RESUMO
At mucosal surfaces, epithelial cells provide a structural barrier and an immune defense system. However, dysregulated epithelial responses can contribute to disease states. Here, we demonstrated that epithelial cell-intrinsic production of interleukin-23 (IL-23) triggers an inflammatory loop in the prevalent oral disease periodontitis. Epithelial IL-23 expression localized to areas proximal to the disease-associated microbiome and was evident in experimental models and patients with common and genetic forms of disease. Mechanistically, flagellated microbial species of the periodontitis microbiome triggered epithelial IL-23 induction in a TLR5 receptor-dependent manner. Therefore, unlike other Th17-driven diseases, non-hematopoietic-cell-derived IL-23 served as an initiator of pathogenic inflammation in periodontitis. Beyond periodontitis, analysis of publicly available datasets revealed the expression of epithelial IL-23 in settings of infection, malignancy, and autoimmunity, suggesting a broader role for epithelial-intrinsic IL-23 in human disease. Collectively, this work highlights an important role for the barrier epithelium in the induction of IL-23-mediated inflammation.
Assuntos
Interleucina-23 , Periodontite , Humanos , Células Epiteliais , Inflamação , Receptor 5 Toll-Like/metabolismoRESUMO
Specific interbacterial adhesion, termed coaggregation, is well established for three early colonizers of the plaque biofilm: streptococci, actinomyces, and veillonellae. However, little is known about interactions of other early colonizers and about the extent of interactions within the bacterial community from a single host. To address these gaps, subject-specific culture collections from two individuals were established using an intraoral biofilm retrieval device. Molecular taxonomy (Human Oral Microbe Identification Microarray [HOMIM]) analysis of biofilm samples confirmed the integrity and completeness of the collections. HOMIM analysis verified the isolation of Streptococcus gordonii and S. anginosus from only one subject, as well as isolation of a previously uncultivated streptococcal phylotype from the other subject. Strains representative of clonal diversity within each collection were further characterized. Greater than 70% of these streptococcal strains from each subject coaggregated with at least one other coisolate. One-third of the strains carry a known coaggregation mediator: receptor polysaccharide (RPS). Almost all nonstreptococcal isolates coaggregated with other coisolates. Importantly, certain Rothia strains demonstrated more coaggregations with their coisolated bacteria than did any Streptococcus or Actinomyces strain, and certain Haemophilus isolates participated in twice as many. Confocal microscopy of undisturbed biofilms showed that Rothia and Haemophilus each occur in small multispecies microcolonies. However, in confluent high-biomass regions, Rothia occurred in islands whereas Haemophilus was distributed throughout. Together, the data demonstrate that coaggregation networks within an individual's oral microflora are extensive and that Rothia and Haemophilus can be important initiators of cell-cell interactions in the early biofilm.IMPORTANCE Extensive involvement of specific interbacterial adhesion in dental plaque biofilm formation has been postulated based on in vitro coaggregation between oral bacteria from culture collections that are not subject specific. In the present study, subject-specific culture collections were obtained from early plaque biofilm of two volunteers, and coaggregations within each culture collection were assayed. Coaggregations, several of which involved a coaggregation-mediating cell surface molecule known from well-studied streptococci, were widespread. Unexpectedly, the little-studied organisms Haemophilus and Rothia participated in the greatest numbers of interactions with community members; these two organisms showed different distributions within the undisturbed biofilm. The data show that coaggregation networks encompass most organisms within the biofilm community of each individual, and they indicate prominent participation of organisms such as Haemophilus and Rothia in early plaque biofilm formation.
