The cytolethal distending toxin of Aggregatibacter actinomycetemcomitans inhibits macrophage phagocytosis and subverts cytokine production
Introduction
The periodontal pathogen Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis. This bacterium, or its products contributes to destruction of the tooth-supporting alveolar bone by triggering a host immune response [1]. Furthermore, there is evidence suggesting that the oral cavity is a microbial reservoir for various systemic infections. A. actinomycetemcomitans can cross the mucosal epithelial barrier, accessing the bloodstream, and has also been detected in atheroma plaques, bacteremia, pericarditis, septicemia, pneumonia and infectious arthritis [2]. It is the most prevalent member of the HACEK group, comprised of Gram-negative facultative bacteria associated with endocarditis [3].
A. actinomycetemcomitans expresses a variety of potential virulence factors, including the cytolethal distending toxin (Cdt), which is composed by three subunits: CdtA, CdtB and CdtC [4]. The enzymatically active CdtB subunit, shares structural and functional homology with mammalian deoxyribonucleaseI (DNase I) [5]. The CdtA and CdtC subunits are required for binding the holotoxin to the plasma membrane of target cells. CdtB-CdtC dimers enters the cells [6] and CdtB then, translocates to the nucleus where it induces DNA lesions [7]. This DNA damage leads to cell cycle arrest at the G2/M interphase of target cells [6], [8], [9], [10]. In addition, CdtB contains a phosphatidylinositol-3,4,5-triphosphate phosphatase activity, leading to cell cycle arrest and apoptosis in some types of cells [11]. Cdt is produced by a variety of Gram-negative mucosa-associated pathogens [12], and data suggest that this toxin may affect interactions between the bacterium and the host immune system in chronic diseases. The Campylobacter jejuni CdtB null mutant leads to a less persistent colonization in mice, and results in an attenuated inflammatory effect when compared to the wild type [13]. A recent study demonstrated that the exposure to sublethal doses of Cdt lead to phenotypic propriety of malignancy of epithelial cells and fibroblasts, without alterations in cell cycle distribution or decrease of cell viability [14]. Sublethal doses of Cdt were shown to induce the production of IL-1β, IL-6 and IL-8 by human monocytes [15]. Furthermore, a recombinant Aa(r)Cdt up-regulated the expression of receptor activator of NFκB ligand (RANKL) in gingival/periodontal ligament and Jurkat T cells [16], [17], [18].
Phagocytic cells of the innate immune system, such as macrophages, act to eliminate microbial pathogens from the bloodstream and tissues. Once recognized, a microbe is engulfed by the phagocyte, triggering the secretion of pro-inflammatory cytokines and chemokines, recruiting additional immune cells and activating the adaptive arm of the immune system. Macrophages activated by microorganisms and their components can be differentiated as M1 (classic activation), producing pro-inflammatory cytokines, such as IL-1, TNFα and IL-12, driving the response to lymphocytes T helper 1 (Th1) type, while the alternative activated macrophages (M2) induce the production of regulatory cytokine such as IL-10 and lead to a Th2 response [19]. Evidence that phagocytic cells could be targeted by Cdt came from studies showing that purified toxin from Haemophilus ducreyi and C. jejuni induced apoptosis in non-proliferating dendritic cells (DCs) and macrophages [20], [21]. Moreover, recombinant Escherichia coli supernatant containing Aa(r)Cdt inhibited the production of NO by murine macrophages [22] and proliferating and non-proliferating U937 macrophages were distinctly affected by AaCdt [23]. However, the specific activities of Cdt for these cell types were not fully understood. Thus, this study aimed to test the hypothesis that Cdt would affect macrophage activation, leading to changes in phagocytosis and production of NO and cytokines, consequently altering the host response in A. actinomycetemcomitans infected cells.
Section snippets
Cell culture
Raw 264.7 murine macrophages were grown in Dulbecco’s modified Eagle’s medium [DMEM (Gibco, Grand Island, NY)] supplemented with 10% fetal calf serum (FCS)and sodium bicarbonate (2.2 g/ml). Penicillin (1664 U/ml) and streptomycin (745 U/ml) were added to cultures and incubated at 37 °C with 5% CO2 in a fully humidified atmosphere.
