Suppression of pathogenicity of Porphyromonas gingivalis by newly developed gingipain inhibitors

Arg-gingipain (Rgp) and Lys-gingipain (Kgp) are cysteine proteinases produced by Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases. Here we show a series of small peptide analogs able to inhibit either Rgp or Kgp, which are synthesized on the basis of the cleavage site specificity of human salivary histatins by each enzyme. Among this series of compounds, carbobenzoxy-Lys-Arg-CO-Lys-N-(CH2)2 (KYT-1) and carbobenzoxy-Glu(NHN(CH3)Ph)-Lys-CO-NHCH2Ph (KYT-36) were found to be the most potent inhibitors of Rgp and Kgp, respectively, with Ki values of 10(-11) to 10(-10) M order. Both inhibitors exhibited slight or no inhibition on mammalian proteinases such as trypsin and cathepsins B, L, and H. All of the virulence induced by the culture supernatant of P. gingivalis tested, including the degradation of various host proteins such as human type I collagen, immunoglobulins, fibronectin, and fibrinogen, disruption of the bactericidal activity of polymorphonuclear leukocytes, and enhancement of the vascular permeability, were strongly inhibited by a combined action of both inhibitors. The functions essential for the bacterium to grow and survive in the periodontal pocket, such as coaggregation and acquisition of amino acids, were also strongly inhibited by the combined action of both inhibitors. The disruption of the adhesion and viability of human fibroblasts and hemagglutination by the organism were strongly suppressed by a single use of KYT-1. These results thus indicate that the newly developed KYT-1 and KYT-36 both should provide a broader application in studies of this important class of enzymes and facilitate the development of new approaches to periodontal diseases.     

Suppression of gingival inflammation induced by Porphyromonas gingivalis in rats by leupeptin

In this study, we developed a procedure to produce gingivitis in rats by inoculation of Porphyromonas gingivalis and studied the contribution of the bacterial cysteine proteinases, Arg-gingipain (Rgp) and Lys-gingipain (Kgp), to the pathology in the gingiva. To adhere the bacterium to periodontal tissues, a cotton thread was inserted between the first and second molar of right maxillary sites of rats. Rats in group A were administered with vehicle alone after bacterial (strain W83) inoculation. In group B, the bacteria were inoculated in combination with leupeptin, a potent inhibitor of Rgp and Kgp, and then leupeptin alone was administered the week after. Rats in group C were administered leupeptin for 6 weeks after bacteria inoculation. All left maxillary gingiva in three groups showed no inflammatory changes. Right maxillary gingiva of group A showed most of the clinical landmarks of gingivitis. Leupeptin exhibited only a little inhibitory effect on this gingivitis in group B, whereas it had a strong inhibitory effect on the inflammation in group C. These results suggest that P. gingivalis-induced gingivitis is attributable to Rgp and Kgp and that leupeptin is more effective in the late phase than the early stage of gingivitis.     

Exploring the Sn binding pockets in gingipains by newly developed inhibitors: structure-based design, chemistry, and activity

Arg-gingipains (Rgps) and Lys-gingipain (Kgp) are cysteine proteinases secreted by Porphyromonas gingivalis, the major pathogen implicated in periodontal disease. Gingipains are essential to the bacterium for its virulence and survival, and development of inhibitors targeting these proteins provides an approach to treat periodontal diseases. Here, we present the first example of structure-based design of gingipains inhibitors, with the use of the crystal structure of RgpB and the homology model of Kgp. Chloromethyl ketones were selected as suitable compounds to explore the specificity of the Sn binding region of both enzymes. Three series of inhibitors bearing Arg or Lys at P1 and different substituents at P2 and P3 were designed, synthesized, and tested. High potency (k(obs)/[I] approximately 10(7) M(-1) s(-1)) was achieved for small ligands, such as the dipeptide analogues. The detailed analysis of Sn binding pockets revealed the molecular basis of inhibitory affinity and provided insight into the structure-activity relationship.     