Assuntos
Bactérias/isolamento & purificação , Aderência Bacteriana , Fenômenos Fisiológicos Bacterianos , Biofilmes , Placa Dentária/microbiologia , Adulto , Bactérias/classificação , Bactérias/genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
UNLABELLED: Although saliva is widely recognized as a primary source of carbon and nitrogen for growth of the dental plaque biofilm community, little is known about how different oral bacteria utilize specific salivary components. To address this question, 32 strains representing 16 genera commonly isolated from early plaque biofilms were compared for growth over two transfers in stimulated (by chewing Parafilm) whole saliva that was stabilized by heat treatment and dialysis. The cell densities, measured by quantitative PCR (qPCR), ranged from â¼1 × 10(6) to 1 × 10(7)/ml for strains of Streptococcus gordonii, Streptococcus oralis, and Streptococcus mitis and one strain of Streptococcus sanguinis Strains of Streptococcus mutans, Gemella haemolysans, and Granulicatella adiacens reached â¼1 × 10(5) to 1 × 10(6)/ml. In contrast, little or no growth was noted for three other strains of S. sanguinis, as well as for strains of Streptococcus parasanguinis, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus sobrinus, Actinomyces spp., Abiotrophia defectiva, and Rothia dentocariosa SDS-PAGE, lectin blotting, and two-dimensional gel electrophoresis of saliva from cultures of S. gordonii, S. oralis, and S. mitis revealed species-specific differences in the degradation of basic proline-rich glycoproteins (PRG). In contrast, saliva from cultures of other bacteria was indistinguishable from control saliva. Species-dependent differences in the utilization of individual host sugars were minor. Thus, differences in salivary glycan foraging between oral species may be important to cross-feeding and cooperation between organisms in dental plaque biofilm development. IMPORTANCE: Bacteria in the mouth use saliva for nutrition. How each of the many types of bacteria uses saliva is not clear. We show that a major protein in saliva, called PRG, is an important nutrition source for certain bacteria but not for others. PRG has many sugar molecules linked in chains, but the sugar is not available for bacteria until the chains are degraded. The bacteria that can grow by digesting this protein break the sugar chains into parts which not only support their own growth but could also be available to support the growth of those bacteria that cannot use the intact protein.
Assuntos
Bactérias/metabolismo , Glicoproteínas/metabolismo , Prolina/metabolismo , Saliva/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Humanos , Saliva/química , Saliva/metabolismoRESUMO
UNLABELLED: The growth of the oral commensal Streptococcus gordonii in saliva may depend on a number of glycoside hydrolases (GHs), including three cell wall-anchored proteins that are homologs of pneumococcal ß-galactosidase (BgaA), ß-N-acetylglucosaminidase (StrH), and endo-ß-N-acetylglucosaminidase D (EndoD). In the present study, we introduced unmarked in-frame deletions into the corresponding genes of S. gordonii DL1, verified the presence (or absence) of the encoded proteins on the resulting mutant strains, and compared these strains with wild-type strain DL1 for growth and glycan foraging in saliva. The overnight growth of wild-type DL1 was reduced 3- to 10-fold by the deletion of any one or two genes and approximately 20-fold by the deletion of all three genes. The only notable change in the salivary proteome associated with this reduction of growth was a downward shift in the apparent molecular masses of basic proline-rich glycoproteins (PRG), which was accompanied by the loss of lectin binding sites for galactose-specific Erythrina cristagalli agglutinin (ECA) and mannose-specific Galanthus nivalis agglutinin (GNA). The binding of ECA to PRG was also abolished in saliva cultures of mutants that expressed cell surface BgaA alone or together with either StrH or EndoD. However, the subsequent loss of GNA binding was seen only in saliva cocultures of different mutants that together expressed all three cell surface GHs. The findings indicate that the growth of S. gordonii DL1 in saliva depends to a significant extent on the sequential actions of first BgaA and then StrH and EndoD on N-linked glycans of PRG. IMPORTANCE: The ability of oral bacteria to grow on salivary glycoproteins is critical for dental plaque biofilm development. Little is known, however, about how specific salivary components are attacked and utilized by different members of the biofilm community, such as Streptococcus gordonii. Streptococcus gordonii DL1 has three cell wall-anchored glycoside hydrolases that are predicted to act on host glycans. In the present study, we introduced unmarked in-frame deletions in the corresponding genes, verified the presence (or absence) of encoded proteins on the resulting mutant strains, and compared these strains with wild-type DL1 for growth and glycan foraging in saliva. The results indicate that the growth of S. gordonii DL1 depends to a significant extent on sequential action of these cell surface GHs on N-linked glycans of basic proline-rich salivary glycoproteins, which appears to be an essential first step in salivary glycan foraging.
