Cloning, sequencing, and expression of the Escherichia coli cytolethal distending toxin genes

Abstract

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.

Comparison of polymerase chain reaction systems for detection of different cdt genes in Escherichia coli strains

“…All the existing 11 PCR systems report high specificity and accuracy for typing of cdt genes, but have not been used widely for epidemiological studies in different regions (Pickett et al 1994;Clark et al 2002;Pandey et al 2003;Toth et al 2003;Bielaszewska et al 2004). Using the multiplex PCR system developed by Toth et al (2003) we have shown that it is sensitive and specific for detection of all different cdt genes, i.e.…”

“…The Cdt A and Cdt C polypeptides constitute the heterodimeric subunit (Lara-Tejero and Galan 2001; Lee et al 2003) that is required for CDT binding to target cells. Five different E. coli cdt sequences have been reported, cdt-I (Scott and Kaper 1994), cdt-II (Pickett et al 1994), cdt-III (Peres et al 1997), cdt-IV (Toth et al 2003) and cdt-V (Bielaszewska et al 2004).…”

Reconstitution and Purification of Cytolethal Distending Toxin of Actinobacillus actinomycetemcomitans

“…As indicated by Shenker et ai, the N-terminal sequence of A. actinomycetemcomitans CdtB is Asn-Leu-Ser-Asp-Phe-Lys-Val-Ala-Thr-Trp-Asn-Leu-Gln-Gly-Ser (21). No N-terminal sequence of the CdtA protein from any bacterium has been determined but a mature CdtA is suggested to be a lipoprotein (26) with the cysteine residue at position + I (corresponding to Cys-16 of CdtA in A. actinomycetemcomitans) as a Nterminal residue (II,14,15,24). A molecular mass of the A. actinomycetemcomitans CdtA from Cys-16 to Asn-222 is calculated as 22.9 kDa.…”

“…Originally, COT was discovered in Escherichia coli OI28:H-as a novel cell distending and cytolethal toxin (8). The cdtABC genes encoding COT were first identified in E. coli OI28:H-and 086:H34 (15,19). Successively, the cdtABC gene homologs have been identified in Shigella dysenteriae (12), Campylobacter jejuni Abbreviations: CBD, chitin binding domain; COT, cytolethal distending toxin; OTT, dithiothreitol; GST, glutathione S-transferase; UP, localized juvenile periodontitis; NiH-SFF, Ni H_ chelated Sepharose Fast Flow.…”

“…The cloned cdtABC genes are composed of the successive cdtA, cdtB, and cdtC genes, and their products, CdtA, CdtB, and CdtC are novel proteins (17). Genetic studies have shown that all the cdtA, cdtB, and cdtC genes are required for the complete CDT activities (3,II,15,16,24,27). Unfortunately, the active CDT has not successfully been purified (17) and hence the structure and the nature of the CDT holotoxin have not yet been understood.…”