Cell-cell Interactions Between Oral Actinomyces And Other Bacteria

口腔放线菌与其他细菌之间的细胞间相互作用

• 批准号: 7593352
• 负责人: PAUL E KOLENBRANDER
• 金额: $ 99.58万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593352
• 关键词: Acetic Acid; Acetic Acids; Actinobacteria class; Actinomyces; Actinomyces naeslundii; Adherence; Antibodies; Bacteria; Bacterial Adhesins; Binding; Biological Assay; Biological Models; Biomass; Carbohydrates; Carbon Dioxide; Catabolism; Cell Communication; Cells; Characteristics; Chemicals; Chemistry; Clinical Protocols; Communication; Communities; Community Developments; Complex; Condition; Data; Dental; Dental Enamel; Dental Pellicle; Dental Plaque; Dental caries; Detection; Development; Ecosystem; Enzymes; Epithelial Cells; Evolution; Exhibits; Fermentation; Fingerprint; Food Webs; Genes; Genetic; Genomics; Goals; Growth; Health; Hour; Human; Human Development; Hydrogen; Institutes; Laboratories; Lactic acid; Little' s Disease; Mediating; Medical; Metabolic; Methods; Microbial Biofilms; Modeling; Molecular; Molecular Biology; Mouth Diseases; Nutritional; Oral; Oral cavity; Organism; Pattern; Periodontal Diseases; Polysaccharides; Process; Production; Pyruvic Acid; Range; Reporting; Role; Saliva; Salivary; Shapes; Signal Transduction; Signaling Molecule; Site; Source; Streptococcus; Streptococcus oralis; Structure; Surface; System; Thinking; Time; Universities; VAI-2; Veillonella; cell type; commensal microbes; directed evolution; interest; member; microbial; microbial community; microorganism interaction; mutant; mutualism; oral bacteria; oral biofilm; oral commensal; organic acid; rRNA Genes; receptor; restoration; spatial relationship; spatiotemporal; sugar; tooth surface

During the development of human oral biofilm communities, microbial interactions are thought to drive the spatial arrangement within bacterial communities. Such communities on enamel form supragingival dental plaque. These intimate interactions are facilitated by physical interactions called coaggregations, which are specific adherences of genetically distinct partner cells that bind to one another to form multicellular networks such as the multispecies communities of human dental plaque. In this reporting period, we used chemically pure 4,5-dihydroxy-2,3-pentanedione (DPD) that was supplied by our colleague Dr. Martin Semmelhack in the Department of Chemistry at Princeton University to show that picomolar concentrations of this diffusible signal molecule are optimal for mediating mutualistic interactions between two oral bacterial species. DPD, a product of the LuxS enzyme in the catabolism of S-ribosylhomocysteine, spontaneously cyclizes to form autoinducer 2 (AI-2). ). AI-2 was proposed by our colleague Dr. Bonnie Bassler in the Department of Molecular Biology and the Howard Hughes Medical Institute at Princeton University to be a universal signal molecule mediating inter-species communication among bacteria. We reported that mutualistic and abundant biofilm growth in flowing saliva of two coaggregation-partner human oral commensal bacteria, Actinomyces naeslundii T14V and Streptococcus oralis 34, is dependent upon production of AI-2 by S. oralis 34. A luxS mutant of S. oralis 34 was constructed which did not produce AI-2. Unlike wild-type dual-species biofilms, A. naeslundii T14V and an S. oralis 34 luxS mutant did not exhibit mutualism and generated only sparse biofilms which contained a 10-fold lower biomass of each species. Restoration of AI-2 levels by chemical (synthetic AI-2 in the form of DPD) or genetic complementation re-established the mutualistic growth and high biomass characteristic for the wild-type dual-species biofilm. Significantly, in this natural two-species system, the optimal DPD concentration is 100- to 1000-fold lower than the detection limit of the currently accepted AI-2 bioassay, indicating that only a very low concentration of AI-2 is required for these organisms to conduct AI-2-signaled inter-species communication. We demonstrated for the first time that picomolar concentrations of the universal inter-species signal AI-2 mediate mutualistic interactions among members of a natural dual-species community. These studies verify that AI-2 is a bona-fide inter-species signal, and its concentration is critical for mutualism between two species of oral bacteria grown under conditions that are representative of the human oral cavity. The role of diffusible signaling molecules in establishing these early communities remains a topic of much interest in my laboratory. The initial colonizers of tooth surfaces are a specific subset of the oral microflora. Of these bacteria, those that colonize the clean enamel surface independently of other bacteria possess mechanisms for attachment to the acquired salivary pellicle covering the enamel and the ability to metabolize salivary components as the sole nutritional source. Alternatively, some bacterial species participate in consortia that are able to attach to enamel and establish as an initial community requiring metabolic interactions among their members. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Our hypothesis is that the initial colonization of tooth surfaces is a repeatable and selective process with certain bacterial species predominating in the nascent biofilm. In this reporting period, we used molecular methods and a retrievable enamel chip model (clinical protocol D-98-0116) to characterize the microbial diversity of early dental biofilms in three subjects. Five hundred and thirty-one 16S-rRNA-gene sequences were analyzed, and 97 distinct phylotypes were identified. The study showed that microbial community composition was statistically different among subjects and that more than two-thirds of the bacterial species in the initial dental plaque were unique to each of the three subjects in the study. A set of ten species was in common in the three subjects, and this result promoted the idea that initial colonization occurred with a subset of species particularly adapted to community development on otherwise unoccupied enamel surface. Repetitive and distinctive community composition within subjects was observed and supported our hypothesis that the initial colonization of tooth surfaces was a repeatable and selective process with certain bacterial species predominating in the nascent biofilm. Metabolic cooperation among bacteria may be important to the establishment of stable oral biofilm communities, and food webs could be set up through this cooperation. Streptococci make up 60-90% of supragingival plaque biomass in the first 24 hours of colonization; they catabolize carbohydrates to short-chain organic acids such as lactic acid and pyruvic acid. Veillonellae constitute as much as 5% of initial plaque biomass, but are unable to catabolize sugars. They rely on the fermentation of organic acids to propionic and acetic acids, carbon dioxide, and hydrogen. Thus, a rudimentary food web could exist whereby veillonellae depend upon organic acids produced by streptococci. Few data exist on proximity of veillonellae and streptococci in plaque. We hypothesized that cell-cell recognition between veillonellae and streptococci was important in supragingival plaque formation, and we set out to examine this in a human model system. The retrievable enamel chip model (clinical protocol D-98-0116) was used to investigate site-specific isolation of veillonellae and streptococci. In this reporting period, we discovered veillonellae isolates bearing adhesins that specifically recognize Type-G receptor polysaccharide (G-RPS) on streptococci. We showed that, between four and eight hours into plaque development, the dominant strains of Veillonella changed in their phenotypic characteristics (coaggregation and antibody reactivity) as well as in their genotypic characteristics (16S-RNA gene sequences and strain-level genomic fingerprint patterns). This succession was 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. The discovery of G-RPS-recognizing veillonellae is the first example of oral bacteria specifically coaggregating with G-RPS-bearing streptococci. These results suggest that these two kinds of bacteria will be found at the same sites in the oral cavity and offer an example of evolution-directed mixed-species communities. Our long-range goal is to understand the molecular mechanisms of cellular communication and their relationship to the spatiotemporal development and establishment of dental plaque and colonization of the host epithelial cells.

