RESUMO
Recently, Veillonella infantium was isolated from tongue biofilm of a Thai child and established as a novel Veillonella species. In this study, a species-specific primer was designed to identify V. infantium on the basis of the sequence of the 70â¯kDa heat shock protein (dnaK) gene of Veillonella infantium JCM 31738T (= TSD-88T). The primer pair generated a specific PCR (Polymerase Chain Reaction) product specific for V. infantium, but not for other oral Veillonella species. This specific primer pair could detect dnaK even from 1â¯pg of genomic DNA extracted from the V. infantium type strain. To validate the primer pair, a number of strains of Veillonella species were isolated from tongue biofilm of 3 Japanese children, DNA was isolated from each strain, and PCR was performed using species-specific primers. All oral Veillonella species except V. infantium were identified by one-step PCR method reported previously. Four kinds of Veillonella species were detected in these subjects. V. rogosae was detected in all subjects and the most predominant species with an average prevalence of 82%. However, V. infantium was detected in 2 of 3 subjects and it was the second most predominant species of oral Veillonella detected in these subjects with an average prevalence of 9.4%. V. infantium appears to coexist with other oral Veillonella species in tongue biofilm. This species-specific primer pair established in this study could be useful to detect V. infantium and support the study of Veillonella for oral health in the future.
Assuntos
Primers do DNA/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Proteínas de Choque Térmico HSP70/genética , Veillonella/isolamento & purificação , Proteínas de Bactérias/genética , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Filogenia , Reação em Cadeia da Polimerase , Especificidade da Espécie , Veillonella/classificação , Veillonella/genéticaRESUMO
BACKGROUND: Successful commensal bacteria have evolved to maintain colonization in challenging environments. The oral viridans streptococci are pioneer colonizers of dental plaque biofilm. Some of these bacteria have adapted to life in the oral cavity by binding salivary α-amylase, which hydrolyzes dietary starch, thus providing a source of nutrition. Oral streptococcal species bind α-amylase by expressing a variety of amylase-binding proteins (ABPs). Here we determine the genotypic basis of amylase binding where proteins of diverse size and function share a common phenotype. RESULTS: ABPs were detected in culture supernatants of 27 of 59 strains representing 13 oral Streptococcus species screened using the amylase-ligand binding assay. N-terminal sequences from ABPs of diverse size were obtained from 18 strains representing six oral streptococcal species. Genome sequencing and BLAST searches using N-terminal sequences, protein size, and key words identified the gene associated with each ABP. Among the sequenced ABPs, 14 matched amylase-binding protein A (AbpA), 6 matched amylase-binding protein B (AbpB), and 11 unique ABPs were identified as peptidoglycan-binding, glutamine ABC-type transporter, hypothetical, or choline-binding proteins. Alignment and phylogenetic analyses performed to ascertain evolutionary relationships revealed that ABPs cluster into at least six distinct, unrelated families (AbpA, AbpB, and four novel ABPs) with no phylogenetic evidence that one group evolved from another, and no single ancestral gene found within each group. AbpA-like sequences can be divided into five subgroups based on the N-terminal sequences. Comparative genomics focusing on the abpA gene locus provides evidence of horizontal gene transfer. CONCLUSION: The acquisition of an ABP by oral streptococci provides an interesting example of adaptive evolution.
Assuntos
Amilases/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Genômica , Streptococcus/genética , Adaptação Biológica , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/classificação , DNA Bacteriano/isolamento & purificação , Transferência Genética Horizontal , Humanos , Modelos Moleculares , Boca/microbiologia , Filogenia , Estrutura Terciária de Proteína , Saliva/enzimologia , Alinhamento de Sequência , Análise de Sequência de Proteína , Simbiose , alfa-Amilases/metabolismoRESUMO
Streptococcus gordonii, a primary colonizer of the tooth surface, interacts with salivary α-amylase via amylase-binding protein A (AbpA). This enzyme hydrolyzes starch to glucose, maltose, and maltodextrins that can be utilized by various oral bacteria for nutrition. Microarray studies demonstrated that AbpA modulates gene expression in response to amylase, suggesting that the amylase-streptococcal interaction may function in ways other than nutrition. The goal of this study was to explore the role of AbpA in gene regulation through comparative transcriptional profiling of wild-type KS1 and AbpA(-) mutant KS1ΩabpA under various environmental conditions. A portion of the total RNA isolated from mid-log-phase cells grown in 5% CO2 in (i) complex medium with or without amylase, (ii) defined medium (DM) containing 0.8% glucose with/without amylase, and (iii) DM containing 0.2% glucose and amylase with or without starch was reverse transcribed to cDNA and the rest used for RNA sequencing. Changes in the expression of selected genes were validated by quantitative reverse transcription-PCR. Maltodextrin-associated genes, fatty acid synthesis genes and competence genes were differentially expressed in a medium-dependent manner. Genes in another cluster containing a putative histidine kinase/response regulator, peptide methionine sulfoxide reductase, thioredoxin protein, lipoprotein, and cytochrome c-type protein were downregulated in KS1ΩabpA under all of the environmental conditions tested. Thus, AbpA appears to modulate genes associated with maltodextrin utilization/transport and fatty acid synthesis. Importantly, in all growth conditions AbpA was associated with increased expression of a potential two-component signaling system associated with genes involved in reducing oxidative stress, suggesting a role in signal transduction and stress tolerance.
