A New Functional Site W115 in CdtA Is Critical for Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin

Abstract

Aggregatibacter actinomycetemcomitans, a specific pathogen of localized aggressive periodontitis, produces a cytolethal distending toxin (CDT) that arrests eukaryotic cells irreversibly in G0/G1 or G2/M phase of the cell cycle. Although structural studies show that the aromatic patch region of CdtA plays an important role in its biological activity, the functional sites of CdtA have not been firmly established. In this study, site-specific mutagenesis strategy was employed for cdtA point mutations construction so as to examine the contributions of individual amino acids to receptor binding and the biological activity of holotoxin. The binding ability was reduced in CdtAY181ABC holotoxin and the biological function of CDT was not weaken in CdtAY105ABC, CdtAY125ABC, CdtAF109ABC and CdtAS106NBC holotoxin suggesting that these sites were not critical to CDT. But the binding activity and cell cycle arrest ability of holotoxin complexes were inhibited in CdtAW115GBC. And this site did not affect the holotoxin assembly by size exclusion chromatography. Therefore, W115 might be a critical site of CdtA binding ability. These findings suggest that the functional sites of CdtA are not only in the aromatic patch region. W115, the new functional site is critical for receptor binding and cell cycle arrest, which provides potential targets for pharmacological disruption of CDT activity.

Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins

“…ducreyi (Hd-CDT) and Aa-CDT defined critical roles for a patch of aromatic residues on CdtA and a hydrophobic groove formed between CdtA and CdtC [ 14 , 46 , 49 , 50 ]. Mutation of other regions of CdtA or CdtC also led to inactive holotoxins, further supporting roles for both subunits [ 14 , 33 , 37 , 46 , 49 , 51 ]. In contrast, CtdBC heterodimers derived from A .…”

Bacterial genotoxins

“…Indeed, mutations of the aromatic patch or leading to groove alteration do not change the stability of the ternary complex, but completely or partially abolished the ability of the toxin to bind and cause cell cycle arrest [5,49]. Point mutation analysis of the Aact cdtA gene has further identified two residues localized outside the hydrophobic patch (Thr 41 and Trp 115) that are required to mediate binding to the surface of the target cells and induction cell cycle arrest, without altering the formation of the trimeric complex [50,51]. It is conceivable that these mutations stabilize the putative binding site at the interface of the CdtA and CdtC subunits, contributing to the binding efficiency of the holotoxin.…”

Inflammatory and Bone Remodeling Responses to the Cytolethal Distending Toxins

“…Yet, there are indications that the glycosphingolipid GM3 can act as one [ 22 , 23 ]. The receptor binding capacity of CDT is attributed to the aromatic aminoacids of the CdtA subunit [ 24 , 25 ], but not restricted to these [ 26 ].…”