leukotoxin and Periodontitis

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.

Topics: Aggregatibacter actinomycetemcomitans; Cell Death; Cell Membrane; Exotoxins; Host-Pathogen Interactions; Humans; Immunity, Humoral; Lymphocyte Function-Associated Antigen-1; Lymphocytes; Monocytes; Neutrophils; Periodontitis; Protein Binding; Virulence

Periodontitis is mankind's most common chronic inflammatory disease. One severe form of periodontitis is localized aggressive periodontitis (LAP), a condition to which individuals of African origin demonstrate an increased susceptibility. The main causative organism of this disease is Actinobacillus actinomycetemcomitans. A member of the Pasteurellaceae, A. actinomycetemcomitans produces a number of interesting putative virulence factors including (a) an RTX leukotoxin that targets only neutrophils and monocytes and whose action is influenced by a novel type IV secretion system involved in bacterial adhesion; (b) the newly discovered toxin, cytolethal distending toxin (CDT); and (c) a secreted chaperonin 60 with potent leukocyte-activating and bone resorbing activities. This organism also produces a plethora of proteins able to inhibit eukaryotic cell cycle progression and proteins and peptides that can induce distinct forms of proinflammatory cytokine networks. A range of other proteins interacting with the host is currently being uncovered. In addition to these secreted factors, A. actinomycetemcomitans is invasive with an unusual mechanism for entering, and traveling within, eukaryotic cells. This review focuses on recent advances in our understanding of the molecular and cellular pathogenicity of this fascinating oral bacterium.

Topics: Actinobacillus Infections; Aggregatibacter actinomycetemcomitans; Bacterial Adhesion; Bacterial Toxins; Chaperonin 60; Exotoxins; Genome, Bacterial; Humans; Periodontitis; Virulence

The purpose of this study was to determine to what extent the presence or absence of periodontal pathogens can distinguish between subjects with chronic and aggressive periodontitis.. A systematic review of cross sectional and longitudinal studies providing microbiological data both from patients with chronic periodontitis (ChP) and aggressive periodontitis (AgP) at a subject level. Strict inclusion criteria were applied. The presence or absence of five microorganisms was selected as primary study parameters: Actinobacillus actinomycetemcomitans (AA), Porphyromonas gingivalis (PG), Prevotella intermedia (PI), Bacteroides forsythus (BF), and Campylobacter rectus (CR).. The presence or absence of AA could be evaluated in 11 papers. In seven papers the presence or absence of PG could be analysed. Subject specific data on PI were available from six studies. Two studies could be used regarding the presence or absence of BF, and two regarding CR. Sensitivity and specificity of every microbiological test were individually calculated for each selected study, assuming that the clinical diagnosis of AgP or ChP was the true status the tests attempted to detect. AgP was considered to be the condition of interest and ChP was considered equivalent to 'non-AgP'. Receiver Operator Characteristic (ROC) diagrams were constructed using these data. ROC diagrams indicated the limited discriminatory ability of all of the test parameters to identify subjects with AgP. An additional assessment showed that the highly leukotoxic variant of AA was uniquely associated with patients suffering from aggressive periodontitis. However, in a high proportion of patients diagnosed with AgP the presence of this variant could not be detected.. The presence or absence of AA, PG, PI, BF or CR could not discriminate between subjects with AgP from those with ChP.

Topics: Acute Disease; Aggregatibacter actinomycetemcomitans; Bacteroides; Campylobacter; Chronic Disease; Exotoxins; Humans; Periodontitis; Porphyromonas gingivalis; Prevotella intermedia; ROC Curve; Sensitivity and Specificity

The humoral arm of the immune system provides protection from many medically significant pathogens. The antigenic epitopes of the pathogens which induce these responses, and the subsequent characteristics of the host response, have been extensively documented in the medical literature, and in many cases have resulted in the development and implementation of effective vaccines or diagnostic tests. There is a substantial body of literature on the humoral immune response in periodontal disease, which is targeted at micro-organisms present within periodontal pockets. However, the significance and specificity of the immune response in periodontal disease have proved difficult to elucidate, due to the large number of potential pathogens in the plaque biofilm and the apparent commensal nature of many of these opportunistic pathogens. This review addresses our current knowledge of the approaches and strategies which have been used to elucidate and examine the concept of immunodominant antigens in medical infections and, more recently, periodontal disease. An identification/understanding of the immunodominant antigens would be informative with respect to: (i) the relative importance of the implicated pathogens, (ii) new approaches to immunological diagnosis, (iii) specific bacterial virulence determinants, (iv) natural protective responses, and (v) the selection of potential vaccine candidate antigens. We conclude that immunodominance of antigens in periodontal disease may be relevant to our understanding of periodontal disease pathogenesis, but due to the complexity and diversity of the 'pathogenic microbial ecology', it is currently an enigmatic topic requiring a multidisciplinary approach linking clinical, microbiological, and immunological investigations. We also conclude, after assessing the literature available on the topic of immunodominance, that it is a term that, if used, must be clearly defined and understood, since it is often used loosely, leading to a general misinterpretation by readers of oral and medical literature.

Topics: Aggregatibacter actinomycetemcomitans; Animals; Antigens, Bacterial; Bacterial Outer Membrane Proteins; Exotoxins; Fimbriae, Bacterial; Humans; Immunodominant Epitopes; Lipopolysaccharides; Periodontitis; Porphyromonas gingivalis; Virulence

Mechanical debridement results in a shift of the bacterial composition in the periodontal pocket on the species level. It is unknown, however, whether a clonal change within a species could lead to the emergence of strains with different levels of virulence. Therefore, in the present study, the genetic variability of Actinobacillus actinomycetemcomitans was assessed and strains identified which were associated with periodontal disease progression following periodontal therapy, i.e., refractory periodontitis. Twenty adult patients with untreated periodontitis and subgingival colonization of A. actinomycetemcomitans were randomly assigned to receive full-mouth scaling alone or scaling with an adjunctive antimicrobial therapy. Both groups received supportive periodontal therapy at 3, 6, 9, 12, 18, and 24 months. Subgingival plaque samples were taken at every visit; venous blood was obtained at 24 months only. A. actinomycetemcomitans isolates were typed by the RAPD method, and antibody reactivity against outer membrane proteins was assessed by immunoblot analysis. Eleven distinct RAPD patterns were found in 18 patients completing the study. All patients harbored only one A. actinomycetemcomitans genotype, and within each patient this genotype persisted throughout the 24-month observation period. No differences in the expression of antibody reactivity against outer membrane proteins were found between strains isolated at baseline and at 24 months. Three genotypes were associated with reduced survival rates of teeth without probing attachment loss of 2 mm or more. The results indicated that (i) most patients harbored only one A. actinomycetemcomitans genotype; (ii) the genotype persisted following therapy; and (iii) only some genotypes were associated with refractory periodontitis.

Topics: Adult; Aggregatibacter actinomycetemcomitans; Amoxicillin; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Bacterial Typing Techniques; Chronic Disease; Clone Cells; Dental Plaque; Disease Progression; DNA Fingerprinting; DNA, Bacterial; Exotoxins; Female; Genetic Variation; Humans; Male; Metronidazole; Middle Aged; Penicillins; Periodontal Pocket; Periodontitis; Random Amplified Polymorphic DNA Technique; Survival Analysis; Virulence

The facultative bacterium Aggregatibacter actinomycetemcomitans (Aa) is strongly associated with periodontitis and is occasionally found in periodontally healthy subjects. We aimed to determine the prevalence of salivary Aa among patients with either periodontitis Grade B (periodontitis-B) or Grade C (periodontitis-C), periodontally healthy controls (HCs), and to determine if systemic antibodies against Aa or its virulence factor leukotoxin A (LtxA) may serve as biomarkers that reveal the oral presence of the bacterium and discriminate subjects with periodontitis-C, periodontitis-B, or no periodontitis from each other.. Serum and unstimulated saliva samples were collected from patients with periodontitis-C (n = 27), patients with periodontitis-B (n = 34), and HCs (n = 28). Serum level of immunoglobulin G antibodies to fragmented whole Aa and to LtxA were quantified using a bead-based assay. Aa was identified in saliva using quantitative polymerase chain reaction (qPCR). All analyses were adjusted for age, sex, and current smoking status.. Aa was present in saliva from 11% of HCs, in 32% of patients with periodontitis-B (P = 0.04 versus HCs), and in 37% of patients with periodontitis-C (P = 0.02 versus HCs). Serum antibodies to fragments of Aa associated significantly with periodontitis-C (P = 0.03), while serum anti-LtxA antibodies associated with both periodontitis-B and periodontitis-C (P = 0.002 and P = 9×10. Aa is highly prevalent in saliva of patients with periodontitis-B or periodontitis-C. Systemic immunoglobulin G antibodies against LtxA distinguish patients with periodontitis, regardless of grade, from HCs, while their quantity reflects the concurrent bacterial burden in the oral cavity.

Topics: Aggregatibacter actinomycetemcomitans; Antibodies, Bacterial; Biomarkers; Exotoxins; Humans; Periodontitis

We and others have previously shown that epigallocatechin gallate (EGCg) inhibits the activity of an important virulence factor, leukotoxin (LtxA), produced by the oral bacterium Aggregatibacter actinomycetemcomitans, suggesting the potential use of this molecule as an anti-virulence strategy to treat periodontal infections. Here, we sought to better understand the effects of EGCg on toxin secretion and A. actinomycetemcomitans pathogenicity in a co-culture model.. We used a quantitative immunoblot assay to determine the concentrations of LtxA in the bacterial supernatant and on the bacterial cell surface. Using a co-culture model, consisting of A. actinomycetemcomitans and THP-1 cells, we studied the impact of EGCg-mediated changes in LtxA secretion on the toxicity of A. actinomycetemcomitans.. EGCg increased production of LtxA and changed the localization of secreted LtxA from the supernatant to the surface of the bacterial cells. In the co-culture model, a single low dose of EGCg did not protect host THP-1 cells from A. actinomycetemcomitans-mediated cytotoxicity, but a multiple dosing strategy had improved effects.. Together, these results demonstrate that EGCg has important, but complicated, effects on toxin secretion and activity; new dosing strategies and comprehensive model systems may be required to properly develop these anti-virulence activities.