在人类口腔生物膜群落的发展过程中，微生物的相互作用被认为驱动了细菌群落的空间排列。这些菌落在牙釉质上形成牙菌斑。这些亲密的相互作用是通过称为共聚集的物理相互作用来促进的，这是遗传上不同的伴侣细胞相互结合形成多细胞网络的特定粘附，例如人类牙菌斑的多物种群落。在本报告期间，我们使用由普林斯顿大学化学系的同事Martin Semmelhack博士提供的化学纯4,5-二羟基-2,3-戊二酮（DPD）来表明，这种扩散信号分子的皮摩尔浓度对于介导两种口腔细菌之间的互惠相互作用是最佳的。DPD是s -核糖体同型半胱氨酸分解代谢过程中LuxS酶的产物，可自发环化形成自诱导剂2 （AI-2）。。AI-2是由我们的同事，普林斯顿大学分子生物系和霍华德休斯医学研究所的Bonnie Bassler博士提出的，是一种介导细菌间物种间交流的通用信号分子。我们报道了两种共同聚集的人类口腔共生细菌——纳斯lundii放线菌T14V和口腔链球菌34——在流动的唾液中相互作用和丰富的生物膜生长依赖于口腔链球菌34产生AI-2。构建了不产生AI-2的口腔链球菌34 luxS突变体。与野生型双种生物膜不同，a . naeslundii T14V和S. oral 34luxs突变体不表现出相互作用，只产生稀疏的生物膜，每个物种的生物量低10倍。通过化学（以DPD形式合成AI-2）或遗传互补恢复AI-2水平，重新建立了野生型双物种生物膜的共生生长和高生物量特征。值得注意的是，在这个天然的两种系统中，DPD的最佳浓度比目前公认的AI-2生物测定的检测限低100- 1000倍，这表明这些生物只需要非常低的AI-2浓度就可以进行AI-2信号的种间通信。我们首次证明了皮摩尔浓度的普遍物种间信号AI-2介导自然双物种群落成员之间的相互作用。这些研究证实，AI-2是一种真正的物种间信号，其浓度对于在具有代表性的人类口腔条件下生长的两种口腔细菌之间的相互作用至关重要。扩散信号分子在建立这些早期群落中的作用仍然是我实验室非常感兴趣的话题。