Fitness profiling of Streptococcus gordonii in oral microenvironments

戈登链球菌是唾液覆盖的牙齿表面生物膜的初始定殖者。 这些先锋定殖者及其胞外产物形成了含有宿主口腔成分的底物 随后的定植细菌可以附着在这些细菌上。因此，戈登氏链霉菌在 通过宿主和细菌粘附素受体等机制建立成熟的口腔生物膜 代谢和细胞间信号的相互作用。建立和维护健康的口腔 微生物组需要这些生态相互作用的良好平衡。的工作假说 建议的研究是鉴定戈登氏链霉菌对物种生存和增殖至关重要的基因 在协同或对抗条件下，将提供对有利于 或不利于口腔健康的状态。这个探索性项目的目标是使用全基因组的方法来 在选定的体外模型条件下鉴定和检测与戈登氏链霉菌适合度相关的基因 那些在口腔里的。我们提出了一种最先进的TN-Seq方法来构建一个饱和的文库 一种广泛使用的口腔共生模型--转座子基因组在戈登链球菌Challis株中的插入 链球菌。文库的有用性将通过在测试条件下的增长来确认，该条件将允许 鉴定插入了对生长和健康至关重要的基因的突变体。突变体具有 在这一过程中，赋予适合性的基因的破坏将减少种群中的频率 实验；相反，文库中插入非必需基因的突变将保持在 频率相等或增加的。从整个种群中恢复的DNA将在#年末收集 实验并通过高通量的染色体DNA测序与输入文库进行比较 位于集成转座子的两侧。中断，因此，较少或无功能的基因将被识别 转座子侧翼核苷酸序列与参考戈登链霉菌基因组序列的比较。 每个突变的恢复频率将被用来计算每个基因在 实验条件。将研究的条件包括生物膜形成和浮游条件。 在存在全人类唾液、血清和双物种培养的情况下对定植很重要 口腔放线菌和纤毛因子，代表已被证明促进或 分别抑制戈登葡萄球菌在生物膜中的定植。被确定为正在发挥作用的选定基因的作用 通过等位基因交换构建特定的突变株，将进一步检验在适应性方面的潜在作用。 以及它们的补体，以验证它们在戈登葡萄球菌生物被膜过程中的生存和增殖中的作用 发展。这些研究将为未来细菌的相互作用和生存提供重要的基础 学习。了解有利于共生口腔微生物区系的基因的作用将提供必要的 对预防可能导致口腔疾病的微生物失调的见解。

Streptococcus gordonii is an initial colonizer of the biofilm that forms on the saliva-coated tooth surface. These pioneer colonizers and their extracellular products form a substratum with host oral components to which subsequent colonizing bacteria can attach. Accordingly, S. gordonii plays a pivotal role in the establishment of the mature oral biofilm through mechanisms such as host and bacterial adhesin-receptor, metabolic, and intercellular signaling interactions. The establishment and maintenance of a healthy oral microbiome requires a favorable balance of these ecological interactions. The working hypothesis of the proposed studies is that identification of S. gordonii genes essential for species survival and proliferation under synergistic or antagonistic conditions will provide essential insights into community dynamics that favor or disfavor a state of oral health. The goal of this exploratory project is to use a genome-wide approach to identify and examine genes involved in S. gordonii fitness under selected in vitro model conditions relevant to those in the oral cavity. We propose a state-of-the-art, Tn-Seq approach to construct a saturated library of transposon genome insertions in S. gordonii strain Challis, a widely-used model of commensal oral streptococci. The usefulness of the library will be confirmed by growth under test conditions that will allow identification of mutants with insertions in genes that are essential for growth and fitness. Mutants with disruptions of genes conferring fitness will decrease in frequency in the population during the course of the experiment; in contrast, mutants in the library with insertions in nonessential genes will be maintained at an equal or increased frequency. Recovered DNA from the entire population will be collected at the end of experiment and compared to the input library via a high throughput sequencing of the chromosomal DNA flanking the integrated transposon. Interrupted and hence, less or non- functional genes will be identified by comparison of the transposon-flanking nucleotide sequence to the reference S. gordonii genome sequence. The frequency of recovery of each mutant will be used to calculate the fitness of each gene under the experimental conditions. Conditions to be studied will include biofilm formation and planktonic conditions important for colonization in presence of whole human saliva, serum, and dual species cultures with Actinomyces oris and Filifactor alocis, representing bacterial species that have been shown to promote or impede, respectively, S. gordonii colonization in biofilms. The roles of selected genes identified as playing potential roles in fitness will be further examined by construction of specific mutant strains by allelic exchange and their complements to verify their roles in the survival and proliferation of S. gordonii during biofilm development. These studies will provide an important foundation for future bacterial interaction and survival studies. Understanding the roles of genes that favor the commensal oral microbiota will provide essential insights into preventing the microbial dysbiosis that can lead to oral disease.