Assuntos
Acetilglucosaminidase/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/enzimologia , Saliva/microbiologia , Streptococcus gordonii/enzimologia , Streptococcus gordonii/crescimento & desenvolvimento , beta-Galactosidase/metabolismo , Acetilglucosaminidase/genética , Proteínas de Bactérias/genética , Membrana Celular/genética , Placa Dentária/microbiologia , Humanos , Streptococcus gordonii/genética , Streptococcus gordonii/isolamento & purificação , beta-Galactosidase/genéticaRESUMO
The oral bacterial microbiome encompasses approximately 700 commonly occurring phylotypes, approximately half of which can be present at any time in any individual. These bacteria are largely indigenous to the oral cavity; this limited habitat range suggests that interactions between the various phylotypes, and between the phylotypes and their environment, are crucial for their existence. Molecular cataloging has confirmed many basic observations on the composition of the oral microbiome that were formulated well before ribosomal RNA-based systematics, but the power and the scope of molecular taxonomy have resulted in the discovery of new phylotypes and, more importantly, have made possible a level of bacterial community analysis that was unachievable with classical methods. Bacterial community structure varies with location within the mouth, and changes in community structure are related to disease initiation and disease progression. Factors that influence the formation and the evolution of communities include selective adherence to epithelial or tooth surfaces, specific cell-to-cell binding as a driver of early community composition, and interorganismal interaction leading to alteration of the local environment, which represents the first step on the road to oral disease. A comprehensive understanding of how these factors interact to drive changes in the composition of the oral microbial community can lead to new strategies for the inhibition of periodontal diseases and dental caries.
Assuntos
Microbiota/fisiologia , Boca/microbiologia , Bactérias/classificação , Fenômenos Fisiológicos Bacterianos , Biofilmes , Cárie Dentária/prevenção & controle , Humanos , Consórcios Microbianos/fisiologia , Doenças Periodontais/prevenção & controleRESUMO
In periodontitis, a common chronic inflammatory condition, gram-negative-rich bacterial biofilms trigger, in susceptible individuals, perpetuating inflammation that results in extensive tissue damage of tooth supporting structures. To delineate immune cell-dependent mechanisms whereby bacterial challenge drives persistent destructive inflammation in periodontitis and other inflammatory diseases, we studied involved tissues ex vivo and investigated host cell responses to the periodontal pathogen Porphyromonas gingivalis, in vitro. Diseased lesions were populated by abundant Th17 cells, linked to infection, chronic inflammation/autoimmunity and tissue pathology. In vitro, P. gingivalis, particularly the more virulent strain W83, stimulated myeloid antigen presenting cells (APC) to drive Th17 polarization. Supernatants from myeloid APC exposed to P. gingivalis were capable of enhancing Th17 but not Th1 polarization. P. gingivalis favored the generation of Th17 responses by stimulating the production of Th17 related cytokines IL-1ß, IL-6 and IL-23, but not Th1 related IL-12. By inducing NFκB activation, P. gingivalis promoted IL-1ß, IL-6 and IL-12p40 production, but not IRF3 phosphorylation, connected to generation of the IL-12p35 chain, ultimately restricting formation of the intact IL-12 molecule. Promotion of Th17 lineage responses was also aided by P. gingivalis proteases, which appeared to differentially degrade pivotal cytokines. In this regard, IL-12 was largely degraded by P. gingivalis, whereas IL-1ß was more resistant to proteolysis. Our data unveil multiple pathways by which P. gingivalis may orchestrate chronic inflammation, providing insights into interventional strategies.
