Cell-cell Interactions Between Oral Actinomyces And Other Bacteria

人类口腔生物膜群落发展的核心是驱动细菌群落空间排列的微生物相互作用。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。口腔链球菌和放线菌之间的相互作用主导了最初牙菌斑的发展。在本报告期间，我们使用DNA微阵列鉴定了戈登链球菌与纳氏放线菌共聚集反应的基因。在共聚集体中，23个基因的表达改变了3倍，其中包括9个参与精氨酸生物合成和运输的基因。利用化学定义的生长培养基评估了哥氏酵母合成精氨酸的能力。在单培养条件下，链球菌精氨酸的生物合成效率低下，链球菌在低精氨酸条件下不能有氧生长。然而，在含有共聚集体的双种培养中，在低精氨酸条件下，gordonii生长到高细胞密度。没有共聚集的等效共培养在培养9小时后出现明显的共聚集时才显示出生长。有或没有A. naeslundii的arh突变体都不能在低精氨酸环境中生长，这表明精氨酸的生物合成对于聚集诱导的链球菌生长是必不可少的。利用定量RT-PCR技术发现，当外源精氨酸耗尽后，单培养3 h后，金花酵母中argC、argG和pyrAb的表达显著上调（10 ~ 100倍）。未诱导共聚集的共培养也表现出类似的调控。而与naeslundii共聚集后1 h内，尽管精氨酸含量丰富，但金刺草中argC、argG和pyrAb的表达却部分上调，而精氨酸含量减少时，mRNA水平并未进一步升高。因此，A. naeslundii稳定了S. gordonii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的情况下实现了有氧生长。细菌之间的代谢合作可能对口腔生物膜初始群落的重复和独特的群落组成很重要，通过这种合作可以建立食物网。混合物种共聚集体之间的交流机制是我实验室非常感兴趣的课题。

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.