Functional Analysis of a Digestive-Tract Microbiome During Dietary Changes

描述（由申请人提供）：在人类中，消化道微生物群是由数百种物种组成的复杂群落。这种微生物组为人类提供了一系列惊人的生化过程，远远超过了动物基因组编码的生化过程。随着动物成熟或改变饮食，微生物组的组成可能会发生变化。这种变化如何影响微生物组的整体生理及其对宿主的贡献尚不清楚。从人类微生物组项目获得的信息产生了丰富的基因组信息，并提供了对可能功能的深入了解，但缺乏来自具有天然微生物组的动物的功能数据。微生物组的复杂性阻碍了识别微生物组表达的基因，即元转录组。本申请研究了一种天然存在的简单微生物组，该微生物组存在于药用水蛭的消化道中。两种细菌在微生物群落中占主导地位，其中一种是可用的遗传工具。在水蛭从茧中孵化出来后的前两周内，以及它们食用血餐后，微生物组的组成发生了变化。如果血液被部分溶解，则作为微生物组的次要成员的梭菌物种变得非常丰富。大规模平行测序将用于监测随着动物成熟和改变饮食，微生物组的组成和元转录组的变化。这将分别使用qPCR和qRT-PCR进行确认。这种分析允许人们确定在特定条件下特定等位基因或菌株是否更丰富。使用gfp融合物，将确定表达基因的细胞的确切位置。环境参数，如电子受体，糖和短链脂肪酸将被测量。因为细菌存在于浮游期和小菌落期，所以可以通过使用FACS分选的细胞的Illumina测序来确定比较小生境特异性基因表达。这项工作将使用适用于更复杂的人类系统的简单系统来确定新的原则，并应用随着测序通量和计算能力的增加而变得更可行的技术来用于高度复杂的系统。

DESCRIPTION (provided by applicant): In humans, the digestive-tract microbiota is a complex community composed out of hundreds of species. This microbiome provides humans with an amazing repertoire of biochemical processes that far exceed those encoded by the animal's genome. As an animal matures or switches diets the microbiome can change in composition. How this change affects the overall physiology of the microbiome and its contribution to the host is poorly understood. The information gained from the Human Microbiome Project has produced a wealth of genome information and is providing insight into possible functions, but functional data from animals with their native microbiome are lacking. The sheer complexity of the microbiome has hindered identifying the genes that the microbiome expresses, the metatranscriptome. This application investigates a naturally occurring simple microbiome that resides in the digestive tract of the medicinal leech. Two bacteria dominate the microbial community and for one genetic tools are available. The composition of the microbiome changes during the first two weeks after the leeches hatch from the cocoon and also after they consume a blood meal. If the blood was partially lysed, a Clostridium species that is minor member of the microbiome became very abundant. Massively parallel sequencing will be used to monitor changes in the composition and in the metatranscriptome of the microbiome as the animals mature and change diet. This will be confirmed using qPCR and qRT-PCR, respectively. This analysis allows one to determine if under specific conditions a particular allele or strain is more abundant. Using gfp-fusions, the exact location of the cells expressing the genes will be determined. Environmental parameter such as electron acceptors, sugars and short chained fatty acids will be measured. Because the bacteria are present both in pelagic and microcolony phases, compare niche specific gene expression can be determined by using Illumina sequencing of FACS sorted cells. This work will determine new principles using a simple system that are applicable to the more complex human system and apply technologies that will become more feasible to use in highly complex systems as the sequencing throughput and computational power increase.