Assuntos
Infecções por Bacteroidaceae/imunologia , Periodontite Crônica/imunologia , Interações Hospedeiro-Patógeno , Porphyromonas gingivalis/imunologia , Células Th17/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/microbiologia , Infecções por Bacteroidaceae/microbiologia , Células Cultivadas , Periodontite Crônica/microbiologia , Meios de Cultivo Condicionados/farmacologia , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Subunidade p35 da Interleucina-12/imunologia , Subunidade p35 da Interleucina-12/metabolismo , Subunidade p40 da Interleucina-12/imunologia , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Interleucina-23/imunologia , Interleucina-23/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Células Mieloides/metabolismo , Células Mieloides/microbiologia , NF-kappa B/genética , NF-kappa B/imunologia , Fosforilação , Proteólise , Transdução de Sinais , Células Th17/efeitos dos fármacos , Células Th17/metabolismoRESUMO
Shear-enhanced adhesion, although not observed for fimbria-mediated adhesion of oral Actinomyces spp., was noted for Hsa-mediated adhesion of Streptococcus gordonii to sialic acid-containing receptors, an interaction implicated in the pathogenesis of infective endocarditis.
Assuntos
Aderência Bacteriana/fisiologia , Boca/microbiologia , Actinomyces/patogenicidade , Actinomyces/fisiologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/fisiologia , Aderência Bacteriana/genética , Fenômenos Biomecânicos , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Endocardite Bacteriana/etiologia , Fímbrias Bacterianas/fisiologia , Genes Bacterianos , Hemaglutininas Virais , Humanos , Técnicas In Vitro , Mutação , Reologia , Streptococcus gordonii/genética , Streptococcus gordonii/patogenicidade , Streptococcus gordonii/fisiologia , Dente/microbiologiaRESUMO
Most microorganisms in nature live in multispecies communities attached to a substratum-biofilms. Within these communities, organismal interaction is spatiotemporally defined. Because biofilms exist at an interface, their environment is characterized by gradients of nutrients that encourage spatial and metabolic diversity within the population. Oral bacterial biofilms were among the first human-associated biofilms to have been extensively investigated. They are diverse in species, and that diversity reflects the range of habitats within the oral cavity. Oral bacterial communities can be studied in vitro and in vivo. These studies have yielded information on interorganismal interactions and the developmental patterns within the communities. The wealth of information on these communities, coupled with their accessibility in their natural state, firmly establishes them as paradigm systems in biofilm research.
Assuntos
Biofilmes , Placa Dentária/microbiologia , Aderência Bacteriana/fisiologia , Biodiversidade , Biofilmes/classificação , Biofilmes/crescimento & desenvolvimento , Biomassa , Gengiva/microbiologia , Humanos , Simbiose/fisiologiaRESUMO
This study evaluated the impact of mechanically stimulated saliva on initial bacterial colonization. Interaction between oral bacteria and both unstimulated and stimulated saliva was examined in vitro by laying labeled bacteria over SDS-PAGE-separated salivary proteins. The effects of chewing on in vivo biofilm, microbial composition, and spatial arrangement were examined in two human volunteers using an intraoral stent containing retrievable enamel chips. In vitro experiments showed that bacterial binding to proteins from stimulated saliva was lower than that to proteins from unstimulated saliva. Lack of binding activity was noted with Streptococcus mutans and Lactobacillus casei. Human Oral Microbe Identification Microarray (HOMIM) analyses revealed a consistent chewing-related increase in the binding of Streptococcus anginosus and Streptococcus gordonii. Immunofluorescence microscopy demonstrated the presence of multi-species colonies and cells bearing different serotypes of the coaggregation-mediating streptococcal cell-surface receptor polysaccharides (RPS). Differences in bacterial colonization were noted between the two volunteers, while the type 4 RPS-reactive serotype was absent in one volunteer. Cells reacting with antibody against Rothia or Haemophilus were prominent in the early biofilm. While analysis of the data obtained demonstrated inter-individual variations in both in vitro and in vivo bacterial binding patterns, stimulating saliva with multiple orosensory stimuli may modulate oral bacterial colonization of tooth surfaces.
