Aggregatibacter Actinomycetemcomitans Serotypes Research Articles

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.

Antimicrobial Effect of Leukocyte- and Platelet-Rich Fibrin Against Aggregatibacter actinomycetemcomitans Mono-Multiserotypes Model

Leukocyte- and platelet-rich fibrin is a widely used platelet concentrate for periodontal surgery procedures. Many benefits are described regarding its use, such as antimicrobial properties. The objective of this study was to evaluate the antimicrobial effects of the different zones of this platelet concentrated against the most prevalent serotypes of Aggregatibacter actinomycetemcomitans in an in vitro mono-multiserotype model. Three patients who were treated at a School of Dentistry in the city where the researchers reside, were included. Modified direct contact method tests and results were analyzed using multivariate logistic regression analysis. In the modified direct contact method test, a decrease in bacterial count was found at time 1, but at time 2, the count increased for all serotypes and zones of L-PRF. It can be noted that the areas with more cellular content in leukocytes and platelet-rich fibrin are the areas with the most antimicrobial powe r. This platelet concentrate would have better results with serotype c. At time point 2, it is likely to act as a growth promoter of A. actinomycetemcomitans.

Antimicrobial effect of Casiopeinas® copper- and ruthenium-based compounds on Aggregatibacter actinomycetemcomitans and in vitro cell viability onto osteoblasts cells.

The present study aims to evaluate the antimicrobial property of Casiopeinas® copper- and ruthenium-based compounds against Aggregatibacter actinomycetemcomitans serotype b (ATCC® 43,718™), as well as the cytotoxicity on an osteoblasts cell line of both compounds. The antibacterial effect of the copper-based compounds (CasII-gly, CasIII-ia) and the ruthenium-based compound (RuN-6) at four different concentrations was evaluated as the inhibition ratio of the bacterial growth after 48h under anaerobic conditions, and the cell viability was measured through resazurin assay. The copper- and ruthenium-based compounds used for this assay were (CasII-gly, CasIII-ia, and RuN-6), showing inhibitory activity between 39 and 62% compared to the antibiotic employed as control 66%. Cell viability was established between 61 and 96%. Casiopeinas® and ruthenium showed dose and time dependent, inhibitory activity on A. actinomycetemcomitans, and low toxicity on cells (osteoblast) underexposure. The compound CasII-gly showed the best antimicrobial effect, and it could be considered a possible antimicrobial agent in periodontal therapy.

Antibacterial properties in-vitro of Mexican serviceberry extracts against dental biofilm species

BACKGROUND: The Mexican serviceberry, Malacomeles denticulata, have been used as a successful oral therapy by Mexican communities without enough scientific support. OBJECTIVE: To evaluate the M. denticulata extracts with selective antibacterial properties over dental biofilm bacteria. METHODS: Fruit, Leaf, and Stem of M. denticulata extracts were evaluated with micro-broth dilution method using ATCC bacteria. OD600 values had compared against each positive control (T-student-test). Anaerobically viability had confirmed by Colony-Forming-Units. Thin-Layer-Chromatography was used to identify the number of compounds and phytochemicals to identify secondary metabolites of the selected extracts. RESULTS: Streptococcus mutans showed Minimum-Bactericidal-Concentrations_(MBC) at 30 mg/mL to Fruit, Leaf, and Stem extracts. Periodontal-pathogens Aggregatibacter actinomycetemcomitans serotype b_(MBC = 30 mg/mL_NS); Fusobacterium nucleatum subsp. nucleatum_(MBC = 30 mg/mL_p<0.05); Parvimonas micra_(MBC = 15 mg/mL_NS); Porphyromonas gingivalis_(MBC = 30 mg/mL_NS); and Prevotella intermedia_(MBC = 3.75 mg/mL_NS) presented higher sensitivity to Leaf-Methanol, than the primary colonizers. Phytochemicals showed positive results to anthraquinones, coumarins, flavonoids, saponins, saponins steroids/triterpenoids, steroids/triterpenes, and tannins/phenols. CONCLUSION: We suggest the natural extracts of fruit and leaf of the Mexican serviceberry for the preventive use over the oral cariogenic or periodontal biofilm species, by their selective antibacterial properties against pathogenic species evaluated in-vitro, and due to the presence of antibacterial secondary metabolites identified as flavonoids and saponins of M. denticulata leaf extracts.

