Dataset Information

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 CdtA(Y181A)BC holotoxin and the biological function of CDT was not weaken in CdtA(Y105A)BC, CdtA(Y125A)BC, CdtA(F109A)BC and CdtA(S106N)BC 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 CdtA(W115G)BC. 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.

SUBMITTER: Li L

PROVIDER: S-EPMC3670888 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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A new functional site W115 in CdtA is critical for Aggregatibacter actinomycetemcomitans cytolethal distending toxin.

Li Lu L Ding Cheng C Duan Jun-lan JL Yang Mi-fang MF Sun Ying Y Wang Xiao-qian XQ Xu Yan Y

PloS one 20130603 6

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 mutatio ...[more]

PMID: 23755273

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Project description:Background and objectivesThe cytolethal distending toxin (Cdt) is a highly conserved exotoxin that are produced by a number of Gram negative bacteria, including Aggregatibacter actinomycetemcomitans, and affects mammalian cells by inhibiting cell division and causing apoptosis. A complete cdt-operon is present in the majority of A. actinomycetemcomitans, but the proportion of isolates that lack cdt-encoding genes (A, B and C) varies according to the population studied. The objectives of this study were to examine serotype, Cdt-genotype, and Cdt-activity in isolates of A. actinomycetemcomitans collected from an adolescent West African population and to examine the association between the carrier status of A. actinomycetemcomitans and the progression of attachment loss (AL).Materials and methodsA total of 249 A. actinomycetemcomitans isolates from 200 Ghanaian adolescents were examined for serotype and cdt-genotype by PCR. The activity of the Cdt-toxin was examined by DNA-staining of exposed cultured cells and documented with flow cytometry. The periodontal status of the participants was examined at baseline and at a two-year follow-up.ResultsPresence of all three cdt-encoding genes was detected in 79% of the examined A. actinomycetemcomitans isolates. All these isolates showed a substantial Cdt-activity. The two different cdt-genotypes (with and without presence of all three cdt-encoding genes) showed a serotype-dependent distribution pattern. Presence of A. actinomycetemcomitans was significantly associated with progression of AL (OR = 5.126; 95% CI = [2.994-8.779], p<0.001).ConclusionA. actinomycetemcomitans isolated from the Ghanaian adolescents showed a distribution of serotype and cdt-genotype in line with results based on other previously studied populations. Presence of A. actinomycetemcomitans was significantly associated with disease progression, in particular the b serotype, whereas the association with disease progression was not particularly related to cdt-genotype, and Cdt-activity.

Project description:The cytolethal distending toxin (Cdt) of Actinobacillus actinomycetemcomitans is an atypical A-B-type toxin consisting of a heterotrimer composed of the cdtA, cdtB, and cdtC gene products. The CdtA and CdtC subunits form two heterogeneous ricin-like lectin domains which bind the holotoxin to the target cell. Point mutations were used to study CdtC structure and function. One (mutC216(F97C)) of eight single-amino-acid replacement mutants identified yielded a gene product that failed to form biologically active holotoxin. Based on the possibility that the F97C mutation destabilized a predicted disulfide, targeted mutagenesis was used to examine the contribution of each of four cysteine residues, in two predicted disulfides (C96/C107 and C135/C149), to CdtC activities. Cysteine replacement mutations in two predicted disulfides (C136/C149 and C178/C197) in CdtA were also characterized. Flow cytometry and CHO cell proliferation assays showed that changing either C96 or C149 in CdtC to alanine abolished the biological activity of holotoxin complexes. However, replacing C107 or C135 in CdtC and any of the four cysteines in CdtA with alanine or serine resulted in only partial or no loss of holotoxin activity. Changes in the biological activities of the mutant holotoxins correlated with altered subunit binding. In contrast to elimination of the B chain of ricin, the elimination of intrachain disulfides in CdtC and CdtA by genetic replacement of cysteines destabilizes these subunit proteins but not to the extent that cytotoxicity is lost. Reduction of the wild-type holotoxin did not affect cytotoxicity, and the reduced form of wild-type CdtA exhibited a statistically significant increase in binding to ligand. A diminished role for intrachain disulfides in stabilizing CdtA and CdtC may have clinical relevance for the A. actinomycetemcomitans Cdt. The cdt gene products secreted by this pathogen assemble and bind to target cells in periodontally involved sites, which are decidedly reduced environments in the human oral cavity.

Dataset Information

A new functional site W115 in CdtA is critical for Aggregatibacter actinomycetemcomitans cytolethal distending toxin.

Publications

A new functional site W115 in CdtA is critical for Aggregatibacter actinomycetemcomitans cytolethal distending toxin.

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