Porphyromonas gingivalis mediated periodontal disease and atherosclerosis: disparate diseases with commonalities in pathogenesis through TLRs

Toll-like receptors (TLRs) are a group of pathogen-associated molecular pattern receptors, which play an important role in innate immune signaling in response to microbial infection. It has been demonstrated that TLRs are differentially up regulated in response to microbial infection and chronic inflammatory diseases such as atherosclerosis. Furthermore hyperlipidemic mice deficient in TLR2, TLR4, and MyD88 signaling exhibit diminished inflammatory responses and decreased atherosclerosis. Accumulating evidence has implicated specific infectious agents including the periodontal disease pathogen Porphyromonas gingivalis in the progression of atherosclerosis. Evidence in humans suggesting that periodontal infection predisposes to atherosclerosis is derived from studies demonstrating that the periodontal pathogen P. gingivalis resides in the wall of atherosclerotic vessels and seroepidemiological studies demonstrating an association between pathogen-specific IgG antibodies and atherosclerosis. We have established that the inflammatory signaling pathways that P. gingivalis utilizes is dependent on the cell type and this specificity clearly influences innate immune signaling in the context of local and distant chronic inflammation induced by this pathogen. We have demonstrated that P. gingivalis requires TLR2 to induce oral inflammatory bone lose in mice. Furthermore, we have demonstrated that P. gingivalis infection accelerates atherosclerosis in hyperlipidemic mice with an associated increase in expression of TLR2 and TLR4 in atherosclerotic lesions. Our recent work with P. gingivalis has demonstrated the effectiveness of specific intervention strategies (immunization) in the prevention of pathogen-accelerated atherosclerosis. Improved understanding of the mechanisms driving infection, and chronic inflammation during atherosclerosis may ultimately provide new targets for therapy.     

Cysteine proteinases in arthritis and inflammation

Summary Balanced enzymatic activity is one of the keystones of physiology and pathophysiology. In organisms, the up- and downregulation of proteolytic enzymes reflects physiological adaptation as well as the presence or development of disease. Advances in molecular biology have revealed numerous aspects in the field of cysteine proteinase research. Several novel members of this growing family of proteinases, such as cathepsins H, S, and O, have been found to participate in inflammatory and arthritic diseases. Interestingly, the majority of the data addressing the involvement of cysteine proteinases in pathophysiological pathways are the result of intensive research on two diseases, periodontal disease and rheumatoid arthritis, which share distinct features of cysteine-proteinase-modulated inflammation and matrix degradation.     

Recent approaches for the treatment of periodontitis

Periodontal disease is a localised inflammatory response caused by the infection of a periodontal pocket arising from the accumulation of subgingival plaque. Periodontal disease has been considered as a possible risk factor for other systemic diseases such as cardiovascular diseases and pre-term low birth weight infants. Advances in understanding the aetiology, epidemiology and microbiology of periodontal pocket flora have revolutionised the therapeutic strategies for the management of periodontal disease progression. This review summarises the recent developments in the field of intra-pocket drug delivery systems and identifies areas where further research may lead to a clinically effective intra-pocket delivery system.     

Recent developments of cathepsin inhibitors and their selectivity

Cathepsins play an important role in the degradation of host connective tissues, the generation of bioactive proteins and antigen processing. They have been implicated in osteoporosis, muscular dystrophy, rheumatoid arthritis, bronchitis, emphysema, viral infection, cancer metastasis and neurodegenerative diseases, such as Alzheimer’s disease and Huntington’s disease. Recently, increased interest in cathepsin inhibitors has been generated with potential therapeutic targets, such as cathepsin K or cathepsin L for osteoporosis and cathepsin S for immune modulation. Of the 53 patents assessed in this review, granted between March 1998 and February 2001, there were 40 patents related to cysteine proteinase inhibitors, 7 related to aspartic proteinase inhibitors and 6 related to serine proteinase inhibitors. Of the 40 patents, 14 disclosed the novel compounds that were more selective against cathepsin K or cathepsin S than cathepsin B and cathepsin L. The compounds, showing experimental evidences, were evaluated and their biological activities in animal models determined. However, only 4 patents presented significant results in vivo. These patents may be a basis for promoting further evaluation and developing second generation cathepsin inhibitors.     

