ABSTRACT
Periodontitis is an exemplar of dysbiosis associated with the coordinated action of multiple members within the microbial consortium. The polymicrobial synergy and dysbiosis hypothesis proposes a dynamic host-microbiome balance, with certain modulators capable of disrupting eubiosis and driving shifts towards dysbiosis within the community. However, these factors remain to be explored. We established a Porphyromonas gingivalis- or Aggregatibacter actinomycetemcomitans-modified subgingival microbiome model and 16S rRNA sequencing revealed that P. gingivalis and A. actinomycetemcomitans altered the microbiome structure and composition indicated by α and ß diversity metrics. P. gingivalis increased the subgingival dysbiosis index (SDI), while A. actinomycetemcomitans resulted in a lower SDI. Furthermore, P. gingivalis-stimulated microbiomes compromised epithelium function and reduced expression of tight junction proteins, whereas A. actinomycetemcomitans yielded mild effects. In conclusion, by inoculating P. gingivalis, we created dysbiotic microcosm biofilms in vitro resembling periodontitis-related subgingival microbiota, exhibiting enhanced dysbiosis and impaired epithelium integrity.
Subject(s)
Microbiota , Periodontitis , Humans , Porphyromonas gingivalis , Aggregatibacter actinomycetemcomitans/genetics , RNA, Ribosomal, 16S/genetics , DysbiosisABSTRACT
Aggregatibacter actinomycetemcomitans is an opportunistic Gram-negative periodontopathogen strongly associated with periodontitis and infective endocarditis. Recent evidence suggests that periodontopathogens can influence the initiation and progression of oral squamous cell carcinoma (OSCC). Herein we aimed to investigate the effect of A. actinomycetemcomitans-derived extracellular vesicles (EVs) on OSCC cell behavior compared with EVs from periodontopathogens known to associate with carcinogenesis. EVs were isolated from: A. actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; Porphyromonas gingivalis; Fusobacterium nucleatum; and Parvimonas micra. The effect of EVs on primary and metastatic OSCC cells was assessed using cell proliferation, apoptosis, migration, invasion, and tubulogenesis assays. A. actinomycetemcomitans-derived EVs reduced the metastatic cancer cell proliferation, invasion, tubulogenesis, and increased apoptosis, mostly in CDT- and LPS O-antigen-dependent manner. EVs from F. nucleatum impaired the metastatic cancer cell proliferation and induced the apoptosis rates in all OSCC cell lines. EVs enhanced cancer cell migration regardless of bacterial species. In sum, this is the first study demonstrating the influence of A. actinomycetemcomitans-derived EVs on oral cancer in comparison with other periodontopathogens. Our findings revealed a potential antitumorigenic effect of these EVs on metastatic OSCC cells, which warrants further in vivo investigations.
Subject(s)
Aggregatibacter actinomycetemcomitans , Apoptosis , Cell Proliferation , Extracellular Vesicles , Mouth Neoplasms , Aggregatibacter actinomycetemcomitans/genetics , Extracellular Vesicles/metabolism , Mouth Neoplasms/microbiology , Mouth Neoplasms/pathology , Humans , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Movement , Fusobacterium nucleatum/physiology , Carcinoma, Squamous Cell/microbiology , Carcinoma, Squamous Cell/pathology , Porphyromonas gingivalis/geneticsABSTRACT
The most common type of periodontal disease is chronic periodontitis, an inflammatory condition caused by pathogenic bacteria in subgingival plaque. The aim of our study was the development of a real-time PCR test as a diagnostic tool for the detection and differentiation of five periodontopathogenic bacteria, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola, in patients with periodontitis. We compared the results of our in-house method with the micro-IDent® semiquantitative commercially available test based on the PCR hybridization method. DNA was isolated from subgingival plaque samples taken from 50 patients and then analyzed by both methods. Comparing the results of the two methods, they show a specificity of 100% for all bacteria. The sensitivity for A. actinomycetemcomitans was 97.5%, for P. gingivalis 96.88%, and for P. intermedia 95.24%. The sensitivity for Tannerella forsythia and T. denticola was 100%. The Spearman correlation factor of two different measurements was 0.976 for A. actinomycetemcomitans, 0.967 for P. gingivalis, 0.949 for P. intermedia, 0.966 for Tannerella forsythia, and 0.917 for T. denticola. In conclusion, the in-house real-time PCR method developed in our laboratory can provide information about relative amount of five bacterial species present in subgingival plaque in patients with periodontitis. It is likely that such a test could be used in dental diagnostics in assessing the efficacy of any treatment to reduce the bacterial burden.