Topics: Aggregatibacter actinomycetemcomitans; Anti-Bacterial Agents; Bacterial Toxins; Catechin; Coculture Techniques; Dose-Response Relationship, Drug; Exotoxins; Humans; Periodontitis; Virulence

Topics: Aggregatibacter actinomycetemcomitans; Animals; Bacterial Adhesion; Biofilms; Durapatite; Exotoxins; Lactic Acid; Macaca mulatta; Male; Microbiota; Mouth; Periodontitis

Catechins, polyphenols derived from tea leaves, have been shown to have antibacterial properties, through direct killing of bacteria as well as through inhibition of bacterial toxin activity. In particular, certain catechins have been shown to have bactericidal effects on the oral bacterium, Aggregatibacter actinomycetemcomitans, as well as the ability to inhibit a key virulence factor of this organism, leukotoxin (LtxA). The mechanism of catechin-mediated inhibition of LtxA has not been shown.. In this work, we studied the ability of six catechins to inhibit LtxA-mediated cytotoxicity in human white blood cells, using Trypan blue staining, and investigated the mechanism of action using a combination of techniques, including fluorescence and circular dichroism spectroscopy, confocal microscopy, and surface plasmon resonance.. We found that all the catechins except (-)-catechin inhibited the activity of this protein, with the galloylated catechins having the strongest effect. Pre-incubation of the toxin with the catechins increased the inhibitory action, indicating that the catechins act on the protein, rather than the cell. The secondary structure of LtxA was dramatically altered in the presence of catechin, which resulted in an inhibition of toxin binding to cholesterol, an important initial step in the cytotoxic mechanism of the toxin.. These results demonstrate that the catechins inhibit LtxA activity by altering its structure to prevent interaction with specific molecules present on the host cell surface.. Galloylated catechins modify protein toxin structure, inhibiting the toxin from binding to the requisite molecules on the host cell surface.

Topics: Aggregatibacter actinomycetemcomitans; Bacterial Toxins; Catechin; Cell Membrane; Cell Survival; Cholesterol; Circular Dichroism; Exotoxins; Humans; Leukocytes; Membrane Fluidity; Microscopy, Confocal; Periodontitis; Protein Structure, Secondary; Surface Plasmon Resonance; THP-1 Cells

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA-treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA-treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA-induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.

Topics: Aggregatibacter actinomycetemcomitans; Cell Death; Cell Line; Epithelial Cells; Exotoxins; Fibroblasts; Gingiva; Glycine; Humans; Leukocyte Elastase; Neutrophils; Periodontitis; Sulfonamides; Virulence Factors

A relationship between rheumatoid arthritis (RA) and periodontitis has been suggested from findings that individuals with RA are prone to have advanced periodontitis and vice versa. In search of possible common pathogenetic features of these two diseases, we investigated the presence of citrullinated proteins and expression of endogenous peptidylarginine deiminases (PAD2 and PAD4), in periodontal tissue of individuals with periodontitis and healthy controls, in relation to the periodontal pathogens Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), producing leukotoxin as virulence factor. These two oral bacteria have been suggested to be linked to anti-citrullinated protein antibodies in patients with RA.. Gingival tissue biopsies were obtained from 15 patients with periodontitis and 15 individuals without periodontal disease. Presence of CD3-positive lymphocytes, citrullinated proteins, PAD2, PAD4, P. gingivalis as well as A. actinomycetemcomitans and Mannheimia haemolytica produced leukotoxins were analysed by immunohistochemistry, followed by triple-blind semi-quantitative analysis. Mann-Whitney and Fisher's exact tests were used to analyse differences between groups. PADI2 and PADI4 mRNA levels were assessed by RT-qPCR and analysed using Wilcoxon signed rank test.. Increased staining of citrullinated proteins was observed in gingival connective tissue from subjects with periodontitis (80%, 12/15) compared to healthy gingival tissue (27%, 4/15), whereas no differences were observed in gingival epithelium. There was also an increased staining of the citrullinating enzymes PAD2 and PAD4 in gingival connective tissue of patients with periodontitis whereas similar levels of PAD2 and PAD4 were observed in the gingival epithelium of the two groups. Similarly, the mRNA levels of PADI2 and PADI4 were also increased in the gingival tissue of patients with periodontitis compared to healthy controls. Furthermore, presence of P. gingivalis and leukotoxins was comparable in both epithelium and connective tissue, from the different investigated individuals with and without periodontitis, and there were no correlations between the presence of periodontal pathogens and the expression of citrullinated proteins or PAD enzymes.. Chronic gingival inflammation is associated with increased local citrullination and PAD2 and PAD4 expression in periodontitis. The increased citrullination and PAD2 and PAD4 expression in periodontitis were, however, independent of the presence of periodontal pathogen P. gingivalis and A. actinomycetemcomitans leukotoxin.

Topics: Adult; Aggregatibacter actinomycetemcomitans; Arthritis, Rheumatoid; Citrullination; Exotoxins; Gingiva; Humans; Inflammation; Lymphocytes; Middle Aged; Periodontitis; Porphyromonas gingivalis; Protein-Arginine Deiminases; RNA, Messenger

Varying cytokine responses of human gingival epithelial cells (HGECs) by Aggregatibacter actinomycetemcomitans subtypes have been found. Most studies have used reference strains, whereas a few has evaluated the cytokine expression in response to clinical subtypes of this bacterial species. This study aimed to examine whether there was any difference in cytokine responses of HGECs stimulated with cell wall extract (CWE) from A. actinomycetemcomitans subtypes included clinical strains from Thai adult periodontitis, various serotypes and non-serotypeable strains, strains from deep or shallow pockets, and reference serotype strains. Totally 50 clinical strains and 7 reference strains of A. actinomycetemcomitans were analyzed for the expression of IL-1β, IL-6, IL-8, and TNF-α mRNAs in HGECs by real time-PCR, and the IL-8 concentrations in cell-free supernatant measured using ELISA. An in vitro effect of released IL-8 on neutrophil migration was examined using transwell chambers. Result showed that among four cytokines studied, IL-8 mRNA was highly up-regulated by both clinical and reference strains. Serotype f revealed the highest expression compared to other serotypes. The JP2-like leukotoxin promoter gene and non-serotypeable (NS1 and NS2) demonstrated lower IL-8 responses compared to serotypeable strains, and IL-8 responses upon stimulation with clinical strains from deep pockets were also significantly lower than those isolated from shallow pockets (P < 0.01). Our findings suggest that the clinical isolates of A. actinomycetemcomitans associating with deep pockets, JP2-like leukotoxin promoter gene, NS1, and NS2 may interfere neutrophil function via minimal and immunosuppressing IL-8 responses, which may enhance their survival and virulence.

Topics: Aggregatibacter actinomycetemcomitans; Cell Movement; Cell Wall; Cells, Cultured; Exotoxins; Gingiva; Gingival Pocket; Humans; Interleukin-8; Neutrophils; Periodontitis; Promoter Regions, Genetic; RNA, Messenger

Leukotoxin (Ltx) expressed by Aggregatibacter actinomycetemcomitans is a powerful exotoxin, which can cause imbalance in host response. Immunoreactivity to Ltx is a marker for presence of leukotoxic A. actinomycetemcomitans, a presence that may modify the disease pattern of colonized individuals. The aim of the present study is to examine presence of systemic immunoreactivity to A. actionmycetemcomitans Ltx with respect to clinical and inflammatory findings in individuals with or without periodontitis (N = 88).. Periodontal status was examined in patients with severe periodontitis (n = 49) and healthy controls (n = 39), and patients received periodontal treatment. Systemic biomarkers associated with inflammation and infections as well as clinical parameters were analyzed at baseline and 3 and 6 months after treatment.. Presence of immunoreactivity against Ltx was associated with impaired remission of disease after periodontal treatment. This immunoreactivity was also significantly associated with increased systemic levels of A. actinomycetemcomitans-specific immunoglobulins and increasing age.. Presence and levels of systemic immunoreactivity against A. actinomycetemcomitans Ltx are associated with decreased remission after otherwise successful periodontal treatment.

Topics: Adult; Aggregatibacter actinomycetemcomitans; Antibodies, Neutralizing; Biomarkers; Case-Control Studies; Exotoxins; Female; Humans; Male; Middle Aged; Periodontitis; Sweden

Aggregatibacter actinomycetemcomitans is the primary etiologic agent of localized aggressive periodontitis. In vitro, it can undergo fimbriated rough to nonfimbriated smooth phenotypic transition, accompanied by an increase in invasive ability and a decrease in adhesive ability. No opposite direction phenotypic transition was reported. To better understand its pathogenicity, the authors studied the morphological changes of nonfimbriated smooth strains induced by growth environmental humidity. Transmission electron microscopy was used to identify fimbriae expression change. It was found that the lower medium humidity, the more fimbriae reexpressed. In conclusion, the smooth strain of A. actinomycetemcomitans can reexpress the fimbriae in lower humidity environment.