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Meios de Cultura/metabolismo , Perfilação da Expressão Gênica , alfa-Amilases Salivares/metabolismo , Amido/metabolismo , Streptococcus gordonii/efeitos dos fármacos , Streptococcus gordonii/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Deleção de Genes , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNARESUMO
Proteins in human saliva are thought to modulate bacterial colonization of the oral cavity. Yet, information is sparse on how salivary proteins interact with systemic pathogens that transiently or permanently colonize the oral environment. Staphylococcus aureus is a pathogen that frequently colonizes the oral cavity and can cause respiratory disease in hospitalized patients at risk. Here, we investigated salivary protein binding to this organism upon exposure to saliva as a first step toward understanding the mechanism by which the organism can colonize the oral cavity of vulnerable patients. By using fluorescently labeled saliva and proteomic techniques, we demonstrated selective binding of major salivary components by S. aureus to include DMBT1(gp-340), mucin-7, secretory component, immunoglobulin A, immunoglobulin G, S100-A9, and lysozyme C. Biofilm-grown S. aureus strains bound fewer salivary components than in the planctonic state, particularly less salivary immunoglobulins. A corresponding adhesive component on the S. aureus surface responsible for binding salivary immunoglobulins was identified as staphylococcal protein A (SpA). However, SpA did not mediate binding of nonimmunoglobulin components, including mucin-7, indicating the involvement of additional bacterial surface adhesive components. These findings demonstrate that a limited number of salivary proteins, many of which are associated with various aspects of host defense, selectively bind to S. aureus and lead us to propose a possible role of saliva in colonization of the human mouth by this pathogen.
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Interações Hospedeiro-Patógeno , Saliva/microbiologia , Proteínas e Peptídeos Salivares/metabolismo , Staphylococcus aureus/metabolismo , Humanos , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Ligação Proteica , Saliva/imunologia , Proteínas e Peptídeos Salivares/imunologia , Proteína Estafilocócica A/metabolismo , Staphylococcus aureus/imunologiaRESUMO
α-Amylase-binding streptococci (ABS) are a heterogeneous group of commensal oral bacterial species that comprise a significant proportion of dental plaque microfloras. Salivary α-amylase, one of the most abundant proteins in human saliva, binds to the surface of these bacteria via specific surface-exposed α-amylase-binding proteins. The functional significance of α-amylase-binding proteins in oral colonization by streptococci is important for understanding how salivary components influence oral biofilm formation by these important dental plaque species. This review summarizes the results of an extensive series of studies that have sought to define the molecular basis for α-amylase binding to the surface of the bacterium as well as the biological significance of this phenomenon in dental plaque biofilm formation.