Subject(s)
Periodontitis , Porphyromonas gingivalis , Real-Time Polymerase Chain Reaction , Humans , Real-Time Polymerase Chain Reaction/methods , Periodontitis/microbiology , Periodontitis/diagnosis , Porphyromonas gingivalis/isolation & purification , Porphyromonas gingivalis/genetics , Aggregatibacter actinomycetemcomitans/isolation & purification , Aggregatibacter actinomycetemcomitans/genetics , Treponema denticola/isolation & purification , Treponema denticola/genetics , Male , Female , Tannerella forsythia/isolation & purification , Tannerella forsythia/genetics , Sensitivity and Specificity , Prevotella intermedia/isolation & purification , Prevotella intermedia/genetics , Middle Aged , Adult , DNA, Bacterial/genetics , Dental Plaque/microbiology , Bacteria/genetics , Bacteria/isolation & purification , Bacteria/classificationABSTRACT
To appraise the literature on the prevalence of the JP2 clone of Aggregatibacter actinomycetemcomitans (A.a.) and on its association with presence and progression of periodontitis in different populations. A systematic search of the literature was conducted in Medline, Embase and Cochrane Library for studies reporting data on detection of the JP2 clone of A.a. A total of 56 papers were included in the review, from an initial search of 685 titles. Studies were carried out in populations with a mean age of 26.34 years (range 6.24-53.85 years). Just over 16% of the overall population assessed (n = 13 751) had the JP2 clone detected. Meta-analyses included 16 studies and 1775 patients, and revealed an association between detection of the JP2 clone and diagnosis of periodontitis (RR = 1.86, 95% 1.43-2.42) from saliva and plaque, with high heterogeneity (I2 = 85%, p < .00001). Meta-analyses included 5 studies and 616 patients, and revealed an association between baseline detection of the JP2 clone and onset of periodontitis over 2 to 5 years (RR = 4.12, 95% 2.42-7.00), with high heterogeneity (I2 = 81%, p < .0003). From the overall risk of bias score, 29 papers were judged as low risk of bias, whilst the remaining papers were judged to have an overall medium or high risk of bias. Detection of the JP2 clone of A.a. in subgingival plaque and saliva samples is associated with increased odds of diagnosis of periodontitis and may be able to predict onset of periodontitis. This systematic review provides clear evidence that in certain populations, the JP2 clone of A.a. is associated with early-onset periodontitis. Furthermore, detection of this bacterium seems to be predictive of disease onset.
Subject(s)
Aggressive Periodontitis , Dental Plaque , Humans , Child , Adolescent , Young Adult , Adult , Middle Aged , Aggregatibacter actinomycetemcomitans/genetics , Exotoxins , Dental Plaque/microbiology , Clone CellsABSTRACT
AIM: To explore whether adjunctive antibiotics can relevantly influence long-term microbiota changes in stage III-IV periodontitis patients. MATERIALS AND METHODS: This is a secondary analysis of a randomized clinical trial on periodontal therapy with adjunctive 500 mg amoxicillin and 400 mg metronidazole or placebo thrice daily for 7 days. Subgingival plaque samples were taken before and 2, 8, 14 and 26 months after mechanical therapy. The V4-hypervariable region of the 16S rRNA gene was sequenced with Illumina MiSeq 250 base pair paired-end reads. Changes at the ribosomal sequence variant (RSV) level, diversity and subgingival-microbial dysbiosis index (SMDI) were explored with a negative binomial regression model and non-parametric tests. RESULTS: Overall, 50.2% of all raw reads summed up to 72 RSVs (3.0%) that were generated from 163 stage III-IV periodontitis patients. Of those, 16 RSVs, including Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans, changed significantly over 26 months because of adjunctive systemic antibiotics. SMDI decreased significantly more in the antibiotic group at all timepoints, whereas the 2-month differences in alpha and beta diversity between groups were not significant at 8 and 14 months, respectively. CONCLUSIONS: Mechanical periodontal therapy with adjunctive antibiotics induced a relevant and long-term sustainable change towards an oral microbiome more associated with oral health.