Comparative Analysis of Cytokine Expression in Oral Keratinocytes and THP-1 Macrophages in Response to the Most Prevalent Serotypes of Aggregatibacter actinomycetemcomitans.

Background: Periodontitis is a chronic inflammatory disease associated with a dysbiotic biofilm. Many pathogens have been related with its progression and severity, one of which is Aggregatibacter actinomycetemcomitans, a Gram-negative bacteria with seven serotypes (a–g) according with the structure of its LPS, with serotype b defined as the most virulent compared with the other serotypes. The aim of this study was to evaluate the response of oral keratinocytes and macrophages to A. actinomycetemcomitans. Methods: Oral keratinocytes (OKF6/TERT2) and macrophages (THP-1) were infected with A. actinomycetemcomitans serotypes a, b and c. The expression of IL-1β, IL-6, IL-8, IL-18, TNF-α, MMP-9, RANKL, TLR-2, TLR-4, TLR-6, thymic stromal lymphopoietin (TSLP), and ICAM-1 was evaluated by qPCR at 2 and 24 h after infection. Results: An increase in the expression of these molecules was induced by all serotypes at both times of infection, with macrophages showing higher levels of expression at 24 h compared to epithelial cells in which the highest levels were observed in the first hours after infection. Conclusions: Keratinocytes and macrophages contribute to the inflammation in periodontitis from the early stages of infection, producing the first waves of cytokines, acting as the first signal for professional immune cell recruitment and modulation of more specific immune responses.

Associations of Antibodies Targeting Periodontal Pathogens With Subclinical Coronary, Carotid, and Peripheral Arterial Atherosclerosis in Rheumatoid Arthritis.

Both periodontal disease and cardiovascular disease (CVD) are overrepresented in rheumatoid arthritis (RA). This study was undertaken to investigate the contribution of periodontal pathogens to CVD in RA. RA patients underwent assessments of coronary artery calcification (CAC), carotid intima-media thickness and plaque, and ankle-brachial index via computed tomography, ultrasound, and Doppler ultrasound, respectively. Sera were assayed for antibodies targeting Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans serotype B (Aa), and Aa-derived leukotoxin A (LtxA). Associations of antibodies against these periodontal pathogens with measures of atherosclerosis were explored using generalized linear models. Among 197 RA patients, anti-Pg was detected in 72 patients (37%), anti-Aa in 41 patients (21%), and anti-LtxA in 84 patients (43%). Adjusting for relevant confounders and reported tooth loss, the mean CAC score was 90% higher in those with anti-Aa and/or anti-LtxA compared with those without either antibody (19 units versus 10 units; P = 0.033). The adjusted odds of CAC ≥100 units were 2.23-fold higher in those with anti-Aa and/or anti-LtxA compared with those without either antibody (P = 0.040). Anti-Aa and/or anti-LtxA seropositivity was significantly associated with all other assessed measures of atherosclerosis except carotid plaque. Anti-Pg was not associated with any measure of atherosclerosis. Higher swollen joint count was associated with CAC exclusively in the group with anti-Aa and/or anti-LtxA. Immunoreactivity against Aa and/or its major virulence factor LtxA was associated with atherosclerosis in multiple vascular beds of RA patients and amplified the effect of swollen joints on coronary atherosclerosis, suggesting a role for treatment/prevention of periodontal disease in the prevention of CVD in RA.

Characterization of Aggregatibacter actinomycetemcomitans Serotype b Strains with Five Different, Including Two Novel, Leukotoxin Promoter Structures.

The JP2 genotype of A. actinomycetemcomitans, serotype b has attracted much interest during the past three decades due to its close association with periodontitis in young individuals and the enhanced expression of a leukotoxin (LtxA). A typical feature of this genotype is a 530-base pair (bp) deletion in the ltxCABD promoter region controlling leukotoxin expression. In the present work, we have characterized serotype b strains with four additional promoter types. Two novel types have been recognized, that is, one with a 230-bp deletion and one with a 172-bp duplication. Moreover, a strain with a 640-bp deletion and three strains with a full-length promoter, including the type strain Y4, were included in the present study. The seven strains were characterized by multi locus sequence typing (MLST) and arbitrarily primed polymerase chain reaction (PCR) and assessed for LtxA production. MLST showed that the strains with the non-JP2-like deletions represented distinct monophyletic groups, whereas the JP2 strain, HK1651, represented a separate branch. LtxA production was high in all three strains with a promoter deletion, whereas the other four strains showed significantly lower levels. It can be concluded that the genetic characterization and determination of LtxA production of A. actinomycetemcomitans isolates from individuals with periodontitis can contribute to the identification of novel virulent genotypes of this bacterium.

