Pilus PilA of the naturally competent HACEK group pathogen Aggregatibacter actinomycetemcomitans stimulates human leukocytes and interacts with both DNA and proinflammatory cytokines.

Each HACEK group pathogen, which can cause infective endocarditis, expresses type IVa pili. The type IVa major pilin PilA plays a role in bacterial colonization, virulence, twitching motility, and the uptake of extracellular DNA. The type IV prepilin homolog PilA of the periodontal pathogen A. actinomycetemcomitans (AaPilA) is linked to DNA uptake and natural competence. Our aim was to investigate the virulence properties and immunogenic potential of AaPilA. Since Neisseria meningitidis PilE, which shares sequence similarity with AaPilA, participates in sequestering host cytokines, we examined the ability of AaPilA to interact with various cytokines. Moreover, we investigated the structural characteristics of AaPilA with molecular modeling. AaPilA was conserved among A. actinomycetemcomitans strains. One of the 18 different natural variants, PilAD7S, is present in naturally competent strains. This variant interacted with DNA and bound interleukin (IL)-8 and tumor necrosis factor (TNF)-α. Specific anti-AaPilA antibodies were present in A. actinomycetemcomitans-positive periodontitis patient sera, and the production of reactive oxygen species from human neutrophils was less effectively induced by the ΔpilA mutant than by the wild-type strains. However, AaPilA did not stimulate human macrophages to produce proinflammatory cytokines, nor was it cytotoxic. The results strengthen our earlier hypothesis that the DNA uptake machinery of A. actinomycetemcomitans is involved in the sequestration of inflammatory cytokines. Furthermore, AaPilA stimulates host immune cells, such as B cells and neutrophils, making it a potential virulence factor.

Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake.

Naturally competent bacteria acquire DNA from their surroundings to survive in nutrient-poor environments and incorporate DNA into their genomes as new genes for improved survival. The secretin HofQ from the oral pathogen Aggregatibacter actinomycetemcomitans has been associated with DNA uptake. Cytokine sequestering is a potential virulence mechanism in various bacteria and may modulate both host defense and bacterial physiology. The objective of this study was to elucidate a possible connection between natural competence and cytokine uptake in A. actinomycetemcomitans. The extramembranous domain of HofQ (emHofQ) was shown to interact with various cytokines, of which IL-8 exhibited the strongest interaction. The dissociation constant between emHofQ and IL-8 was 43 nM in static settings and 2.4 µM in dynamic settings. The moderate binding affinity is consistent with the hypothesis that emHofQ recognizes cytokines before transporting them into the cells. The interaction site was identified via crosslinking and mutational analysis. By structural comparison, relateda type I KH domain with a similar interaction site was detected in the Neisseria meningitidis secretin PilQ, which has been shown to participate in IL-8 uptake. Deletion of hofQ from the A. actinomycetemcomitans genome decreased the overall biofilm formation of this organism, abolished the response to cytokines, i.e., decreased eDNA levels in the presence of cytokines, and increased the susceptibility of the biofilm to tested ß-lactams. Moreover, we showed that recombinant IL-8 interacted with DNA. These results can be used in further studies on the specific role of cytokine uptake in bacterial virulence without interfering with natural-competence-related DNA uptake.

A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1ß and interleukin-8.

Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1ß. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1ß and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI- mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1ß in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1ß internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

Environmental stimuli shape biofilm formation and the virulence of periodontal pathogens.

Periodontitis is a common inflammatory disease affecting the tooth-supporting structures. It is initiated by bacteria growing as a biofilm at the gingival margin, and communication of the biofilms differs in health and disease. The bacterial composition of periodontitis-associated biofilms has been well documented and is under continual investigation. However, the roles of several host response and inflammation driven environmental stimuli on biofilm formation is not well understood. This review article addresses the effects of environmental factors such as pH, temperature, cytokines, hormones, and oxidative stress on periodontal biofilm formation and bacterial virulence.

Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1ß. Some bacterial species can alter their physiological properties as a result of sensing IL-1ß. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1ß sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1ß through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1ß than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1ß, but this binding was not specific, as a control protein for IL-1ß also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1ß-binding surface-exposed lipoprotein that may be part of the bacterial IL-1ß-sensing system.

