Probing oral pathogen interactions in picoliter-scale enclosures

描述（由申请人提供）：在人类口腔内，细菌细胞间的通讯和复杂的多物种相互作用调节微生物基因表达，影响口腔生物膜的生态平衡和人类健康。许多细菌使用小信号分子进行交流，这一过程称为群体感应（QS）。基本的 QS 模型指出，细菌使用信号分子“计算”其细胞数量，并根据种群密度调节群体行为。人口腔内这些群体信号的浓度可能受到多种环境因素的影响，例如人口规模和唾液或牙菌斑间隙液的流量，但目前尚不完全了解哪些因素调节 QS。该提案旨在表征影响 QS 的参数，并检查三种口腔细菌的高密度、小群体中的相互作用：戈登链球菌、伴放线聚集菌 (Aa) 和变形链球菌。戈氏链球菌是一种共生的机会性病原体，定植于 Aa（局部侵袭性牙周炎的病原体）和变形链球菌（人类龋齿的主要病原体）附近。当前的微生物学研究几乎专门研究比人类口腔中正常情况大得多的规模的微生物通讯和相互作用。我们建议使用我们为研究较小群体规模（d104 细胞）而开发的新方法来改变探测微生物相互作用的方法。通过这项技术，可以在由交联蛋白制成的皮升大小的陷阱中捕获单个细菌。在这些多孔细菌“龙虾陷阱”内，细菌快速生长到所需的细胞数量，并封闭在用户定义的三维几何结构中。该提案的目标是确定大种群的表型是否与小种群相关，并确定物种的空间分布如何影响多微生物相互作用。我们计划检查陷阱内小群落中两种先前表征的多微生物相互作用：S. gordonii H2O2 产生介导对宿主先天免疫的 Aa 抗性；戈氏链球菌蛋白酶的产生抑制变形链球菌中细菌素的表达。我们将使用 GFP 转录报告菌株和荧光活/死染色剂来探索皮升大小的群落中的这些相互作用。该项目的目标与国家牙科和颅面研究所的使命一致：该提案旨在提高对口腔病原体的传播和多物种相互作用的理解，此外还将通过引入一种新颖、有用的技术来研究与人类口腔感染和疾病相关的种群规模，从而推动口腔微生物学领域的发展。 公共卫生相关性：当前的微生物学研究几乎专门检查比人类口腔中正常情况大得多的细菌。我们建议改变探测微生物相互作用的方法，使用一种监测 1 到 10,000 个细菌种群大小的技术。借助这项先进技术，我们将研究与疾病相关的细菌细胞间通讯以及与人类口腔相关的种群规模的多物种相互作用。

DESCRIPTION (provided by applicant): Within the human mouth, bacterial cell-cell communication and complex multispecies interactions regulate microbial gene expression, affecting the ecological balance of oral biofilms and human health. Many bacteria communicate using small signaling molecules, a process termed quorum sensing (QS). The basic QS model states that bacteria 'count' their cell numbers using signaling molecules, and modulate group behavior based upon population density. The concentration of these quorum signals within the human mouth is likely affected by multiple environmental factors, such as population size and flow of saliva or crevicular fluid across dental plaque, however it is not yet fully understood which factors regulate QS. This proposal aims to characterize the parameters that affect QS, and examine interactions in highly dense, small populations of three species of oral bacteria: Streptococcus gordonii, Aggregatibacter actinomycetemcomitans (Aa), and Streptococcus mutans. S. gordonii is a commensal and opportunistic pathogen, and colonizes adjacent to both Aa, the etiologic agent of localized aggressive periodontitis, and S. mutans, the primary causative agent of dental caries in humans. Current microbiology studies almost exclusively examine microbial communication and interactions on scales much larger than normally occur in the human mouth. We propose a change in the approach for probing microbial interactions using a novel methodology we developed to study smaller population sizes (d104 cells). With this technology, a single bacterium can be captured within a picoliter-sized trap made of cross-linked protein. Within these porous bacterial "lobster traps," bacteria grow rapidly to a desired cell number enclosed in a three-dimensional user-defined geometry. The goal of this proposal is to determine whether phenotypes of large populations are relevant in small populations, and to determine how spatial distribution of species affects polymicrobial interactions. We plan to examine two previously characterized polymicrobial interactions in small communities within the traps: S. gordonii H2O2 production mediating Aa resistance to host innate immunity; and S. gordonii protease production inhibiting bacteriocin expression in S. mutans. We will probe for these interactions in picoliter-sized communities using GFP transcriptional reporters strains, and fluorescent live/dead stains. The goals of this project are in line with the mission of the National Institute of Dental and Craniofacial Research: the proposal aims to improve the understanding of communication and multispecies interactions of oral pathogens, and furthermore will advance the field of oral microbiology by introducing a novel, useful technique for studying population sizes relevant to infections and disease in the human mouth. PUBLIC HEALTH RELEVANCE: Current microbiology studies almost exclusively examine bacteria on scales much larger than normally occur in the human mouth. We propose a change in the approach for probing microbial interactions using a technique that monitors population sizes from 1 to 10,000 bacteria. With this advanced technology, we will study disease related bacterial cell-cell communication and multispecies interactions in population sizes relevant to those in the human oral cavity.