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Biofilmes/crescimento & desenvolvimento , Proteínas e Peptídeos Salivares/metabolismo , Streptococcus/fisiologia , alfa-Amilases/metabolismo , Humanos , Ligação Proteica , Streptococcus/crescimento & desenvolvimentoRESUMO
BACKGROUND: Porphyromonas gingivalis (hereafter "Pg") is an oral pathogen that has been hypothesized to act as a keystone driver of inflammation and periodontal disease. Although Pg is most readily recovered from individuals with actively progressing periodontal disease, healthy individuals and those with stable non-progressing disease are also colonized by Pg. Insights into the factors shaping the striking strain-level variation in Pg, and its variable associations with disease, are needed to achieve a more mechanistic understanding of periodontal disease and its progression. One of the key forces often shaping strain-level diversity in microbial communities is infection of bacteria by their viral (phage) predators and symbionts. Surprisingly, although Pg has been the subject of study for over 40 years, essentially nothing is known of its phages, and the prevailing paradigm is that phages are not important in the ecology of Pg. RESULTS: Here we systematically addressed the question of whether Pg are infected by phages-and we found that they are. We found that prophages are common in Pg, they are genomically diverse, and they encode genes that have the potential to alter Pg physiology and interactions. We found that phages represent unrecognized targets of the prevalent CRISPR-Cas defense systems in Pg, and that Pg strains encode numerous additional mechanistically diverse candidate anti-phage defense systems. We also found that phages and candidate anti-phage defense system elements together are major contributors to strain-level diversity and the species pangenome of this oral pathogen. Finally, we demonstrate that prophages harbored by a model Pg strain are active in culture, producing extracellular viral particles in broth cultures. CONCLUSION: This work definitively establishes that phages are a major unrecognized force shaping the ecology and intra-species strain-level diversity of the well-studied oral pathogen Pg. The foundational phage sequence datasets and model systems that we establish here add to the rich context of all that is already known about Pg, and point to numerous avenues of future inquiry that promise to shed new light on fundamental features of phage impacts on human health and disease broadly. Video Abstract.
Assuntos
Bacteriófagos , Doenças Periodontais , Humanos , Bacteriófagos/genética , Porphyromonas gingivalis/genética , Prófagos/genética , Sequência de BasesRESUMO
Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in S. gordonii CH1 cells treated with native amylase relative to those treated with denatured amylase. An abpA-deficient strain of S. gordonii exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed abpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment.
Assuntos
Amilases/metabolismo , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Vias Biossintéticas/genética , Ácidos Graxos/biossíntese , Expressão Gênica , Streptococcus gordonii/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas e Peptídeos Salivares/metabolismo , Streptococcus gordonii/genéticaRESUMO
The genus Veillonella is a common and abundant member of the oral microbiome. It includes eight species, V. atypica, V. denticariosi, V. dispar, V. infantium, V. nakazawae, V. parvula, V. rogosae and V. tobetusensis. They possess important metabolic pathways that utilize lactate as an energy source. However, the overall metabolome of these species has not been studied. To further understand the metabolic framework of Veillonella in the human oral microbiome, we conducted a comparative pan-genome analysis of the eight species of oral Veillonella. Analysis of the oral Veillonella pan-genome revealed features based on KEGG pathway information to adapt to the oral environment. We found that the fructose metabolic pathway was conserved in all oral Veillonella species, and oral Veillonella have conserved pathways that utilize carbohydrates other than lactate as an energy source. This discovery may help to better understand the metabolic network among oral microbiomes and will provide guidance for the design of future in silico and in vitro studies.
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Fimbriae, lipopolysaccharide (LPS), and extracellular polymeric substance (EPS) all contribute to biofilm formation by the periodontopathogen Aggregatibacter actinomycetemcomitans. To understand how individual biofilm determinants respond to changing environmental conditions, the transcription of genes responsible for fimbria, LPS, and EPS production, as well as the translation of these components, was determined in rough (Rv) and isogenic smooth (Sv) variants of A. actinomycetemcomitans cultured in half-strength and full-strength culture medium under anaerobic or aerobic conditions, and in iron-supplemented and iron-chelated medium. The transcription of tadV (fimbrial assembly), pgaC (extracellular polysaccharide synthesis), and orf8 or rmlB (lipopolysaccharide synthesis) was measured by real-time PCR. The amounts of fimbriae, LPS, and EPS were also estimated from stained sodium dodecyl sulfate-polyacrylamide gels and verified by Western blotting and enzyme-linked immunoadsorbent assay using specific antibodies. Each gene was significantly upregulated in the Rv compared to in the Sv. The transcription of fimbrial, LPS, and EPS genes in the Rv was increased approximately twofold in cells cultured in full-strength medium under anaerobic conditions compared to that in cells cultured under aerobic conditions. Under anaerobic conditions, the transcription of fimbrial and EPS enzymes was elevated in both Rv and Sv cells cultured in half-strength medium, compared to that in full-strength medium. Iron chelation also increased the transcription and translation of all biofilm determinants compared to their expression with iron supplementation, yet the quantity of biofilm was not significantly changed by any environmental perturbation except iron limitation. Thus, anaerobic conditions, nutrient stress, and iron limitation each upregulate known biofilm determinants of A. actinomycetemcomitans to contribute to biofilm formation.
