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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：
7733896
负责人：
PAUL E KOLENBRANDER
金额：
$ 94万
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/7733896
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 DNA Microarray format 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 Range 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微生物相互作用，其驱动细菌群落内的空间排列。釉质上的这些群落形成龈上牙菌斑。这些密切的相互作用是由称为凝聚的物理相互作用促进的，凝聚是遗传上不同的伴侣细胞的特异性粘附，这些细胞相互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初始牙菌斑的发展。在本报告期内，我们使用了DNA微阵列，以确定戈登链球菌的基因调节反应共聚集与内氏放线菌。共聚集体中23个基因的表达变化>3倍，包括9个参与精氨酸生物合成和转运的基因。S.使用化学成分确定的生长培养基评估Gordonii合成精氨酸的能力。在单一培养中，链球菌精氨酸的生物合成是低效的，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种培养物中，S。戈登氏菌在低精氨酸条件下生长至高细胞密度。没有共聚集体的等同共培养物显示没有生长，直到共聚集体是明显的，这发生在孵育9小时后。argH突变体不能在低精氨酸条件下生长。naeslundii，表明精氨酸的生物合成是必不可少的凝聚诱导的链球菌生长。使用定量RT-PCR，argC，argG和pyrAb的表达在S.当外源精氨酸耗尽时，生长3小时后的戈登氏菌单培养物。没有诱导共聚集的共培养物表现出类似的调节。但在与A. naeslundii中argC、argG和pyrAb的表达。尽管精氨酸丰富，但戈登氏菌的mRNA水平部分上调，并且当精氨酸减少时，mRNA水平没有进一步增加。因此，A. naeslundii稳定S. Gordonii精氨酸生物合成基因在共聚集体中的表达，并且当外源精氨酸受限时能够有氧生长。细菌之间的代谢合作可能是重要的重复和独特的社区组成的初始口腔生物膜社区，食物网可以通过这种合作建立。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课题。牙齿表面的最初殖民者是口腔微生物菌群的特定子集。在这些细菌中，独立于其他细菌而定殖于清洁釉质表面的那些细菌具有附着于覆盖釉质的获得性唾液膜的机制，并且具有代谢唾液组分作为唯一营养源的能力。我们通过使用流动装置和sorbarod过滤器（纸包裹的紧密堆积的纤维束：圆柱体直径10 mm;长度20 mm）模拟体内环境，其中唾液用于营养。实现了高细胞密度，我们评估了链球菌-放线菌群落产生通用信号分子自诱导物-2（AI-2）的能力。口腔链球菌34和内氏放线菌T14 V作为单菌种和双菌种生物膜生长。 48 h后，叉指状S. oralis 34和A. Naeslundii T14 V含有3.2 × 109个细胞：比单一物种生物膜多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，生物膜中的细胞密度为细胞数/mL）。在1-h和48-h S之间观察到浓度比降低10倍以上。oralis34-A. Naeslundii T14 V生物膜表明，AI-2的峰值产生发生在群落发育的早期，随后是非常低的稳态水平。特定浓度的AI-2似乎是必不可少的口腔黏膜生物膜社区的启动。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。