Expression of the Cytolethal Distending Toxin (Cdt) Operon inActinobacillus actinomycetemcomitans:Evidence That the CdtB Protein Is Responsible for G2 Arrest of the Cell Cycle in Human T Cells

Abstract

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several Gram-negative bacteria. In this study, we report that the cdt locus in A. actinomycetemcomitans is composed of five open reading frames, designated orf1, orf2, cdtA, cdtB, and cdtC. The deduced amino acid sequences of the five open reading frames are highly conserved among A. actinomycetemcomitans strains 652, Y4, 29522, and HK1651. There is also strong homology with the Cdt proteins of Haemophilus ducreyi (87–91%), but only partial homology with that of Campylobacter jejuni and Escherichia coli (29–48%). Analysis of A. actinomycetemcomitans mRNA by RT-PCR suggests that the two small open reading frames upstream of cdtA are coexpressed with cdtA, cdtB, and cdtC. We next utilized a series of plasmids that express various combinations of the cdt genes to determine their requirement for expression of immunoinhibitory activity. Cell extracts of E. coli transformed with each of the plasmids were tested for their capacity to induce G2 arrest in the cell cycle of PHA-activated human T cells. These experiments suggest that expression of cdtB alone is sufficient to induce G2 arrest in human T cells, but do not exclude the possibility that cdtC also contributes to cell cycle arrest. The implications of our results with respect to the function of the individual Cdt proteins are discussed.

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

“…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).…”

Cytolethal Distending Toxin Family Members Are Differentially Affected by Alterations in Host Glycans and Membrane Cholesterol

“…To test whether host factors required for intoxication are shared between different CDTs, we set out to quantify the level of sensitivity to Ec-, Hd-, Aa-, and Cj-CDTs in a series of diverse cell lines. We intoxicated Chinese hamster ovary (CHO-K1) and HeLa cells, which represent epithelial cells commonly used for CDT studies, as well as T-cells (OT-1) and macrophage-like cells (RAW 264.7 and IC-21), which are proposed to be important targets for CDTs in vivo (35). In addition, we intoxicated 3T3 fibroblasts and Y-1 adrenal cells, which were reported to be resistant to Ec-, Hd-, and Cj-CDTs (16, 36 -38).…”

Genomic distribution and functions of uptake signal sequences in Actinobacillus actinomycetemcomitans

“…Some USS-free regions were found to harbour rRNA operons and loci for ribosomal proteins (Smith et al, 1999) and genomic islands ) (see also http://www.oralgen.lanl.gov/), whereas others contain putative virulence factors of A. actinomycetemcomitans [e.g. matrix binding protein EmaA (Mintz, 2004), cytolethal distending toxin (Mayer et al, 1999;Shenker et al, 2000;Sugai et al, 1998), fimbria/Tad locus (Haase et al, 1999;Inouye et al, 1990;Ishihara et al, 1997;Kachlany et al, 2000;Planet et al, 2003) and leukotoxin (Kolodrubetz et al, 1989;Kraig et al, 1990;Lally et al, 1989)]. …”