Aggressive Forms Of Periodontitis Research Articles

Leukotoxin A Production and Release by JP2 and Non-JP2 Genotype Aggregatibacter actinomycetemcomitans in Relation to Culture Conditions

Aggressive forms of periodontitis, especially in young patients, are often associated with an increased proportion of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans of the microbiota of the affected periodontal sites. One of the virulence factors of A. actinomycetemcomitans is a leukotoxin (LtxA) that induces a pro-inflammatory cell death process in leukocytes. A. actinomycetemcomitans exhibits a large genetic diversity and different genotypes vary in LtxA production capacity. The genotype JP2 is a heavy LtxA producer due to a 530-base pair deletion in the promoter for the toxin genes, and this trait has been associated with an increased pathogenic potential. The present study focused on the production and release of LtxA by different A. actinomycetemcomitans genotypes and serotypes under various growth conditions. Four different strains of this bacterium were cultured in two different culture broths, and the amount of LtxA bound to the bacterial surface or released into the broths was determined. The cultures were examined during the logarithmic and the early stationary phases of growth. The JP2 genotype exhibited the highest LtxA production among the strains tested, and production was not affected by the growth phase. The opposite was observed with the other strains. The composition of the culture broth had no effect on the growth pattern of the tested strains. However, the abundant release of LtxA from the bacterial surface into the culture broth was found in the presence of horse serum. Besides confirming the enhanced leucotoxicity of the JP2 genotype, the study provides new data on LtxA production in the logarithmic and stationary phases of growth and the effect of media composition on the release of the toxin from the bacterial membrane.

Green Synthesis of Bacopa monnieri-Mediated Magnesium Oxide Nanoparticles and Analysis of Their Antimicrobial, Antioxidant, and Cytotoxic Properties.

Background The management of aggressive forms of periodontal disease has become an issue of concern due to the emergence of bacterial resistance. Nanoparticles (NPs) have emerged as a potential therapeutic agent with a multitude of biological functions. The green synthesis of these NPs is more eco-friendly than conventional methods. The present study aimed at the green synthesis of magnesium oxide nanoparticles using Bacopa monnieri (bMgO NPs) and its antibacterial, antioxidant, and cytotoxic analysis. Materials and methods Magnesium oxide NPs were green synthesized using B. monnieri extract using a wet chemical method. The resultant bMgO NPs were assessed for antibacterial activity against Staphylococcus aureus and Escherichia coli. Antioxidant activity was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and the hydrogen peroxide (H2O2) assay. Cytotoxicity was assessed using zebrafish viability on treatment with bMgO NPs. Results Compared to the antibiotic standard, the green synthesized bMgO NPs showed good antibacterial properties against S. aureus and E. coli. It also showed excellent antioxidant activity and biocompatibility. Conclusion The bMgO NPs have great potential as a local drug delivery agent and should be further explored for their antibacterial and antioxidant properties in vivo.

Analysis of oral microbiome diversity in Alzheimer’s disease patients from Kazakhstan

AbstractBackgroundRecent studies have suggested that periodontal disease and alterations in the oral microbiome may be associated with cognitive decline and the development of Alzheimer’s disease (AD). Here, we report a pilot case‐control study of oral microbiota diversity in AD patients in comparison with healthy seniors from the Central Asian region.MethodWe have characterized the bacterial taxonomic composition of the oral microbiome from AD patients (n = 69) compared to the healthy group (n = 72) using 16S ribosomal RNA sequencing.ResultOur analysis showed that the oral microbiome of AD has a higher microbial diversity. More importantly, differences across taxonomic groups are distinguished by the diversity of opportunistic flora in the AD group. We identified differences between phyla and genera in bacterial abundance, including a decrease in the Firmicutes/Bacteroidetes ratio in the microbiome of AD participants. LEfSe allowed determining an enrichment at the Genus Pseudomonas, Prevotella_9, UCG_002, Esherichia_Shigella, Mogibacterium, Ralstonia, and Bergeyella in the AD group. A significant increase in the genus Mogibacterium, which is also associated with an aggressive form of periodontitis and type 2 diabetes mellitus, was revealed. The genus Ralstonia are commensal representatives of the oral microbiome, but with a reduced immune system they exhibit pathogenic properties and are associated with a higher risk of mortality in hematological malignancies and hereditary disorders of hematopoiesis. One of the least studied Bergeyella spp, which is a representative of the oral microbiome of dogs, cats, and pigs, therefore, it is isolated on contact and is associated with the risk of preterm birth, as well as the development of endocarditis in a weakened body.ConclusionOur results confirm that the oral microbiome is a potential target for therapeutic intervention in AD. However, a comprehensive picture of the full composition of the oral microbiome in patients with AD requires further investigation.

