Joseph M. DiRienzo

Functional studies of the recombinant subunits of a cytolethal distending holotoxin

Cytolethal distending toxin (CDT) is a multicomponent bacterial holotoxin that targets most eukarytotic cells causing distension and cell cycle arrest. A number of diverse pathogenic bacterial species associated with diarrhoea, chancroid, chronic hepatitis and periodontal disease produce a CDT. Synthesis of the holotoxin is directed by the expression of three genes, cdtA, cdtB and cdtC. Although the product of the CdtB gene was previously identified as a type I deoxyribonuclease, the functions of the cdtA and cdtC products have not been characterized. Using the periodontal pathogen, Actinobacillus actinomycetemcomitans, we demonstrate that the recombinant product of the CdtA gene binds to the surface of Chinese hamster ovary (CHO) cells. This protein did not induce distension or cytotoxicity when introduced into the cytosol using a lipid‐based protein delivery system. Recombinant CdtB and CdtC proteins failed to bind to CHO cells. However, the delivery of either CdtB or CdtC into the cytosol resulted in the characteristic pattern of distension followed by cell death. Based on these results, it appears that the CdtA protein subunit alone is responsible for anchoring the holotoxin to the cell surface. The CdtC subunit, in concert with CdtB, contributes to the cytotoxic activities of the holotoxin. The specific mechanism of CdtC cytotoxicity is currently unknown.

Characterization of point mutations in the cdtA gene of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans

The Cdt is a family of gram‐negative bacterial toxins that typically arrest eukaryotic cells in the G0/G1 or G2/M phase of the cell cycle. The toxin is a heterotrimer composed of the cdtA, cdtB and cdtC gene products. Although it has been shown that the CdtA protein subunit binds to cells in culture and in an enzyme‐linked immunosorbent assay (CELISA) the precise mechanisms by which CdtA interacts with CdtB and CdtC has not yet been clarified. In this study we employed a random mutagenesis strategy to construct a library of point mutations in cdtA to assess the contribution of individual amino acids to binding activity and to the ability of the subunit to form biologically active holotoxin. Single unique amino acid substitutions in seven CdtA mutants resulted in reduced binding of the purified recombinant protein to Chinese hamster ovary cells and loss of binding to the fucose‐containing glycoprotein, thyroglobulin. These mutations clustered at the 5′‐ and 3′‐ends of the cdtA gene resulting in amino acid substitutions that resided outside of the aromatic patch region and a conserved region in CdtA homologues. Three of the amino acid substitutions, at positions S165N (mutA81), T41A (mutA121) and C178W (mutA221) resulted in gene products that formed holotoxin complexes that exhibited a 60% reduction (mutA81) or loss (mutA121, mutA221) of proliferation inhibition. A similar pattern was observed when these mutant holotoxins were tested for their ability to induce cell cycle arrest and to convert supercoiled DNA to relaxed and linear forms in vitro. The mutations in mutA81 and mutA221 disrupted holotoxin formation. The positions of the amino acid substitutions were mapped in the Haemophilus ducreyi Cdt crystal structure providing some insight into structure and function.

Localization of Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Subunits during Intoxication of Live Cells

ABSTRACT The cytolethal distending toxin (Cdt), produced by some clinically important Gram-negative bacterial species, is related to the family of AB-type toxins. Three heterologous proteins (CdtA, CdtB, and CdtC) and a genotoxin mode of action distinguish the Cdt from others in this toxin class. Crystal structures of several species-specific Cdts have provided a basis for predicting subunit interactions and functions. In addition, empirical studies have yielded significant insights into the in vivo interactions of the Cdt subunits. However, there are still critical gaps in information about the intoxication process. In this study, a novel protein tagging technology was used to localize the subunits in Chinese hamster ovary cells (CHO-K1). A tetracysteine motif was engineered in each subunit, and in subunits with mutations in predicted functional domains, to permit detection with the fluorescein arsenical hairpin binding (FlAsH) dye Lumio green. Live-cell imaging, in conjunction with confocal microscopy, was used to capture the locations of the individual subunits in cells intoxicated, under various conditions, with hybrid heterotrimers. Using this approach, we observed the following. (i) The CdtA subunit remains on the cell surface of CHO cells in association with cholesterol-containing and cholesterol-depleted membrane. (ii) The CdtB subunit is exclusively in the cytosol and, after longer exposure times, localizes to the nucleus. (iii) The CdtC subunit is present on the cell surface and, to a greater extent, in the cytosol. These observations suggest that CdtC, but not CdtA, functions as a chaperone for CdtB entry into cells.