Topics: Aggregatibacter actinomycetemcomitans; Culture Media; Exotoxins; Fimbriae, Bacterial; Humans; Humidity; Microscopy, Electron, Transmission; Periodontitis; Phenotype; Polymerase Chain Reaction; RNA, Bacterial

The aim of this study was to characterize the main periodontal bacterial species in Down syndrome (DS) patients with and without periodontitis.. This cross-sectional study involved 75 DS patients, 45 with and 30 without periodontitis. Informed consent, health and dental questionnaires and periodontitis diagnosis were performed PCR and LAMP assays were performed on subgingival dental plaque sample.. Tannerella forsythia was the most frequent bacteria detected in the group with and without periodontitis (95.5 and 63.3%) followed by Treponema denticola (88.8 and 50%) and Porphyromonas gingivalis (53.3 and 25% respectively). There were statistical differences between groups (p < 0.05). Pg fimA type I was the most frequent Porphyromonas gingivalis genotype. Two different sets of primers (Aa-F/Aa-R and ltx3/ltx4) were used to detect Aggregatibacter actinomycetemcomitans and different frequencies were obtained, (68% and 14.6% respectively), they had a weak correlation (Cohen Kappa = 0.16). After sequencing of PCR products, ltx3/ltx4 showed more specificity. JP2 clone of A. actinomycetemcomitans was not detected in any sample.. The composition of oral biofilm is fundamental for the development of periodontal disease independently of immunological alterations associated with DS. The frequency of detection of A. actinomycetemcomitans reported in the literature has a wide range, because the primers and probes applied

Topics: Aggregatibacter actinomycetemcomitans; Bacterial Toxins; Bacteroides; Biofilms; Cross-Sectional Studies; Dental Plaque; DNA Primers; DNA, Bacterial; Down Syndrome; Exotoxins; Female; Fimbriae Proteins; Genotype; Humans; Male; Microbial Consortia; Periodontal Attachment Loss; Periodontal Pocket; Periodontitis; Periodontium; Pili, Sex; Porphyromonas gingivalis; Tooth Loss; Treponema denticola; Young Adult

To examine the genetic diversity of Aggregatibacter actinomycetemcomitans in Thai adults.. Subgingival plaque samples from 453 subjects were analysed for A. actinomycetemcomitans serotypes, the presence of the high leukotoxin-producing JP2 clone and cytolethal distending toxin genes (cdtABC) using the polymerase chain reaction technique. In subjects who were positive for cdtABC, restriction fragment length polymorphism analysis was used to identify a single nucleotide polymorphism (SNP) in the cdtB gene at amino acid position 281. The extent and severity of periodontal disease were compared between subjects harbouring different A. actinomycetemcomitans genotypes.. Eighty six subjects (19%) were positive for A. actinomycetemcomitans. The JP2 clone was not detected. Serotype c was the most prevalent (57%), followed by serotypes a (33%) and b (7%). Among A. actinomycetemcomitans-positive subjects, 27% were positive for cdtABC. All cdtABC-positive subjects possessed the SNP in the cdtB, which is involved with increased toxin activity. The presence of A. actinomycetemcomitans, but not a specific genotype, was significantly related to increased probing depth and periodontal attachment loss.. Our results confirm the previous findings that genotype distribution of A. actinomycetemcomitans varies between ethnic groups. However, no clear relationship between a specific genotype and periodontal conditions was observed.

Topics: Adult; Aggregatibacter actinomycetemcomitans; Analysis of Variance; Bacterial Toxins; Chi-Square Distribution; Cross-Sectional Studies; Dental Plaque; Exotoxins; Female; Humans; Male; Middle Aged; Molecular Epidemiology; Periodontal Attachment Loss; Periodontitis; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Serotyping; Thailand

Periodontal disease is an inflammatory condition caused by bacterial infections that result in loss of the tooth supporting tissue. The periodontal pathogens produce virulence factors with capacity to affect the host immune response. Aggregatibacter actinomycetemcomitans is a periodontal pathogen that produces a leukotoxin that specifically affects human leukocytes. The aims of the present study were to examine the presence and function of systemic antibodies to the leukotoxin.. One hundred and ninety-seven middle-aged (57 ± 5 years) Swedes with well-documented periodontal status and medical factors related to cardiovascular diseases were studied. These data have been published previously. The serum samples were examined for the presence of leukotoxin antibodies by western blot and the capacity to neutralize leukotoxicity in an activity assay with leukotoxin and cultured leukemic cells.. The results showed a high prevalence (57%) of antibodies against A. actinomycetemcomitans leukotoxin in the analyzed population. These antibodies were correlated with leukotoxin neutralizing capacity as well as with the ELISA titers of A. actinomycetemcomitans-specific IgA and IgG. In addition, high levels of leukotoxin antibodies were correlated with increasing age, but not with periodontal disease parameters or cardiovascular risk factors.. Systemic antibodies against A. actionmycetemcomitans leukotoxin were common in this adult Swedish population. These antibodies might contribute to limit the systemic effects of the infection.

Topics: Aggregatibacter actinomycetemcomitans; Alveolar Bone Loss; Antibodies, Bacterial; Bacterial Toxins; Blotting, Western; Case-Control Studies; Cell Line, Tumor; Cytotoxins; Dental Plaque Index; Exotoxins; Humans; Hypertension; Immunity, Humoral; Immunoglobulin A; Immunoglobulin G; Immunosuppressive Agents; Leukemia, Monocytic, Acute; Middle Aged; Myocardial Infarction; Periodontal Index; Periodontitis

Although certain serotypes of Aggregatibacter actinomycetemcomitans are associated more with aggressive periodontitis than are other serotypes, the correlation between distinct lineages and virulence traits in this species is poorly understood. This study aimed to evaluate the polymorphism of genes encoding putative virulence factors of clinical isolates, and to correlate these findings with A. actinomycetemcomitans serotypes, genotypes and periodontal status of the hosts.. Twenty-six clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Genotyping was performed using pulse-field gel electrophoresis. Polymorphisms in the genes encoding leukotoxin, Aae, ApaH and determinants for serotype-specific O polysaccharide were investigated.. The isolates were classified into serotypes a-f, and exhibited three apaH genotypes, five aae alleles and 25 macrorestriction profiles. Two serotype b isolates (7.7%), obtained from Brazilian patients with aggressive periodontitis, were associated with the highly leukotoxic genotype; these isolates showed identical fingerprint patterns and aae and apaH genotypes. Serotype c, obtained from various periodontal conditions, was the most prevalent among Brazilian isolates, and isolates were distributed in two aae alleles, but formed a genetically distinct group based on apaH analysis. Cluster analysis showed a close relationship between fingerprinting genotypes and serotypes/apaH genotypes, but not with aae genotypes.. Apart from the deletion in the ltx promoter region, no disease-associated markers were identified. Non-JP2-like strains recovered from individuals with periodontal disease exhibited considerable genetic variation regarding aae/apaH genotypes, serotypes and XhoI DNA fingerprints.

Topics: Actinobacillus Infections; Adhesins, Bacterial; Aggregatibacter actinomycetemcomitans; Aggressive Periodontitis; Alleles; Bacterial Outer Membrane Proteins; Bacterial Toxins; Base Pairing; Chronic Periodontitis; Deoxyribonucleases, Type II Site-Specific; DNA Fingerprinting; Exotoxins; Genetic Variation; Genotype; Humans; O Antigens; Periodontal Index; Periodontal Pocket; Periodontitis; Periodontium; Polymorphism, Genetic; Promoter Regions, Genetic; Serotyping; Virulence Factors

For many years, attention has been given to the oral bacterium Aggregatibacter actinomycetemcomitans, as a species possibly implicated in the etiology of aggressive periodontitis in adolescents. One of the major virulence factors of A. actinomycetemcomitans is the leukotoxin which is able to kill important cells of the immune system. As demonstrated in population genetic analyses, the population structure of A. actinomycetemcomitans is mainly clonal with evolutionary lineages corresponding to the serotypes. A particular highly leukotoxic clone (JP2) of serotype b has been discovered. The JP2 clone, with an estimated origin some 2400 years ago, is found to be highly conserved, based on analyses of a collection of JP2 clone strains collected through more than 20 years from individuals of diverse origin and living geographically widespread. Despite demonstration of minor evolutionary changes within the genome of JP2 clone strains of A. actinomycetemcomitans, the JP2 clone strains constitute a unique clonal type, the characteristics of which include a 530 basepair deletion in the leukotoxin operon implicated in the enhanced leukotoxic activity of the clone. Mapping of the geographic occurrence of the JP2 clone of A. actinomycetemcomitans has revealed that its colonization is largely restricted to individuals of African descent. Characteristic mutations, which allow JP2 clone isolates from the Mediterranean region to be distinguished from isolates from West Africa, including the Cape Verde islands, suggest that the JP2 clone initially emerged as a distinct genotype in the Mediterranean region of Africa and subsequently spread to West Africa, from where it might have been transferred to the American continent during the transatlantic slave trade. The finding of a sustained selective colonization of individuals of African descent, despite geographical separation from the African continent for centuries, suggests that the JP2 clone might have a distinct host tropism. Further studies are needed to elucidate the reasons for the apparent selective colonization of the Mediterranean and Western African populations. The JP2 clone of A. actinomycetemcomitans appears to play a prominent role in the etiology of aggressive periodontitis compared to other clonal types of the species. While A. actinomycetemcomitans, in general, is considered an opportunistic pathogen of the resident oral microbiota, the JP2 clone has features similar to those of an exogenous pathogen. Clonal

Topics: Actinobacillus Infections; Adolescent; Africa, Northern; Africa, Western; Aggregatibacter actinomycetemcomitans; Antigens, Bacterial; Black People; Evolution, Molecular; Exotoxins; Genotype; Humans; Mutation; Opportunistic Infections; Periodontitis

Aggregatibacter actinomycetemcomitans is an oral pathogen causing localized aggressive periodontitis (LAP). Recently, we characterized for the first time a quinol peroxidase (QPO) that catalyzes peroxidase activity using quinol in the respiratory chain of A. actinomycetemcomitans for the reduction of hydrogen peroxide. In the present study, we characterized the phenotype of a QPO null mutant. The QPO null mutant shows an oxidative stress phenotype, suggesting that QPO plays a certain role in scavenging endogenously generated reactive oxygen species. Notably, we discovered that the QPO null mutant exhibits a production defect of leukotoxin (LtxA), which is a secreted bacterial toxin and is known to target human leukocytes and erythrocytes. This result suggests that QPO would be considered as a potential drug target to inhibit the expression of LtxA from A. actinomycetemcomitans for the treatment and prevention of LAP.