Assuntos
Biofilmes , Boca/microbiologia , Saliva , Humanos , Streptococcus/classificação , Streptococcus/isolamento & purificação , Streptococcus/fisiologiaRESUMO
Background: Molecular taxonomic assignments in oral microbial communities have been made using probe-matching approaches, but never compared to those obtained by more readily accepted tree-based approaches. Objective: To compare community composition profiles obtained from a probe-matching approach (HOMINGS) to those from a closed-ended tree-based approach (QIIME using the eHOMD database). Design: HOMINGS and QIIME were used for parallel analysis of ten mock community samples, and of 119 supragingival plaque samples from ecologically unique sites (sound tooth surfaces in healthy subjects, sound tooth surfaces in patients with primary Sjögren's Syndrome, and carious lesions in Sjögren's Syndrome patients). Linear discriminant analysis Effective Size (LEfSe) was used to identify discriminating taxa among the natural plaque samples. Results: Community composition profiles of all samples were congruent between the two analysis aproaches. Alpha and beta diversity of the natural plaque communities were likewise similar. Communities from pSS patients and those from individuals with normal salivary flow differed in alpha and beta diversity. Both classification approaches yielded differences in composition predicted for samples from these subject cohorts, and discriminating taxa were similar between approaches. Conclusions: A direct comparison demonstrates that HOMINGS is largely equivalent to the tree-based approach as implemented here.
RESUMO
Human oral bacteria interact with their environment by attaching to surfaces and establishing mixed-species communities. As each bacterial cell attaches, it forms a new surface to which other cells can adhere. Adherence and community development are spatiotemporal; such order requires communication. The discovery of soluble signals, such as autoinducer-2, that may be exchanged within multispecies communities to convey information between organisms has emerged as a new research direction. Direct-contact signals, such as adhesins and receptors, that elicit changes in gene expression after cell-cell contact and biofilm growth are also an active research area. Considering that the majority of oral bacteria are organized in dense three-dimensional biofilms on teeth, confocal microscopy and fluorescently labeled probes provide valuable approaches for investigating the architecture of these organized communities in situ. Oral biofilms are readily accessible to microbiologists and are excellent model systems for studies of microbial communication. One attractive model system is a saliva-coated flowcell with oral bacterial biofilms growing on saliva as the sole nutrient source; an intergeneric mutualism is discussed. Several oral bacterial species are amenable to genetic manipulation for molecular characterization of communication both among bacteria and between bacteria and the host. A successful search for genes critical for mixed-species community organization will be accomplished only when it is conducted with mixed-species communities.
Assuntos
Bactérias/metabolismo , Bactérias/patogenicidade , Homosserina/análogos & derivados , Boca/microbiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Antígenos de Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Homosserina/metabolismo , Humanos , Lactonas/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Streptococcus/metabolismo , Streptococcus/patogenicidadeRESUMO
Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.
Assuntos
Aderência Bacteriana , Biofilmes , Placa Dentária/microbiologia , Streptococcus gordonii/crescimento & desenvolvimento , Streptococcus oralis/crescimento & desenvolvimento , Veillonella/crescimento & desenvolvimento , Esmalte Dentário/microbiologia , Genes Bacterianos , Genes de RNAr , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Filogenia , Polissacarídeos Bacterianos/metabolismo , Pontos Quânticos , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Streptococcus gordonii/genética , Streptococcus gordonii/metabolismo , Streptococcus oralis/genética , Streptococcus oralis/metabolismo , Veillonella/genética , Veillonella/metabolismoRESUMO
The oral cavity is home to unique resident microbial communities whose interactions with host immunity are less frequently studied than those of the intestinal microbiome. We examined the stimulatory capacity and the interactions of two oral bacteria, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum), on Dendritic Cell (DC) activation, comparing them to the effects of the well-studied intestinal microbe Escherichia coli (E. coli). Unlike F. nucleatum and E. coli, P. gingivalis failed to activate DCs, and in fact silenced DC responses induced by F. nucleatum or E. coli. We identified a variant strain of P. gingivalis (W50) that lacked this immunomodulatory activity. Using biochemical approaches and whole genome sequencing to compare the two substrains, we found a point mutation in the hagA gene. This protein is though to be involved in the alteration of the PorSS/gingipain pathway, which regulates protein secretion into the extracellular environment. A proteomic comparison of the secreted products of the two substrains revealed enzymatic differences corresponding to this phenotype. We found that P. gingivalis secretes gingipain(s) that inactivate several key proinflammatory mediators made by DCs and/or T cells, but spare Interleukin-1 (IL-1) and GM-CSF, which can cause capillary leaks that serve as a source of the heme that P. gingivalis requires for its survival, and GM-CSF, which can cause epithelial-cell growth. Taken together, our results suggest that P. gingivalis has evolved potent mechanisms to modulate its virulence factors and dampen the innate immune response by selectively inactivating most proinflammatory cytokines.