Deciphering the toxicological role of Porphyromonas gingivalis derived endotoxins in liver diseases

Periodontitis is a most prevalent and infectious multifactorial inflammatory disease and is characterized by the progressive destruction of the tooth-supporting tissues. Porphyromonas gingivalis, a Gram‑negative oral anaerobe, mainly causes periodontitis and it is one of the most important risk factors responsible for aggravation of existing systemic diseases. Several experimental and clinical studies have shown the positive association between periodontitis and different forms of liver disease. Periodontal diseases increase the prevalence of non-alcoholic fatty liver diseases and cirrhosis. Infected periodontium and pathogens in the periodontal microenvironments release pathogen-associated molecular patterns such as peptidoglycan, lipopolysaccharides, gingipain, fimbria, bacterial DNA, etc, and damage-associated molecular patterns such as interleukins-1α, β, − 8, and galectin-3, etc. These virulence factors and cytokines enter the bloodstream, disseminate into the whole body, and induce a variety of systemic pathological effects, including liver diseases (steatosis and fibrosis). Maintaining oral hygiene by scaling and root planning significantly improves liver damage in patients with periodontitis. Dentists and physicians should have more awareness in understanding the bidirectional nature of the relationship between oral and systemic diseases. Importantly, periodontitis condition aggravates simple fatty liver into fibrotic disease and therefore, the aim of this review is to understand the possible link between periodontitis and liver diseases.     

Periodontal diseases and rheumatoid arthritis: a coincident model for therapeutic intervention?

There are remarkable similarities in the pathogenesis of periodontal diseases and rheumatoid arthritis. The mechanisms that drive antigen induced sequelae of oxidative stress are discussed in this review. A poorly modulated inflammatory response drives both diseases resulting in oxidative stress induced tissue injury. Immune complex formation in response to the periodontal pathogen Porphyromonas gingivalis triggering the production of ROS in both gingivae and synovium of RA patients has been reported. Elevated antibody levels to several periodontal pathogens in RA patients has implications on both RA and periodontal diseases. Periodontal patients are challenged individuals representing a multifactorial aetiopathogenesis with potential for therapeutic intervention in the context of free radical damage. Subjects with moderate to severe periodontal bone loss are significantly more likely than healthy individuals to have several co-existing systemic conditions resulting in ROS mediated damage. There is potential for dual induction of periodontal disease by existing inflammatory mechanisms of systemic diseases rather than exacerbation of low grade inflammation only; emphasizing the relevance of reducing inflammatory burden for disease control. Therapeutic strategies based on disease mechanisms include combined low dose non-steroidal anti-inflammatory drugs and doxycycline for synergistic reduction of matrix metalloproteinase activity in periodontal tissues and RA; sub-optimal dosing with CMT-8 and a biphosphonate clodronate to reduce pathologically elevated levels of MMPs, elastase and to restore alveolar bone in experimental periodontits demonstrating dual applications. Therapeutic interventions relevant to both diseases discussed in this review, have scope for a double hit in periodontal patients with co-existing RA and vice versa.     

Porphyromonas gingivalis, Periodontal pathogen, lipopolysaccharide induces angiogenesisvia extracellular signal-regulated kinase 1/2 activation in human vascular endothelial cells

