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Cytolethal distending toxin (CDT) is a bacterial genotoxin that causes host cell cycle arrest and death. We previously employed a Saccharomyces cerevisiae model with inducible expression of the CDT catalytic subunit from Aggregatibacter actinomycetemcomitans, Aa CdtB, and showed that a wide variety of host factors play a role in facilitating the activity of CdtB. Our observation that a yeast H2B mutant defective in chromatin condensation was partially resistant to CdtB implies that chromatin structure may affect CDT function. In this study, we identified host chromatin regulatory genes required for CdtB cytotoxicity. We found that the deletion of HTZ1 or certain subunits of SWR, INO80, and SIR complexes increased cellular resistance to CdtB. We hypothesized that CdtB may interact with Htz1 or the chromatin, but immunoprecipitation experiments failed to detect physical interaction between CdtB and Htz1 or the chromatin. However, we observed reduced nuclear localization of CdtB in several mutants, suggesting that impaired nuclear translocation may, at least partly, explain the mechanisms of CdtB resistance. In addition, mutations in chromatin regulatory genes induce changes in the global gene expression profile, and these may indirectly affect CdtB toxicity. Our results suggest that decreased expression of endoplasmic reticulum (ER)-Golgi transport-related genes that may be involved in CdtB transport and/or increased expression of DNA repair genes may contribute to CdtB resistance. These results suggest that the functions of chromatin regulators may contribute to the activity of CDT in host cells.

Aggregatibacter actinomycetemcomitans is an oral pathogen causing periodontal disease and bacterial endocarditis. It produces cytolethal distending toxin (CDT) that could damage mammalian cells and tissues. CDT is a tripartite protein toxin composed of CdtA, CdtB, and CdtC. We have previously indicated that CdtA is a lipoprotein and that the proteolytic processing of CdtA is important for biogenesis and secretion of CDT holotoxin. Here, we established an in vitro processing assay of CdtA and investigated the interactions of CdtA with other Cdt subunits. This assay demonstrated that incubation of membrane-bound CdtA (MCdtA), CdtB, and CdtC immediately generated a processed form of CdtA (CdtA'), which is recovered from the soluble fraction. In contrast, incubation of soluble membrane-unbound CdtA with CdtB and CdtC did not yield any CdtA'. Furthermore, incubation of CdtC with MCdtA was enough to induce rapid processing of MCdtA, whereas CdtB alone was unable to induce the processing. Coimmunoprecipitation demonstrated that CdtA' and CdtC formed a complex. Furthermore, subsequent addition of CdtB to this reaction mixture resulted in complete CDT holotoxin complex. The cytolethal distending activity assay demonstrated that CDT complex containing CdtA' showed far stronger cytotoxicity than that containing CdtA. Collectively, our data suggest that CDT holotoxin formation in vivo is a sequential event: interaction of MCdtA and CdtC induces proteolytic processing of MCdtA, and the released CdtA' forms a complex with CdtC. Subsequent binding of CdtB to the CdtA'/CdtC complex results in CDT holotoxin formation., (Copyright © 2018 American Society for Microbiology.)

Background: Aggregatibacter actinomycetemcomitans is one of the etiological pathogens implicated in the onset of periodontal disease. This pathogen produces cytolethal distending toxin (CDT) that acts as a genotoxin to induce cell cycle arrest and cellular distension in cultured cell lines. Therefore, CDT is a possible virulence factor; however, the in vivo activity of CDT on periodontal tissue has not been explored. Here, CDT was topically applied into the rat molar gingival sulcus; and the periodontal tissue was histologically and immunohistochemically examined., Materials and Methods: Recombinant purified A. actinomycetemcomitans CDT was applied to gingival sulcus of male Wistar rats and tissue samples were immunohistochemmically examined., Results: One day after application, infiltration of neutrophils and dilation of blood vessels in the gingival connective tissue were found. At day three, desquamation and detachment of cells in the junctional epithelium was observed. This abrasion of junctional epithelium was not observed in rats treated with mutated CDT, in which a His274Ala mutation is present in the CdtB subunit. This indicates the tissue abrasion may be caused by the genotoxicity of CdtB. Expression of the proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, was significantly suppressed using CDT treatment in the junctional epithelium and gingival epithelium., Conclusion: Using the rat model, these data suggest CDT intoxication induces cell cycle arrest and damage in periodontal epithelial cells in vivo., (© 2011 John Wiley & Sons A/S.)

Introduction: The current detection methods for periodontopathogens mainly use polymerase chain reactions. However, there are few methods available for visualizing the bacteria that impact on patients with periodontal disease for use in health education. The purpose of this study was to develop a specific detection method to visualize periodontopathogenic bacteria., Methods: Fluorescently-labeled oligonucleotide probes directed to specific 16S ribosomal RNA (rRNA) sequences of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were synthesized. Cultured individual bacterial species were fixed with 4% paraformaldehyde and smeared on glass slides. Fluorescein isothiocyanate-labeled oligonucleotide probes were hybridized under stringent conditions with smeared whole cells, and then probe specificity was investigated by epifluorescence microscopy., Results: Comparatively long (50-mer) oligonucleotide probes for P. gingivalis and A. actinomycetemcomitans were designed. These probes clearly hybridized with 16S rRNA of the target species in situ and single bacterial cells were detectable visually. The probes exhibited no cross-hybridization against the additional organisms that were closely related to the target species., Conclusions: The fluorescence in situ hybridization technique is a specific and reliable method by which to visually identify the target organisms. The oligonucleotide probes designed in this study will be useful for detecting P. gingivalis and A. actinomycetemcomitans populations.

We examined the cytotoxicity in culture medium of Actinobacillus actinomycetemcomitans against the human monocyte-macrophage-like cell line U937 using the trypan blue exclusion test and WST-1 test. We found that A. actinomycetemcomitans Y4 showed the highest cytotoxic activity among the three different serotype strains and the cytotoxic effects of both bacterial cells and culture supernatants in A. actinomycetemcomitans Y4 were stronger on phorbol-12-myristate 13-acetate (PMA)-induced U937 cells than uninduced U937 cells. Morphological changes in PMA-induced U937 cells treated with culture supernatants differed from those treated with leukotoxin, and a difference in the susceptibility to 56 degrees C heat treatment was found between culture supernatants and leukotoxin. The cytotoxic activity by WST-1 was determined more rapidly and strongly than that by trypan blue assay. These findings suggested that the cytotoxic effect of A. actinomycetemcomitans was influenced by the differentiation of U937 cells and may be more potent on the respiratory chain than the cell membrane.

We re-evaluated several antibiotics including newer ones, for their in vitro killing activity, as well as their inhibitory activity, against clinical isolates of periodontopathic bacteria. Tetracyclines were active against Porphyromonas gingivalis, and were highly active against Prevotella intermedia, but demonstrated only a low killing activity against Actinobacillus actinomycetemcomitans. Rokitamycin, a new macrolide, and clindamycin were highly active against P. gingivalis and P. intermedia, but showed very weak killing activity against A. actinomycetemcomitans. Quinolones demonstrated excellent bactericidal activity against A. actinomycetemcomitans, and good inhibitory and bactericidal activity against P. gingivalis and P. intermedia. Metronidazole had an activity almost equivalent to quinolones against P. gingivalis and P. intermedia; but it was the least active against A. actinomycetemcomitans.