Evolution of the Serotypes of aggregatibacter Actinomycetemcomitans In Relation to Aggressive Periodontitis and Geographic Origin of Individuals – A Review of the Literature

Evolution of the Serotypes of aggregatibacter Actinomycetemcomitans In Relation to Aggressive Periodontitis and Geographic Origin of Individuals – A Review of the Literature

Aggregatibacter actinomycetemcomitans serotypes associate with periodontal and coronary artery disease status.

We investigated the association between the Aggregatibacter actinomycetemcomitans serotypes, periodontal status and coronary artery disease (CAD). The study population included 497 patients who underwent coronary angiography, and clinical oral examination. Quantitative polymerase chain reaction assays were designed to identify the serotypes from saliva samples. Aggregatibacter actinomycetemcomitans serotype frequencies were as follows: serotype "c" 35.7%, "b" 28.6%, "a" 26.2%, "e" 7.1%, "d" 2.4% and "f" 0%. The subjects with a detectable serotype had less teeth and higher bleeding on probing than those with no serotype. Serotypes "b" and "c" associated with periodontal probing depths and periodontal inflammatory burden. The saliva and subgingival bacterium quantities and serum antibody levels against A.actinomycetemcomitans were highest in patients harbouring serotype "c." Serotypes "b" and "c" were most frequent (59.3%) in patients with CAD (p=.040), and they associated with the risk of stable CAD with an odds ratio of 2.67 (95% confidence interval 1.06-7.44). Also, the severity of CAD (p=.018) associated with serotypes "b" and "c." Aggregatibacter actinomycetemcomitans serotypes "b" and "c" associate with both periodontal and CAD status. Detectable serotypes associate with the quantity and the serology of the bacterium emphasizing both local and systemic effect of the A.actinomycetemcomitans serotypes.

Serotypes of Aggregatibacter actinomycetemcomitans in relation to periodontal status and assessment of leukotoxin in periodontal disease: A clinico-microbiological study.

Context:Aggregatibacter actinomycetemcomitans (A.a) serotypes may add some important information of the pathogenetic background of periodontal infections. A.a leukotoxin is an important virulence factor in the pathogenesis of periodontal disease and its rate of progression. When compared to minimally leukotoxic strains, variants of A.a highly leukotoxic strains produce 10–20 times more leukotoxin.Aims:The aim of the present study was to detect serotypes a, b, c, d, and e of A.a its leukotoxin and find its correlation with periodontal status.Settings and Design:Microbiological analysis and cross-sectional study.Materials and Methods:A total of 80 subjects (40 chronic periodontitis and 40 aggressive periodontitis) in the age range of 14–55 years were selected. Subgingival plaque samples were collected and checked for the presence of A.a. Following isolation of the organism, detection of the serotypes and leukotoxin assessment was done.Statistical Analysis Used:The proportions of A.a were calculated using descriptive statistics in terms of percentage. Chi-square test was used to find association between serotype, leukotoxin, and periodontal disease in individual group.Results:Out of 80 plaque samples, 45% tested positive for A.a. serotype b was detected in 33.33%, whereas serotype e in 8.33% samples and serotype c in 2.77% samples. Serotypes a and d were not detected in any of the samples. A combination of serotypes was seen in 47.22% of the sites. Of these 76.47% showed a combination of 2 serotypes, while 23.52%showed a combination of 3 serotypes. 8.33% showed untypable serotype. All samples had low-toxic variants of A.a.Conclusions:Serotype b and serotype e were predominant in chronic periodontitis, and serotype b was predominant in aggressive periodontitis. An association could be present between serotype and periodontal disease.