Evaluation of the humoral immune response to the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans Y4 in subjects with localized aggressive periodontitis

INTRODUCTION Cytolethal distending toxin (Cdt) is potentially one of several virulence factors of Aggregatibacter actinomycetemcomitans, the prime etiological agent of localized aggressive periodontitis (LAP). Little is known regarding the Cdt-specific antibody response in humans. The current study is a quantitative and qualitative evaluation of the toxin-specific antibody response in a cohort of LAP patients and age-, race- and sex-matched controls. METHODS Ninety-five subjects provided a total of 692 serum samples. Sera were analysed by enzyme-linked immunosorbent assays to determine the titers of antibody against the intact Cdt holotoxin as well as the individual subunit proteins (CdtA, CdtB, and CdtC). Neutralization of growth inhibition mediated by Cdt was evaluated in a modified colony-forming assay using Chinese hamster ovary cells. RESULTS Fourteen of the 95 subjects exhibited significant serum Cdt-binding activity. There were no differences in the percentages of seropositive individuals or in the mean antibody titers between the control and LAP groups. Binding activity was detected against each of the three Cdt subunit proteins in all of the positive samples. Neutralization of Cdt-mediated growth inhibition was detected in samples from all of the seropositive subjects (range 20-75%). CONCLUSIONS Cdt, a recently identified A. actinomycetemcomitans virulence factor, is capable of inducing a neutralizing antibody response indicating that the toxin is produced during natural infection of humans. The failure of a vast majority (20 of 23) of the LAP subjects to mount a significant anti-Cdt response may in part explain their relative susceptibility to the disease.

Association of pharyngitis with oral antibiotic use for the treatment of acne: A cross sectional and prospective cohort study

OBJECTIVE To prospectively evaluate the association between antibiotics used to treat acne and pharyngitis. DESIGN Cross-sectional and 9-month prospective cohort. SETTING Urban university setting. PARTICIPANTS University students. INTERVENTION Participants were asked to fill out a survey form, were swabbed for culture, and had a visual examination for acne. MAIN OUTCOME MEASURE Report of pharyngitis. RESULTS In the cross-sectional study, 10 of the 15 students receiving oral antibiotics for acne reported an episode of pharyngitis in the previous 30 days, whereas 47 of the 130 students not receiving oral antibiotics, but who had acne, reported an episode of pharyngitis in the prior month. The unadjusted odds ratio (OR) (95% CI) associating current oral antibiotic use in acne patients with a self-reported episode of pharyngitis was 3.53 (95% CI, 1.14-10.95). In the cohort study, there were 358 female and 218 male participants; 36 (6.2%) received oral antibiotics for acne during the study, and 96 (16.6%) received topical antibiotics for acne. Using mixed model logistic regression, the OR was 4.34 (95% CI, 1.51-12.47) associating oral antibiotic use with pharyngitis. Less than 1% of participants were colonized by group A streptococcus, which was not associated with pharyngitis. CONCLUSIONS Our studies show that that the odds of reporting pharyngitis is more than 3 times baseline in patients receiving oral antibiotics for acne vs those who are not receiving oral antibiotics. The true clinical importance of these findings needs to be evaluated further by prospective studies, but this finding is not associated with group A streptococcus.

Cytolethal Distending Toxin Damages the Oral Epithelium of Gingival Explants

The cytolethal distending toxin (Cdt), expressed by the periodontal pathogen Aggregatibacter actinomycetemcomitans, inhibits the proliferation of cultured epithelial cells by arresting the cell cycle. The gingival epithelium is an early line of defense against microbial assault. When damaged, bacteria collectively gain entry into underlying connective tissue where microbial products can affect infiltrating inflammatory cells, leading to the destruction of the attachment apparatus. Histological evaluation of rat and healthy human gingival tissue exposed ex vivo to the Cdt for 36 and 18 hours, respectively, revealed extensive detachment of the keratinized outer layer and distention of spinous and basal cells in the oral epithelium. Treated human tissue also exhibited disruption of rete pegs and dissolution of cell junctions. Cells in the connective tissue appeared unaffected. Primary gingival epithelial cells, but not gingival fibroblasts, isolated from the same healthy human tissue were cell-cycle-arrested when treated with the toxin. These findings provide new evidence that the Cdt severely damages the oral epithelium, ex vivo, by specifically targeting epithelial cells, in situ. The Cdt shows preferential targeting of the epithelium as opposed to connective tissue in animal and human gingival explant models. Abbreviations: cytolethal distending toxin (Cdt), connective tissue (CT), 4′,6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), junctional epithelium (JE), oral epithelium (OE), rete pegs (RP), sulcular epithelium (SE)

Role of aromatic amino acids in receptor binding activity and subunit assembly of the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans.