Topics: Actinobacillus Infections; Aggregatibacter actinomycetemcomitans; Bacterial Proteins; Exotoxins; Humans; Hydrogen Peroxide; Hydroquinones; Mutation; Oxidative Stress; Periodontitis; Peroxidase

beta2-Glycoprotein I (beta 2GPI) is important in the suppression of coagulation, and antibodies against TLRVYK peptides on the beta 2GPI molecule are related to thrombosis. According to the Swiss-Prot database, Aggregatibacter actinomycetemcomitans leukotoxin c has sequences (SIRVYK) that are homologous to the TLRVYK peptides. The aim of this study was to investigate the effects of A. actinomycetemcomitans infection on the antibody response against SIRVYK peptides in patients with periodontitis.. Serum immunoglobulin G (IgG) antibody and IgG subclass antibody titers against SIRVYK or TLRVYK peptides were measured by enzyme-linked immunosorbent assay in 46 patients with aggressive periodontitis (eight with localized disease, 38 with generalized disease), 28 patients with chronic periodontitis, and 20 periodontally healthy subjects. The presence of A. actinomycetemcomitans in plaque and saliva samples was determined using polymerase chain reaction.. The level of anti-SIRVYK antibodies was significantly higher in patients who were A. actinomycetemcomitans-positive than in A. actinomycetemcomitans-negative patients (P < 0.05) in the chronic periodontitis group. A similar trend was found in the antibody response to TLRVYK peptide; however, no statistically significant difference was seen between A. actinomycetemcomitans-positive and -negative patients. The A. actinomycetemcomitans-positive patients displayed significantly higher levels of anti-SIRVYK IgG2 and IgG3 antibodies than A. actinomycetemcomitans-negative patients (P < 0.05 and P < 0.05, respectively). The level of IgG2 was highest among the four IgG subclasses and it predominantly increased in patients who were A. actinomycetemcomitans-positive. Anti-TLRVYK antibody levels were significantly correlated with anti-SIRVYK IgG antibody levels.. The results suggest that A. actinomycetemcomitans infection may elicit anti-SIRVYK IgG antibodies and modify the anti-TLRVYK antibody response in patients with periodontitis by molecular mimicry with beta2GPI.

Topics: Adult; Aggregatibacter actinomycetemcomitans; Antibodies, Bacterial; Anticoagulants; Bacterial Toxins; beta 2-Glycoprotein I; Chronic Disease; Dental Plaque; Exotoxins; Gingival Hemorrhage; Humans; Immunoglobulin G; Middle Aged; Molecular Mimicry; Peptide Fragments; Periodontal Pocket; Periodontitis; Periodontium; Polymerase Chain Reaction; Saliva

Aggregatibacter (Actinobacillus) actinomycetemcomitans forms a leukotoxin that selectively lyses primate neutrophils, monocytes and triggers apoptosis in promyeloic cells and degranulation of human neutrophils. Recently, we showed that the leukotoxin causes activation of caspase-1 and abundant secretion of bio-active IL-1beta from human macrophages. In this study, we show that high levels of IL-beta correlated with a high proportion of A. actinomycetemcomitans in clinical samples from a patient with aggressive periodontitis. To determine the relative contribution of leukotoxin to the overall bacteria-induced IL-1beta secretion, macrophages were isolated from peripheral blood and exposed to different concentrations of live A. actinomycetemcomitans strains with either no, low or high production of leukotoxin. Cell lysis and levels of IL-1beta, IL-6, TNF-alpha and caspase-1 were measured by ELISA and flow cytometry. Leukotoxin was the predominant cause of IL-1beta secretion from macrophages, even in the A. actinomycetemcomitans strain with low leukotoxin production. Macrophages exposed to non-leukotoxic bacteria accumulated cytosolic pro-IL-1beta, which was secreted by a secondary exposure to leukotoxic bacteria. In conclusion, the present study shows for the first time that A. actinomycetemcomitans-induced IL-1beta secretion from human macrophages in vitro is mainly caused by leukotoxin.

Topics: Adult; Aggregatibacter actinomycetemcomitans; Bacterial Proteins; Caspase 1; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Exotoxins; Flow Cytometry; Humans; Interleukin-1beta; Interleukin-6; Macrophages; Periodontitis; Tumor Necrosis Factor-alpha

Most Aggregatibacter actinomycetemcomitans strains express relatively low levels of leukotoxin, encoded by the orfA-ltxCABD operon. However, several strains isolated from patients with localized aggressive periodontitis are hyperleukotoxic and transcribe the ltx operon at high levels. These strains possess a copy of IS1301 in the ltx promoter and previous studies have suggested that the presence of the insertion sequence increases ltx transcription by uncoupling a cis-acting negative regulator of ltx expression from the basal elements of the ltx promoter. However, we now report that replacing IS1301 with an equal length of random sequence has little effect on transcriptional activity of the ltx promoter, suggesting that the physical displacement of the negative regulatory element does not contribute to the hyperleukotoxic phenotype of IS1301-containing strains. Instead, we show that a -10-like element upstream of the transposase ORF of IS1301 is required for increased transcriptional activity of the ltx promoter. Site-specific mutation of the -10 sequence, or reversing the orientation of IS1301 relative to the basal ltx promoter elements, reduced transcriptional activity to levels exhibited by the native ltx promoter. However, no increase in transcription was observed when IS1301 was recombinantly inserted into a ltx promoter that contained a truncated copy of orfA, suggesting that an intact orfA may also be required for IS1301-mediated induction of ltxCABD. Therefore, to determine if orfA functions as a regulator of ltx expression, three independent ltx-promoter-lacZ-reporter constructs containing frameshift mutations in orfA were analysed. Each exhibited significantly lower expression of beta-galactosidase than the control reporter with intact orfA. In addition, OrfA protein was shown, by mobility shift electrophoresis, to interact with the ltx promoter at or downstream of the -35 sequence. These results suggest that a potential transposase promoter and the OrfA polypeptide may modulate leukotoxin expression in hyperleukotoxic A. actinomycetemcomitans strains containing IS1301.

Topics: Actinobacillus Infections; Aggregatibacter actinomycetemcomitans; Base Sequence; beta-Galactosidase; DNA Transposable Elements; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Exotoxins; Frameshift Mutation; Gene Expression Regulation, Bacterial; Genes, Regulator; Genes, Reporter; Humans; Molecular Sequence Data; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Operon; Periodontitis; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Transcription, Genetic

Actinobacillus actinomycetemcomitans is associated with localized aggressive periodontitis, a disease characterized by rapid loss of the alveolar bone surrounding the teeth. Receptor activator of NF-kappaB Ligand (RANKL) and osteoprotegerin (OPG) are two molecules that regulate osteoclast formation and bone resorption. RANKL induces osteoclast differentiation and activation, whereas OPG blocks this process by acting as a decoy receptor for RANKL. The purpose of this study was to investigate the effect of A. actinomycetemcomitans on the expression of RANKL and OPG in human gingival fibroblasts and periodontal ligament cells. RANKL mRNA expression was induced in both cell types challenged by A. actinomycetemcomitans extract, whereas OPG mRNA expression remained unaffected. Cell surface RANKL protein was also induced by A. actinomycetemcomitans, whereas there was no change in OPG protein secretion. A cytolethal distending toxin (Cdt) gene-knockout strain of A. actinomycetemcomitans did not induce RANKL expression, in contrast to its wild-type strain. Purified Cdt from Haemophilus ducreyi alone, or in combination with extract from the A. actinomycetemcomitans cdt mutant strain, induced RANKL expression. Pretreatment of A. actinomycetemcomitans wild-type extract with Cdt antiserum abolished RANKL expression. In conclusion, A. actinomycetemcomitans induces RANKL expression in periodontal connective tissue cells. Cdt is crucial for this induction and may therefore be involved in the pathological bone resorption during the process of localized aggressive periodontitis.

Topics: Aggregatibacter actinomycetemcomitans; Bacterial Toxins; Bone Resorption; Carrier Proteins; Cells, Cultured; Exotoxins; Fibroblasts; Gene Expression Regulation; Gingiva; Glycoproteins; Humans; Lipopolysaccharides; Membrane Glycoproteins; Osteoprotegerin; Periodontal Ligament; Periodontitis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger

This study characterized Actinobacillus actinomycetemcomitans isolates from young Chinese aggressive periodontitis patients.. Subgingival plaque samples (two/subject) were collected from diseased subjects < 25 years old (n = 9, mean age 21.1 +/- 1.6 years) and age-matched periodontitis-free controls (n = 47, mean age 22.0 +/- 1.1 years). Selective and anaerobic culture were used. The serotype, leukotoxin gene (ltx) operon promoter and the cytolethal distending toxin (cdt) genes complex of the A. actinomycetemcomitans isolates were investigated. Effects of the isolates on non-keratinizing periodontal ligament epithelial cells monolayer were studied.. Diseased subjects had significantly higher full-mouth bleeding score (p = 0.002) and total viable counts from plaque samples (7.2 x 10(6) vs. 2.1 x 10(5) CFU/paperpoint, p < 0.005). A. actinomycetemcomitans was isolated from 67%/56% or 6%/4% of diseased or controls subject/sites, respectively (p < 0.001). The proportion of A. actinomycetemcomitans isolatable from aggressive periodontitis or periodontitis-free associated subgingival plaque was low (0.7% vs. 0.1%, p < 0.02). The serotype of the isolates was characterized. All isolates possessed 652-like ltx gene promoter and all but one serotype c isolate from a diseased patient had intact cdtABC genes. That particular strain appeared to confer the least cellular damages on periodontal ligament epithelial monolayer compared to others.. This preliminary study confirmed the notion of increased prevalence and quantity of A. actinomycetemcomitans associated with aggressive periodontitis in young patients. The overall ltx promoter and cdt characteristics of the A. actinomycetemcomitans isolates, however, were similar among the diseased and control groups. A strain lacking the cdtABC gene appeared to be less damaging to a periodontal ligament epithelial cell model. Further studies therefore are warranted to clarify the pathogenic role and potentials of A. actinomycetemcomitans in aggressive periodontitis.

Topics: Actinobacillus Infections; Adult; Aggregatibacter actinomycetemcomitans; Animals; Bacterial Toxins; Case-Control Studies; Exotoxins; Humans; Periodontitis; Swine

Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis secrete several potent virulence factors and are known to be two of the major periodontal pathogens. In the present case-control study, the systemic immunoreactivity to A. actinomycetemcomitans exotoxins, cytolethal distending toxin (Cdt) and leukotoxin was analyzed in adult subjects with periodontitis and in periodontally healthy controls. Furthermore, systemic immunoreactivity to P. gingivalis was analyzed in these subjects. Reactivity to the A. actinomycetemcomitans toxins was determined in bioassays that quantified neutralizing antibodies, and P. gingivalis antibodies were detected by enzyme-linked immunosorbent assay (ELISA). The results showed a significantly enhanced immunoreactivity to P. gingivalis in the subjects with periodontitis, while the reactivity to A. actinomycetemcomitans leukotoxin showed no significant difference between patients and controls. However, combined immunoreactivity to leukotoxin and Cdt was more prevalent in the subjects with periodontitis than in the controls. In addition, immunoreactivity to leukotoxin correlated to periodontitis in men but not in women. In conclusion, data from the present study indicate that immunoreactivity to P. gingivalis is frequent in adult periodontitis, while the role of A. actinomycetemcomitans seems to be more complex and depends on gender of the infected subject as well as the virulence of the bacteria.