Assuntos
Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Porphyromonas gingivalis/imunologia , Animais , Antibiose , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citocinas/análise , Citocinas/metabolismo , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Escherichia coli/genética , Feminino , Fusobacterium/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1/análise , Interleucina-1/metabolismo , Lectinas/química , Lectinas/genética , Lectinas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Porphyromonas gingivalis/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/microbiologiaRESUMO
The usual context for genome-genome interactions is DNA-DNA interactions, but the manifestation of the genome is the cell. Here we focus on cell-cell interactions and relate them to the process of building multi-species biofilm communities. We propose that dental plaque communities originate as a result of intimate interactions between cells (genomes) of different species and not through clonal growth of genetically identical cells. Although DNA exchange might occur between cells within these communities, we limit our opinions to discussions of the spatiotemporal and metabolic relationships that exist here. We believe the multi-species interactions occurring during the early stages of biofilm formation determine the species composition and nature of the mature biofilm. The human oral cavity provides easy access to natural biofilms on a retrievable enamel chip, which is an excellent model for the study of genome-genome interactions.
Assuntos
Biofilmes/crescimento & desenvolvimento , Placa Dentária/microbiologia , Genoma Bacteriano , Ecossistema , Variação Genética , Humanos , Modelos Biológicos , Recombinação GenéticaRESUMO
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.
Assuntos
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
BACKGROUND: Strains of viridans group streptococci that initiate colonization of the human tooth surface typically coaggregate with each other and with Actinomyces naeslundii, another member of the developing biofilm community. These interactions generally involve adhesin-mediated recognition of streptococcal receptor polysaccharides (RPS). The objective of our studies is to understand the role of these polysaccharides in oral biofilm development. METHODS: Different structural types of RPS have been characterized by their reactions with specific antibodies and lectin-like adhesins. Streptococcal gene clusters for RPS biosynthesis were identified, sequenced, characterized and compared. RPS-producing bacteria were detected in biofilm samples using specific antibodies and gene probes. RESULTS: Six different types of RPS have been identified from representative viridans group streptococci that coaggregate with A. naeslundii. Each type is composed of a different hexa- or heptasaccharide repeating unit, the structures of which contain host-like motifs, either GalNAcbeta1-3Gal or Galbeta1-3GalNAc. These motifs account for RPS-mediated recognition, whereas other features of these polysaccharides are more closely associated with RPS antigenicity. The RPS-dependent interaction of S. oralis with A. naeslundii promotes growth of these bacteria and biofilm formation in flowing saliva. Type specific differences in RPS production have been noted among the resident streptococcal floras of different individuals, raising the possibility of RPS-based differences in the composition of oral biofilm communities. CONCLUSION: The structural, functional and molecular properties of streptococcal RPS support a recognition role of these cell surface molecules in oral biofilm formation.