Subject(s)
Microbiota , Periodontitis , Humans , Anti-Bacterial Agents/therapeutic use , Anti-Bacterial Agents/pharmacology , RNA, Ribosomal, 16S , Periodontitis/drug therapy , Amoxicillin/therapeutic use , Metronidazole/therapeutic use , Porphyromonas gingivalis/genetics , Microbiota/genetics , Aggregatibacter actinomycetemcomitans/geneticsABSTRACT
OBJECTIVE: This investigation explored oral-gut microbial signatures with potential to distinguish among periodontal conditions. BACKGROUND DATA: The interplay between the oral and gut microbiomes may be a critical pathway linking periodontal diseases and systemic inflammatory disorders. The mechanisms by which oral microorganisms translocate to the gut and cause microbial dysbiosis, favoring an inflammatory state, are still unknown. As a first approach, characterization of oral-gut microbial profiles associated with periodontal health and diseases can provide insights on such mechanisms of etiology and pathogenesis. METHODS: Fecal and saliva samples from individuals with periodontal health (PH, 8), gingivitis (GG, 17), and periodontitis (PD, 24) were analyzed for their microbial composition by 16S rRNA gene sequencing. Microbial taxa were compared and correlated to periodontal parameters. Multivariate discriminant analysis (MDA) was carried out to identify profiles related to health and disease. RESULTS: Few significant differences in oral-gut taxa were detected among clinical groups, although increase in fecal Fusobacterium nucleatum ss vincentii and salivary Aggregatibacter actinomycetemcomitans, Parvimonas micra, and Fretibacterium sp. HMT358 were strongly correlated with deep pockets and inflammation (p < .01). Over 50% of the fecal microbiota comprised microorganisms shared between oral and gut sites, whereas oral taxa were detected in approximately 9%, particularly enriched in GG fecal samples (p = .04). Trends for lower fecal richness and higher salivary diversity in PD compared to PH were observed. MDA was able to classify correctly 82% of the patients into the clinical groups. Main classifiers of periodontitis were high BMI, older age, and enrichment of oral-fecal Leptotrichia sp. HMT4, Peptostreptococcus stomatis, Dialister invisus, and a novel Lautropia sp. HMTC89-like organism. CONCLUSION: Within the limitations of an exploratory investigation, specific profiles of oral-gut taxa, including known and potential novel organisms, combined with social-demographic features were able to discriminate individuals with periodontal diseases in this study population.
Subject(s)
Gastrointestinal Microbiome , Microbiota , Periodontal Diseases , Periodontitis , Humans , RNA, Ribosomal, 16S/genetics , Microbiota/genetics , Periodontitis/diagnosis , Periodontitis/microbiology , Aggregatibacter actinomycetemcomitans/genetics , Periodontal Diseases/microbiologyABSTRACT
The reported prevalence of periodontitis in children and adolescents varies considerably between populations globally. This cross-sectional study compares clinical and microbiological findings on 83 Somali immigrants and 96 non-Somali children aged 10-17 years old living in Trollhättan, Sweden. The clinical examination included registration of bleeding on probing, plaque, and calculus on incisors and first molars. The distance between cemento-enamel junction and bone level was measured on bitewing radiographs. Pooled microbiological samples (1 µL) were taken from the mesial surface of 16, 11, 31, 36, and analyzed by culture and real-time polymerase chain reaction for seven periodontal associated bacterial species. The Somali participants had poorer oral hygiene and more bleeding, plaque, and calculus. Ten of the Somali but none of the non-Somali participants showed periodontal breakdown (radiographical bone loss > 3 mm), corresponding to a prevalence of 12% (95% CI: 5.9, 21.0%). The presence of A. actinomycetemcomitans was almost exclusively associated with Somali participants. Further, the JP2 clone was found in five Somalis (including two periodontitis cases) confirming the association of this clone with African populations. The Somali group showed significantly higher frequencies and numbers of Porphyromonas gingivalis and Treponema denticola, implying a mature and adult type of subgingival microbiota.
Subject(s)
Calculi , Dental Plaque , Periodontitis , Adolescent , Adult , Aggregatibacter actinomycetemcomitans/genetics , Child , Cross-Sectional Studies , Dental Plaque/microbiology , Humans , Periodontitis/microbiology , Porphyromonas gingivalis , Real-Time Polymerase Chain Reaction , Somalia , Sweden/epidemiologyABSTRACT
Aggregatibacter actinomycetemcomitans is a major periodontal pathogen that was identified firstly in actinomycotic lesions and later in advanced forms of periodontal diseases as well as in oral cavity of healthy subjects. The particular pathogenicity of this specie makes it a target for extensive studies both at fundamental and practical scales. The current advances in experimental and clinical research related to this bacterium focus the light on epidemiologic features, virulence, and invasiveness aspects as well as on identification challenges, bacterial susceptibility, and anti-virulence strategies. The present chapter provide to scientists and periodontal researchers a comprehensive overview on the main advances made in this field with a special focus on epidemiologic dissemination, microbial diagnosis, virulence factors and clinical implementations of such progress.