Profiling of Aggregatibacter actinomycetemcomitans Serotypes B and C and the Genotypes in Periodontal Health and Disease

Background:A. actinomycetemcomitans is prevalent in periodontitis but is found in some periodontally healthy individuals as well. Certain serotypes of the organism have shown to determine severity of the disease. The distribution of serotype and genotype is affected by geographic and ethnic variation. Therefore, the present study was aimed to detect serotypes b & c of A. actinomycetemcomitans and the genotypes and find its correlation with periodontal status. Materials and Methods: A total of 75 subjects (25 aggressive periodontitis, 25 chronic periodontitis and 25 periodontally healthy) in age range of 14-55 yrs were included. Subgingival plaque samples were collected and checked for the presence of A. actinomycetemcomitans. Following isolation of the organism, detection of the serotype b or c was done by multiplex PCR. Genotyping of A. actinomycetemcomitans was done by arbitrarily primed PCR(polymerase chain reaction). Results: Out of 75 plaque samples, 35(46.66%) tested positive for A. actinomycetemcomitans. Serotype c was detected in 19/35 (54.28%), whereas serotype b alone was not detected in any of the samples. Two samples were positive for both the serotypes (b and c) (5.71%) and 14 (40%) were untypeable. 14 different arbitrarily primed PCR genotypes were obtained among 35 A. actinomycetemcomitans isolates. Conclusion: Serotype c was predominant in periodontally diseased as well as periodontally healthy individuals. An association could be present between genotype – serotype and genotype – periodontal status.

The effect of capsicum frutescens-l to transient receptor potential vinaloid-1, toll like receptors (tlr-4) and interleukin 1 beta (il-1β) on periodontitis

Objective: Indonesia has many kinds of useful herbs, which are often used as species such as chili, pepper or cayenne pepper (Capsicum frutescens L). Previous study showed topical capsaicin can be used as a therapy to cure pain due to inflammation. Small concentrations of capsaicin can attenuate cytokines in the inflammatory process. Capsaicin studies in animal model showed activation of the transient receptor potential vanilloid-1. Capsaicin can decrease various cytokines such as IL-6, IL-12, IL-1β, and increase IL-10. Capsaicin is a natural agonist for transient receptor potential vanilloid-1. The aim of this study is to prove the effect of capsaicin on transient receptor potential vanilloid-1 expression and TNF-α and TLR-4 toward pain.Material and Methods: An experimental laboratory research used animal model Wistar male rats (Rattus novegicus) induced with Aggregatibacter actinomycetemcomitans serotype b and green chili extract (Capsicum frutescens L.) with a dose of 0.0912 mg/kg/day was applied to surface of the gingiva on maxillary first molar for 7 days. An immunohistochemical examination was conducted to see the density of transient receptor potential vanilloid-1, and the expression of TLR-4 and IL-1β in the mucosal tissues of the oral cavity.Results: There were significant differences in the applications of Capsicum frutescens L. with increasing of TRPV ligand-1 and IL-1β (p < 0.05), while the TLR-4 (p > 0.05) showed a significant difference to TRPV-1 and IL-1β. There is no significant difference to TLR-4.Conclusion: Capsaicin can increase TRPV-1 and decrease IL-1β but did not affect the TLR-4. Capsaicin can be used to decrease pain and accelerate healing process in periodontitis.

The cagE gene sequence as a diagnostic marker to identify JP2 and non-JP2 highly leukotoxic Aggregatibacter actinomycetemcomitans serotype b strains

ABSTRACTAggregatibacter actinomycetemcomitans is associated with aggressive forms of periodontitis, and with systemic infections such as endocarditis. The cagE gene encodes a ≈39-kDa putative exotoxin expressed by A. actinomycetemcomitans. The prevalence of cagE, and its significance in periodontal disease, has not yet been thoroughly evaluated. In the present study, the role of the cagE gene as a diagnostic marker has been investigated.We have used PCR, and whole genome sequencing data to determine the prevalence of cagE in A. actinomycetemcomitans based on analysis of (i) 249 isolates, collected and cultivated in a Ghanaian longitudinal cohort study, (ii) a serotype b collection of 19 strains, and (iii) the 36 A. actinomycetemcomitans genomes available in the NCBI database.Whereas cagE was absent in the other serotypes, this gene sequence was linked to a virulent and highly leukotoxic group of serotype b strains, including both JP2 and non-JP2 genotypes.We therefore propose that cagE has a potential to be used as a PCR-based gene marker for the identification of a virulent and highly leukotoxic group of serotype b strains, including both JP2 and non-JP2 genotypes. This might be of importance in the risk assessment of the development of periodontal attachment loss in young individuals.

