Cell-cell Interaction--Oral Actinomyces &Other Bacteria

Dental &Craniofacial Research

Investigators

Linked publications & trials

Abstract

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.