Cell-cell Interaction--Oral Actinomyces & Other Bacteria

在人类口腔生物膜群落的发展过程中，微生物的相互作用被认为驱动了细菌群落的空间排列。这些菌落在牙釉质上形成牙菌斑。在本报告期间，我们研究了扩散信号分子在微生物群落发展中的作用。gordonii链球菌和非典型微孔菌是牙菌斑生物膜的两种早期定植成员，它们被认为参与了代谢通讯；gordonii发酵碳水化合物形成乳酸，乳酸是V.异型菌的首选发酵底物。α -淀粉酶是一种s.o donii酶，参与细胞内储存的糖原加工成可发酵的碳水化合物。我们发现，在琼脂板共培养这些生物的过程中，发生了一个信号事件，导致S. gordonii α -淀淀酶编码基因amyB的表达增加。以人唾液为唯一营养物的共聚焦扫描激光显微镜（在三维空间上收集荧光细胞的图像信息）显示，V. atypica引起S. gordonii增加PamyB-？gfp （amyB启动子引导编码绿色荧光蛋白的基因表达）转录融合在一个空间分辨的方式。这种转录融合报告系统通过产生绿色荧光蛋白（GFP）来响应适当的信号，在共聚焦激光显微镜下可以检测到。在该开放体系中，只有含有异型弧菌的混合种微菌落链球菌表达绿色荧光蛋白；邻近的godonii菌落缺乏异型弧菌不表达GFP。在含有gordonii和V. atypica的封闭系统（如生长培养基的培养瓶）中，流式细胞术分析（绘制细胞群中每个细胞的荧光水平并绘制细胞的数值分布）显示含有PamyB-？gfp报告质粒的平均荧光水平比未与异型弧菌孵育的戈登弧菌高20倍。因此，在一个封闭的系统中，一个可扩散的信号可以积累超过所需的阈值，种间信号介导基因表达的变化。我们提供的证据表明，在开放系统中，如在天然口腔生物膜中占主导地位的开放系统中，物种之间的扩散信号被设计为在短距离内发挥作用，大约为一微米。这些数据支持我们的假设，即牙菌斑群落起源于不同物种细胞之间的密切相互作用，而不是基因相同的细胞克隆生长。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（如多物种群落）的特定粘附。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。

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. In this reporting period, we examined the role of diffusible signaling molecules in the development of microbial communities. Streptococcus gordonii and Veillonella atypica, two early colonizing members of the dental plaque biofilm, have been postulated to participate in metabolic communication; S. gordonii ferments carbohydrates to form lactic acid, which is a preferred fermentation substrate for V. atypica. Alpha-amylase is an S. gordonii enzyme involved in the processing of stored intracellular glycogen to make fermentable carbohydrates. We found that, during agar-plate co-culture of these organisms, a signaling event occurs that results in increased expression of the S. gordonii alpha-amylase-encoding gene amyB. Confocal scanning laser microscopy (collects image information on fluorescent cells in three dimensions) of co-culture flowcell-grown biofilms using human saliva as the sole nutrient showed that V. atypica caused S. gordonii to increase expression of a PamyB-?gfp (amyB promoter directing expression of the gene encoding the green fluorescent protein) transcriptional fusion in a spatially resolved fashion. This transcriptional fusion reporter system responds to a proper signal by producing green fluorescent protein (GFP), which is detectable in the confocal laser microscope. In this open system, only those streptococci in mixed-species microcolonies containing V. atypica expressed green fluorescent protein; nearby S. gordonii colonies that lacked V. atypica did not express GFP. In a closed system (such as a culture flask of growth medium) containing S. gordonii and V. atypica, flow cytometric analysis (plots the level of fluorescence of each cell in a population of cells and plots the numerical distribution of the cells) showed that S. gordonii containing the PamyB-?gfp reporter plasmid exhibited mean fluorescence levels 20-fold higher than did S. gordonii that had not been incubated with V. atypica. Thus, in a closed system where a diffusible signal can accumulate above a required threshold, interspecies signaling mediates a change in gene expression. We provided evidence that, in open systems like those that predominate in natural oral biofilms, diffusible signals between species are designed to function over short distances, on the order of one micrometer. These data support our hypothesis that dental plaque communities initiate through intimate interactions between cells of different species and not by clonal growth of genetically identical cells. 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 multispecies communities. The role of diffusible signaling molecules in establishing these early communities remains a topic of much interest in my laboratory. In nature, bacteria exist in multispecies communities. We are continuing our in vivo studies on initial colonizing bacteria of human tooth enamel biofilms. Our hypothesis is that these initial colonizing bacteria are in mixed-species communities. The hypothesis can be investigated by staining these bacteria with fluorescently labeled antibodies that react with bacterial surface molecules. We have been routinely using these fluorophore-conjugated antibodies coupled with confocal laser microscopy (gives multi-focal plane images of communities in situ). This gives the spatial arrangement of specific kinds of cells within the community, and we have been using this procedure in conjunction with our retrievable enamel chip in vivo model. In this reporting period, we initiated our investigation of the feasibility of using quantum dot (QD; also called nanocrystal)-conjugated antibodies to locate early-colonized bacteria on the surface of enamel pieces for the purpose of micromanipulating the entire community (approximately 5 to 10 cells of different species) for study of metabolic mutualism, signaling, regulation of gene expression, and physical interactions. We compared the use of QD-conjugated antibodies and our routinely used fluorophore-conjugated antibodies. QDs have several attractive properties that make them a promising tool for biofilm imaging, however few direct comparisons between QD-antibody conjugates and traditional fluorophore-antibody conjugates have been made. Specifically, we made a direct comparison in which primary immunofluorescence labeling of bacterial cells in biofilms by QD-antibody conjugates is compared with that by Alexa Fluor?-antibody conjugates. Alexa Fluor? conjugates have been used previously and routinely for study of intra- and interspecies interactions in oral biofilms in my laboratory. We used a commercially available QD preparation that was offered as ?ready-for-conjugation? to antibody. We conjugated it to affinity-purified immunoglobulin-G from rabbit serum raised against whole cells of the oral bacterial strain Streptococcus gordonii DL1. The QD-antibody conjugates formed a stable colloidal suspension. First, the ability of the conjugates to stain streptococcal cells from standard planktonic bacterial cultures was assessed using epifluorescence microscopy; under these conditions, QD conjugates displayed identical resolution and better bleach resistance than did Alexa Fluor? conjugates. We next evaluated the conjugates for staining of streptococcal cells grown as biofilms. The biofilms were grown in flowcells using 25% saliva as the sole carbon source. Fluorescence was detected using confocal laser microscopy. Under these conditions, both the QD conjugates and the Alexa Fluor? conjugates yielded high single-cell resolution (bacterial cell surface definition); however a four-fold higher concentration of the QD conjugates compared to Alexa Fluor? conjugates was required. Our results indicated that QD conjugates performed better than did Alexa Fluor? conjugates in certain applications such as prolonged exposure to excitation using epifluorescence. QD-conjugated antibodies do not bleach and, thus, they are essential for locating and micromanipulating a community for further study, a time-consuming procedure. One community has been micromanipulated, and the members of the community grew on saliva-based agar. We are continuing this research program of investigating metabolic mutualism, signaling by diffusible molecules, genetic regulation and coaggregation. 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.