Cell-cell Interactions Between Oral Actinomyces And Othe

口腔放线菌与其他细胞之间的相互作用

• 批准号: 7317794
• 负责人: PAUL E KOLENBRANDER
• 金额: --
• 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/7317794
• 关键词: Cell; cell; Interactions; Between; Oral

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，微生物相互作用被认为驱动细菌群落内的空间排列。牙釉质上的此类群落形成龈上牙菌斑。这些亲密的相互作用是由称为共聚集的物理相互作用促进的，共聚集是遗传上不同的伙伴细胞的特定粘附，这些细胞彼此结合形成多细胞网络，例如人类牙菌斑的多物种群落。在本报告期内，我们使用了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)，结果表明，这种可扩散信号分子的皮摩尔浓度最适合介导两种口腔细菌物种之间的互惠相互作用。 DPD 是 S-核糖基高半胱氨酸分解代谢中 LuxS 酶的产物，自发环化形成自诱导剂 2 (AI-2)。 ）。 AI-2 是由普林斯顿大学分子生物学系和霍华德休斯医学研究所的同事 Bonnie Bassler 博士提出的，它是介导细菌间种间通讯的通用信号分子。我们报道了两种共聚集伙伴人类口腔共生细菌（内氏放线菌 T14V 和口腔链球菌 34）在流动唾液中的互利和丰富的生物膜生长依赖于口腔链球菌 34 产生的 AI-2。构建了口腔链球菌 34 的 luxS 突变体，该突变体不产生 AI-2。与野生型双物种生物膜不同，内氏放线菌 T14V 和口链球菌 34 luxS 突变体不表现出互利共生，仅产生稀疏的生物膜，其中每个物种的生物量低 10 倍。通过化学（DPD 形式的合成 AI-2）或基因互补恢复 AI-2 水平，重新建立了野生型双物种生物膜的互利生长和高生物量特征。值得注意的是，在这种天然的两物种系统中，最佳 DPD 浓度比目前接受的 AI-2 生物测定的检测限低 100 至 1000 倍，这表明这些生物体只需要非常低浓度的 AI-2 即可进行 AI-2 信号的种间通讯。我们首次证明了通用种间信号 AI-2 的皮摩尔浓度可介导自然双物种群落成员之间的互利相互作用。这些研究证实 AI-2 是一种真正的种间信号，其浓度对于在代表人类口腔的条件下生长的两种口腔细菌之间的共生至关重要。可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。 牙齿表面的最初定殖者是口腔微生物群的特定子集。在这些细菌中，那些独立于其他细菌在干净的牙釉质表面定殖的细菌具有附着于覆盖牙釉质的后天唾液膜的机制以及将唾液成分代谢为唯一营养来源的能力。或者，一些细菌物种参与能够附着在牙釉质上并建立为需要其成员之间代谢相互作用的初始群落的群落。初始微生物群的表征是了解群落成员之间相互作用的第一步，群落成员之间的相互作用塑造了随后的生物膜发展。我们的假设是，牙齿表面的初始定植是一个可重复的选择性过程，某些细菌种类在新生生物膜中占主导地位。在本报告期内，我们使用分子方法和可回收的牙釉质芯片模型（临床方案 D-98-0116）来表征三个受试者早期牙齿生物膜的微生物多样性。分析了 531 个 16S-rRNA 基因序列，并鉴定了 97 种不同的系统发育型。研究表明，受试者之间的微生物群落组成存在统计学差异，并且初始牙菌斑中超过三分之二的细菌种类对于研究中的三名受试者都是独特的。这三个受试者中有十个物种是共同的，这一结果促进了这样一种观点，即最初的定殖发生在特别适合在其他未被占据的牙釉质表面上群落发展的物种子集上。观察到受试者体内重复且独特的群落组成，并支持我们的假设，即牙齿表面的初始定植是一个可重复和选择性的过程，某些细菌种类在新生生物膜中占主导地位。 细菌之间的代谢合作对于建立稳定的口腔生物膜群落可能很重要，并且可以通过这种合作建立食物网。在定植的前 24 小时内，链球菌占龈上菌斑生物量的 60-90%；它们将碳水化合物分解代谢为短链有机酸，例如乳酸和丙酮酸。韦荣球菌占初始斑块生物量的 5%，但无法分解代谢糖。它们依靠有机酸发酵成丙酸、乙酸、二氧化碳和氢气。因此，可能存在一个基本的食物网，韦永球菌依赖于链球菌产生的有机酸。关于斑块中韦荣球菌和链球菌的接近程度的数据很少。我们假设韦荣球菌和链球菌之间的细胞间识别对于龈上菌斑形成很重要，并且我们着手在人体模型系统中对此进行检查。可回收的牙釉质芯片模型（临床方案 D-98-0116）用于研究韦荣球菌和链球菌的位点特异性分离。在本报告期内，我们发现了含有粘附素的韦永菌分离株，可特异性识别链球菌上的 G 型受体多糖 (G-RPS)。我们发现，在斑块形成的 4 到 8 小时内，韦荣球菌的优势菌株的表型特征（共聚集和抗体反应性）以及基因型特征（16S-RNA 基因序列和菌株水平基因组指纹模式）发生了变化。这种演替与混合物种细菌菌落的发展相协调。在自然生物膜发育过程中，群落结构的变化可能会非常迅速地发生，我们认为这一过程可能会影响该生态系统内的进化。识别 G-RPS 的韦永球菌的发现是口腔细菌与携带 G-RPS 的链球菌特异性共聚集的第一个例子。这些结果表明，这两种细菌将在口腔的相同部位发现，并提供了进化导向的混合物种群落的例子。我们的长期目标是了解细胞通讯的分子机制及其与牙菌斑时空发育和建立以及宿主上皮细胞定植的关系。