Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.

Non-conventional therapeutics for oral infections.

As our knowledge of host-microbial interactions within the oral cavity increases, future treatments are likely to be more targeted. For example, efforts to target a single species or key virulence factors that they produce, while maintaining the natural balance of the resident oral microbiota that acts to modulate the host immune response would be an advantage. Targeted approaches may be directed at the black-pigmented anaerobes, Porphyromonas gingivalis and Prevotella intermedia, associated with periodontitis. Such pigments provide an opportunity for targeted phototherapy with high-intensity monochromatic light. Functional inhibition approaches, including the use of enzyme inhibitors, are also being explored to control periodontitis. More general disruption of dental plaque through the use of enzymes and detergents, alone and in combination, shows much promise. The use of probiotics and prebiotics to improve gastrointestinal health has now led to an interest in using these approaches to control oral disease. More recently the potential of antimicrobial peptides and nanotechnology, through the application of nanoparticles with biocidal, anti-adhesive and delivery capabilities, has been explored. The aim of this review is to consider the current status as regards non-conventional treatment approaches for oral infections with particular emphasis on the plaque-related diseases.

Identification of organic acids in Cichorium intybus inhibiting virulence-related properties of oral pathogenic bacteria.

The low molecular mass (LMM) extract of Cichorium intybus var. silvestre (red chicory) has been shown to inhibit virulence-linked properties of oral pathogens including Streptococcus mutans, Actinomyces naeslundii and Prevotella intermedia. In the present study HPLC-DAD-ESI/MS(2) was used to investigate the compounds contained in this extract for their anti-virulence activity. The extract contained a number of components, including oxalic, succinic, shikimic and quinic acids, which interfere with the growth and virulence traits (i.e., biofilm formation, adherence to epithelial cells and hydroxyapatite) of oral pathogens involved in gingivitis and tooth decay. Succinic and quinic acid seem to be the most potent, mainly by interfering with the ability of oral pathogens to form biofilms (either through inhibition of their development or promotion of their disruption). Our findings suggest that one or more of these compounds may modulate plaque formation in vivo, which is a prerequisite for the development of both caries and gingivitis.

Hemolytic activity of Prevotella intermedia and Prevotella nigrescens strains: influence of abiotic factors in solid and liquid assays.

The influence of growth medium, hemin and menadione, blood source and atmosphere of incubation on the expression of hemolytic activity of 25 strains of Prevotella intermedia and Prevotella nigrescens was evaluated. The best hemolytic activity was observed for samples of both species growing in brain heart infusion agar and incubated in Brewer-like anaerobic jars for 48 h. Hemolysis was less intense and occurred later in the presence of hemin and menadione in solid media. beta-Hemolysis was detected for medium supplemented with horse or human blood and alpha-hemolysis was observed when sheep blood was used. These results suggesting some specificity for the hemolytic activity were also observed in liquid assays in which sheep erythrocytes were found to be resistant to hemolysis while horse and human cells where lysed. In liquid assays, the hemolytic activity of all studied strains remained stable in the pH range of 6.0 to 8.5 and was not altered by iron-scavenging compounds or atmosphere of incubation. The phenomenon of hot/cold hemolysis was ruled out as the mechanism of action of P. intermedia and P. nigrescens hemolysin.

Identification of hemolytic activity in Prevotella intermedia.

Hemolysin production was measured in strains of Prevotella intermedia. Zones of beta-hemolysis were detected on agar plates supplemented with either sheep, rabbit or human erythrocytes. A standard tube assay was performed on cell suspensions of the organism to measure hemolytic activity, which was found to be dose dependent, eliminated by heat treatment, and saturable with increasing concentrations of blood. Growth-phase experiments suggested that hemolysin production was increased during logarithmic growth and was reduced during stationary phase. Cell fractionation, performed on several strains of P. intermedia, localized the activity in the outer membrane and in cell vesicles. The biological implication of this study is that P. intermedia, by virtue of its hemolytic activity, is capable of liberating the hemoglobin from erythrocytes, thereby acquiring an essential nutrient, iron, for its metabolism.