Subject(s)
Periodontal Diseases , Periodontitis , Aggregatibacter actinomycetemcomitans/genetics , Humans , Periodontal Diseases/microbiology , Periodontitis/microbiology , Virulence Factors/geneticsABSTRACT
Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis resulting in premature tooth loss in adolescents. Tooth adherence and biofilm persistence are prerequisites for survival in the oral domain. Here, using a rhesus monkey model, 16S rRNA sequencing, and weighted network analysis, we assessed colonization of A. actinomycetemcomitans variants and ascertained microbial interactions in biofilm communities. Variants in A. actinomycetemcomitans leukotoxin (ltx) were created, labeled, inoculated, and compared with their progenitor strain for in vivo colonization. Samples of tooth-related plaque were assessed for colonization at baseline and after debridement and inoculation of labeled strains. Null, minimal, and hyper-Ltx-producing strains were created and assessed for hydroxyapatite binding and biofilm formation in vitro. Ltx-hyperproducing strains colonized with greater prevalence and at higher levels than wild type or ltx mutants (P = 0.05). Indigenous and inoculated A. actinomycetemcomitans strains that attached were associated with lactate-producing species (i.e., Leptotrichia, Abiotrophia, and Streptoccocci). A. actinomycetemcomitans was found at 0.13% of the total flora at baseline and at 0.05% 4 wk after inoculation. In vivo data were supported by in vitro results. We conclude that hyper-Ltx production affords these strains with an attachment advantage providing a foothold for competition with members of the indigenous microbiota. Increased attachment can be linked to ltx gene expression and up-regulation of adherence-associated genes. Growth of attached A. actinomycetemcomitans in vivo was enhanced by lactate availability due to consorting species. These associations provide A. actinomycetemcomitans with the constituents required for its colonization and survival in the complex and competitive oral environment.
Subject(s)
Aggregatibacter actinomycetemcomitans/pathogenicity , Mouth/microbiology , Periodontitis/microbiology , Aggregatibacter actinomycetemcomitans/genetics , Aggregatibacter actinomycetemcomitans/metabolism , Aggregatibacter actinomycetemcomitans/physiology , Animals , Bacterial Adhesion/drug effects , Biofilms , Durapatite/pharmacology , Exotoxins/genetics , Exotoxins/metabolism , Lactic Acid/metabolism , Macaca mulatta , Male , MicrobiotaABSTRACT
PURPOSE: To investigate the global prevalence of the JP2 genotype of Aggregatibacter actinomycetemcomitans (Aa). METHODS: A comprehensive electronic search of databases, PUBMED, MEDLINE, EMBASE, BIOSIS, and SCOPUS, was conducted up to August 2021. All published articles and studies were considered, excluding animal studies, editorials, personal opinions, letters to editor, conference abstracts, posters, and those studies without full text. The primary objective of this systematic review was to determine the presence of the JP2 genotype of Aa in the world population. RESULTS: A total of 295 articles were identified, of which 62 were preselected, and 51 were finally included in this review. Due to variable study designs and high heterogeneity, a meta-analysis was not conducted. A total of 9744 subjects were screened for the presence of the JP2 genotype of Aa worldwide, and only 621 cases were found positive. CONCLUSIONS: A relatively high presence of JP2 genotype of Aa was found in subjects from South America, North America, and Africa. There were no studies estimating the presence of the JP2 genotype of Aa in the Oceania region. The heterogeneity and quality of the included publications suggest that caution should be exercised when interpreting the data and that there remains an important need for additional evidence. CLINICAL RELEVANCE: Periodontitis is a highly prevalent inflammatory oral disorder with substantial aesthetic, functional, and psychological implications for patients. The JP2 genotype of Aa is implicated in the pathogenesis of periodontitis. To the best of our knowledge, there is a lack of systematic reviews estimating the presence of the JP2 genotype of Aa in the global population. We identified a relatively high presence of the JP2 genotype of Aa in specific geographic areas of the world, and we propose that cross-sectional and longitudinal studies are lacking in the Oceania region and need to be conducted to estimate the presence of the JP2 genotype of Aa in this region.