Differential Virulence of Aggregatibacter actinomycetemcomitans Serotypes Explained by Exoproteome Heterogeneity.

Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative bacterial pathogen associated with periodontitis and nonoral diseases like rheumatoid arthritis and Alzheimer´s disease. Aa isolates with the serotypes a, b, and c are globally most prevalent. Importantly, isolates displaying these serotypes have different clinical presentations. While serotype b isolates are predominant in severe periodontitis, serotypes a and c are generally encountered in mild periodontitis or healthy individuals. It is currently unknown how these differences are reflected in the overall secretion of virulence factors. Therefore, this study was aimed at a comparative analysis of exoproteomes from different clinical Aa isolates with serotypes a, b, or c by mass spectrometry, and a subsequent correlation of the recorded exoproteome profiles with virulence. Overall, we identified 425 extracellular proteins. Significant differences in the exoproteome composition of isolates with different serotypes were observed in terms of protein identification and abundance. In particular, serotype a isolates presented more extracellular proteins than serotype b or c isolates. These differences are mirrored in their virulence in infection models based on human salivary gland epithelial cells and neutrophils. Remarkably, serotype a isolates displayed stronger adhesive capabilities and induced more lysis of epithelial cells and neutrophils than serotype b or c isolates. Conversely, serotype c isolates showed relatively low leukotoxicity, while provoking NETosis to similar extents as serotype a and b isolates. Altogether, we conclude that the differential virulence presentation by Aa isolates with the dominant serotypes a, b, or c can be explained by their exoproteome heterogeneity. IMPORTANCE Periodontitis is an inflammatory disease that causes progressive destruction of alveolar bone and supporting tissues around the teeth, ultimately resulting in tooth loss. The bacterium Aggregatibacter actinomycetemcomitans (Aa) is a prevalent causative agent of periodontitis, but this oral pathogen is also associated with serious extraoral diseases like rheumatoid arthritis and Alzheimer's disease. Clinical Aa isolates are usually distinguished by serotyping, because of known serotype-specific differences in virulence. Aa with serotype b is associated with aggressive forms of periodontitis, while isolates with serotypes a or c are usually encountered in cases of mild periodontitis or healthy individuals. The molecular basis for these differences in virulence was so far unknown. In the present study, we pinpoint serotype-specific differences in virulence factor production by clinical Aa isolates. We consider these findings important, because they provide new leads for future preventive or therapeutic approaches to fight periodontitis and associated morbidities.

Carriage of the JP2 Genotype of Aggregatibacter actinomycetemcomitans by Periodontitis Patients of Various Geographic Origin, Living in Sweden.

The JP2 genotype of Aggregatibacter actinomycetemcomitans serotype b is associated with aggressive forms of periodontitis and was initially identified as affecting adolescents in North and West Africa. The dissemination of this genotype follows the migration routes and can today be detected in samples from periodontitis patients in a high number of countries. In the present study, we aim to describe findings of the JP2 genotype A. actinomycetemcomits in a clinical laboratory at the Dental School, Odontology, Umeå University, Sweden. The findings of JP2 carriers are documented during a 21-year period, and the age and geographic origin of the sampled individuals are described. In addition, the collected JP2 isolates were separated into North or West African origin by analyses of the presence of a point mutation in the hbpA2 pseudogene of the bacterium. In a total of 2296 sampled individuals during this period in this Swedish population of periodontitis patients, 32 JP2 carriers were detected by cultivation and PCR. The geographic background of these individuals was diverse, including sixteen with African origin, ten with a Swedish origin and six additional ones with a non-African origin. The JP2 genotypes of A. actinomycetemcomitans were mainly isolated from young individuals (<35 years of age), and seven out of the 32 isolates were of a West African origin based on the sequence of hbpA2. We conclude that the JP2 genotype of A. actinomycetemcomitans can be detected world-wide in subgingival plaque samples from adolescents affected by periodontitis.

