Probing oral pathogen interactions in picoliter-scale enclosures

描述(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的表达，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，这一过程被称为群体感应(QS)。基本的QS模型指出，细菌使用信号分子来“计算”它们的细胞数量，并根据种群密度来调节群体行为。这些法定信号在人类口腔内的浓度可能受到多种环境因素的影响，如人口大小和唾液或沟液通过牙菌斑的流量，然而，目前还不完全清楚哪些因素调节QS。这项建议旨在表征影响QS的参数，并研究三种口腔细菌：戈登链球菌、伴生放线杆菌(AA)和变形链球菌的高密度、小种群之间的相互作用。戈登链霉菌是一种共生性和机会性的病原体，与引起局部侵袭性牙周炎的致病菌AA和引起人类龋齿的变形链球菌相邻。目前的微生物学研究几乎完全是在比通常在人类口腔中发生的规模大得多的范围内研究微生物的交流和相互作用。我们建议改变探索微生物相互作用的方法，使用我们开发的一种新的方法来研究较小的种群数量(d104细胞)。利用这项技术，可以在皮升大小的由交联蛋白质制成的陷阱中捕获单个细菌。在这些多孔性的细菌“龙虾陷阱”内，细菌迅速生长到所需的细胞数量，这些细胞被包围在用户定义的三维几何图形中。这项建议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菌相互作用。我们计划在陷阱内的小群落中检测两种先前描述的多微生物相互作用：戈登链球菌产生H_2O_2介导对宿主天然免疫的AA抵抗；戈登链球菌产生蛋白酶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/死染色在皮升大小的社区中探索这些相互作用。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之间的交流和多物种相互作用的了解，并通过引入一种新的、有用的技术来研究与人类口腔感染和疾病相关的种群大小，进一步推动口腔微生物学领域的发展。

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.