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Proteínas de Bactérias/biossíntese , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Pasteurellaceae/fisiologia , Polissacarídeos Bacterianos/biossíntese , RNA Mensageiro/biossíntese , Animais , Perfilação da Expressão Gênica , Humanos , RNA Bacteriano/biossíntese , Estresse FisiológicoRESUMO
BACKGROUND: A comparison of the salivary microbiome of non-diabetic and diabetic cohorts having periodontal health, gingivitis and periodontitis could reveal microbial signatures unique to each group that will increase understanding of the role of oral microbiota in the pathogenesis of disease, and assist with diagnosis and risk assessment for both periodontal disease and diabetes. METHODS: A group of individuals diagnosed with type 2 diabetes (T2D) was compared with a group without T2D. For both the diabetic and non-diabetic cohorts, three subgroups were established: periodontal health, gingivitis, and periodontitis. Salivary DNA was extracted (n = 146), polymerase chain reaction was performed to amplify 16S rRNA hypervariable region V3-V4, and constructed libraries were sequenced and subjected to bioinformatic and statistical analyses. RESULTS: Microbiome analysis resulted in 88 different genus level operational taxonomic units (OTUs) for differential abundance testing. Results were largely described by two trends. Trend 1 showed OTUs that increased in abundance with increasing periodontal disease, and in diabetics relative to non-diabetics. Trend 1 OTUs comprised a mix of primarily anaerobic commensals and potential periodontopathogens. Trend 2 was driven primarily by genera that decreased in abundance in those with diabetes relative to those without diabetes, which included other anaerobes associated with periodontal disease. Overall, oral microbial diversity decreased in diabetics and increased with progression of periodontal disease compared with periodontally healthy controls. CONCLUSION: Although select microbiota increased in both diabetes and periodontal disease progression, these genera decreased in co-existing diabetes and periodontal disease. These findings suggest that the genera abundance continues to change with additional stress imposed by co-existing conditions.
Assuntos
Diabetes Mellitus Tipo 2 , Microbiota , Doenças Periodontais , Periodontite , Adulto , Humanos , RNA Ribossômico 16SRESUMO
The oral commensal bacterium Streptococcus gordonii interacts with salivary amylase via two amylase-binding proteins, AbpA and AbpB. Based on sequence analysis, the 20-kDa AbpA protein is unique to S. gordonii, whereas the 82-kDa AbpB protein appears to share sequence homology with other bacterial dipeptidases. The aim of this study was to verify the peptidase activity of AbpB and further explore its potential functions. The abpB gene was cloned, and histidine-tagged AbpB (His-AbpB) was expressed in Escherichia coli and purified. Its amylase-binding activity was verified in an amylase ligand binding assay, and its cross-reactivity was verified with an anti-AbpB antibody. Both recombinant His-AbpB and partially purified native AbpB displayed dipeptidase activity and degraded human type VI collagen and fibrinogen, but not salivary amylase. Salivary amylase precipitates not only AbpA and AbpB but also glucosyltransferase G (Gtf-G) from S. gordonii supernatants. Since Streptococcus mutans also releases Gtf enzymes that could also be involved in multispecies plaque interactions, the effect of S. gordonii AbpB on S. mutans Gtf-B activity was also tested. Salivary amylase and/or His-AbpB caused a 1.4- to 2-fold increase of S. mutans Gtf-B sucrase activity and a 3- to 6-fold increase in transferase activity. An enzyme-linked immunosorbent assay verified the interaction of His-AbpB and amylase with Gtf-B. In summary, AbpB demonstrates proteolytic activity and interacts with and modulates Gtf activity. These activities may help explain the crucial role AbpB appears to play in S. gordonii oral colonization.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Streptococcus gordonii/enzimologia , Amilases/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Colágeno Tipo VI/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Escherichia coli/genética , Fibrinogênio/metabolismo , Expressão Gênica , Glucosiltransferases/metabolismo , Humanos , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Streptococcus gordonii/genética , Especificidade por SubstratoRESUMO
BACKGROUND: Ventilator-associated pneumonia (VAP) is a leading cause of morbidity and mortality in patients hospitalized in intensive care units. Recent studies suggest that dental plaque biofilms serve as a reservoir for respiratory pathogens. The goal of this study was to determine the genetic relationship between strains of respiratory pathogens first isolated from the oral cavity and later isolated from bronchoalveolar lavage fluid from the same patient undergoing mechanical ventilation with suspected VAP. METHODS: Plaque and tracheal secretion samples were obtained on the day of hospital admission and every other day thereafter until discharge from the intensive care unit from 100 patients who underwent mechanical ventilation. Bronchoalveolar lavage was performed for 30 patients with suspected VAP. Pulse-field gel electrophoresis and multilocus sequence typing were used to determine the genetic relatedness of strains obtained from oral, tracheal, and bronchoalveolar lavage samples. RESULTS: Isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter species, and enteric species recovered from plaque from most patients were indistinguishable from isolates recovered from bronchoalveolar lavage fluid (i.e., had >95% similarity of pulse-field gel electrophoresis patterns). Nearly one-half of the Pseudomonas strains showed identical genetic profiles between patients, which suggested a common environmental source of infection. CONCLUSIONS: Respiratory pathogens isolated from the lung are often genetically indistinguishable from strains of the same species isolated from the oral cavity in patients who receive mechanical ventilation who are admitted to the hospital from the community. Thus, dental plaque serves as an important reservoir for respiratory pathogens in patients who undergo mechanical ventilation.
Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Técnicas de Tipagem Bacteriana , Líquido da Lavagem Broncoalveolar/microbiologia , Placa Dentária/microbiologia , Pneumonia Associada à Ventilação Mecânica/microbiologia , Adulto , Idoso , Bactérias/genética , Análise por Conglomerados , Impressões Digitais de DNA , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Respiração Artificial , Análise de Sequência de DNA , Traqueia/microbiologiaRESUMO
Veillonella species are known to contribute to the formation of early oral biofilms and tend to be prevalent in people with poor oral hygiene status. Here, we report the draft genome sequences of 4 oral Veillonella strains that were established recently as novel species.
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[This corrects the article DOI: 10.1371/journal.pone.0172647.].
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BACKGROUND: There is emerging evidence linking diabetes with periodontal disease. Diabetes is a well-recognized risk factor for periodontal disease. Conversely, pro-inflammatory molecules released by periodontally-diseased tissues may enter the circulation to induce insulin resistance. While this association has been demonstrated in adults, there is little information regarding periodontal status in obese children with and without type 2 diabetes (T2D). We hypothesized that children with T2D have higher rates of gingivitis, elevated salivary inflammatory markers, and an altered salivary microbiome compared to children without T2D. METHODS: Three pediatric cohorts ages 10-19 years were studied: lean (normal weight-C), obese (Ob), and obese with T2D (T2D). Each subject completed an oral health survey, received a clinical oral examination, and provided unstimulated saliva for measurement of inflammatory markers and microbiome analysis. RESULTS: The diabetes group was less likely to have had a dental visit within the last six months. Body mass index (BMI) Z-scores and waist circumference/height ratios were similar between Ob and T2D cohorts. The number of carious lesions and fillings were similar for all three groups. The gingival index was greater in the T2D group compared to the Ob and C groups. Although salivary microbial diversity was minimal between groups, a few differences in bacterial genus composition were noted. CONCLUSIONS: Obese children with T2D show a trend toward poorer oral health compared to normal weight and obese children without T2D. This study characterizes the salivary microbiome of children with and without obesity and T2D. This study supports a modest link between T2D and periodontal inflammation in the pediatric population.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/complicações , Microbiota , Obesidade Infantil/metabolismo , Obesidade Infantil/microbiologia , Glândulas Salivares/metabolismo , Glândulas Salivares/microbiologia , Adolescente , Biomarcadores/metabolismo , Estudos de Casos e Controles , Criança , Estudos Transversais , Feminino , Humanos , Inflamação/metabolismo , Masculino , Obesidade Infantil/sangue , Obesidade Infantil/complicações , Adulto JovemRESUMO
BACKGROUND: Prolonged induction of excessive levels of inflammatory mediators contributes to the pathogenesis of chronic disease states, such as periodontitis. It is thus important to develop safe and effective anti-inflammatory strategies for therapeutic reasons. In this study, we determined the ability of aqueous extracts from elder flower (Sambucus nigra) to inhibit the proinflammatory activity of major virulence factors from the periodontal pathogens Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. METHODS: Monocytes/macrophages or neutrophils were incubated with whole cells of P. gingivalis, A. actinomycetemcomitans, or purified components thereof (lipopolysaccharide and fimbriae) in the absence or presence of elder flower extract and were assayed for cytokine production, integrin activation, or induction of the oxidative burst. RESULTS: The elder flower extract was found to potently inhibit all proinflammatory activities tested. Investigation of the underlying mechanisms revealed that the anti-inflammatory extract inhibited activation of the nuclear transcription factor kappaB and of phosphatidylinositol 3-kinase. CONCLUSION: The elder flower extract displays useful anti-inflammatory properties that could be exploited therapeutically for the control of inflammation in human periodontitis.