Connections between Exoproteome Heterogeneity and Virulence in the Oral Pathogen Aggregatibacter actinomycetemcomitans.

ABSTRACTAggregatibacter actinomycetemcomitans is a Gram-negative bacterial pathogen associated with severe periodontitis and nonoral diseases. Clinical isolates of A. actinomycetemcomitans display a rough (R) colony phenotype with strong adherent properties. Upon prolonged culturing, nonadherent strains with a smooth (S) colony phenotype emerge. To date, most virulence studies on A. actinomycetemcomitans have been performed with S strains of A. actinomycetemcomitans, whereas the virulence of clinical R isolates has received relatively little attention. Since the extracellular proteome is the main bacterial reservoir of virulence factors, the present study was aimed at a comparative analysis of this subproteome fraction for a collection of R isolates and derivative S strains, in order to link particular proteins to the virulence of A. actinomycetemcomitans with serotype b. To assess the bacterial virulence, we applied different infection models based on larvae of the greater wax moth Galleria mellonella, a human salivary gland-derived epithelial cell line, and freshly isolated neutrophils from healthy human volunteers. A total number of 351 extracellular A. actinomycetemcomitans proteins was identified by mass spectrometry, with the S strains consistently showing more extracellular proteins than their parental R isolates. A total of 50 known extracellular virulence factors was identified, of which 15 were expressed by all investigated bacteria. Importantly, the comparison of differences in exoproteome composition and virulence highlights critical roles of 10 extracellular proteins in the different infection models. Together, our findings provide novel clues for understanding the virulence of A. actinomycetemcomitans and for development of potential preventive or therapeutic avenues to neutralize this important oral pathogen.IMPORTANCE Periodontitis is one of the most common inflammatory diseases worldwide, causing high morbidity and decreasing the quality of life of millions of people. The bacterial pathogen Aggregatibacter actinomycetemcomitans is strongly associated with aggressive forms of periodontitis. Moreover, it has been implicated in serious nonoral infections, including endocarditis and brain abscesses. Therefore, it is important to investigate how A. actinomycetemcomitans can cause disease. In the present study, we applied a mass spectrometry approach to make an inventory of the virulence factors secreted by different clinical A. actinomycetemcomitans isolates and derivative strains that emerged upon culturing. We subsequently correlated the secreted virulence factors to the pathogenicity of the investigated bacteria in different infection models. The results show that a limited number of extracellular virulence factors of A. actinomycetemcomitans have central roles in pathogenesis, indicating that they could be druggable targets to prevent or treat oral disease.

The assessment of a role of muramyl peptides in the treatment of patients with aggressive form of periodontitis

Was to determine the effect of the drug based on the composition of muramyl peptides isolated from the cell walls of gram-negative bacteria on the production of α-defensins and the detectability of Porphyromonas gingivalis in patients with an aggressive form of periodontitis. 60 patients aged 28 to 40 years with an aggressive form of periodontitis were randomized into two equal groups, the main and control. In both groups, patients were removed dental deposits and taught the rules of oral hygiene, followed by three-fold control. In the main group, 200 micrograms of the drug based on the composition of muramyl peptides were additionally administered intramuscularly daily for 7 days. Initially and after 7, 21, 90 days, the level of human neutrophil peptides (hnp1-3) in blood serum and periodontal pockets was determined by the enzyme immunoassay, as well as the presence of P. gingivalis in periodontal pockets by polymerase chain reaction (PCR). In patients of the control group, the concentration of hnp1-3 in periodontal pockets and blood serum did not change significantly during the entire study. The use of PM in the main group caused an increase in the local and systemic levels of hnp1-3, which correlated with a persistent decrease in the detectability of P. gingivalis. The drug based on the composition of muramyl peptides of the cell wall of gram-negative bacteria potentiates the eradication of P. gingivalis by stimulating the production of hnp1-3 in patients with an aggressive form of periodontitis.