Interleukin-1ß is internalised by viable Aggregatibacter actinomycetemcomitans biofilm and locates to the outer edges of nucleoids.

The opportunistic pathogen Aggregatibacter actinomycetemcomitans causes periodontitis, which is a biofilm infection that destroys tooth-supportive tissues. Interleukin (IL)-1ß, a central proinflammatory cytokine of periodontitis, is an essential first line cytokine for local inflammation that modulates the cell proliferation and anti-pathogen response of human gingival keratinocytes. Previously, we demonstrated that A. actinomycetemcomitans biofilms bind IL-1ß; however, whether this binding is an active process is not known. In this study, we showed for the first time with immuno-electron microscopy that viable bacterial biofilm cells internalised IL-1ß when co-cultured with an organotypic mucosa. Decreased biofilm viability hindered the ability of biofilm to sequester IL-1ß and caused IL-1ß leakage into the culture medium. In some A. actinomycetemcomitans cells, intracellular IL-1ß localized to the outer edges of the nucleoids. We identified the DNA-binding protein HU as an IL-1ß interacting protein with mass spectroscopy and showed the interaction of recombinant HU and IL-1ßin vitro using enzyme-linked immunosorbent assay (ELISA). Close contact with a viable A. actinomycetemcomitans biofilm decreased the proliferation and apoptosis of human gingival keratinocytes as demonstrated using Ki-67 and the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining, respectively. Our results suggest that viable A. actinomycetemcomitans biofilms may disturb the critical first steps of local inflammation in periodontitis by binding and internalising IL-1ß. The interaction of IL-1ß with conserved HU provides a potential mechanism for shaping bacterial gene expression.

Fusobacterium nucleatum biofilm induces epithelial migration in an organotypic model of dento-gingival junction.

BACKGROUND: Effects of Fusobacterium nucleatum (F. nucleatum) biofilm on epithelial cell proliferation, apoptotic cell death, and basement membrane constituent collagen IV production were examined in an organotypic dento-epithelial (OD-E) model. METHODS: The OD-E model was constructed by seeding keratinocytes on fibroblast-containing collagen gels and by placing tooth pieces on top. A 3-day-old biofilm either a laboratory strain (American Type Culture Collection [ATCC] 25586) or a clinical strain (Anaerobe Helsinki Negative [AHN] 9508) of F. nucleatum was placed on the top of the model. The coculture was incubated ≤24 hours. The expression and localization of Ki-67, caspase-3, and collagen IV were examined by immunohistochemistry. RESULTS: Hematoxylin and eosin staining showed epithelial migration and lateral sprouting into the connective tissue matrix in F. nucleatum OD-E cocultures. The proliferation pattern of the in vitro dento-epithelial junction was changed. In controls without bacterial challenge, the Ki-67 expression was abundant in the cells attached to the tooth, whereas in F. nucleatum biofilm-treated cultures, the Ki-67-expressing cells were more often in the connective tissue-facing side of the epithelium. An apoptotic marker caspase-3 was expressed in controls and in F. nucleatum laboratory strain ATCC cocultures throughout the epithelium, in contrast to cultures treated with F. nucleatum clinical strain AHN, in which caspase-3 was absent. Collagen IV stainings were negative in both controls and F. nucleatum cocultures. CONCLUSION: F. nucleatum biofilm coculture with OD-E model causes lateral sprouting of the epithelium with an altered epithelial proliferation pattern, resembling the histologic changes seen in vivo in the early pathogenesis of periodontal disease.

Trimeric form of intracellular ATP synthase subunit ß of Aggregatibacter actinomycetemcomitans binds human interleukin-1ß.

Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1ß, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1ß receptor has been identified, current knowledge of the bacterial IL-1ß sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1ß than inactive ones. The effect of IL-1ß on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1ß to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1ß, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1ß and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1ß slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1ß. Our results suggest that IL-1ß might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit ß interacted with IL-1ß, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1ß during inflammation.