Subject(s)
Aggregatibacter actinomycetemcomitans , Aggregatibacter actinomycetemcomitans/genetics , Cross-Sectional Studies , Genotype , Humans , Periodontitis/microbiology , PrevalenceABSTRACT
Periodontal disease is a frequent pathology worldwide, with a constantly increasing prevalence. For the optimal management of periodontal disease, there is a need to take advantage of actual technology to understand the bacterial etiology correlated with the pathogenic mechanisms, risk factors and treatment protocols. We analyzed the scientific literature published in the last 5 years regarding the recent applications of mRNA analysis in periodontal disease for the main known bacterial species considered to be the etiological agents: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Tannerella forsythia. We identified new pathogenic mechanisms, therapeutic target genes and possible pathways to prevent periodontal disease. The mRNA analysis, as well as the important technological progress in recent years, supports its implementation in the routine management of periodontal disease patients.
Subject(s)
Aggregatibacter actinomycetemcomitans , Periodontal Diseases , Aggregatibacter actinomycetemcomitans/genetics , Humans , Periodontal Diseases/microbiology , Porphyromonas gingivalis/genetics , RNA, Messenger/genetics , Tannerella forsythia/genetics , Treponema denticolaABSTRACT
Background and Objectives: Candida albicans can be detected in subgingival sites of patients with periodontitis. However, the association between oral Candida albicans and periodontitis has not been fully elucidated in Japanese adults. The aim of this study is to clarify the relationship between oral Candida albicans infection/co-infection of oral C. albicans and Porphyromonas gingivalis and periodontitis among middle-aged and older Japanese people. Materials and Methods: Eighty-six patients (mean age 70.4 years) who visited the Hiroshima University Hospital from April to September 2021 were investigated in this study. Oral swab samples were collected from the tongue surface. C. albicans and P. gingivalis DNA was detected by real-time PCR using specific DNA primer sets. C. albicans-positive participants were classified into two groups according to the presence or absence of intron insertion of C. albicans DNA by PCR analysis. Results: C. albicans was detected in 22 (25.6%) of the 86 patients. Patients in their 80s recorded a higher C. albicans-positive rate (35.3%) compared with other participants. However, there was no significant association between the C. albicans positivity rate and clinical parameters such as sex, age, systemic disease, denture use, or oral health status. Of the 22 C. albicans-positive participants, 10 participants (45.5%) had C. albicans with intron insertion; 70% of participants who had C. albicans with intron insertion exhibited ≥6 mm probing depth. C. albicans/P. gingivalis co-infection was found in 12 patients (14%). Importantly, binomial logistic regression analysis revealed that C. albicans/P. gingivalis co-infection was significantly associated with ≥6 mm periodontal pockets with bleeding on probing (p = 0.02). Conclusions: Co-infection of C. albicans and P. gingivalis is involved in active periodontitis in middle-aged and older people.
Subject(s)
Coinfection , Periodontitis , Adult , Aged , Aggregatibacter actinomycetemcomitans/genetics , Candida albicans/genetics , DNA , Humans , Japan/epidemiology , Middle Aged , Periodontitis/complications , Porphyromonas gingivalis/geneticsABSTRACT
Among the main bacteria implicated in the pathology of periodontal disease, Aggregatibacter actinomycetemcomitans (Aa) is well known for causing loss of periodontal attachment and systemic disease. Recent studies have suggested that secreted extracellular RNAs (exRNAs) from several bacteria may be important in periodontitis, although their role is unclear. Emerging evidence indicates that exRNAs circulate in nanosized bilayered and membranous extracellular vesicles (EVs) known as outer membrane vesicles (OMVs) in gram-negative bacteria. In this study, we analyzed the small RNA expression profiles in activated human macrophage-like cells (U937) infected with OMVs from Aa and investigated whether these cells can harbor exRNAs of bacterial origin that have been loaded into the host RNA-induced silencing complex, thus regulating host target transcripts. Our results provide evidence for the cytoplasmic delivery and activity of microbial EV-derived small exRNAs in host gene regulation. The production of TNF-α was promoted by exRNAs via the TLR-8 and NF-κB signaling pathways. Numerous studies have linked periodontal disease to neuroinflammatory diseases but without elucidating specific mechanisms for the connection. We show here that intracardiac injection of Aa OMVs in mice showed successful delivery to the brain after crossing the blood-brain barrier, the exRNA cargos increasing expression of TNF-α in the mouse brain. The current study indicates that host gene regulation by microRNAs originating from OMVs of the periodontal pathogen Aa is a novel mechanism for host gene regulation and that the transfer of OMV exRNAs to the brain may cause neuroinflammatory diseases like Alzheimer's.-Han, E.-C., Choi, S.-Y., Lee, Y., Park, J.-W., Hong, S.-H., Lee, H.-J. Extracellular RNAs in periodontopathogenic outer membrane vesicles promote TNF-α production in human macrophages and cross the blood-brain barrier in mice.