Oleoresins and naturally occurring compounds of Copaifera genus as antibacterial and antivirulence agents against periodontal pathogens

Invasion of periodontal tissues by Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans can be associated with aggressive forms of periodontitis. Oleoresins from different copaifera species and their compounds display various pharmacological properties. The present study evaluates the antibacterial and antivirulence activity of oleoresins obtained from different copaifera species and of ten isolated compounds against two causative agents of periodontitis. The following assays were performed: determination of the minimum inhibitory concentration (MIC), determination of the minimum bactericidal concentration (MBC), and determination of the antibiofilm activity by inhibition of biofilm formation and biofilm eradication tests. The antivirulence activity was assessed by hemagglutination, P. gingivalis Arg-X and Lis-X cysteine protease inhibition assay, and A. actinomycetemcomitans leukotoxin inhibition assay. The MIC and MBC of the oleoresins and isolated compounds 1, 2, and 3 ranged from 1.59 to 50 μg/mL against P. gingivalis (ATCC 33277) and clinical isolates and from 6.25 to 400 μg/mL against A. actinomycetemcomitans (ATCC 43717) and clinical isolates. About the antibiofilm activity, the oleoresins and isolated compounds 1, 2, and 3 inhibited biofilm formation by at least 50% and eradicated pre-formed P. gingivalis and A. actinomycetemcomitans biofilms in the monospecies and multispecies modes. A promising activity concerning cysteine protease and leucotoxin inhibition was also evident. In addition, molecular docking analysis was performed. The investigated oleoresins and their compounds may play an important role in the search for novel sources of agents that can act against periodontal pathogens.

The assessment of aggressive periodontitis at Kuantan, Pahang: A retrospective study

Aim: Aggressive periodontitis (AgP) is a less common but rapidly destructive form of periodontitis. Literature is scarce regarding the prevalence and characteristics of the disease in Malaysia. This study was conducted to assess the prevalence of AgP and its correlation with sociodemographic, risk factors, and clinical presentations. Materials and Methods: This retrospective cross-sectional descriptive study of AgP includes patients who had attended the IIUM Dental Clinic from the year 2014 up to June 2017. The data regarding the characteristics of AgP were extracted from patients’ case records systematically using a standard data extraction form. Descriptive analysis, independent t-test, and χ2 test were conducted using IBM SPSS, version 24.0 software. Results: Of 262 periodontitis cases, 11 were confirmed to have AgP, giving a prevalence of 4.2%. Seven of them had the generalized form of AgP with a mean (±SE) age of 36.4 (±6.99) years. Eight of them were female, the majority were Malay, and four of them claimed of having familial aggregation. The localized form had significantly deeper pocket depth of at least 6 mm. However, the percentage of bleeding on probing and plaque scores were significantly higher in the generalized form of the disease (P < 0.05). First molars showed the highest frequency of tooth loss and clinical attachment loss. Conclusion: The prevalence rate of AgP in a specified population in Kuantan was found at 4.2%, affecting younger age group compared to chronic periodontitis. Age, the percentage of bleeding on probing, plaque score, and deep pockets were significantly associated with the types of AgP.

Interleukin Gene Variability and Periodontal Bacteria in Patients with Generalized Aggressive Form of Periodontitis.

