Cell-cell Interactions Between Oral Actinomyces And Other Bacteria

人类口腔生物膜群落发展的核心是微生物之间的相互作用，这种相互作用推动了细菌群落的空间排列。牙釉质上的这种群落形成了牙周上的牙菌斑。这些亲密的相互作用是由称为共聚体的物理相互作用促进的，共聚体是遗传上不同的伙伴细胞之间的特定黏附，相互结合形成多细胞网络，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最初的牙菌斑形成。在本报告期间，我们使用DNA微阵列来鉴定戈登链球菌与奈斯伦迪放线菌共聚集时受调控的基因。23个基因的表达在共聚体中变化了3倍，其中包括9个与精氨酸生物合成和运输有关的基因。用一种化学定义的生长介质评估了戈登链霉菌合成精氨酸的能力。在单一培养条件下，链球菌精氨酸生物合成效率低下，在低精氨酸条件下不能有氧生长。然而，在含有共聚体的双物种培养中，戈登葡萄球菌在低精氨酸条件下生长到高细胞密度。无共聚体的等量共培养细胞在培养9h后才有明显的共聚体生长。一个argh突变体不能在低精氨酸中生长，无论有没有奈氏链球菌，这表明精氨酸的生物合成对于共聚集诱导的链球菌的生长是必不可少的。定量RT-PCR结果显示，在外源精氨酸耗尽的情况下，生长3h的戈登螺旋藻ARGC、ARGG和PYRAb的表达显著上调(10~100倍)。没有诱导共聚集的共培养显示出类似的调节。然而，在与奈氏拟青霉共聚集后的1h内，虽然含有丰富的精氨酸，但高登螺旋藻的ARGC、ARGG和PYRAb的表达却部分上调，而当精氨酸减少时，其表达水平不再进一步增加。因此，内氏假单胞菌稳定了戈登氏链霉菌精氨酸生物合成基因在共聚体中的表达，并在外源精氨酸受到限制的情况下实现了有氧生长。细菌之间的代谢合作对初始口腔生物膜群落的重复和独特的群落组成可能是重要的，通过这种合作可以建立食物网。在我的实验室里，混合物种共聚体之间的交流机制是一个非常有趣的话题。 牙齿表面的初始定殖者是口腔微生物区系中的一个特殊子集。在这些细菌中，那些独立于其他细菌在清洁的牙釉质表面定居的细菌具有附着在覆盖在牙釉质上的后天唾液膜上的机制，并具有将唾液成分代谢为唯一营养来源的能力。我们使用流动装置和索巴德过滤器(用纸包裹的紧密包装的纤维：圆柱体直径10 mm，长度20 mm)和唾液来模拟体内环境。我们获得了高细胞密度，并评估了链球菌-放线菌群落产生通用信号分子自动诱导器-2(AI-2)的能力。口腔共生菌口腔链球菌34和奈氏放线菌T14V分别作为单菌种和双菌种生物膜进行培养。48h后，交叉型口腔沙门氏菌和内氏锥虫T14V双种生物膜群落的细胞数为3.2×109个，是单种生物膜的5倍。然而，这些48h的双物种生物膜表现出最低的AI-2浓度比(AI-2浓度以纳米分子/生物膜中的L为单位，细胞密度以生物膜中的细胞数/毫升为单位)。口腔沙门氏菌34-A在1小时和48小时之间的浓度比下降了10倍以上。Nesludii T14V生物膜表明，AI-2的生产高峰出现在社区发展的早期，随后是一个非常低的稳定水平。特定浓度的AI-2似乎对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。

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.