Porphyromonas gingivalis is a major periodontal pathogen. The lipopolysaccharide (LPS) secreted from P. gingivalis is implicated in the initiation and progression of periodontitis. Aberrant angiogenesis is often associated with lesion formation in chronic periodontitis. In this study, we report that P. gingivalis LPS activates angiogenic cascade, migration, invasion and tube formation in human umbilical vein endothelial cells (HUVECs). Furthermore, P. gingivalis LPS potently stimulated in vivo neovascularization in chick chorioallantoic membrane (CAM) and the mouse Matrigel plug assay. P. gingivalis LPS had no effect on the expression of vascular endothelial growth factor (VEGF) or its receptor, Flk-1, implying that P. gingivalis LPS-induced angiogenesis may result from its direct action on endothelial cells. P. gingivalis LPS evoked activation of the mitogen-activated protein kinase ERK1/2 in HUVECs, which is closely linked to angiogenesis. Taken together, these results strongly suggest P. gingivalis LPS plays an important role in the pathological angiogenesis for periodontal diseases, such as periodontitis.     

Targeted antimicrobial activity of a specific IgG-SMAP28 conjugate against Porphyromonas gingivalis in a mixed culture

Antimicrobial peptides coupled to a ligand, receptor or antibody for a specific pathogenic bacteria could be used to develop narrow-spectrum pharmaceuticals with 'targeted' antimicrobial activity void of adverse reactions often associated with the use of broad-spectrum antibiotics. To assess the feasibility of this approach, in this study sheep myeloid antimicrobial peptide (SMAP) 28 was linked to affinity- and protein G-purified rabbit immunoglobulin G (IgG) antibodies specific to the outer surface of Porphyromonas gingivalis strain 381. The selective activity of the P. gingivalis IgG-SMAP28 conjugate was then assessed by adding it to an artificially generated microbial community containing P. gingivalis, Aggregatibacter actinomycetemcomitans and Peptostreptococcus micros. The specificity of the P. gingivalis IgG-SMAP28 conjugate in this mixed culture was concentration-dependent. The conjugate at 50 microg protein/mL lacked specificity and killed P. gingivalis, A. actinomycetemcomitans and P. micros. The conjugate at 20 microg protein/mL was more specific and killed P. gingivalis. This is an initial step to develop a selective antimicrobial agent that can eliminate a specific periodontal pathogen, such as P. gingivalis, from patients with periodontal disease without harming the normal commensal flora.     

Aggregatibacter actinomycetemcomitans Leukotoxin: A Powerful Tool with Capacity to Cause Imbalance in the Host Inflammatory Response

Aggregatibacter actinomycetemcomitans has been described as a member of the indigenous oral microbiota of humans, and is involved in the pathology of periodontitis and various non-oral infections. This bacterium selectively kills human leukocytes through expression of leukotoxin, a large pore-forming protein that belongs to the Repeat in Toxin (RTX) family. The specificity of the toxin is related to its prerequisite for a specific target cell receptor, LFA-1, which is solely expressed on leukocytes. The leukotoxin causes death of different leukocyte populations in a variety of ways. It activates a rapid release of lysosomal enzymes and MMPs from neutrophils and causes apoptosis in lymphocytes. In the monocytes/macrophages, the toxin activates caspase-1, a cysteine proteinase, which causes a proinflammatory response by the activation and secretion of IL-1β and IL-18. A specific clone (JP2) of A. actinomycetemcomitans with enhanced leukotoxin expression significantly correlates to disease onset in infected individuals. Taken together, the mechanisms by which this toxin kills leukocytes are closely related to the pathogenic mechanisms of inflammatory disorders, such as periodontitis. Therapeutic strategies targeting the cellular and molecular inflammatory host response in periodontal diseases might be a future treatment alternative.     

Antimicrobial activity of tannin components from Vaccinium vitis-idaea L

Reactive oxygen species have been implicated as important pathological mediators in many clinical disorders, including periodontal disease. As a possible alternative for the treatment of periodontal disease, the antimicrobial activity of six tannins isolated from Vaccinium vitis-idaea L., with confirmed antioxidant activity, were assayed by the agar dilution method against selected periodontal pathogens, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia. The results showed that epicatechin-(4beta-->8)-epicatechin-(4beta-->8, 2beta-->O-->7)-catechin had strong antimicrobial activity against P. gingivalis and P. intermedia, but not A. actinomycetemcomitans. The other tannins tested did not show antimicrobial activity. We conclude that tannins isolated from V. vitis-idaea L. with antimicrobial activity could potentially be used for the treatment of periodontal disease.     

