Functional studies of the recombinant subunits of a cytolethal distending holotoxin

Abstract

Summary Cytolethal distending toxin (CDT) is a multicomponent bacterial holotoxin that targets most eukarytotic cells causing distension and cell cycle arrest. A number of diverse pathogenic bacterial species associated with diarrhoea, chancroid, chronic hepatitis and periodontal disease produce a CDT. Synthesis of the holotoxin is directed by the expression of three genes, cdtA, cdtB and cdtC. Although the product of the CdtB gene was previously identified as a type I deoxyribonuclease, the functions of the cdtA and cdtC products have not been characterized. Using the periodontal pathogen, Actinobacillus actinomycetemcomitans, we demonstrate that the recombinant product of the CdtA gene binds to the surface of Chinese hamster ovary (CHO) cells. This protein did not induce distension or cytotoxicity when introduced into the cytosol using a lipid‐based protein delivery system. Recombinant CdtB and CdtC proteins failed to bind to CHO cells. However, the delivery of either CdtB or CdtC into the cytosol resulted in the characteristic pattern of distension followed by cell death. Based on these results, it appears that the CdtA protein subunit alone is responsible for anchoring the holotoxin to the cell surface. The CdtC subunit, in concert with CdtB, contributes to the cytotoxic activities of the holotoxin. The specific mechanism of CdtC cytotoxicity is currently unknown.

Differential effect of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans on co-cultures of human oral cells

“…A recombinant clone containing the A. actinomycetemcomitans cdt locus, E. coli BL21(DE3)(pET15bcdt), has been constructed and characterized previously (Mao & DiRienzo, 2002). For preparation of CDT-containing extracts, the recombinant clone was grown in Luria-Bertani medium containing 100 ìg ampicillin ml À1 at 37 8C with vigorous shaking.…”

“…The holotoxin from this bacterium is assembled from the products of three genes (cdtA, cdtB and cdtC) that have homologues in at least five other pathogenic Gram-negative bacterial genera (Sugai et al, 1998;Mayer et al, 1999;Mao & DiRienzo, 2002). Eukaryotic cells that are sensitive to the toxin are usually arrested at the G 0 /G 1 or G 2 /M phase of the growth cycle (Comayras et al, 1997;Whitehouse et al, 1998;Cortes-Bratti et al, 1999;Shenker et al, 1999).…”

Genome-wide Expression Analyses of Campylobacter jejuni NCTC11168 Reveals Coordinate Regulation of Motility and Virulence by flhA

“…Our studies demonstrate that cdtC is also independently regulated by 28 . This subunit may have some activity on its own, since injection of purified CdtC from Actinobacillus actinomycetemcomitans into the host cytosol caused distention and cell death (57), although transfection experiments with cdtC in C. jejuni did not demonstrate any changes in cell morphology (58).…”

A CdtA-CdtC Complex Can Block Killing of HeLa Cells by Haemophilus ducreyi Cytolethal Distending Toxin

“…The protein products of the individual cdt genes of H. ducreyi are most similar (i.e., Ͼ90% identity) to those of Actinobacillus actinomycetemcomitans (18). The calculated molecular weights of the mature form of these three proteins are as follows: CdtA, 22,855; CdtB, 28,955; and CdtC, 18,356. There is now considerable evidence to indicate that the CDT holotoxin, at least for H. ducreyi (9), Campylobacter jejuni (14), and A. actinomycetemcomitans (1,17,27), is comprised of CdtA, CdtB, and CdtC bound together to form a tripartite toxin. Determination of the function of the individual cdt gene products has proven challenging, however.…”

“…Determination of the function of the individual cdt gene products has proven challenging, however. The CdtB protein from several different bacterial pathogens has been shown to possess DNase activity (7,8,10,13,17) when introduced into or expressed within eukaryotic cells, and there seems to be a reasonable consensus that a functional CdtB molecule is essential for expression of toxicity by CDT (15). This property of DNase activity inherent in the CdtB protein is consistent with reports of CDT causing arrest of different cell types in the G 2 /M or G 1 and G 2 phases of the cell cycle (3,5,10,15,24,29,30).…”