Aggregatibacter actinomycetemcomitans Biofilm Reduces Gingival Epithelial Cell Keratin Expression in an Organotypic Gingival Tissue Culture Model.

Epithelial cells express keratins, which are essential for the structural integrity and mechanical strength of the cells. In the junctional epithelium (JE) of the tooth, keratins such as K16, K18, and K19, are expressed, which is typical for non-differentiated and rapidly dividing cells. The expression of K17, K4, and K13 keratins can be induced by injury, bacterial irritation, smoking, and inflammation. In addition, these keratins can be found in the sulcular epithelium and in the JE. Our aim was to estimate the changes in K4, K13, K17, and K19 expression in gingival epithelial cells exposed to Aggregatibacter actinomycetemcomitans. An organotypic gingival mucosa and biofilm co-culture was used as a model system. The effect of the biofilm after 24 h was assessed using immunohistochemistry. The structure of the epithelium was also studied with transmission electron microscopy (TEM). The expression of K17 and K19, as well as total keratin expression, decreased in the suprabasal layers of epithelium, which were in close contact with the A. actinomycetemcomitans biofilm. The effect on keratin expression was biofilm specific. The expression of K4 and K13 was low in all of the tested conditions. When stimulated with the A. actinomycetemcomitans biofilm, the epithelial contact site displayed a thick necrotic layer on the top of the epithelium. The A. actinomycetemcomitans biofilm released vesicles, which were found in close contact with the epithelium. After A. actinomycetemcomitans irritation, gingival epithelial cells may lose their resistance and become more vulnerable to bacterial infection.

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.

Extracellular citrullination inhibits the function of matrix associated TGF-ß.

In inflammatory arthritis peptidyl arginine deiminase (PAD) enzymes can citrullinate arginine residues in extracellular matrix (ECM) proteins, such as collagens and fibronectin. This may lead to the generation of anti-citrullinated protein antibodies, important diagnostic markers in rheumatoid arthritis. In addition, the citrullination may directly affect protein function. Based on structural analysis, we found that most ECM-associated growth factors (GFs) have arginine residues in their receptor recognition sites. Thus, they are potential functional targets of extracellular citrullination. To examine this further, we focused on the citrullination of transforming growth factor-ßs (TGF-ß), well-known ECM-associated GFs. PAD-treatment of CHO-LTBP1 cell derived matrix, rich with TGF-ß, decreased the level of TGF-ß activity as detected by HaCaT and MLEC-PAI-1/Lu reporter cells. Additional experiments indicated that PAD-treatment inhibits the integrin-mediated TGF-ß activation since PAD-treatment decreased the binding of integrin αVß6 ectodomain as well as integrin-mediated spreading of MG-63 and HaCaT cells to ß1-latency associated peptide (TGF-ß1 LAP). The citrullination of the RGD site, an important integrin recognition motif, was confirmed by mass spectrometry. Furthermore, the citrullination of active TGF-ß1 inhibited its binding to recombinant TGF-ß receptor II, and prevented its ability to activate TGF-ß signaling. Thus, extracellular PAD activity can affect the function of ECM-associated growth factors by different mechanisms. Importantly, the citrullination of both latent and active TGF-ß has the potency to regulate the inflammatory process.