Subject(s)
Bacterial Outer Membrane/metabolism , Blood-Brain Barrier/metabolism , Extracellular Vesicles/genetics , Macrophages/metabolism , Periodontal Diseases/metabolism , RNA, Small Untranslated/genetics , Tumor Necrosis Factor-alpha/metabolism , Aggregatibacter actinomycetemcomitans/chemistry , Aggregatibacter actinomycetemcomitans/genetics , Animals , Extracellular Vesicles/metabolism , Gene Expression Profiling , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Male , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Periodontal Diseases/microbiology , Periodontal Diseases/pathology , RNA, Bacterial/genetics , U937 CellsABSTRACT
AIM: We investigated the association between the Aggregatibacter actinomycetemcomitans serotypes, periodontal status and coronary artery disease (CAD). MATERIALS AND METHODS: 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. RESULTS: 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." CONCLUSIONS: 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.
Subject(s)
Aggregatibacter actinomycetemcomitans/genetics , Coronary Artery Disease/microbiology , Periodontal Diseases/microbiology , Aged , Aggregatibacter actinomycetemcomitans/isolation & purification , Coronary Angiography , Coronary Artery Disease/diagnostic imaging , Female , Gingiva/microbiology , Humans , Male , Middle Aged , Polymerase Chain Reaction , Saliva/microbiology , SerogroupABSTRACT
The periodontal pathogen, Aggregatibacter actinomycetemcomitans is extremely sensitive to even a mildly acidic pH resulting from metabolic acids secreted during growth, losing viability rapidly as the pH goes below 6.0. Cells grown at high glucose concentration grow fast but rapidly lose viability. However, if the cells are grown at low glucose concentration, the pH of the growth medium first decreases slowly for about 24 h and then starts to increase. This increase of pH is indicative of cell death since the spontaneous rise of pH due to the presence of bicarbonate can no longer be opposed by secreted metabolic acids. By monitoring these pH changes on a petri dish, a method was developed to screen for sugar transport-deficient mutants from a library of transposon insertion mutants. Isolation of a mannose phosphotransferase mutant strain is described. The mutant cells were found to be more viable and for a longer period of time than wild-type cells both in high and low glucose concentrations due to slower metabolism and less acid secreted. This observation highlights the concern that spontaneous mutations in the sugar transport genes may be selected for in patients due to increased viability of the mutant cells especially in a biofilm.
Subject(s)
Aggregatibacter actinomycetemcomitans/metabolism , Bacterial Proteins/genetics , Monosaccharide Transport Proteins/genetics , Periodontal Diseases/microbiology , Aggregatibacter actinomycetemcomitans/genetics , Bacterial Proteins/metabolism , Gene Library , Humans , Monosaccharide Transport Proteins/metabolism , Mutation , Sugars/metabolismABSTRACT
Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are strongly associated with periodontitis, and their evaluations are relevant to understand their role in the etiology and progression of periodontal diseases. In this study, the qualitative and quantitative detection of A. actinomycetemcomitans and F. nucleatum, as well as their genetic diversity, were evaluated in individuals with gingivitis, chronic periodontitis and periodontally healthy. In addition, the biotyping, serotyping, and prevalence of the ltx and cdt genes in A. actinomycetemcomitans were also determined. Subgingival biofilms obtained from gingivitis (70), periodontitis (75) and healthy (95) individuals were analyzed by cultures and PCR. Bacterial typing and presence of ltx and cdt genes in A. actinomycetemcomitans were also verified. DNA from A. actinomycetemcomitans and F. nucleatum was detected respectively, in 65.7% and 57.1% of gingivitis, 80% and 68% of periodontitis, and 57.8% and 37.8% of healthy. A. actinomycetemcomitans from gingivitis were biotypes I, II, IV, V, and X, and serotypes a, c, and e. In periodontitis, biotypes II, VI, and X, and serotypes a, b, and c were found. In healthy subjects, biotypes II and X, and serotypes b and c were found. The LTX and ltxA were observed in strains from gingivitis and periodontitis pockets. Subsequently, our data also showed no direct relationship between ltxA gene expression and leukotoxin gene 530-bp presence. On the other hand, cdt gene predominated during the inflammatory disease process. Our results strongly support a role of A. actinomycetemcomitans and F. nucleatum in advanced stage of periodontal disease.
