin had no effect on activity. In addition, mutants containing deletions of conserved regions of CdtA and CdtC were able to bind to the surface of HeLa cells but were not able to participate in holotoxin assembly. Finally, both Cdt mutant subunits were able to effectively compete with CDT holotoxin in the HeLa cell binding assay.

Cytolethal distending toxin (CDT) from the diarrheagenic bacteriumCampylobacter jejuni was shown to cause a rapid and specific cell cycle arrest in HeLa and Caco-2 cells. Within 24 h of treatment, CDT caused HeLa cells to arrest with a 4N DNA content, indicative of cells in G2 or early M phase. Immunofluorescence studies indicated that the arrested cells had not entered M phase, since no evidence of tubulin reorganization or chromatin condensation was visible. CDT treatment was also shown to cause HeLa cells to accumulate the inactive, tyrosine-phosphorylated form of CDC2. These results indicated that CDT treatment results in a failure to activate CDC2, which leads to cell cycle arrest in G2. This mechanism of action is novel for a bacterial toxin and provides a model for the generation of diarrheal disease byC. jejuni and other diarrheagenic bacteria that produce CDT.

A limited number of Escherichia coli isolates which produce an apparently novel toxin, termed cytolethal distending toxin (CDT), have been reported. The toxic activity produced by these strains causes certain cultured cell lines to become slowly distended and then disintegrate. DNA was isolated from the CDTproducing E. coli strain, 9142-88, and cloned into a cosmid vector. Plasmid DNA from a toxin-positive transductant was further subcloned until a plasmid with a 4-kb insert which still encoded the toxin activity was obtained. Nucleotide sequencing of a portion of this insert revealed the presence of three adjacent open reading frames. Further subcloning and deletion analysis suggested that the products of all three open reading frames may be required for toxin activity. Minicell experiments identified the products of all three open reading frames. The three proteins had predicted sizes of 27,753, 29,531, and 19,938 Da, and all three appeared to have strong consensus leader sequences. None of the three predicted proteins had significant homology to known proteins.