RESUMO
A bacterial etiology of rheumatoid arthritis (RA) has been suspected since the beginnings of modern germ theory. Recent studies implicate mucosal surfaces as sites of disease initiation. The common occurrence of periodontal dysbiosis in RA suggests that oral pathogens may trigger the production of disease-specific autoantibodies and arthritis in susceptible individuals. We used mass spectrometry to define the microbial composition and antigenic repertoire of gingival crevicular fluid in patients with periodontal disease and healthy controls. Periodontitis was characterized by the presence of citrullinated autoantigens that are primary immune targets in RA. The citrullinome in periodontitis mirrored patterns of hypercitrullination observed in the rheumatoid joint, implicating this mucosal site in RA pathogenesis. Proteomic signatures of several microbial species were detected in hypercitrullinated periodontitis samples. Among these, Aggregatibacter actinomycetemcomitans (Aa), but not other candidate pathogens, induced hypercitrullination in host neutrophils. We identified the pore-forming toxin leukotoxin A (LtxA) as the molecular mechanism by which Aa triggers dysregulated activation of citrullinating enzymes in neutrophils, mimicking membranolytic pathways that sustain autoantigen citrullination in the RA joint. Moreover, LtxA induced changes in neutrophil morphology mimicking extracellular trap formation, thereby releasing the hypercitrullinated cargo. Exposure to leukotoxic Aa strains was confirmed in patients with RA and was associated with both anticitrullinated protein antibodies and rheumatoid factor. The effect of human lymphocyte antigen-DRB1 shared epitope alleles on autoantibody positivity was limited to RA patients who were exposed to Aa These studies identify the periodontal pathogen Aa as a candidate bacterial trigger of autoimmunity in RA.
Assuntos
Aggregatibacter actinomycetemcomitans , Anticorpos Antiproteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Citrulina/química , Infecções por Pasteurellaceae/imunologia , Periodontite/microbiologia , Adulto , Artrite Reumatoide/microbiologia , Autoantígenos/química , Estudos de Casos e Controles , Doença Crônica , Ensaios Clínicos como Assunto , Feminino , Cadeias HLA-DRB1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Periodontite/imunologia , Estudos ProspectivosRESUMO
Growth of oral bacteria in situ requires adhesion to a surface because the constant flow of host secretions thwarts the ability of planktonic cells to grow before they are swallowed. Therefore, oral bacteria evolved to form biofilms on hard tooth surfaces and on soft epithelial tissues, which often contain multiple bacterial species. Because these biofilms are easy to study, they have become the paradigm of multispecies biofilms. In this Review we describe the factors involved in the formation of these biofilms, including the initial adherence to the oral tissues and teeth, cooperation between bacterial species in the biofilm, signalling between the bacteria and its role in pathogenesis, and the transfer of DNA between bacteria. In all these aspects distance between cells of different species is integral for oral biofilm growth.
Assuntos
Biofilmes , Metagenoma , Boca/microbiologia , Humanos , Doenças Periodontais/microbiologia , Saliva/microbiologiaRESUMO
Streptococci are the primary component of the multispecies oral biofilm known as supragingival dental plaque; they grow by fermentation of sugars to organic acids, e.g., lactic acid. Veillonellae, a ubiquitous component of early plaque, are unable to use sugars; they ferment organic acids, such as lactate, to a mixture of shorter-chain-length acids, CO(2), and hydrogen. Certain veillonellae bind to (coaggregate with) streptococci in vitro. We show that, between 4 and 8 hours into plaque development, the dominant strains of Veillonella change in their phenotypic characteristics (coaggregation and antibody reactivity) as well as in their genotypic characteristics (16S RNA gene sequences as well as strain level fingerprint patterns). This succession is coordinated with the development of mixed-species bacterial colonies. Changes in community structure can occur very rapidly in natural biofilm development, and we suggest that this process may influence evolution within this ecosystem.
Assuntos
Biofilmes/crescimento & desenvolvimento , Boca/microbiologia , Veillonella/fisiologia , Placa Dentária/microbiologia , Gengiva/microbiologia , Humanos , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Veillonella/classificação , Veillonella/genética , Veillonella/crescimento & desenvolvimentoRESUMO
The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.