Bacteria strains and growth conditions
A. actinomycetemcomitans D7S-SA is a spontaneously occurring non-fimbriated (smooth) derivative of the serotype a clinical isolate D7S1 [24], [25]. Strain D7S-SA CHE001 is
Cdt affects phagocytosis by Raw 264.7 macrophages
The percentage of viable internalized A. actinomycetemcomitans D7S-SA was significantly lower that of its isogenic Cdt-deficient strain D7S-SA CHE001 after 2 h of co-culture with Raw 246.7 cells (Fig. 1A). These data were confirmed by qPCR (Fig. 1B).
No viable intracellular bacteria were observed after 20 h of co-culture with the wild-type and mutant A. actinomycetemcomitans strains. However, a higher percentage of intracellular non-viable bacterial of the Cdt-deficient mutant cells were detected
Discussion
Macrophages play a central role in inflammatory reactions initiated by pathogenic bacteria. Many pathogens have evolved specific mechanisms to avoid, alter, or disable the antimicrobial effects of macrophages. Our previous data indicated that AaCdt would modulate macrophages function by inhibiting NO production [31].
The present study provides evidence that Cdt may impair phagocytosis by Raw 264.7 cells. Data were obtained not only by comparing phagocytosis in co-cultures with the Cdt-deficient
Conclusion
Taken together, our data indicated that the presence of Cdt may be an important strategy used by A. actinomycetemcomitans to thwart an effective immune response during a infection, leading to a decrease in phagocytosis. Additionally, A. actinomycetemcomitans Cdt modulates the immune response indirectly by manipulating cytokines production by macrophages.
Acknowledgements
This work was supported by FAPESP Grants: 09/54178-0 and 09/54849-1, NIDCR Grant R01 DE12212 (CC) and an USPHS Grant DE012593 (JMD) from the National Institutes of Health.
References (53)
- et al.
Actinobacillus actinomycetemcomitans endocarditis
Clin Microbiol Infect
(2004) - et al.
Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin
Cytokine
(2005) - et al.
Interactions of Haemophilus ducreyi and purified cytolethal distending toxin with human monocyte-derived dendritic cells, macrophages and CD4+ T cells
Microbes Infect
(2004) - et al.
Nitric oxide production by a murine macrophage cell line (RAW264.7 cells) stimulated with Aggregatibacter actinomycetemcomitans surface-associated material
Anaerobe
(2011) - et al.
Aggregatibacter actinomycetemcomitans targets NLRP3 and NLRP6 inflammasome expression in human mononuclear leukocytes
Cytokine
(2012) - et al.
IL-1-induced receptor activator of NF-kappa B ligand in human periodontal ligament cells involves ERK-dependent PGE2 production
Bone
(2005) Cytokines that promote periodontal tissue destruction
J Periodontol
(2008)- et al.
Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in nonoral infections
Periodontol
(2000) - et al.
Identification of a cytolethal distending toxin gene locus and features of a virulence-associated region in Actinobacillus actinomycetemcomitans
Infect Immun
(1999) - et al.
DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest
Mol Microbiol
(2000)
Localization of Aggregatibacter actinomycetemcomitans cytolethal distending toxin subunits during intoxication of live cells
Infect Immun
CdtA, CdtB, and CdtC form a tripartite complex that is required for cytolethal distending toxin activity
Infect Immun
Chronic exposure to the cytolethal distending toxins of Gram-negative bacteria promotes genomic instability and altered DNA damage response
Cell Microbiol
Campylobacter jejuni cytolethal distending toxin promotes DNA repair responses in normal human cells
Infect Immun
Carbohydrate-binding specificity of the Escherichia coli cytolethal distending toxin CdtA-II and CdtC-II subunits
Infect Immun
A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5-triphosphate phosphatase activity
J Immunol
Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages
Microbiology
Characterisation of cytolethal distending toxin (CDT) mutants of Campylobacter jejuni
J Med Microbiol
Chronic exposure to the cytolethal distending toxins of Gram-negative bacteria promotes genomic instability and altered DNA damage response
Cell Microbiol
Recombinant Actinobacillus actinomycetemcomitans cytolethal distending toxin proteins are required to interact to inhibit human cell cycle progression and to stimulate human leukocyte cytokine synthesis
Infect Immun
Cytolethal distending toxin upregulates RANKL expression in Jurkat T-cells. APMIS
APMIS: Acta Pathol., Microbiol., Immunol. Scand.
