Immune response to cytolethal distending toxin of Aggregatibacter actinomycetemcomitans in periodontitis patients

Abstract

Background and objective: Cytolethal distending toxin (CDT) is a genotoxin produced by Aggregatibacter actinomycetemcomitans. In spite of its association with pathogenesis, little is known about the humoral immune response against the CDT. This study aimed to test whether subgingival colonization and humoral response to A. actinomycetemcomitans would lead to a response against CDT.

Material and methods: Sera from periodontally healthy, localized and generalized aggressive periodontitis and chronic periodontitis subjects (n = 80) were assessed for immunoglobulin G titers to A. actinomycetemcomitans serotypes a/b/c and to each CDT subunit (CdtA, CdtB and CdtC) by ELISA. A. actinomycetemcomitans subgingival levels and neutralization of CDT activity were also analyzed.

Results: Sera from 75.0% localized and 81.8% generalized aggressive periodontitis patients reacted to A. actinomycetemcomitans. A response to serotype b was detected in localized (66.7%) and generalized aggressive periodontitis (54.5%). Reactivity to A. actinomycetemcomitans correlated with subgingival colonization (R = 0.75, p < 0.05). There was no correlation between A. actinomycetemcomitans colonization or response to serotypes and the immunoglobulin G response to CDT subunits. Titers of immunoglobulin G to CdtA and CdtB did not differ among groups; however, sera of all generalized aggressive periodontitis patients reacted to CdtC. Neutralization of CDT was not correlated with levels of antibodies to CDT subunits.

Conclusion: Response to CdtA and CdtB did not correlate with the periodontal status of the subject in the context of an A. actinomycetemcomitans infection. However, a response to CdtC was found in sera of generalized but not of localized aggressive periodontitis subjects. Differences in response to CdtC between generalized and localized aggressive periodontitis subjects indicate that CDT could be expressed differently by the infecting strains. Alternatively, the antibody response to CdtC could require the colonization of multiple sites.

Fig. 1

Cytolethal distending toxin activity in CHO cells when reconstituted toxin was added to the cells. Reconstituted toxin formed by different amounts (in μg) of CDT subunits A, B and C was added to 240 cells, and the number of CFU was counted after 7 d of incubation. Filled bars represent cells inoculated with reconstituted CDT holotoxin. Open bars represent controls (Neg control, negative control without CDT addition, and non-reconstituted toxin). *p < 0.001 (ANOVA–Tukey) compared with negative control.

Fig. 2

Distribution of sera IgG levels from subjects with different periodontal conditions against A. actinomycetemcomitans serotypes a (A), b (B) and c (C) based on cut-off levels for reactivity after O.D. corrected values (F_serotype) obtained by ELISA. Non-reactive subjects: OD corrected values below 0. Sera dilution 1:20 000. Statistically significant differences between groups are indicated as *p < 0.01 and **p < 0.001 (ANOVA– Tukey). White boxes represent the outlier values (outside the cut-off, determined by the statistic program Statgraphics) and the crosses represent the means of the values.

Fig. 3

Distribution of serum IgG levels from subjects with different periodontal conditions for CdtA (A), CdtB (B) and CdtC (C), based on cut-off levels for reactivity after O.D. corrected values (F_Cdt). Sera dilution 1:150. Statistically significant differences when compared with healthy subjects are indicated as *p < 0.05 (ANOVA–Tukey). White boxes represent the outlier values (outside the cut-off, determined by the statistic program Statgraphics) and the crosses represent the means of the values.

Fig. 4

Sera IgG levels to A. actinomycetemcomitans serotype c (sera dilution 1:20 000) and to CdtC (sera dilution 1:150) in generalized aggressive periodontitis subjects based on cut-off levels for reactivity after O.D. corrected values (F_serotype c). *p < 0.001 (Spearman rank correlation).