ABSTRACT The periodontal pathogen Aggregatibacter actinomycetemcomitans produces a cytolethal distending toxin (Cdt) that inhibits the proliferation of oral epithelial cells. Structural models suggest that the CdtA and CdtC subunits of the Cdt heterotrimer form two putative lectin domains with a central groove. A region of CdtA rich in heterocyclic amino acids (aromatic patch) appears to play an important role in receptor recognition. In this study site-specific mutagenesis was used to assess the contributions of aromatic amino acids (tyrosine and phenylalanine) to receptor binding and CdtA-CdtC assembly. Predominant surface-exposed aromatic residues that are adjacent to the aromatic patch region in CdtA or are near the groove located at the junction of CdtA and CdtC were studied. Separately replacing residues Y105, Y140, Y188, and Y189 with alanine in CdtA resulted in differential effects on binding related to residue position within the aromatic region. The data indicate that an extensive receptor binding domain extends from the groove across the entire face of CdtA that is oriented 180° from the CdtB subunit. Replacement of residue Y105 in CdtA and residues Y61 and F141 in CdtC, which are located in or at the periphery of the groove, inhibited toxin assembly. Taken together, these results, along with the lack of an aromatic amino acid-rich region in CdtC similar to that in CdtA, suggest that binding of the heterotoxin to its cell surface receptor is mediated predominantly by the CdtA subunit. These findings are important for developing strategies designed to block the activity of this prominent virulence factor.

The cytolethal distending toxin of Aggregatibacter actinomycetemcomitans inhibits macrophage phagocytosis and subverts cytokine production.

Aggregatibacter actinomycetemcomitans is an important periodontal pathogen that can participate in periodontitis and other non-oral infections. The cytolethal distending toxin (Cdt) is among the virulence factors produced by this bacterium. The Cdt is also secreted by several mucosa-associated Gram-negative pathogens and may play a role in perpetuating the infection by modulating the immune response. Although the toxin targets a wide range of eukaryotic cell types little is known about its activity on macrophages which play a key part in alerting the rest of the immune system to the presence of pathogens and their virulence factors. In view of this, we tested the hypothesis that the A. actinomycetemcomitans Cdt (AaCdt) disrupts macrophage function by inhibiting phagocytic activity as well as affecting the production of cytokines. Murine macrophages were co-cultured with either wild-type A. actinomycetemcomitans or a Cdt(-) mutant. Viable counts and qPCR showed that phagocytosis of the wild-type strain was significantly reduced relative to that of the Cdt(-) mutant. Addition of recombinant Aa(r)Cdt to co-cultures along with the Cdt(-) mutant diminished the phagocytic activity similar to that observed with the wild type strain. High concentrations of Aa(r)Cdt resulted in decreased phagocytosis of fluorescent bioparticles. Nitric oxide production was modulated by the presence of Cdt and the levels of IL-1β, IL-12 and IL-10 were increased. Production of TNF-α did not differ in the co-culture assays but was increased by the presence of Aa(r)Cdt. These data suggest that the Cdt may modulate macrophage function in A. actinomycetemcomitans infected sites by impairing phagocytosis and modifying the pro-inflammatory/anti-inflammatory cytokine balance.

Cell Junction Remodeling in Gingival Tissue Exposed to a Microbial Toxin

The gingival epithelium plays a key role in protecting the supporting structures of the teeth from bacteria and their products. In ex vivo experiments, we recently showed that the cytolethal distending toxin (Cdt) from the periodontal pathogen Aggregatibacter actinomycetemcomitans causes extensive damage to gingival tissue. Morphological changes included detachment of the keratinized outer layer, distention of spinous and basal cells in the oral epithelium, disruption of rete pegs, and apparent dissolution of cell junctions. Adherens junctions (zonula adherens) are essential for maintaining barrier function and integrity of gingival epithelium. Therefore, immunohistochemical and RT-PCR analyses of human gingival explants (HGX) and human gingival epithelial cells (HGEC) were utilized for a closer examination of the effects of the Cdt on E-cadherin, the key membrane component of adherens junctions. Although there was some variability among tissue donors, exposure of gingival tissue or isolated epithelial cells to the toxin generally resulted in a pronounced increase in the expression and cytosolic distribution of E-cadherin, accompanied by an increase in levels of the intracellular scaffolding proteins β-catenin and β-actin. These results indicate that the Cdt induced substantial remodeling of adherens junctions, with a potential impact on the barrier function of gingival epithelium. Abbreviations: cytolethal distending toxin (Cdt), 4′,6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), transepithelial resistance (TER).

Breaking the Gingival Epithelial Barrier: Role of the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin in Oral Infectious Disease

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is part of the HACEK group that causes infective endocarditis, a constituent of the oral flora that promotes some forms of periodontal disease and a member of the family of species that secrete a cytolethal distending toxin (Cdt). The family of bacteria that express the cdt genes participate in diseases that involve the disruption of a mucosal or epithelial layer. In vitro studies have shown that human gingival epithelial cells (HGEC) are native targets of the Cdt that typically induces DNA damage that signals growth arrest at the G2/M interphase of the cell cycle. The gingival epithelium is an early line of defense in the oral cavity against microbial assault. When damaged, bacteria collectively gain entry into the underlying connective tissue where microbial products can affect processes and pathways in infiltrating inflammatory cells culminating in the destruction of the attachment apparatus of the tooth. One approach has been the use of an ex vivo gingival explant model to assess the effects of the Cdt on the morphology and integrity of the tissue. The goal of this review is to provide an overview of these studies and to critically examine the potential contribution of the Cdt to the breakdown of the protective gingival barrier.