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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

基本信息

批准号：
7967015
负责人：
PAUL E KOLENBRANDER
金额：
$ 67.92万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
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/7967015
关键词：
Actinobacteria class Actinomyces Actinomyces naeslundii Adherence Aerobic Anabolism Arginine Bacteria Binding Caliber Cell Communication Cell Count Cell Density Cells Chemicals Coculture Techniques Communication Communities Community Developments Complex Culture Media DNA Microarray Chip Dental Enamel Dental Pellicle Dental Plaque Dental caries Development Devices Environment Epithelial Cells Exhibits Fiber Food Webs Genes Goals Growth Health Human Human Development Laboratories Length Little&apos s Disease Mediating Messenger RNA Metabolic Microbial Biofilms Molecular Mouth Diseases Nature Nutritional Oral Paper Periodontal Diseases Production Regulation Reporting Reverse Transcriptase Polymerase Chain Reaction Role Saliva Salivary Signal Transduction Signaling Molecule Source Streptococcus Streptococcus gordonii Streptococcus oralis Surface VAI-2 cell type commensal microbes in vivo Model interest microbial microorganism interaction mutant nutrition oral bacteria oral biofilm oral commensal oral streptococci response spatial relationship spatiotemporal tooth surface

项目摘要

Central to the development of human oral biofilm communities are microbial interactions that 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. Interactions among oral streptococci and actinomyces dominate initial dental plaque development. In this reporting period, we used a DNA microarray to identify Streptococcus gordonii genes regulated in response to coaggregation with Actinomyces naeslundii. Expression of 23 genes changed >3-fold in coaggregates, including nine genes involved in arginine biosynthesis and transport. The capacity of S. gordonii to synthesize arginine was assessed using a chemically defined growth medium. In monoculture, streptococcal arginine biosynthesis was inefficient and streptococci could not grow aerobically in low arginine. In dual-species cultures containing coaggregates, however, S. gordonii grew to high cell density in low arginine. Equivalent co-cultures without coaggregates showed no growth until coaggregation was evident, which occurred after 9 h of incubation. An argH mutant was unable to grow in low arginine with or without A. naeslundii, indicating that arginine biosynthesis was essential for coaggregation-induced streptococcal growth. Using quantitative RT-PCR, expression of argC, argG and pyrAb was strongly (10- to 100-fold) up-regulated in S. gordonii monocultures after 3 h growth when exogenous arginine was depleted. Co-cultures without induced coaggregation showed similar regulation. However, within 1 h after coaggregation with A. naeslundii, expression of argC, argG and pyrAb in S. gordonii was partially up-regulated although arginine was plentiful, and mRNA levels did not increase further when arginine was diminished. Thus, A. naeslundii stabilizes S. gordonii expression of arginine biosynthesis genes in coaggregates and enables aerobic growth when exogenous arginine is limited. Metabolic cooperation among bacteria may be important to the repetitive and distinctive community composition of initial oral biofilm communities, and food webs could be set up through this cooperation. The mechanisms of communication among mixed-species coaggregates is 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 possess the ability to metabolize salivary components as the sole nutritional source. We modelled the in vivo environment by using a flow device and sorbarod filters (paper-wrapped sheaf of tightly packed fibers: cylinder 10 mm diameter; 20 mm length) with saliva for nutrition. High cell densities were achieved, and we evaluated the ability of a streptococcus-actinomyces community to produce the universal signaling molecule autoinducer-2 (AI-2). Oral commensal bacteria Streptococcus oralis 34 and Actinomyces naeslundii T14V were grown as single-species and dual-species biofilms. After 48 h, dual-species biofilm communities of interdigitated S. oralis 34 and A. naeslundii T14V contained 3.2x109 cells: 5-fold more than single-species biofilms. However, these 48-h dual-species biofilms exhibited the lowest AI-2 concentration ratio (AI-2 concentration as nanomoles/L in the biofilm to cell density as cell number/mL of the biofilm). The more than 10-fold decrease in concentration ratio seen between 1-h and 48-h S. oralis 34-A. naeslundii T14V biofilms suggests that peak production of AI-2 occurs early in community development and is followed by a very low steady-state level. Specific concentrations of AI-2 appear to be essential for the initiation of oral commensal biofilm 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.

人类口腔生物膜群落发展的核心是驱动细菌群落内空间排列的微生物相互作用。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。口腔链球菌和放线菌之间的相互作用主导着牙菌斑的初始发育。在本报告期内，我们使用 DNA 微阵列来识别响应内氏放线菌共聚集而调节的戈登链球菌基因。共聚集体中 23 个基因的表达变化超过 3 倍，其中包括涉及精氨酸生物合成和运输的 9 个基因。使用化学成分确定的生长培养基评估戈登沙门氏菌合成精氨酸的能力。在单一培养中，链球菌精氨酸生物合成效率低下，链球菌不能在低精氨酸条件下有氧生长。然而，在含有共聚集体的双物种培养物中，戈登沙门氏菌在低精氨酸中生长至高细胞密度。不含共聚集体的等效共培养物显示出不生长，直到共聚集明显为止，共聚集在孵育 9 小时后发生。 argH 突变体在有或没有内氏放线菌的情况下都无法在低精氨酸中生长，表明精氨酸生物合成对于共聚集诱导的链球菌生长至关重要。使用定量 RT-PCR，当外源精氨酸耗尽时，S. gordonii 单一培养物在生长 3 小时后，argC、argG 和pyrAb 的表达强烈上调（10 至 100 倍）。没有诱导共聚集的共培养物显示出类似的调节。然而，在与内氏放线菌共聚集后1小时内，尽管精氨酸充足，但戈氏沙门氏菌中argC、argG和pyrAb的表达部分上调，并且当精氨酸减少时，mRNA水平并没有进一步增加。因此，内氏放线菌稳定了戈登沙门氏菌在共聚集体中精氨酸生物合成基因的表达，并在外源精氨酸有限时能够有氧生长。细菌之间的代谢合作对于初始口腔生物膜群落的重复且独特的群落组成可能很重要，并且可以通过这种合作建立食物网。混合物种共聚集体之间的通讯机制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而定植于清洁牙釉质表面的细菌具有附着于覆盖牙釉质的后天唾液膜的机制，并具有将唾液成分代谢为唯一营养源的能力。我们通过使用流动装置和 sorbarod 过滤器（用纸包裹的紧密堆积的纤维束：圆柱体直径 10 毫米；长度 20 毫米）和唾液营养来模拟体内环境。获得了高细胞密度，并且我们评估了链球菌-放线菌群落产生通用信号分子自诱导剂-2 (AI-2) 的能力。口腔共生细菌口腔链球菌 34 和内氏放线菌 T14V 作为单物种和双物种生物膜生长。 48 小时后，叉指型 S.oralis 34 和 A. naeslundii T14V 的双物种生物膜群落含有 3.2x109 个细胞：是单物种生物膜的 5 倍。然而，这些48小时的双物种生物膜表现出最低的AI-2浓度比（生物膜中的AI-2浓度为纳摩尔/L，细胞密度为生物膜的细胞数/mL）。 S.oralis 34-A 在 1 小时和 48 小时之间观察到浓度比下降超过 10 倍。 naeslundii T14V 生物膜表明 AI-2 的峰值产生发生在群落发育早期，随后是非常低的稳态水平。 AI-2 的特定浓度似乎对于口腔共生生物膜群落的启动至关重要。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的关系。