Assuntos
Aggregatibacter actinomycetemcomitans/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Flores , Mediadores da Inflamação/antagonistas & inibidores , Extratos Vegetais/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , Sambucus nigra , Aggregatibacter actinomycetemcomitans/imunologia , Animais , Antígeno CD11b/efeitos dos fármacos , Antígenos CD18/efeitos dos fármacos , Fímbrias Bacterianas/imunologia , Humanos , Interleucinas/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , NF-kappa B/antagonistas & inibidores , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Porphyromonas gingivalis/imunologia , Explosão Respiratória/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fatores de Virulência/antagonistas & inibidoresRESUMO
Fresh clinical isolates of the periodontal pathogen Actinobacillus actinomycetemcomitans have an adherent, rough colony morphology that transforms into a minimally adherent, smooth colony phenotype during successive in vitro passage. The objectives of this study were: (1) to compare biofilm formation of the rough (RVs) and smooth variants (SVs) of several strains of A. actinomycetemcomitans grown under various environmental conditions and (2) to examine the dynamics of biofilm formation. A microtitre plate biofilm assay was used to evaluate biofilm formation of strains grown in broth with modified salt concentration and pH, and to evaluate the effect of pre-conditioning films. Scanning electron microscopy (SEM) was used to monitor microscopic changes in morphology. Dynamics of biofilm formation were measured in a flowcell monitored by confocal microscopy. The RVs generally produced greater biofilm than the SVs. However, medium-dependent differences in biofilm formation were evident for some rough/smooth pairs. The RVs were more tolerant to changes in salt and pH, and more resistant to chlorhexidine than the SVs. Horse serum virtually eliminated, and saliva significantly reduced, biofilm formation by the SVs in contrast to the RVs. SEM revealed no alteration in morphology with change of environment. In a flowcell, the RVs produced towers of microcolonies anchored by a small contact area, whereas the SVs produced an open architecture of reduced height. After 7 days in a flowcell, the rough to smooth phenotype transition could be demonstrated. In conclusion, strain, growth medium and conditioning film all affect biofilm formation. The RVs produce biofilms of unique architecture that may serve to protect the bacterium from environmental perturbations.
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Aggregatibacter actinomycetemcomitans/fisiologia , Biofilmes/crescimento & desenvolvimento , Aggregatibacter actinomycetemcomitans/efeitos dos fármacos , Aggregatibacter actinomycetemcomitans/ultraestrutura , Anaerobiose/fisiologia , Técnicas Bacteriológicas/métodos , Biofilmes/efeitos dos fármacos , Clorexidina/farmacologia , Meios de Cultura , Desinfetantes/farmacologia , Concentração de Íons de Hidrogênio , Microscopia Confocal/métodos , Microscopia Eletrônica de Varredura/métodos , Reação do Ácido Periódico de Schiff/métodos , Fenótipo , Saliva/fisiologia , Cloreto de Sódio/farmacologiaRESUMO
Surface display of proteins by sortases in Gram-positive bacteria is crucial for bacterial fitness and virulence. We found a unique gene locus encoding an amylase-binding adhesin AbpA and a sortase B in oral streptococci. AbpA possesses a new distinct C-terminal cell wall sorting signal. We demonstrated that this C-terminal motif is required for anchoring AbpA to cell wall. In vitro and in vivo studies revealed that SrtB has dual functions, anchoring AbpA to the cell wall and processing AbpA into a ladder profile. Solution structure of AbpA determined by NMR reveals a novel structure comprising a small globular α/ß domain and an extended coiled-coil heliacal domain. Structural and biochemical studies identified key residues that are crucial for amylase binding. Taken together, our studies document a unique sortase/adhesion substrate system in streptococci adapted to the oral environment rich in salivary amylase.