DNase I homologous residues in CdtB are critical for cytolethal distending toxin‐mediated cell cycle arrest

Abstract

SummaryCytolethal distending toxins (CDTs) block cell division by arresting the eukaryotic cell cycle at G2/M. Although previously not recognized in standard BLAST searches, a position-specific iterated (PSI) BLAST search of the protein data bank using CDT polypeptides as query sequences indicated that CdtB bears significant position-specific homology to type I mammalian DNases. The PSI-BLAST sequence alignment reveals that residues of DNase I involved in phosphodiester bond hydrolysis (His134 and His252) are conserved in CdtB as well as their respective hydrogen bond pairs (Glu78 and Asp212). CdtB also contains a pentapeptide motif found in all DNase I enzymes. Further, crude CDT preparations possess detectable DNase activity not associated with identical preparations from control cells. Five CdtB mutations in amino acids corresponding to DNase I active site residues were prepared and expressed together with wild-type CdtA and CdtC polypeptides. Mutation in four of the five DNase-specific active site residues resulted in CDT preparations that lacked DNase activity and failed to induce cellular distension or arrest division of HeLa cells. The fifth mutation, Glu86 (Glu78 in DNase I), retained the ability to induce a moderate level of cell cycle arrest and displayed reduced DNase activity relative to wild-type CDT. Together, these data suggest that the CDT holotoxin has intrinsic DNase activity that is associated with the CdtB polypeptide and that this DNase activity may be responsible for the CDT-induced cell cycle arrest.

An N-terminal Segment of the Active Component of the Bacterial Genotoxin Cytolethal Distending Toxin B (CDTB) Directs CDTB into the Nucleus

“…Recently, two research groups indicated independently that CDT has structural homology to human DNase I and suggested that CDTB is an active component of the CDT complex acting as a DNase. In support of this, E. coli CDTB has been demonstrated to possess nicking activity toward purified plasmid in vitro (14). These findings have raised the possibility that CDTB directly damages chromosomal DNA, which results in the onset of phosphorylation of the checkpoint control cascade described above.…”

“…Among them, all but Glu 66 are located in the C-terminal half of the CDTB molecule. Previously, Elwell and Dreyfus demonstrated that His 154 , Asp 229 , Asp 260 , and His 261 of E. coli CDTB are essential for the activity, but Glu 66 is not (14). Taken together with the notion that N-terminal 48 -124 aa plays a role as a domain for nuclear transport, we hypothesized that CDTB forms a two-domain structure composed of the N-terminal domain and C-terminal genotoxic activity domain.…”

“…Taken together, these results strongly suggest that entry of CDTB into the nucleus is necessary for the induction of cytopathic effect by CDT holotoxin. DISCUSSION CDT is the first bacterial protein toxin found to be seemingly active as a DNase in target mammalian cells (14,15). Previous studies suggest that CDTB is a toxic component and that CDTA and -C are carriers of CDTB to target cells (15,28).…”

“…Lara-Tejero and Galan (15) suggested that this difference was because of the difference of the intracellular concentration of CDTB molecule, and they demonstrated cytodistension by microinjecting a very low concentration of C. jejuni CDTB into the cytosol. Elwell and Dreyfus (14), and Lara-Tejero and Galan (15) independently identified that CDTB has significant similarity to the DNase I family. The essential amino acids for metal binding and catalytic domains of the DNase I family are conserved in the CDTB family, and all of them are located at the C terminus of CDTB (14).…”

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

“…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). CDT triggers the block in cell-cycle progression through the action of a DNase I-like nuclease (CdtB) that causes double-strand breaks in the host-cell DNA (Elwell & Dreyfus, 2000;LaraTejero & Galán, 2000;Cortes-Bratti et al, 2001;Frisan et al, 2003;Hassane et al, 2003).…”