Topics: Adult; Aged; Aggregatibacter actinomycetemcomitans; Antibodies, Bacterial; Bacterial Toxins; Case-Control Studies; Cytotoxins; Exotoxins; Female; Humans; Male; Middle Aged; Neutrophils; Periodontal Attachment Loss; Periodontitis; Periodontium; Porphyromonas gingivalis; Sex Factors; Virulence Factors

The use of whole saliva has shown to be promising in detecting Actinobacillus actinomycetemcomitans out of the subgingival environment. The objective of the present study was to evaluate the use of unstimulated saliva in detecting A. actinomycetemcomitans and to compare the subgingival and extracrevicular occurrence of this pathogen in Brazilian subjects with chronic periodontitis.. Sixty-six patients (mean age 38.01 9.28 years) with advanced generalized chronic periodontitis were sampled. Subgingival plaque samples were collected from eight sites per patient representing the two deepest sites of each quadrant. Samples of the mucous surfaces, including dorsal surface of the tongue and cheek, were collected with a sterile swab and placed in a microtube containing a reduced solution. Samples of unstimulated saliva were also collected in sterile tubes and 0.1 ml of whole saliva was diluted in 1 ml of reduced solution. The presence of A. actionomycetemcomitans was established using bacterial culture in trypticase soy bacitracin vancomycin selective media. Polymerase chain reaction (PCR) was used to differentiate highly from minimally leukotoxic strains in patients who presented A. actinomycetemcomitans in at least two sampled sites.. A. actinomycetemcomitans was isolated from 63.63% of subgingival samples, 56.06% of saliva samples, and 45.45% of samples from mucous surfaces. No statistical difference was observed between subgingival and salivary occurrence of the microorganism. Linear regression showed an association between subgingival plaque and saliva (r(2) = 0.897; P = 0.015) and mucous membrane and saliva (r(2) = 0.152; P = 0.024). The same A. actinomycetemcomitans leukotoxic profile was observed in all sampled sites for a given patient.. These results suggest that in advanced periodontitis, unstimulated saliva is representative of pooled subgingival plaque samples and its use is appropriate in the oral detection of A. actinomycetemcomitans.

Topics: Adult; Aggregatibacter actinomycetemcomitans; Chronic Disease; Dental Plaque; Exotoxins; Female; Humans; Linear Models; Male; Middle Aged; Mouth Mucosa; Periodontitis; Polymerase Chain Reaction; Saliva

Previous studies suggest differences between geographically and racially distinct populations in the prevalence of periodontopathic bacteria as well as greater periodontal destruction associated with infection by highly leucotoxic Actinobacillus actinomycetemcomitans. The present study examined these hypotheses in Brazilians with aggressive or chronic periodontitis.. Clinical, radiographical, and microbiological assessments were performed on 25 aggressive periodontitis and 178 chronic periodontitis patients including 71 males and 132 females, 15-69 years of age.. The prevalence of Porphyromonas gingivalis was similar to that of other South American populations. The prevalence of A. actinomycetemcomitans and its highly leucotoxic subgroup was higher in Brazilians. Highly leucotoxic A. actinomycetemcomitans was more prevalent in aggressive periodontitis (chi2=27.83) and positively associated with deep pockets (>6 mm, chi2=18.26) and young age (<29 years, chi2=18.68). Greater mean attachment loss was found in subjects with highly leucotoxic A. actinomycetemcomitans than in subjects with minimally leucotoxic (p=0.0029) or subjects not infected (p=0.0001).. These data support the hypothesis of differences between populations in the prevalence of periodontopathic bacteria and of greater attachment loss in sites infected with highly leucotoxic A. actinomycetemcomitans. Detection of highly leucotoxic A. actinomycetemcomitans in children and adolescents may be a useful marker for aggressive periodontitis.

Topics: Acute Disease; Adolescent; Adult; Aged; Aggregatibacter actinomycetemcomitans; Bacteroides; Brazil; Campylobacter rectus; Chronic Disease; Colony Count, Microbial; Cross-Sectional Studies; Dental Plaque; DNA, Bacterial; Exotoxins; Female; Humans; Logistic Models; Male; Middle Aged; Periodontitis; Porphyromonas gingivalis; Prevalence; Prevotella intermedia

Chronic infections and associated inflammatory markers are suggested risk factors for cardiovascular diseases (CVD) and stroke. The proinflammatory cytokine interleukin (IL)-1beta is suggested to play a role in the regulation of local inflammatory responses in both CVD and periodontitis. The leukotoxin from the periodontal pathogen Actinobacillus actinomycetemcomitans has recently been shown to cause abundant secretion of IL-1beta from macrophages. The aim of the present study was to compare the prevalence of systemic antibodies to A. actinomycetemcomitans leukotoxin in stroke cases (n = 273) and matched controls (n = 546) in an incident case-control study nested within the Northern Sweden MONICA and Vasterbotten Intervention cohorts.. Antibodies to A. actinomycetemcomitans leukotoxin were analyzed in a bioassay with HL-60 cells (leukocytes), purified A. actinomycetemcomitans leukotoxin, and plasma. Plasma samples which inhibited lactate dehydrogenase release from leukotoxin-lysed cells by > or =50% were classified as antibody positive.. Antibodies to A. actinomycetemcomitans leukotoxin were detected in 18.8% of the women and 15.2% of the men. Women with those antibodies had a significantly decreased risk for stroke (OR = 0.28, 95% CI: 0.13-0.59), but not men (OR = 0.88, 95% CI: 0.52-1.51).. The immunoreactivity to A. actinomycetemcomitans leukotoxin correlates negatively with a future stroke in woman, but not in men. Further studies are needed to explain the underlying mechanisms, as well as the biological relevance of this finding.

Topics: Actinobacillus Infections; Aggregatibacter actinomycetemcomitans; Antibodies, Bacterial; Case-Control Studies; Exotoxins; Female; Humans; Male; Middle Aged; Periodontitis; Risk Factors; Seroepidemiologic Studies; Sex Distribution; Stroke

The JP2 clone of Actinobacillus actinomycetemcomitans, a high-leukotoxin-producing strain, characterized by a 530-basepair (bp) deletion in the promoter region of the leukotoxin gene operon and mainly found among individuals with African origin, is associated with localized aggressive periodontitis. The objective of the study was to examine the occurrence of periodontal disease in a Moroccan immigrant family living in Denmark in which the oldest son (14 year) was referred and treated for localized aggressive periodontitis. Further, the potential occurrence of the JP2 clone of A. actinomycetemcomitans in the family was examined. Here we present the clinical, radiographic, and microbiological findings from the family. Clinical and radiographic examination of the other family members revealed that 3 of 5 younger siblings had localized aggressive periodontitis, one had gingivitis and the mother had chronic periodontitis. Despite scaling followed by intensive maintenance therapy several family members, including the sibling with gingivitis, had further attachment loss at the 1-year examination. The JP2 clone of A. actinomycetemcomitans was isolated from subgingival plaque samples from 4 children with periodontitis. In contrast, it was not detected in plaque from the oldest boy, who had been treated for localized aggressive periodontitis by surgery combined with antibiotic therapy. The 4 children with periodontitis and colonized with the JP2 clone were treated by scaling and antibiotic administration. One month later the JP2 clone could still be detected in plaque samples. In conclusion, it is confirmed that members of immigrant families with African origin are potential carriers of the JP2 clone and that those families often have multiple family members with localized aggressive periodontitis. It is proposed that those families are given periodontal examination frequently to benefit from early diagnosis and treatment of the disease.

Topics: Actinobacillus Infections; Adolescent; Adult; Aggregatibacter actinomycetemcomitans; Bacterial Toxins; Base Sequence; Child; Chronic Disease; Denmark; Dental Plaque; Emigration and Immigration; Exotoxins; Female; Follow-Up Studies; Gingivitis; Humans; Male; Morocco; Operon; Periodontitis; Promoter Regions, Genetic; Sequence Deletion

Actinobacillus actinomycetemcomitans, an oral bacterium implicated in the etiology of periodontal diseases, produces a leukotoxin that selectively lyses primate neutrophils and monocytes, the major populations of defense cells in the periodontium. Though lysis requires expression of the receptor lymphocyte function-associated molecule 1 (LFA-1) on the cell surface, not all LFA-1-expressing leukocyte populations are equally susceptible to the toxin. In this study, the susceptibility of human leukocytes to leukotoxin-induced lysis is compared to their expression of LFA-1 and the activity of caspase 1. Cytolysis was determined by the activity of lactate dehydrogenase released from peripheral human leukocytes after 1-h exposure to leukotoxin. Monocytes were lysed at leukotoxin concentrations of > or = 5 ng/ml, while the corresponding values for neutrophils and lymphocytes were approximately 10 times greater. Similar LFA-1 expression was found in all susceptible cell populations irrespective of their degree of sensitivity to the toxin. Exposure of monocytes to leukotoxin increased their caspase 1 activity about fivefold within 10 to 20 min. Presence of the caspase 1 inhibitor Ac-YVAD-CMK significantly blocked the leukotoxin-induced lysis of monocytes only. At sublytic concentrations, leukotoxin induced no apoptotic activity in monocytes, as revealed by the lack of caspase 3 activation and DNA fragmentation. Monocytes are the most lysis-sensitive leukocytes for A. actinomycetemcomitans leukotoxin. Their lysis by this toxin depends on caspase 1 activation and proceeds through a process that differs from classical apoptosis.