The cytolethal distending toxin induces receptor activator of NF-kappaB ligand expression in human gingival fibroblasts and periodontal ligament cells
Infect Immun
Exploring the full spectrum of macrophage activation
Nat Rev Immunol
Intracellular survival of Campylobacter jejuni in human monocytic cells and induction of apoptotic death by cytholethal distending toxin
Infect Immun
Inhibition of interferon-gamma-induced nitric oxide production in endotoxin-activated macrophages by cytolethal distending toxin
Oral Microbiol Immunol
Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces apoptosis in nonproliferating macrophages by a phosphatase-independent mechanism
Infect Immun
Cited by (36)
Helicobacters and cancer, not only gastric cancer?
2022, Seminars in Cancer BiologyCitation Excerpt :CDT also inhibits macrophage phagocytosis and modifies the pro-inflammatory/anti-inflammatory cytokine balance [179]. Moreover, the presence of CDT is meaningful for bacterial proliferation [179], as observed in mice for long-term colonization of H. hepaticus [161,162]. Some cells with persistent DSBs survive the acute phase of CDT intoxication and enter senescence [180,176], a long-term cell cycle arrest.
Oral and intestinal bacterial exotoxins: Potential linked to carcinogenesis
2020, Progress in Molecular Biology and Translational ScienceFunctionality of the Interleukin 8 haplotypes in lymphocytes and macrophages in response to gram-negative periodontopathogens
2019, GeneCitation Excerpt :In contrast, the Hap-2-lymphocytes stimulated with Aa showed higher IL12 gene expression. It is possible that increased expression of TNFA inhibited IL12 gene expression by Aa-stimulated macrophages as part of negative feedback and self-limiting mechanisms controlling cytokine gene expression (Ando-Suguimoto et al., 2014). In infectious conditions, such as periodontal disease, phagocytosis, production of nitric oxide (NO) and oxidative burst with the production of reactive oxygen species (ROS) are important in the removal and killing of pathogens (Trinchieri and Gerosa, 1996).
Role and mechanism of the nod-like receptor family pyrin domain-containing 3 inflammasome in oral disease
2019, Archives of Oral BiologyCitation Excerpt :Thus, it is tempting to speculate that the involvement of K+ efflux and ATP/purinergic receptors 7 signalling by leukotoxin may participate in inflammasome-dependent IL-1β secretion in periodontitis. Cytolethal distending toxin, a family of heat-labile protein cytotoxins, regulates A. actinomycetemcomitans infection site function in macrophages by reducing phagocytosis and interfering with the secretion of pro-inflammatory/anti-inflammatory cytokines (Ando-Suguimoto et al., 2014). A study reported that cytolethal distending toxin activates the NLRP3 inflammasome in human monocytic cell lines, leading to the release of pro-inflammatory cytokines, including IL-1β, IL-6 and tumour necrosis factor-α, within 5 h and IL-18 within 48 h.
Oral pathogenesis of Aggregatibacter actinomycetemcomitans
2017, Microbial PathogenesisCitation Excerpt :These differences illustrate that CDT may be expressed by the infecting strains differently, and anti-cdtC antibody production might require the colonization of multiple sites [70]. Another function of CDT is the disruption of macrophage functions by preventing phagocytic activity and modifying the cytokines' balance [71]. In this process, the production level of cytokine (e.g. IL-1β, IL-10, and IL-12) increases, nitric oxide production is modulated, and the level of TNF-α does not differ by the presence of CDT in macrophage cells [71].