Host genetic predispositions to dysregulated immune response can influence the development of the aggressive form of periodontitis (AgP) through susceptibility to oral dysbiosis and subsequent host-microbe interaction. This case-control study aimed to perform a multilocus analysis of functional variants in selected interleukin (IL) genes in patients with the generalized form of AgP in a homogenous population. Twelve polymorphisms in IL-1 gene cluster, IL-6 and its receptor, IL-10, IL-17A, and IL-18 were determined in 91 AgP patients and 210 controls. Analysis of seven selected periodontal bacteria in subgingival sulci/pockets was performed with a commercial DNA-microarray kit in a subgroup of 76 individuals. The pilot in vitro study included stimulation of peripheral blood monocytes (PBMC) from 20 individuals with periodontal bacteria and measurement of IL-10 levels using the Luminex method. Only the unctional polymorphism IL-10 −1087 A/G (rs1800896) and specific IL-10 haplotypes were associated with the development of the disease (p < 0.05, Pcorr > 0.05). Four bacterial species occurred more frequently in AgP than in controls (p < 0.01, Pcorr < 0.05). Elevated IL-10 levels were found in AgP patients, carriers of IL-10 −1087GG genotype, and PBMCs stimulated by periodontal bacteria (p < 0.05, Pcorr > 0.05). We therefore conclude that a combination of genetic predisposition to the altered expression of IL-10 and the presence of specific periodontal bacteria may contribute to Th1/Th2 balance disruption and AgP development.

Tooth loss in aggressive periodontitis: Results 25 years after active periodontal therapy in a private practice.

To assess tooth loss in patients with aggressive periodontitis (AgP) 10-35years after active periodontal therapy (APT) in a private practice and to detect possible factors influencing tooth loss. In 100 patients with AgP, tooth loss was recorded over a median follow-up period of 25.5years after APT, retrospectively. Patient- and tooth-level factors were assessed with a Cox frailty regression model. Of 2,380 teeth, 227 were lost during a median follow-up time of 25.5years (2.3±3.6 teeth/patient, range 0-17 teeth), resulting in a mean tooth loss rate of 0.09 teeth/patient/year. At patient-level, statistically significant factors for tooth loss were smoking (p=.039) and the baseline diagnosis generalized AgP (p<.001). Influencing factors at tooth-level were location in the maxilla (p=.003), baseline bone loss (p<.001), molars (p<.001) and premolars (p<.001) as well as abutment teeth (p=.009). Tooth loss occurred rarely in patients with AgP treated in a private practice over a long-time period. Annual tooth loss rates are comparable with those described in university settings. Smoking, generalized form of AgP, location/type of tooth, baseline bone loss and abutment status could be detected as factors impacting upon tooth loss.

Papillon lefevre syndrome- A literature review and case report

Papillon-Lefèvre syndrome (PLS) is an extremely rare genetic disorder. This disorder is mostly observed in children from 1-5 years of age. Alteration of CTSC gene which regulates the production of enzyme cathepsin C is responsible for this syndrome. The common characteristics are palmoplantar keratosis and aggressive form of periodontitis. The patient might become completely edentulous in the absence of appropriate and early treatment. Early lose of teeth in young children may have two important concerns- 1. Pychological impact. 2. Prosthetic rehabilitation. Till date, there is no definitive treatment protocol for PLS cases. This article provides a detailed review about the various possible etiologies, clinical features, differential diagnosis and treatment protocols followed for the periodontal management of PLS. This article reports a Case of Papillon Lefèvre syndrome and its management with long term follow up. Keywords: Periodontitis, Papillon Lefevre syndrome.

Genetic Profiling of Aggregatibacter actinomycetemcomitans Serotype B Isolated from Periodontitis Patients Living in Sweden.

The bacterium Aggregatibacter actinomycetemcomitans is associated with aggressive forms of periodontitis and with systemic diseases, such as endocarditis. By assessing a Ghanaian longitudinal adolescent cohort, we earlier recognized the cagE gene as a possible diagnostic marker for a subgroup of JP2 and non-JP2 genotype serotype b A. actinomycetemcomitans strains, associated with high leukotoxicity as determined in a semi-quantitative cell assay. This group of A. actinomycetemcomitans is associated with the progression of attachment loss. In the present work, we used conventional polymerase chain reaction (PCR) and quantitative PCR to perform the cagE genotyping of our collection of 116 selected serotype b A. actinomycetemcomitans strains, collected over a period of 15 years from periodontitis patients living in Sweden. The A. actinomycetemcomitans strains carrying cagE (referred to as cagE+; n = 49) were compared to the cagE-negative strains (n = 67), present at larger proportions in the subgingival plaque samples, and were also much more prevalent in the young (≤35 years) compared to in the old (>35 years) group of patients. Our present results underline the potential use of cagE genotyping in the risk assessment of the development of periodontal attachment loss in Swedish adolescents.