Topics: Actinobacillus Infections; Aggregatibacter actinomycetemcomitans; Bacterial Toxins; Caspase 1; Caspase 3; Caspases; Exotoxins; HL-60 Cells; Humans; In Vitro Techniques; Lymphocyte Function-Associated Antigen-1; Lymphocytes; Monocytes; Neutrophils; Periodontitis

The aim of this study was to determine the prevalence and the structure of the leukotoxin promoter region of Actinobacillus actinomycetemcomitans in an ethnic Chinese population.. Subgingival plaque samples were collected from 42 patients with moderate to advanced periodontitis and 50 periodontally healthy patients. A. actinomycetemcomitans was detected directly from the crude subgingival plaque by PCR using leukotoxin gene specific primers. The presence of A. actinomycetemcomitans was determined by a single 285 bp PCR amplicon.. A. actinomycetemcomitans was found to be present in the subgingival plaque of 68 out of a total of 92 patients examined (74%). 29 out of the 42 periodontitis patients tested were carriers of A. actinomycetemcomitans (69%). Among the periodontally healthy patients studied, 39 out of 50 subjects possessed the bacteria (78%). PCR analysis of the promoter region of the ltx operon revealed that none of the 42 moderate to advanced periodontitis patients examined harboured A. actinomycetemcomitans strains with the JP2-like promoter of the ltx operon, known to enhance leukotoxin expression. 2 out of the 27 advanced periodontitis patients clinically diagnosed as suffering from rapidly progressive periodontitis were found to be carriers of the mildly toxic strain of A. actinomycetemcomitans with the characteristic 652-like promoter.. The high prevalence of A. actinomycetemcomitans, regardless of whether the subgingival samples were analysed from patients with healthy or diseased periodontium suggests that this bacterial species is part of the normal oral flora of ethnic Chinese. Our preliminary results also suggested that subjects who harboured the mildly toxic strain of A. actinomycetemcomitans were potentially susceptible to aggressive forms of periodontitis.

Topics: Actinobacillus Infections; Adult; Aggregatibacter actinomycetemcomitans; Case-Control Studies; China; Dental Plaque; Exotoxins; Female; Gene Expression Regulation, Bacterial; Humans; Male; Middle Aged; Periodontitis; Polymerase Chain Reaction; Prevalence; Singapore

Actinobacillus actinomycetemcomitans leukotoxin is thought to be an important virulence factor in the pathogenesis of localized juvenile and other forms of early-onset periodontitis. Some highly leukotoxic A. actinomycetemcomitans strains produce 10 to 20 times more leukotoxin than other minimally leukotoxic strains. The distribution, clonality, and intrafamilial transmission of highly leukotoxic A. actinomycetemcomitans were examined in order to determine the importance of leukotoxin in the pathogenesis of periodontitis.. The polymerase chain reaction (PCR) was used to differentiate highly leukotoxic from minimally leukotoxic strains in examining 1,023 fresh A. actinomycetemcomitans isolates and strains from our culture collection. These were obtained from 146 subjects including 71 with localized juvenile periodontitis (LJP), 4 with early-onset periodontitis, 11 with post-localized juvenile periodontitis, 41 with adult periodontitis, and 19 periodontally normal subjects. The arbitrarily primed polymerase chain reaction (AP-PCR) analysis of 30 oral isolates from each of 25 subjects was used to determine the intraoral distribution of A. actinomycetemcomitans clones. AP-PCR was also used to examine the transmission of A. actinomycetemcomitans in 30 members of 6 families. The clonality of 41 highly leukotoxic A. actinomycetemcomitans strains was evaluated by both AP-PCR and ribotyping.. Highly leukotoxic A. actinomycetemcomitans was found only in subjects with localized juvenile and early-onset periodontitis. Fifty-five percent of the LJP subjects harbored highly leukotoxic A. actinomycetemcomitans isolates. Seventy-three percent of the A. actinomycetemcomitans isolates in these subjects were highly leukotoxic. Highly leukotoxic A. actinomycetemcomitans infected younger subjects (mean age 13.95 years, range 5 to 28 years) than minimally leukotoxic (mean age 35.47 years, range 6 to 65 years). Most subjects were infected with only one A. actinomycetemcomitans genotype. However, PCR of whole dental plaques and subsequent analysis of up to 130 individual oral isolates suggested a possible shift in A. actinomycetemcomitans over time in that a few subjects harbored both highly leukotoxic and minimally leukotoxic strains. AP-PCR analysis was consistent with intrafamilial A. actinomycetemcomitans transmission. Ribotyping and AP-PCR analysis confirmed a previous report that highly leukotoxic A. actinomycetemcomitans consists of a single clonal type.. This study suggests that localized juvenile and other forms of Actinobacillus-associated periodontitis are primarily associated with the highly leukotoxic clone of A. actinomycetemcomitans.

Topics: Actinobacillus Infections; Adolescent; Adult; Age Factors; Aged; Aggregatibacter actinomycetemcomitans; Aggressive Periodontitis; Bacterial Toxins; Chi-Square Distribution; Child; Child, Preschool; Clone Cells; Cytotoxins; Dental Plaque; Exotoxins; Female; Humans; Male; Middle Aged; Periodontitis; Periodontium

Actinobacillus actinomycetemcomitans strains showing a 530-bp deletion in the promoter region of the leukotoxin gene operon elaborate high amounts of leukotoxin that may play a role in the pathogenesis of periodontal disease. This study used polymerase chain reaction detection to determine the occurrence of the 530-bp deletion in 94 A. actinomycetemcomitans strains from individuals of various ethnic backgrounds. Eleven blacks and one Hispanic subject but no Caucasian or Asian subjects showed the 530-bp deletion in the leukotoxin promoter region, suggesting that the deletion is mainly a characteristic of individuals of African descent. A. actinomycetemcomitans strains exhibiting a deletion in the leukotoxin promoter region occurred both in individuals having severe periodontitis and in adolescents revealing no evidence of destructive periodontal disease.

Topics: Actinobacillus Infections; Adolescent; Adult; Aggregatibacter actinomycetemcomitans; Asian People; Bacterial Toxins; Base Pairing; Black People; Cytotoxins; Ethnicity; Exotoxins; Gene Deletion; Gene Frequency; Hispanic or Latino; Humans; Operon; Periodontal Diseases; Periodontitis; Polymerase Chain Reaction; Promoter Regions, Genetic; White People

Salivary, gingival crevicular fluid and serum-specific immunoglobulin G (IgG)-subclass antibodies to Actinobacillus actinomycetemcomitans leuktoxin were quantified by enzyme-linked immunosorbent assay. Samples were taken from six patients with periodontal pockets > or = 5 mm, harboring A. actinomycetemcomitans in subgingival plaque and from six healthy, sex- and age-matched controls, who did not harbor A. actinomycetemcomitans. In individuals suffering from periodontitis, the median values of specific IgG1- and IgG2-subclass antibodies in saliva, gingival crevicular fluid and serum were, respectively IgG1 147 ng/ml, 5226 ng/ml and 7318 ng/ml and IgG2 4.8 ng/ml, 934 ng/ml and 860 ng/ml. In the patients, specific IgG3 antibodies were detected in one out of six individuals in saliva, in two individuals in gingival crevicular fluid and in five out of six patients in serum with a median value of 561 ng/ml. The median values of specific IgG4 antibodies in saliva, gingival crevicular fluid and serum were below detectable levels. The median values of the total IgG subclasses in saliva and serum were 14622 ng/ml and 10.3 g/l respectively. Individuals with periodontitis had, compared with controls, a higher ratio of specific IgG1 antibodies to total IgG1 in saliva (P < 0.05) and in serum (P < 0.05) and a higher ratio of specific IgG antibodies to total IgG in saliva (P < 0.05) and in serum (P < 0.01). The results show an elevation of both oral and systemic specific antibodies to A. actinomycetemcomitans leukotoxin.

Topics: Adult; Aggregatibacter actinomycetemcomitans; Antibodies, Bacterial; Bacterial Toxins; Exotoxins; Female; Gingival Crevicular Fluid; Humans; Immunoglobulin G; Male; Middle Aged; Periodontitis; Saliva; Statistics, Nonparametric

Actinobacillus actinomycetemcomitans is a major periodontal pathogen which is associated with both early-onset periodontitis and adult cases refractory to conventional periodontal therapy, although the organism has also been shown to be widely distributed among dentate healthy individuals. The observed disease status may be associated with a variation in virulence of different strains or clones. The aim of the present study was to analyse genotype distribution as assessed by an arbitrarily primed polymerase chain reaction (AP-PCR) among 51 isolates of A. actinomycetemcomitans recovered from more than 200 young adult recruits with no or minor periodontal disease. In addition, isolates from 25 periodontitis patients as well as reference strains were genotyped. Primers amplifying (i) a specific sequence in the ltxA region, (ii) a specific 16S rRNA sequence and (iii) sequences in the leukotoxin promoter region were used to verify species identity of the strains. Three random oligonucleotide primers were employed to analyse genomic polymorphisms of the organism by means of PCR. A total of 19 genotypes could be distinguished, which were grouped by cluster analysis into 5 major clusters based on genetic similarity and a complete linkage sort. Whereas 3 clusters assembled A. actinomycetemcomitans genotypes isolated from both healthy subjects and periodontitis patients, one cluster containing 4 different genotypes exclusively comprised isolates from healthy or gingivitis subjects. Another cluster with 2 genotypes consisted of strains originating from periodontitis patients (p < 0.05). One strain characterized by a specific 530 bp deletion in the promoter region of the leukotoxin region was identified in a Ghanese patient with localized juvenile periodontitis. It was concluded that there is considerable clonal diversity of A. actinomycetemcomitans strains isolated from healthy or periodontally diseased subjects, and that genetically closely related groups might be associated with health or disease.