Emerging concepts in periodontal therapy

Conventional periodontal therapy consists of mechanical scaling and root planing, and surgical treatment. This is still the mainstay of periodontal treatment. Adjunctive antimicrobial treatments, both systemic and local delivery, are becoming more sophisticated and useful in the treatment of recurrent periodontitis. Also very promising are adjunctive treatments that modulate the host response and decrease levels of destructive pro-inflammatory cytokines or matrix metalloproteinases. Smoking is a major risk factor for periodontitis and has a profound impact on the progression of periodontal bone and attachment loss. In the interest of improved periodontal health patients should be encouraged to stop smoking. Finally bacterial endotoxins that stimulate the release of pro-inflammatory cytokines can have systemic effects and may lead to pre-term, low birthweight babies, and cardiovascular diseases such as atherosclerosis, myocardial infarction and stroke. Health professionals need to be cognisant of the effect dental health can have on systemic diseases and refer for treatment when appropriate to ensure that optimum oral and systemic health is achieved for their patients.     

Identification of hop polyphenolic components which inhibit prostaglandin E2 production by gingival epithelial cells stimulated with periodontal pathogen

Chronic marginal periodontitis is a destructive inflammatory disease caused by an imbalance between bacterial virulence and host defense ability, resulting in eventual tooth exfoliation. Porphyromonas gingivalis, a major periodontal pathogen, triggers a series of cellular inflammatory responses including the production of prostaglandin E2 (PGE2), which causes periodontal destruction; thus, anti-inflammatory reagents are considered beneficial for periodontal therapy. In the present study, we examined whether hop- and apple-derived polyphenols (HBP and ACT, respectively) inhibit PGE2 production by human gingival epithelial (HGE) cells stimulated with P. gingivalis components. HGE cells were stimulated with P. gingivalis membrane vesicles, and the effects of HBP, ACT and epigallocatechin gallate (EGCg) on PGE2 production by HGE cells were evaluated using an enzyme-linked immunosorbent assay. HBP and EGCg significantly inhibited PGE2 production, whereas ACT did not. By further fractionation steps of HBP to identify the effective components, 3 components of HBP, 2-[(2-methylpropanoyl)-phloroglucinol]1-O-beta-D-glucopyranoside (MPPG), quercetin 3-O-beta-D-glucopyranoside (isoquercitrin), and kaempferol 3-O-beta-glucopyranoside (astragalin), were found to be elements which significantly inhibited cellular PGE2 production. These results suggest that HBP is a potent inhibitor of cellular PGE2 production induced by P. gingivalis, and HBP may be useful for the prevention and attenuation of periodontitis.     

Serum microbial- and host-derived markers of periodontal diseases: a review

Periodontitis is bacterial infection of tooth-supporting tissues leading to inflammation and, subsequently, to loss of teeth. It is one of the most common infections worldwide. Recent studies have shown that periodontal infection may pose a threat to general health by increasing the risk of cardiovascular and lung diseases, and preterm labour. Thus, useful markers of systemic exposure to periodontitis are needed. Markers of periodontitis in serum include those derived directly from periodontopathic pathogens and those originating from the host defence and immune mechanisms. Periodontitis is associated with endotoxemia, which can be directly measured as elevated concentrations of lipopolysaccharide (LPS) in periodontitis patients compared with healthy subjects. Also indirect methods determining endotoxemia, such as elevated concentrations of serum LPS binding protein, soluble CD14, and antibodies to LPS of periodontal pathogens have been reported. Surrogate measures of the host response against periodontal infection, such as matrix metalloproteinases, cytokines, chemokines, inflammation markers, antiphospholipid antibodies, and antibodies to periodontal pathogens, have been used. Of these, however, only antibodies to periodontal pathogens may be seen as specific markers of systemic exposure to periodontopathic pathogens. In this paper we describe and discuss serum markers of periodontitis that have been used for research purposes and/or to support diagnostics. Based on literature review, we encourage research and development of serum screening methods for periodontitis that could be used by general physicians.     