High salivary levels of JP2 genotype of Aggregatibacter actinomycetemcomitans is associated with clinical attachment loss in Moroccan adolescents.

It has previously been shown that the presence of Aggregatibacter actinomycetemcomitans in subgingival plaque is significantly associated with increased risk for clinical attachment loss. The highly leukotoxic JP2 genotype of this bacterium is frequently detected in adolescents with aggressive forms of periodontitis. The aims of the study were to quantify the levels of JP2 and non‐JP2 genotypes of A. actinomycetemcomitans in saliva of Moroccan adolescents with the JP2 genotype earlier detected in the subgingival plaque. The salivary concentrations of inflammatory proteins were quantified and linked to the clinical parameters and microbial findings. Finally, a mouth rinse with leukotoxin‐neutralizing effect was administrated and its effect on the levels the biomarkers and A. actinomycetemcomitans examined. The study population consisted of 22 adolescents that previously were found to be positive for the JP2 genotype in subgingival plaque. Periodontal registration and sampling of stimulated saliva was performed at baseline. A mouth rinse (active/placebo) was administrated, and saliva sampling repeated after 2 and 4 weeks rinse. The salivary levels of JP2 and non‐JP2 were analyzed by quantitative PCR and inflammatory proteins by ELISA. Both the JP2 and the non‐JP2 genotype were detected in all individuals with significantly higher levels of the non‐JP2. Enhanced levels of the JP2 genotype of A. actinomycetemcomitans was significantly correlated to the presence of attachment loss (≥3 mm). Salivary concentrations of inflammatory biomarkers did not correlate to periodontal condition or levels of A. actinomycetemcomitans. The use of active or placebo leukotoxin‐neutralizing mouth rinse did not significantly interfered with the levels of these biomarkers. Saliva is an excellent source for detection of A. actinomycetemcomitans on individual basis, and high levels of the JP2 genotype were significantly associated with the presence of clinical attachment loss.

One-year follow-up of the immune profile in serum and selected sites of generalized and localized aggressive periodontitis

BackgroundThe local and systemic immunological profiles of important inflammatory mediators in the localized (LAgP) and generalized (GAgP) forms of aggressive periodontitis are still unknown, as well as the effect of periodontal therapy on these parameters. The aim of this prospective study was to evaluate clinical and immune responses of patients with AgP undergoing nonsurgical treatment. Material and methodsEighteen patients with GAgP, 10 with LAgP and 10 healthy participants were included in this study. AgP participants were submitted to scaling and root planing plus systemic antibiotics (amoxicillin and metronidazole). At baseline and 1-year follow-up were measured clinical parameters, such as probing depth [PD] and clinical attachment loss [CAL], and the levels of 10 immunological mediators (GM-CSF, M-CSF, MCP-1, ICAM-1, CXCL8, IL-1β, TNF-α, IL-17, IL-4, and IL-10) in the gingival crevicular fluid (GCF) of selected sites [AgP forms: PD ≥ 6 mm or the deepest, bleeding on probing (BoP) and bone loss measured by periapical radiography; healthy individuals: PD ≤ 3 mm, no BoP, no bone loss] and serum. ResultsAfter periodontal treatment both forms of AgP presented a significant reduction of PD and CAL, an increase of GM-CSF, ICAM-1, MCP-1, TNF-α, IL-17, IL-4, and IL-10 in the GCF, as well as of GM-CSF and IL-4 in the serum, and a reduction in the serum concentration of IL-1β. Serum levels of M-CSF, ICAM-1, and MCP-1 remained significantly below those found in healthy individuals in both forms of AgP even after therapy. An increase in the systemic or local levels of MCP-1, ICAM-1 and the anti-inflammatory profile (IL-4, IL-10) was correlated with an improvement in clinical parameters of LAgP patients. Also, a local reduction of IL-1β levels in both forms of AgP was correlated with an increase in the clinical attachment gain. ConclusionNonsurgical periodontal therapy was successful in improving clinical parameters and modulating the immune response in both forms of AgP. However, this therapeutic approach does not seem to affect the deficient level of important serum mediators involved in mechanisms of cell transmigration.

