The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors. Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g., Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway in Vibrio harveyi. Here, we report that the periodontal pathogen Actinobacillus actinomycetemcomitans expresses a homolog of V. harveyi luxS and secretes an AI-2-like signal. Cell-free conditioned medium from A. actinomycetemcomitans or from a recombinant Escherichia coli strain (E. coli AIS) expressing A. actinomycetemcomitans luxS induced luminescence in V. harveyi BB170 >200-fold over controls. AI-2 levels peaked in mid-exponential-phase cultures of A. actinomycetemcomitans and were significantly reduced in late-log-and stationary-phase cultures. Incubation of early-log-phase A. actinomycetemcomitans cells with conditioned medium from A. actinomycetemcomitans or from E. coli AIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide. In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth from E. coli AIS ؊ , a recombinant strain in which luxS was insertionally inactivated. A. actinomycetemcomitans AI-2 also induced expression of afuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition by A. actinomycetemcomitans. Finally, A. actinomycetemcomitans AI-2 added in trans complemented a luxS knockout mutation in P. gingivalis by modulating the expression of the luxS-regulated genes uvrB and hasF in this organism. Together, these results suggest that LuxS-dependent signaling may modulate aspects of virulence and the uptake of iron by A. actinomycetemcomitans and induce responses in other periodontal organisms in mixed-species oral biofilm.

The innate immune response is critical for the epithelial antimicrobial barrier. The human ␤-defensins are small, cationic antimicrobial peptides that are made by epithelial cells and that play a role in mucosal and skin defenses. Human ␤-defensin 1 (hBD-1) is expressed constitutively in epithelial tissues, whereas hBD-2 and hBD-3 are expressed in response to bacterial stimuli or inflammation. Previous studies showed that hBD-2 was induced by Fusobacterium nucleatum cell wall extract without the involvement of the NF-B transcription factors, which typically are associated with innate immunity and inflammation. The goal of this study was to characterize signaling pathways involved in hBD-2 induction in response to commensal and pathogenic bacteria. Cultured human oral and foreskin keratinocytes were treated separately with inhibitors of NF-B, c-Jun N-terminal kinase (JNK), and p38 and then stimulated with oral commensal Streptococcus gordonii, oral pathogens Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, skin commensal Staphylococcus epidermidis, or skin pathogen Streptococcus pyogenes. Different bacteria induced different levels of hBD-2 and in response to the various inhibitors tested, although certain common patterns were observed for commensal-and pathogen-stimulated cells. hBD-2 induction by all bacteria tested was partially or completely blocked by inhibitors of the JNK and p38 pathways. However, in addition, hBD-2 induction by pathogenic bacteria in both oral and foreskin keratinocytes was blocked by inhibitors of NF-B. The results indicate that commensal and pathogenic bacteria utilize different pathways in hBD-2 induction and suggest that epithelial cells from different body sites have common signaling mechanisms to distinguish between commensal and pathogenic bacteria.

Greater levels of dissolved titanium were detected in submucosal plaque around implants with peri-implantitis compared with healthy implants, indicating an association between titanium dissolution and peri-implantitis. Factors triggering titanium dissolution, as well as the role of titanium corrosion in the peri-implant inflammatory process, warrant further investigation.

The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an argininespecific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.Quorum sensing, the density-dependent regulation of gene expression, is widespread among both gram-negative and gram-positive bacteria. Quorum sensing involves the synthesis and detection of extracellular signaling molecules termed autoinducers (AIs) (2, 13). Quorum sensing in gram-negative bacteria was first described for the marine symbiotic organism Vibrio fischeri. The number of acyl homoserine lactone (HSL) AI molecules in a given culture of V. fischeri increases as the cell density increases, and once a critical concentration of AI is reached, a signal transduction cascade that leads to the production of bioluminescence by cells is initiated (15). Components of this system include LuxI, an acyl HSL synthase that directs synthesis of 3-oxo-hexanoyl-HSL (V. fischeri AI-1); AinS, an acyl HSL synthase that catalyzes the synthesis of octanoyl-HSL (V. fischeri AI-2); and LuxR, a transcriptional activator necessary for responses to V. fischeri AI-1 (10). Homologues of the luxI and luxR genes of V. fischeri have been described now for a range of gram-negative bacteria and are responsible for the density-dependent regulation of quite diverse physiological functions (1,2,13,30,41). Light production by Vibrio harveyi is similarly under the control of quorumsensing systems, however the bioluminescence genes are not regulated by homologues of the V. fischeri LuxI and LuxR proteins (3, 4). Rather, in V. harveyi, quorum sensing involves two parallel regulatory systems. Signaling system 1 is dependent on two genes, luxL and luxM, for the synthesis of N-3-hydroxybutanoyl-L-HSL (V. harveyi AI-1), and signal detection is mediated by the sensor kinase LuxN (3,26). LuxM shows sequence homology to V. fischeri AinS (10). Signaling system 2 requires the luxS gene for the synthesis of V. harveyi AI-2, a non-HSL AI, the structure of which is unknown (41, 42). The primary sensor for V. harveyi AI-2 is thought to be LuxP, and the LuxP-AI-2 complex interacts with LuxQ to initiate signal transduction (4, 26). Signals from both LuxN and LuxQ feed i...

Gingival epithelia utilize multiples signaling pathways to regulate innate immune responses to various oral bacteria, but little is understood about how these bacteria alter epithelial epigenetic status. Here we report DNA methyltransferase (DNMT1) and histone deacetylase expressions were decreased in gingival epithelial cells treated with oral pathogen Porphyromonas gingivalis and non-pathogen Fusobacterium nucleatum. Pretreatment with Trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins hBD2 and CCL20 in response to both bacterial challenges. Pretreatment with DNMT inhibitor 5-Azacytidine increased hBD2 and CCL20 expressions in response to F. nucleatum, but not to P. gingivalis. Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs. F. nucleatum. This study provides a new insight into the bacteria-specific innate immune responses via epigenetic regulation.