Subject(s)
Aggregatibacter actinomycetemcomitans/isolation & purification , Fusobacterium nucleatum/isolation & purification , Periodontal Diseases/microbiology , Adult , Aggregatibacter actinomycetemcomitans/classification , Aggregatibacter actinomycetemcomitans/genetics , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Cross-Sectional Studies , Exotoxins/genetics , Exotoxins/metabolism , Female , Fusobacterium nucleatum/classification , Fusobacterium nucleatum/genetics , Genotype , Humans , Male , Middle Aged , Young AdultABSTRACT
Aggregatibacter actinomycetemcomitans (Aa) is a pathogenic bacterium residing in the subgingival plaque biofilm strongly associated with the pathogenesis of periodontitis. The aim of this investigation was to study the protein differential expression of Aa when growing on biofilm compared with planktonic state using proteomic analysis by the 2D-DIGE system. Eighty-seven proteins were differentially expressed during biofilm growth (1.5-fold, p < 0.05), with 13 overexpressed and 37 down-expressed. Those repressed were mainly proteins involved in metabolism, biosynthesis, and transport. The overexpressed proteins were outer membrane proteins (OMPs) and highly immunogenic proteins such as YaeT (OMP), FtsZ, OMP39, OMP18/16, the chaperone GroEL, OMPA, adenylate kinase (Adk), and dihydrolipoamide acetyltransferase. The enrichment fractions of the OMPs from biofilm and planktonic states were obtained, and these proteins were analyzed by Western blotting with human serum from a periodontitis patient and one healthy control. These immunogenic proteins overexpressed in the biofilm may represent candidate virulence factors.
Subject(s)
Aggregatibacter actinomycetemcomitans/genetics , Bacterial Outer Membrane Proteins/genetics , Biofilms/growth & development , Gene Expression Regulation, Bacterial , Plankton/genetics , Virulence Factors/genetics , Adenylate Kinase/genetics , Adenylate Kinase/metabolism , Aggregatibacter actinomycetemcomitans/growth & development , Aggregatibacter actinomycetemcomitans/isolation & purification , Aggregatibacter actinomycetemcomitans/metabolism , Bacterial Outer Membrane Proteins/metabolism , Dihydrolipoyllysine-Residue Acetyltransferase/genetics , Dihydrolipoyllysine-Residue Acetyltransferase/metabolism , Gene Expression Profiling , Gene Ontology , Humans , Metabolic Networks and Pathways/genetics , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Sequence Annotation , Periodontitis/microbiology , Plankton/growth & development , Plankton/metabolism , Two-Dimensional Difference Gel Electrophoresis , Virulence Factors/metabolismABSTRACT
Periodontitis is a microbial infection that destroys the structures that support the teeth. Although it is typically a chronic condition, rapidly progressing, aggressive forms are associated with the oral pathogen Aggregatibacter actinomycetemcomitans One of this bacterium's key virulence traits is its ability to attach to surfaces and form robust biofilms that resist killing by the host and antibiotics. Though much has been learned about A. actinomycetemcomitans since its initial discovery, we lack insight into a fundamental aspect of its basic biology, as we do not know the full set of genes that it requires for viability (the essential genome). Furthermore, research on A. actinomycetemcomitans is hampered by the field's lack of a mutant collection. To address these gaps, we used rapid transposon mutant sequencing (Tn-seq) to define the essential genomes of two strains of A. actinomycetemcomitans, revealing a core set of 319 genes. We then generated an arrayed mutant library comprising >1,500 unique insertions and used a sequencing-based approach to define each mutant's position (well and plate) in the library. To demonstrate its utility, we screened the library for mutants with weakened resistance to subinhibitory erythromycin, revealing the multidrug efflux pump AcrAB as a critical resistance factor. During the screen, we discovered that erythromycin induces A. actinomycetemcomitans to form biofilms. We therefore devised a novel Tn-seq-based screen to identify specific factors that mediate this phenotype and in follow-up experiments confirmed 4 mutants. Together, these studies present new insights and resources for investigating the basic biology and disease mechanisms of a human pathogen.IMPORTANCE Millions suffer from gum disease, which often is caused by Aggregatibacter actinomycetemcomitans, a bacterium that forms antibiotic-resistant biofilms. To fully understand any organism, we should be able to answer: what genes does it require for life? Here, we address this question for A. actinomycetemcomitans by determining the genes in its genome that cannot be mutated. As for the genes that can be mutated, we archived these mutants into a library, which we used to find genes that contribute to antibiotic resistance, leading us to discover that antibiotics cause A. actinomycetemcomitans to form biofilms. We then devised an approach to find genes that mediate this process and confirmed 4 genes. These results illuminate new fundamental traits of a human pathogen.