Gingival crevicular fluid and salivary HIF-1α, VEGF, and TNF-α levels in periodontal health and disease.

Hypoxia-inducible factor-1 alpha (HIF-1α) is expressed as an adaptive response to hypoxia, mediates angiogenesis through the expression of vascular endothelial growth factor (VEGF) and can be induced by tumor necrosis factor-alpha (TNF-α). This study aimed to investigate the gingival crevicular fluid (GCF) and salivary HIF-1α, VEGF, and TNF-α levels in periodontal health and disease. A total of 87 individuals, 20 generalized aggressive periodontitis (G-AgP), 20 chronic periodontitis (CP), 26 gingivitis patients, and 21 periodontally healthy individuals, were included. Clinical periodontal parameters were recorded; GCF and salivary samples were collected; and HIF-1α, VEGF, and TNF-α levels were measured by enzyme-linked immunosorbent assay. Nonparametric tests were used for the statistical analyses. G-AgP and CP groups had significantly higher GCF HIF-1α, VEGF, and TNF-α total amounts than gingivitis and healthy groups (P<0.05). GCF HIF-1α and TNF-α total amounts in gingivitis group were significantly higher than the healthy group (P<0.05). GCF and salivary concentrations of biomarkers were similar in both periodontitis groups (P>0.05). Salivary HIF-1α concentrations in gingivitis group were significantly higher than G-AgP and healthy groups (P<0.05). GCF HIF-1α, VEGF, and TNF-α total amounts were positively correlated with the site-specific clinical periodontal parameters and with each other (P<0.05). HIF-1α is detectable in GCF and saliva of periodontally diseased and healthy individuals, and the GCF levels of the biomarker can be affected by disease status. Increased GCF HIF-1α, VEGF, and TNF-α levels in both chronic and aggressive form of periodontitis might suggest the role of TNF-α/HIF-1α/VEGF pathway in the pathogenesis of periodontal diseases.

Comparison of miRNA expression profiles in individuals with chronic or aggressive periodontitis

MicroRNAs (miRNAs) may play an important role in inflammatory response. However, the involvement of miRNAs in the pathogenesis of periodontitis is unclear. The present study aimed to compare the miRNA expression profiles in individuals with chronic (CP) or aggressive (AP) periodontitis. Eighteen non-smoker individuals (CP=9 and AP=9) without any history of systemic diseases or previous periodontal therapies were selected at the Clinics of Periodontology from the Federal University of Minas Gerais. Gingival tissue samples were collected during the initial periodontal therapy. miRNAs were isolated, and expression patterns of 754 miRNAs were assessed with a quantitative miRNA PCR array. miRNAs expression profiles were compared between CP and AP groups. There were no differences observed in the miRNAs expression profiles between CP and AP (p>0.05). According to the microarray analyses, the most expressed miRNAs in both groups were hsa-miR-1274b, hsa-let-7b-5p, hsa-miR-24-3p, hsa-miR-19b-3p, hsa-miR-720, hsa-miR-126-3p, hsa-miR-17-3p and hsa-miR-21-3p. Findings suggested no differences in miRNAs expression profiles between chronic and aggressive forms of periodontitis. The overexpression of specific miRNAs could provide insights into the pathogenesis of both forms of the disease.

Role of OmpA1 and OmpA2 in Aggregatibacter actinomycetemcomitans and Aggregatibacter aphrophilus serum resistance