Differential human Th22-lymphocyte response triggered by Aggregatibacter actinomycetemcomitans serotypes

ObjectiveIn Aggregatibacter actinomycetemcomitans, different serotypes have been described based on lipopolysaccharide (LPS) antigenicity. When T lymphocytes were stimulated with these serotypes, different patterns of T-helper (Th)1 and Th17-type of immune responses were reported. Recently, two new Th phenotypes have been described and named Th9 and Th22 lymphocytes; however, their role in the pathogenesis of periodontitis remains unclear. This study aimed to investigate the potential Th9 and/or Th22 lymphocyte responses when stimulated with autologous dendritic cells infected with different A. actinomycetemcomitans serotypes. MethodsMonocyte-derived dendritic cells and naïve CD4+ T lymphocytes were obtained from healthy donors and stimulated with different serotypes of A. actinomycetemcomitans at a multiplicity of infection MOI=102 or their purified LPS (10–50ng/ml). The levels for the Th9 and Th22-associated cytokines, as well as the transcription factor master-switch genes implied in their differentiation Spi-B and AhR, were quantified by qPCR and ELISA. ResultsWhen stimulated with the serotype b of A. actinomycetemcomitans, higher levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were detected in dendritic cells, as well as higher levels of IL-22 and AhR were detected in T lymphocytes, when compared with stimulation with the other serotypes. ConclusionsThe serotype b of A. actinomycetemcomitans has a higher capacity of trigger Th22-type of immune response in both dendritic cells and T lymphocytes. These data allow us to suggest that, when the serotype b of A. actinomycetemcomitans is a significant part of the subgingival biofilm, the Th22 polarization might be triggered within the periodontal lesion.

In vitro complement activation, adherence to red blood cells and induction of mononuclear cell cytokine production by four strains of Aggregatibacter actinomycetemcomitans with different fimbriation and expression of leukotoxin.

The periodontal pathogen Aggregatibacter actinomycetemcomitans has been proposed as pro-atherogenic, and complement-mediated adherence to red blood cells (RBCs) may facilitate its systemic spread. We investigated the ability of four strains of A. actinomycetemcomitans with differential expression of leukotoxin A (LtxA) and fimbriae to activate complement, adhere to RBCs and elicit cytokine responses by mononuclear cells (MNCs). Aggregatibacter actinomycetemcomitans serotype b strains HK 921, HK 1651, HK 2092 and HK 2108 were fluorescence-labeled, incubated with human whole blood cells in the presence of autologous serum, and assessed for RBC adherence by flow cytometry and for capacity to induce cytokine production by cytometric bead array analysis. The levels of IgG to A. actinomycetemcomitans serotype b were quantified by ELISA, as was consumption of complement. The JP2 clone variants HK 1651 and, to a lesser extent, HK 2092, consumed complement efficiently, while HK 2108 (= strain Y4) consumed complement poorly. Nonetheless, the four tested strains adhered equally well to RBCs in the presence of autologous serum, without causing RBC lysis. The JP2 clone variant HK 2092, selectively lacking LtxA production, induced higher production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10 by MNCs than did the other three strains, while the four strains induced similar production of IL-12p70. RBCs facilitated the HK 2092-induced production of TNF-α and IL-1β, and IL-6 was enhanced by RBCs, and this facilitation could be counteracted by blockade of complement receptor 3 (CD11b/CD18). Our data suggest that the JP2 clone of A. actinomycetemcomitans, most closely resembled by the variant HK 1651, activates complement well, while strain Y4, represented by HK 2108, activates complement poorly. However, all strains of A. actinomycetemcomitans adhere to RBCs and, when capable of producing LtxA, prevent production of inflammatory cytokines by MNCs. This "immunologically silent" immune adherence may facilitate systemic spread and atherogenesis.

Virulence of Aggregatibacter actinomycetemcomitans serotypes and DGGE subtypes isolated from chronic adult periodontitis in Thailand

A high proportion of non-serotypeable isolates of Aggregatibacter actinomycetemcomitans among Thai periodontitis cases has been previously reported. The aim of this study was to investigate the expression of leukotoxin and toxicity, cytolethal distending toxin (Cdts), and internalization and the killing effect on fibroblasts by A. actinomycetemcomitans subtypes from Thai chronic periodontitis cases. A total of 96 A. actinomycetemcomitans strains from 37 periodontitis cases, previously serotyped with PCR and subtyped with DGGE, were examined for the presence of the ltx gene and cdt genes (cdtBC), and tested for leukotoxin expression, leukotoxicity, internalization, and apoptosis of fibroblast cells. The ltx gene was present in all isolates, while 84.4% showed the cdtBC gene. Two strains with a JP2-like ltx gene with a deletion of 530 bp in the promoter region, serotyped as c, showed virulence of similar magnitude to the JP2 strain. Furthermore, a higher virulence was found in the two non-serotypeable DGGE subtypes, NS1 and NS2, compared with the serotypeable strains (serotype a-f, serotype b and d were absent). Generally, the virulence of strains obtained from deep periodontal pockets was higher than those isolated from shallow non-bleeding pockets. A. actinomycetemcomitans subtypes isolated from adult Thais with chronic periodontitis showed a highly variable virulence, leukotoxin expression, leukotoxicity, internalization and apoptosis of fibroblast, and are regulated both genetically and environmentally.

Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes.

ABSTRACTIn Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the different A. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analyzed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation.Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the different A. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels.Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b.Conclusion A T-lymphocyte response biased towards a Th1- and Th17-pattern of CCL and CCR expression was detected under stimulation with the serotype b of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans serotypes and DGGE subtypes in Thai adults with chronic periodontitis

ObjectiveTo investigate the distribution of Aggregatibacter actinomycetemcomitans serotypes and DGGE subtypes among isolates from Thai chronic periodontitis patients. DesignForty-four adult Thai periodontitis patients were assessed by a full mouth recording for CAL, PPD, and BOP. Seventy-nine strains of A. actinomycetemcomitans were isolated from deep pockets on selective TSBV agar and 17 strains were isolated from shallow pockets. The strains were serotyped using PCR and subtyped using DGGE. ResultsThe prevalence of A. actinomycetemcomitans was 84.1%. Non-serotypeable A. actinomycetemcomitans strains occurred equally frequent as serotypeable (54.5%); serotype a 18.2%, serotype c 15.9%, serotype e 9.1%, and serotype f 11.4%. Serotype b and d were not detected. A JP2 like strain but serotyped as c was isolated from two patients, and another two strains showed an 886bp insertion on the ltx promoter of their A. actinomycetemcomitans isolates. DGGE typing disclosed 16 different subtypes among the non-serotypeable strains. Two of them (NS1 and NS2) were more common (12.7 and 10.1%) among the strains than the other 14 subtypes (˂5.1%). Most patients showed only one subtype (32.4%) but 29.7% had 2 and 3 different subtypes while 8.1% revealed 4 subtypes in one and the same deep pocket. ConclusionThis study showed a greater subtype diversity of A. actinomycetemcomitans predominated by non-serotypeable strains than previously reported in an adult Thai population. It was also revealed for the first time that isolates with a 530bp deletion or 886bp insertion of the ltx promoter were serotyped as serotype c.

T-lymphocyte phenotype and function triggered by Aggregatibacter actinomycetemcomitans is serotype-dependent.

Based on lipopolysaccharide (LPS) antigenicity, different Aggregatibacter actinomycetemcomitans serotypes have been described. Serotype b strains have demonstrated a stronger capacity to trigger cytokine production on dendritic cells (DCs). As DCs regulate the development of T-lymphocyte lineages, the objective of this investigation was to study the response of T lymphocytes after being stimulated with autologous DCs primed with different bacterial strains belonging to the most prevalent serotypes of A. actinomycetemcomitans in humans: a-c. Human DCs were primed with increasing multiplicity of infection (10(-1) -10(2) ) or the purified LPS (10-50 ng/mL) of A. actinomycetemcomitans serotypes a-c and then used to stimulate autologous naïve CD4(+) T lymphocytes. The T-helper (Th) type 1, Th2, Th17 and T-regulatory transcription factors T-bet, GATA-3, RORC2 and Foxp3, which are the master-switch genes implied in their specific differentiation, as well as T-cell phenotype-specific cytokine patterns were quantified by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, the intracellular expression of T-bet/interferon-γ, GATA-3/interleukin-4, RORC2/interleukin-17A and Foxp3/transforming growth factor-β1 was analysed by double staining and flow cytometry. All the A. actinomycetemcomitans serotypes led to T-lymphocyte activation; however, when T lymphocytes were stimulated with DCs primed with the A. actinomycetemcomitans serotype b strain or their purified LPS, higher levels of Th1- and Th17-associated transcription factors and cytokines were detected compared with similar experiments with the other serotypes. These results demonstrate that serotype b of A. actinomycetemcomitans has a higher capacity of trigger Th1 and Th17 phenotype and function and it was demonstrated that their LPS is a more potent immunogen compared with the other serotypes.