Inflammatory mechanisms and redox status in periodontal and cardiometabolic diseases: effects of adjunctive nutritional antioxidants and statins

Periodontal pathogens in plaque biofilm initiate periodontitis, which is influenced by genetic and environmental factors. The resultant pro-oxidant status imposed on the periodontium, exacerbated by episodic hyperinflammatory damage contributes to progression of periodontitis and tooth loss in susceptible subjects. Increasing documentation of bi-directional connections between periodontal and cardiometabolic disorders makes it an intriguing area of therapeutic intervention for mutual benefit. Periodontitis and associated comorbidities demonstrate similar risk markers of inflammation during disease progression. Depending on the extent and severity of the inflammatory response, periodontitis could impact significantly on systemic inflammatory loading and influence the progression of endothelial dysfunction, atherosclerotic plaque instability, dyslipidaemia and insulin resistance. Some of the common mechanisms involved are discussed, relevant to periodontal and cardiometabolic disorders which have been documented as having a bidirectional relationship with periodontal disease progression; abating in response to treatment. Periodontal disease may be a useful marker of a susceptible immune system, or directly affect the progression of systemic diseases due to inflammatory loading. These mechanisms mediated by coordinated actions of cytokines, acute phase proteins, enzymes and their sequelae are addressed in the context of conventional periodontal therapy and its outcome with a modulatory role on metabolic diseases. Applications for the role of nutritional and therapeutic antioxidants as adjuncts in diseases with a distinctly prooxidant profile are discussed. Accurate therapeutic targeting as an adjunct to conventional periodontal treatment in this context, for mutual benefit to subjects with periodontitis and cardiometabolic diseases is a challenge.     

Effect of anti-CD14 antibody on experimental periodontitis induced by Porphyromonas gingivalis lipopolysaccharide

The lipopolysaccharide (LPS) released by Porphyromonas gingivalis, a Gram-negative bacterium found in the periodontal pockets of patients with periodontitis, induces bone resorbing activity in vivo. We previously showed that a receptor for LPS on human gingival fibroblasts and gingival epithelial cells is CD14. In this study, we established a mouse model of experimental periodontitis by applying a P. gingivalis LPS solution to the buccal region of mice. P. gingivalis LPS-induced bone resorption and interleukin-6 production in the gingival tissues were significantly inhibited by pretreatment with anti-CD14 antibody for 5 weeks prior to LPS treatment. This result suggests that anti-CD14 antibody may be usable as a prototype for the development of drugs for the treatment of periodontal disease.     

Porphyromonas gingivalis Gingipains Trigger a Proinflammatory Response in Human Monocyte-derived Macrophages Through the p38α Mitogen-activated Protein Kinase Signal Transduction Pathway

Porphyromonas gingivalis, the major etiologic agent of chronic periodontitis, produces a broad spectrum of virulence factors, including Arg- and Lys-gingipain cysteine proteinases. In this study, we investigated the capacity of P. gingivalis gingipains to trigger a proinflammatory response in human monocyte-derived macrophages. Both Arg- and Lys-gingipain preparations induced the secretion of TNF-α and IL-8 by macrophages. Stimulation of macrophages with Arg-gingipain A/B preparation at the highest concentration was associated with lower amounts of cytokines detected, a phenomenon likely related to proteolytic degradation. The inflammatory response induced by gingipains was not dependent of their catalytic activity since heat-inactivated preparations were still effective. Stimulating macrophages with gingipain preparations was associated with increased levels of phosphorylated p38α MAPK suggesting its involvement in cell activation. In conclusion, our study brought clear evidence that P. gingivalis Arg- and Lys-gingipains may contribute to the host inflammatory response, a critical factor in periodontitis-associated tissue destruction.