ABSTRACTAggregatibacter actinomycetemcomitans and Aggregatibacter aphrophilus belong to the HACEK group of fastidious Gram-negative organisms, a recognized cause of infective endocarditis. A. actinomycetemcomitans is also implicated in aggressive forms of periodontitis. We demonstrated that A. aphrophilus strains, as A. actinomycetemcomitans are ubiquitously serum resistant. Both species encode two Outer membrane protein A paralogues, here denoted OmpA1 and OmpA2. As their respective pangenomes contain several OmpA1 and OmpA2 alleles, they represent potential genotypic markers. A naturally competent strain of A. actinomycetemcomitans and A. aphrophilus, respectively were used to elucidate if OmpA1 and OmpA2 contribute to serum resistance. Whereas OmpA1 was critical for survival of A. actinomycetemcomitans D7SS in 50% normal human serum (NHS), serum resistant ompA1 mutants were fortuitously obtained, expressing enhanced levels of OmpA2. Similarly, OmpA1 rather than OmpA2 was a major contributor to serum resistance of A. aphrophilus HK83. Far-Western blot revealed that OmpA1AA, OmpA2AA, and OmpA1AP can bind to C4-binding protein, an inhibitor of classical and mannose-binding lectin (MBL) complement activation. Indeed, ompA1 mutants were susceptible to these pathways, but also to alternative complement activation. This may at least partly reflect a compromised outer membrane integrity but is also consistent with alternative mechanisms involved in OmpA-mediated serum resistance.

Generation of a recombinant Aggregatibacter actinomycetemcomitans RTX toxin in Escherichia coli

A leukotoxin (LtxA) that is produced by Aggregatibacter actinomycetemcomitans (Aa) is an important virulence determinant in an aggressive form of periodontitis in adolescents. Understanding the function of this protein at the molecular level is critical to elucidating its role in the disease process. To accomplish genetic analysis of the protein structure and relating these observations to toxin function, we have developed an E. coli expression system for the generation and rapid purification of LtxA. Cloning the structural toxin gene, ltxA, from Aa strain JP2 under control of T7 promoter-1 of pCDFDuet-1 vector resulted in expression of a 114 KDa protein which could be easily purified by the presence of a carboxy-terminal engineered double hexahistidine (double–His6) tag and was immunologically reactive with an anti-LtxA monoclonal antibody, but was not cytotoxic. Cloning a second gene, ltxC, an acyltransferase gene, into the vector under control of T7 promoter-2, resulted in expression of the biologically active LtxA. The toxin was extracted from E. coli inclusion bodies, purified by immobilized metal affinity chromatography, and refolded by dialysis. When compared by circular dichroism (CD) spectroscopy analysis, acylated recombinant LtxA has a secondary structure consistent with wt LtxA, while variations in α-helical structure of nonacylated LtxA were observed. No modifications in α-helix were found upon the toxin's binding with liposome-incorporated cholesterol. Our results suggest that pure, biologically active recombinant LtxA can be isolated by a one-step affinity chromatography from E. coli. The toxic and structural properties of the recombinant LtxA are similar to its wt counterpart.

Aggregatibacter actinomycetemcomitans H-NS promotes biofilm formation and alters protein dynamics of other species within a polymicrobial oral biofilm

Aggregatibacter actinomycetemcomitans is a Gram-negative organism, strongly associated with aggressive forms of periodontitis. An important virulence property of A. actinomycetemcomitans is its ability to form tenacious biofilms that can attach to abiotic as well as biotic surfaces. The histone-like (H-NS) family of nucleoid-structuring proteins act as transcriptional silencers in many Gram-negative bacteria. To evaluate the role of H-NS in A. actinomycetemcomitans, hns mutant derivatives of serotype a strain D7S were generated. Characteristics of the hns mutant phenotype included shorter and fewer pili, and substantially lower monospecies biofilm formation relative to the wild type. Furthermore, the D7S hns mutant exhibited significantly reduced growth within a seven-species oral biofilm model. However, no apparent difference was observed regarding the numbers and proportions of the remaining six species regardless of being co-cultivated with D7S hns or its parental strain. Proteomics analysis of the strains grown in monocultures confirmed the role of H-NS as a repressor of gene expression in A. actinomycetemcomitans. Interestingly, proteomics analysis of the multispecies biofilms indicated that the A. actinomycetemcomitans wild type and hns mutant imposed different regulatory effects on the pattern of protein expression in the other species, i.e., mainly Streptococcus spp., Fusobacterium nucleatum, and Veillonella dispar. Gene ontology analysis revealed that a large portion of the differentially regulated proteins was related to translational activity. Taken together, our data suggest that, apart from being a negative regulator of protein expression in A. actinomycetemcomitans, H-NS promotes biofilm formation and may be an important factor for survival of this species within a multispecies biofilm.