Subject(s)
Aggregatibacter actinomycetemcomitans/genetics , Genome, Bacterial , Periodontitis/microbiology , Aggregatibacter actinomycetemcomitans/classification , Aggregatibacter actinomycetemcomitans/isolation & purification , Gene Library , Genetic Fitness , Genomics , Humans , Mouth/microbiology , Mutagenesis, InsertionalABSTRACT
BACKGROUND: The virulence of Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) in any individual depends on the type of strain of this bacterium. To our knowledge, there have been no studies reported in Indian subjects about A. actinomycetemcomitans serotype occurrence, co-existence with herpes virus and the possible influence of such co-existence on periodontal pathology. METHODS: Subjects for this study were a subset of a larger study to identify the prevalence of A. actinomycetemcomitans in chronic periodontitis. A total of 63 subjects (12 periodontally healthy and 51 with chronic periodontitis) who were positive for A. actinomycetemcomitans were serotyped for strain-level identification. The presence of Human Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) was tested in subgingival plaque samples by polymerase chain reaction. RESULTS: All five serotypes a to e were detected. Of the samples analyzed 38.09% harbored a single serotype, 36.5% had two serotypes, 6.3% demonstrated three and 4.7% demonstrated four serotypes. None of the samples showed presence of JP2 strain. Serotypes b, c, and e were most frequently identified in these individuals (46.03%, 36.5% and 38.09% respectively). Presence of serotypes b and c and absence of serotype d was associated with increased PD and CAL. Among 63 samples analyzed, 11 samples had CMV, four samples had EBV and nine samples had both these viruses. The PD and CAL were significantly higher (p = 0.04) when a combination of CMV and one of the serotypes was present indicating a pathological role of the coexistence. CONCLUSION: Multiple serotypes are associated with chronic periodontitis in Indians, however, JP2 strains are not detectable in this cohort. Presence of multiple serotypes and a combination of any serotype with herpesvirus is associated with greater severity of the disease.
Subject(s)
Aggregatibacter actinomycetemcomitans/classification , Periodontal Diseases/microbiology , Periodontal Diseases/virology , Serogroup , Simplexvirus/classification , Adult , Aggregatibacter actinomycetemcomitans/genetics , Aggregatibacter actinomycetemcomitans/isolation & purification , Chronic Periodontitis/epidemiology , Chronic Periodontitis/microbiology , Chronic Periodontitis/virology , Coinfection , Cytomegalovirus , DNA, Bacterial/analysis , DNA, Viral/analysis , Dental Plaque/microbiology , Dental Plaque/virology , Female , Gingiva , Herpesvirus 4, Human , Humans , India , Male , Middle Aged , Pasteurellaceae Infections/microbiology , Periodontal Attachment Loss/microbiology , Periodontal Index , Serotyping , Simplexvirus/genetics , Simplexvirus/isolation & purificationABSTRACT
Gingival junctional epithelial cell apoptosis caused by periodontopathic bacteria exacerbates periodontitis. This pathological apoptosis is involved in the activation of transforming growth factor ß (TGF-ß). However, the molecular mechanisms by which microbes induce the activation of TGF-ß remain unclear. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) activated TGF-ß receptor (TGF-ßR)/smad2 signalling to induce epithelial cell apoptosis, even though Aa cannot bind to TGF-ßR. Additionally, outer membrane protein 29 kDa (Omp29), a member of the Aa Omps family, can induce actin rearrangements via focal adhesion kinase (FAK) signalling, which also plays a role in the activation of TGF-ß by cooperating with integrin. Accordingly, we hypothesized that Omp29-induced actin rearrangements via FAK activity would enhance the activation of TGF-ß, leading to gingival epithelial cell apoptosis in vitro. By using human gingival epithelial cell line OBA9, we found that Omp29 activated TGF-ßR/smad2 signalling and decreased active TGF-ß protein levels in the extracellular matrix (ECM) of cell culture, suggesting the transactivation of TGF-ßR. Inhibition of actin rearrangements by cytochalasin D or blebbistatin and knockdown of FAK or integrinß1 expression by siRNA transfection attenuated TGF-ßR/smad2 signalling activity and reduction of TGF-ß levels in the ECM caused by Omp29. Furthermore, Omp29 bound to fibronectin (Fn) to induce its aggregation on integrinß1, which is associated with TGF-ß signalling activity. All the chemical inhibitors and siRNAs tested blocked Omp29-induced OBA9 cells apoptosis. These results suggest that Omp29 binds to Fn in order to facilitate Fn/integrinß1/FAK signalling-dependent TGF-ß release from the ECM, thereby inducing gingival epithelial cell apoptosis via TGF-ßR/smad2 pathway.