Topics: Actinobacillus Infections; Adolescent; Adult; Aggregatibacter actinomycetemcomitans; Aggressive Periodontitis; Bacterial Toxins; Base Pairing; Clone Cells; Cytotoxins; Exotoxins; Gene Deletion; Genetic Linkage; Genotype; Gingivitis; Humans; Male; Periodontitis; Periodontium; Polymerase Chain Reaction; Polymorphism, Genetic; Promoter Regions, Genetic; RNA, Ribosomal, 16S; Virulence

The expression of leukotoxin varies among Actinobacillus actinomycetemcomitans strains and is dependent in part on the structure of the ltx promoter region. Highly leukotoxic strains, characterized by a 530 base pair (bp) deletion within the ltx promoter, have been associated with juvenile periodontitis in the United States and Europe. In the present study, we analyzed the ltx promoter structure to elucidate whether A. actinomycetemcomitans from Japanese periodontitis patients exhibits the highly toxic phenotype.. Forty-five A. actinomycetemcomitans strains, including 43 clinical isolates, the highly leukotoxic strain JP2, and a minimally leukotoxic strain 652 were used in the study. The ltx promoter structure was analyzed by polymerase chain reaction (PCR), with oligonucleotide primers focusing the ltx promoter region, and nucleotide sequencing. Leukotoxic activity was determined by trypan blue exclusion. Western blotting assay was performed to detect the level of leukotoxin polypeptide.. A 495 bp PCR product was amplified from JP2, a 1025 bp product from 652 and 41 of the clinical isolates, and a 1926 bp product from the remaining two clinical isolates (AaIS1, AaIS2). Sequencing of the 1926 bp PCR fragment showed that it was similar to that of strain 652 but contained an 886 bp region that was identified as an insertion sequence (IS). Both AaIs strains expressed high levels of leukotoxicity, similar to strain JP2. In addition, a mutant (AaIS-) that had lost the IS element expressed a significantly lower level of leukotoxicity compared with AaIS strains. Furthermore, the levels of leukotoxin polypeptide expressed by these strains were consistent with their whole cell leukotoxicity.. A. actinomycetemcomitans clinical strains which were isolated from Japanese periodontitis patients do not possess the 530 bp ltx promoter deletion. The results of this study suggest that a high level of leukotoxin expression correlates with the insertion of the transposable DNA element.

Topics: Adolescent; Adult; Aggregatibacter actinomycetemcomitans; Amino Acid Sequence; Bacterial Toxins; Base Sequence; DNA Transposable Elements; DNA, Bacterial; Exotoxins; Female; Gene Expression Regulation, Bacterial; Humans; Male; Middle Aged; Periodontitis; Polymerase Chain Reaction; Promoter Regions, Genetic; Sequence Analysis, DNA; Sequence Deletion

Actinobacillus actinomycetemcomitans is a major periodontal pathogen which is associated with early-onset and adult periodontitis. The organism has been shown to be widely distributed among dentate, healthy individuals as well. It has been demonstrated that A. actinomycetemcomitans shows great genetic diversity. An especially virulent clone of the organism (JP2-like) with a specific 530-base pair (bp) deletion in the promoter region of the leukotoxin gene has been isolated from localized juvenile periodontitis patients and related subjects of African and African-American origin. The aim of the present study was to examine the presence of this specific clone employing specific oligonucleotide primers in a polymerase chain reaction among 51 isolates of A. actinomycetemcomitans recovered from 201, 18- to 25-year-old recruits with no or minor periodontal disease. In addition, clinical isolates from 37 periodontitis patients were analyzed as well as reference strains ATCC 29524, NCTC 9710, Y4 and JP2. Primers amplifying a specific 285-bp amplification product in the ltxA region of the leukotoxin gene and a specific 547-bp amplification product of 16 S rRNA were used to genetically confirm identification of the organisms. Primers amplifying sequences in the leukotoxin promoter region were used to identify the deletion. Species specific primers definitively identified all A. actinomycetemcomitans isolates. No isolates from army recruits or the reference strains displayed the deletion in the leukotoxin promoter region. However, in the periodontitis group, a 24-year-old individual from Ghana with localized juvenile periodontitis was identified with an intraoral infection with highly toxic A. actinomycetemcomitans (JP2-like). Present results confirm previous observations of absence of a highly toxic clone of A. actinomycetemcomitans among periodontally healthy and diseased Caucasians as well as a possible presence in localized juvenile periodontitis in individuals of African origin.

Topics: Adolescent; Adult; Aggregatibacter actinomycetemcomitans; Aggressive Periodontitis; Bacterial Toxins; Black People; Clone Cells; DNA, Bacterial; Exotoxins; Genes, Bacterial; Humans; Military Personnel; Periodontitis; Polymerase Chain Reaction; Promoter Regions, Genetic; Sequence Deletion; United States; Virulence; White People

The codon usage patterns of 21 genes encompassing 5800 codons from Actinobacillus actinomycetemcomitans were analyzed. A. actinomycetemcomitans genes could be divided into two groups based on their function and G + C content. One group included those genes encoding basic cellular functions. This group displayed an average G + C content of 48%. A second group comprised genes encoding the leukotoxin determinant, an insertion sequence and a plasmid. This group displayed an average G + C content of 36%. These findings suggest that portions of the A. actinomycetemcomitans genome may have been acquired by horizontal gene transfer from one or more distantly related species. We present a table of A. actinomycetemcomitans codon usage. These data may be used to establish standards for computer programs that predict A. actinomycetemcomitans protein coding regions and may be useful in designing degenerate oligonucleotide probes.

Topics: Adolescent; Aggregatibacter actinomycetemcomitans; Bacterial Toxins; Base Composition; Codon; DNA, Bacterial; Exotoxins; Female; Genes, Bacterial; Genetic Code; Humans; Periodontitis

Actinobacillus actinomycetemcomitans has been associated with different forms of periodontitis, particularly with localized juvenile periodontitis (LJP). The bacterium possesses several virulence factors which have been shown to interact with the host immune system. Among these factors, leukotoxin, surface antigens (serotype) and bacteriophages have been suggested directly or indirectly to influence the course of infection. However, few studies have been able to show associations between these factors and periodontal disease, alone or in combination. Thus, the purpose of the present study was to investigate possible correlations between periodontal disease status and selected virulence factors (serotype, presence of bacteriophages, and the presence of a 530 bp deletion in the promoter region of the leukotoxin gene). 36 subjects took part in the study. Serotype c was the most frequently found serotype among periodontally affected subjects, although serotypes a and b were also present. 27 out of 36 strains harbored bacteriophages, and there was strong evidence that some of the bacteriophages were different from the previously characterized phi Aa phage. A. actinomycetemcomitans containing the F-fragment phage were more frequently associated with periodontal disease. Five strains, all serotype b, 3 from LJP patients and 2 from healthy subjects, showed a 530-bp deletion in the promoter region of the leukotoxin gene.

Topics: Actinobacillus Infections; Adolescent; Adult; Aggregatibacter actinomycetemcomitans; Aggressive Periodontitis; Antibodies, Bacterial; Antigens, Bacterial; Antigens, Surface; Bacterial Toxins; Bacteriophages; Base Composition; Child; Cytotoxins; Exotoxins; Gene Deletion; Humans; Immunoblotting; Lysogeny; Middle Aged; Nucleic Acid Hybridization; Periodontitis; Periodontium; Promoter Regions, Genetic; Serotyping; Virulence

Genetic analysis of an Actinobacillus actinomycetemcomitans population consisting of 88 clinically well characterized Finnish isolates performed by multilocus enzyme electrophoresis confirmed that the five serotypes divide into two phylogenetic lineages, one comprising serotypes b and c and one comprising serotypes a, d, and e. There was no association between any subpopulation and the periodontal health status of the subject from whom the isolates originated, suggesting that the role of A. actinomycetemcomitans in periodontitis is largely opportunistic in the population examined. Southern blot analyses of genomic DNA digested with each of the restriction endonucleases MspI, RsaI, and TaqI revealed extremely limited genetic polymorphism of the structural leukotoxin gene, ltxA, and its associated promoter. All isolates hybridized to a 530-bp DNA fragment derived from the promoter region of the leukotoxin gene operon of a minimally leukotoxic A. actinomycetemcomitans strain. Deletion of the 530-bp sequence has been associated with significantly increased toxin production detected among isolates from patients with juvenile periodontitis in North America but was detected neither among the 88 isolates in the present collection analyzed nor among more than 60 strains in another population of northern European A. actinomycetemcomitans isolates analyzed previously.

Topics: Actinobacillus Infections; Adult; Aggregatibacter actinomycetemcomitans; Aggressive Periodontitis; Alleles; Base Sequence; Child; Chromosome Mapping; DNA Primers; DNA, Bacterial; Enzymes; Europe; Exotoxins; Genes, Bacterial; Genetic Variation; Humans; Molecular Sequence Data; Periodontitis; Polymerase Chain Reaction; Polymorphism, Genetic; Promoter Regions, Genetic; Serotyping; Virulence

In a previous population-based study of 3896 7-9-year-old children living in Sweden, it was found that 32 children (0.8%) exhibited radiographic, periodontal bone loss at > or = 2 proximal surfaces of their deciduous teeth. In the present study, 26 of the 32 children were subjected to additional oral and systemic health examination. 20 other children without any radiographic evidence of bone loss in their primary dentition served as referents. None of the cases or the referents were detected to have any systemic disease. The frequency of bleeding and suppuration on probing, radiographic proximal calculus and probing attachment loss was higher among the cases than the referents. Actinobacillus actinomycetemcomitants was found subgingivally in 14 of the cases but in none of the referents. 11 of 22 cases analysed for presence of serum antibodies against A. actinomycetemcomitans leukotoxin were sero-positive compared to none of 7 referents available for analysis. Evaluation of the data from each child revealed wide variations in clinical parameters among the children in the case group. In this group, there were children with deep probing depths, probing attachment loss, suppuration on probing, proximal calculus and presence of subgingival A. actinomycetemcomitans, indicating current periodontitis. However, in the case group there were also children without positive signs of inflammatory disease, similar to the children in the reference group. In fact, the findings suggest that less than half of the number of individuals with > or = 2 proximal sites with bone loss had current periodontitis.

Topics: Aggregatibacter actinomycetemcomitans; Alveolar Bone Loss; Antibodies, Bacterial; Bacterial Toxins; Case-Control Studies; Child; Dental Calculus; Dental Plaque; Exotoxins; Female; Gingival Hemorrhage; Health Status; Humans; Male; Periodontal Attachment Loss; Periodontal Pocket; Periodontitis; Suppuration; Tooth, Deciduous

Actinobacillus actinomycetemcomitans (Aa) has been implicated in most cases of localized juvenile periodontitis and some cases of severe adult periodontitis and refractory periodontitis. The Aa leukotoxin plays an important role in the pathogenesis of Aa associated periodontal disease. Rapid detection of Aa in a periodontal pocket is hampered by the slow growth and fastidious nature of this bacterium. In this study, we developed a rapid, sensitive, nonradioactive polymerase chain reaction (PCR) to identify a unique As sequence directly from subgingival plaque samples. Two oligonucleotide primers derived from DNA sequences of the leukotoxin gene were used in the PCR. The Aa-specific DNA fragments were analyzed by agarose gel electrophoresis and then visualized under 302 nm ultraviolet light after staining with ethidium bromide. In the 12 subgingival plaque samples screened, the Aa-specific sequences were found in five out of nine sites with periodontitis. No Aa-specific sequence was found in three healthy sites. The specificity of the amplified DNA fragments was confirmed by direct DNA sequencing. These results indicated that the PCR technique should assist in the rapid detection of Aa in subgingival plaque samples. Moreover, combined with direct DNA sequencing, this method can be used to study the molecular epidemiology of this periodontal pathogen.

Topics: Actinobacillus Infections; Adult; Aggregatibacter actinomycetemcomitans; Bacterial Toxins; Base Sequence; DNA, Bacterial; Exotoxins; Humans; Male; Molecular Sequence Data; Periodontitis; Polymerase Chain Reaction

Salivary and gingival crevicular fluid antibodies and systemic antibodies were analysed for levels and specificity against Actinobacillus actinomycetemcomitans components. The major reactivity of salivary and serum IgA1 and IgA2 antibodies to the periodontal pathogen A. actinomycetemcomitans was against bands between 14 and 83 kD for IgA1 and bands between 14 and 68 kD for IgA2 in Western blot. In addition to specific binding, there was also a hitherto unrecognized Fc-mediated binding of IgG antibodies to an A. actinomycetemcomitans component around 50 kD. Serum IgG antibodies to A. actinomycetemcomitans leukotoxin displayed the highest median value and only 1 individual showed salivary IgM antibodies in ELISA. Elevated levels of gingival crevicular fluid IgA2 antibodies indicated a local production of IgA from periodontal tissues. Using synthetic peptides, several distinct epitopes on the leukotoxin were recognized by both salivary and serum IgA antibodies.

Topics: Aggregatibacter actinomycetemcomitans; Antibodies, Bacterial; Blotting, Western; Exotoxins; Gingival Crevicular Fluid; Humans; Immunoglobulins; Periodontitis; Saliva

A strategy has been developed to examine the hypothesis that leucotoxin is a critical virulence factor of Actinobacillus actinomycetemcomitans in a non-human primate (Macaca fascicularis). Firstly the leucotoxin gene from A. actinomycetemcomitans was cloned and sequenced. This DNA contained a functional leucotoxin gene, as protein extracts of Escherichia coli with the cloned sequences lysed appropriate human cell lines. The protein encoded by lktA shared at least 42% identity with P. haemolytica leucotoxin and with the alpha-haemolysins from E. coli and A. pleuropneumoniae. The lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be related to the LktC proteins from these other bacteria and with which it shared at least 49% amino acid identity. Despite the overall homology to the other leucotoxins/haemolysins, the LktA from A. actinomycetemcomitans has several unique properties including a very basic pI of 9.7, as compared to pIs approx. 6.2 for lktA proteins in other bacteria. Using the cloned genes as probes produced evidence that a TOX- strain contains the leucotoxin gene but fails to transcribe it at high levels. The second avenue of investigation was to develop methods for examining the humoral immune responses in the monkey to bacterial toxins such as lktA. A. actinomycetemcomitans was detected in subgingival plaque samples from approx. 40% of the animals. A. actinomycetemcomitans comprised less than 1% to 9% of the flora. Most A. actinomycetemcomitans isolates were serotype b and each of the monkeys had serum IgG antibody to A. actinomycetemcomitans serotype b (generally considered to be lktA-producing strains). An ELISA was developed to examine the isotype/subclass distribution, level and avidity of serum antibody in the monkey following parenteral immunization with a prototype bacterial exotoxin (tetanus toxoid). IgG1 and IgG3 antibody predominated over IgG2 and IgG4 after primary immunization. Secondary immunization elicited enriched IgG1 and IgG4 responses. Primary immunization increased avidity indices of IgG to tetanus toxoid from approx. 0.9 (baseline) to a mean of 1.72 and secondary immunization significantly increased the avidity index to 2.56.

Topics: Actinobacillus; Animals; Antibodies, Bacterial; Antibody Specificity; Bacterial Toxins; Base Sequence; Cloning, Molecular; Cytotoxins; DNA, Bacterial; Exotoxins; Female; Genes, Bacterial; Immunization; Macaca fascicularis; Nucleic Acid Hybridization; Periodontitis; RNA, Bacterial; Virulence

Both Bacteroides gingivalis and Actinobacillus actinomycetemcomitans have been implicated in the destruction of periodontal tissues. To understand better the role putative virulence factors from the two bacterial species may play in an infection, the collagenase gene from Bact. gingivalis and the leucotoxin gene from A. actinomycetemcomitans were cloned. As it is intended to generate strains carrying defined mutations in these genes for in vitro and in vivo experiments, extensive restriction mapping and sequence analyses of these clones are being undertaken. Furthermore a conjugation system for Bact. gingivalis and A. actinomycetemcomitans will be established.

Topics: Actinobacillus; Bacterial Toxins; Bacteroides; Cloning, Molecular; Cytotoxins; Exotoxins; Genes, Bacterial; Genomic Library; Humans; Microbial Collagenase; Operon; Periodontitis; Virulence

Strains of A. actinomycetemcomitans (A.a) from juvenile periodontitis patients (JP), adult periodontitis patients (AP), and 14-yr-old healthy children were tested for the correlation between leukotoxin activity and the number of outer membranous vesicles measured in electron micrographs. To determine the potential for connective tissue destruction following the interaction of polymorphonuclear leukocytes (PMN) with the bacteria, the lysosomal release of neutrophil elastase was assessed. The highest potential to kill leukocytes and to release lysosomal elastase from them was observed in the strains isolated from JP patients. No correlation existed between leukotoxic activity and the number of outer membranous vesicles per bacterium when the data from A.a. strains from all sources were combined. Furthermore, no significant differences were found between the numbers of outer membranous vesicles in the three groups tested. The only significant correlation between the number of vesicles and leukotoxicity was found in the A.a. strains derived from the mouths of healthy children.

Topics: Actinobacillus; Adolescent; Adult; Aggressive Periodontitis; Bacterial Toxins; Cell Membrane; Exotoxins; Humans; Microscopy, Electron; Middle Aged; Neutrophils; Pancreatic Elastase; Periodontal Diseases; Periodontitis

We have recently isolated several groups of bacteriophages infecting Actinobacillus actinomycetemcomitans from periodontal lesions in patients with rapidly destructive periodontitis. Bacteriophage infection of these bacteria in these patients was restricted to periodontal pockets showing radiographic evidence of recent bone loss and suggests an association between phage-infected A. actinomycetemcomitans and active periodontal disease. On the basis of the biological activity of bacteriophages we propose a working hypothesis to explain the mechanism by which a phage may increase bacterial virulence in periodontal disease.

Topics: Actinobacillus; Adolescent; Adult; Aged; Aggressive Periodontitis; Bacteriolysis; Bacteriophages; Child; Child, Preschool; Exotoxins; Humans; Middle Aged; Periodontitis; Periodontium; Virulence

Topics: Actinobacillus; Adolescent; Adult; Antibodies, Bacterial; Exotoxins; Female; Humans; Male; Middle Aged; Periodontitis

The promyelocytic HL-60 cell line was examined for susceptibility to leukotoxin from Actinobacillus actinomycetemcomitans. Strains of A. actinomycetemcomitans which caused lysis of human peripheral blood polymorphonuclear leukocytes also lysed HL-60 cells as determined by release of intracellular lactate dehydrogenase. The killing of HL-60 cells by A. actinomycetemcomitans was dose dependent and temperature dependent, reached maximal levels after 45 min of incubation, and was inhibited by rabbit antisera to A. actinomycetemcomitans. Of 100 oral isolates of A. actinomycetemcomitans from 55 subjects, 16% from 11 healthy subjects, 43% from 13 adult periodontitis patients, 75% from 4 insulin-dependent diabetics, 66% from 2 generalized juvenile periodontitis patients, and 55% from 25 localized juvenile periodontitis patients produced leukotoxin. The same subject could harbor both leukotoxin-producing and -nonproducing isolates. The significantly higher proportion of leukotoxin-producing isolates in the disease groups compared with the healthy group is consistent with the hypothesis that leukotoxin from A. actinomycetemcomitans is an important virulence factor in the pathogenesis of certain forms of periodontal disease.

Topics: Actinobacillus; Cell Line; Cytotoxicity, Immunologic; Exotoxins; Hot Temperature; Humans; Immune Sera; L-Lactate Dehydrogenase; Leukemia, Myeloid, Acute; Neutrophils; Periodontitis

Topics: Actinobacillus; Adult; Antigens, Bacterial; Cytotoxicity, Immunologic; Epitopes; Exotoxins; Humans; Immunodiffusion; In Vitro Techniques; L-Lactate Dehydrogenase; Neutrophils; Periodontitis

Topics: Actinobacillus; Animals; Cytotoxicity, Immunologic; Exotoxins; Humans; L-Lactate Dehydrogenase; Neutrophils; Periodontitis; Rabbits

A soluble extract from Actinobacillus actinomycetemcomitans (designated strain Y4) caused dose-dependent cytotoxic changes in PMN isolated from the gingival crevices (C-PMN) of normal adults. When the toxin was preincubated with sera from patients with juvenile periodontitis, there was a significant inhibition of toxic activity. In contrast a variety of other sera from normal subjects with healthy gingiva, and from patients with chronic gingivitis, chronic periodontitis, recurrent herpes labialis, rheumatoid arthritis, or ulcerative colitis enhanced the leukotoxic activity. The neutralization of toxin by serum from patients with juvenile periodontitis was probably due to specific antibodies. Since Actinobacillus actinomycetemcomitans organisms can be frequently identified in subgingival plaque from patients with juvenile periodontitis, the capacity of Y4 toxin to kill C-PMN may contribute to the pathogenesis of this disease.

Topics: Actinobacillus Infections; Adult; Animals; Arthritis, Rheumatoid; Chronic Disease; Colitis, Ulcerative; Crohn Disease; Culture Media; Exotoxins; Gingival Diseases; Herpes Labialis; Humans; Neutralization Tests; Neutrophils; Periodontitis; Rabbits