Role of carbon availability in environmental adaptation by Borrelia burgdorferi

碳有效性在伯氏疏螺旋体环境适应中的作用

• 批准号: 8829534
• 负责人: Linden T Hu
• 金额: $ 21.65万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8829534
• 关键词: Acetylglucosamine; Affect; Animal Model; Arabinose; Bacteria; Biological Assay; Borrelia burgdorferi; Carbohydrates; Carbon; Chitin; Collaborations; Complement; Complex; DNA; Data; Data Analyses; Development; Disadvantaged; Environment; Frequencies; Fructose; Galactose; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Determinism; Genetic Programming; Genetic screening method; Genome; Glucose; Glycerol; Glycogen; Goals; Growth; Homologous Gene; Human; In Vitro; Individual; Infection; Laboratories; Libraries; Life; Lyme Disease; Maltose; Mammals; Mannose; Massive Parallel Sequencing; Metabolic Pathway; Metabolism; Methods; Microbial Biofilms; Monosaccharides; Mus; Mutagenesis; Mutation; Nutrient; Operon; Organism; Pathogenesis; Pathway interactions; Phenotype; Play; Polysaccharides; Positioning Attribute; Production; RNA-Binding Proteins; Recording of previous events; Relative (related person); Repression; Research Personnel; Ribose; Role; Running; Signal Transduction; Source; Speed; System; Techniques; Technology; Testing; Ticks; Toxin; Trehalose; Virulence; Virulence Factors; Xylose; base; chitobiose; detection of nutrient; environmental adaptation; environmental change; exoskeleton; fitness; genetic manipulation; high throughput screening; high throughput technology; in vivo; in vivo Model; mutant; pathogen; pressure; protein expression; public health relevance; research study; screening; sugar; tool; uptake

 DESCRIPTION (provided by applicant): Borrelia burgdorferi, the causative agent of Lyme disease, is a tick-borne bacterial pathogen that must adapt to varying environments in its tick and mammalian hosts. B. burgdorferi has been shown to significantly alter protein expression to adapt to environmental changes, but factors that trigger these changes and the mechanisms behind them are still not well understood. The availability of carbon sources for use by the bacteria differ substantially in mammals, where glucose is the primary carbon source available, and in ticks, where glycogen, trehalose, and chitobiose may be more readily available. For other bacteria, changes in the carbon source being utilized is important in regulating expression of genes involved in carbon metabolism, as well as virulence factors, toxins and other factors important in adapting to specific environments. Whether B. burgdorferi utilizes carbon source availability to adapt to environments is not known; however several B. burgdorferi genes related to carbon utilization have been shown to be critical for survival in specific hosts. Studies to understand the genetic determinants of environmental adaptation by B. burgdorferi have been slowed by a paucity of genetic tools-particularly those amenable to high-throughput screening. Massively parallel sequencing is a rapidly developing technology for the study of bacterial pathogenesis. Dr. Andrew Camilli, a co-investigator on this proposal, has paired massively parallel sequencing with transposon mutagenesis in a strategy called Tn-seq. Tn-seq involves screening of a library against a selective pressure and then sequencing the flanking regions to the transposon en masse, to identify the relative frequency of the mutants before and after selection. Dr. Tao Lin's laboratory has developed the first transposon mutant library in B. burgdorferi. In our preliminary studies, we have successfully employed Tn-seq on B. burgdorferi transposon mutants grown in vitro and in vivo in mice and shown that it is a sensitive and reproducible technique. The screening of subsets of the library in mice revealed that several of the genes likely to be involved in carbon utilization show either increased or decreased fitness for survival in mice. In this proposal, we examine the role of carbon utilization by B. burgdorfer. We will screen our transposon library by Tn-seq after growth in the presence or absence of specific sugars. Based on analysis of this data, which we expect will identify genes involved in uptake and metabolism of specific carbon sources as well as global regulators involved in controlling genetic programs for environmental adaptation, we will select a subset of 5 genes for testing by creation of targeted deletion mutants and complemented mutants. These strains will be tested for survivial in ticks and mice and in competition assays. The development of Tn-seq for application to studies of B. burgdorferi has the potential to greatly accelerate our understanding of the pathogenesis of this organism and the mechanisms by which it adapts to its hosts.

 描述（由申请方提供）：莱姆病病原体伯氏疏螺旋体是一种蜱传细菌病原体，必须适应其蜱和哺乳动物宿主的不同环境。B。已显示伯氏菌显著改变蛋白质表达以适应环境变化，但触发这些变化的因素及其背后的机制仍不清楚。供细菌使用的碳源的可用性在哺乳动物中显著不同，其中葡萄糖是可用的主要碳源，而在蜱中，糖原、海藻糖和壳二糖可能更容易获得。对于其他细菌，所利用的碳源的变化在调节参与碳代谢的基因的表达以及毒力因子、毒素和其他在适应特定环境中重要的因子方面是重要的。是否B. burgdorferi利用碳源的可用性来适应环境是未知的;然而，几个B.与碳利用相关的伯氏螺旋体基因已被证明对于在特定宿主中的存活至关重要。 B研究了解环境适应的遗传决定因素。由于缺乏遗传工具，特别是那些适于高通量筛选的遗传工具，使得Burgdorferi的研究进展缓慢。大规模平行测序是一种快速发展的细菌致病机制研究技术。Andrew Camilli博士是这项提议的共同研究者，他将大规模平行测序与转座子诱变配对，采用了一种名为Tn-seq的策略。Tn-seq涉及针对选择压力筛选文库，然后对转座子基因组的侧翼区域进行测序，以确定选择前后突变体的相对频率。林涛博士的实验室开发出了B中的第一个转座子突变库。burgdorferi。在我们的初步研究中，我们已经成功地将Tn-seq应用于B。Burgdorferi转座子突变体在小鼠体内和体外生长，并显示它是一种灵敏和可重复的技术。在小鼠中筛选文库的子集显示，可能参与碳利用的几个基因显示出小鼠生存适应性的增加或减少。 在这个提议中，我们研究了B的碳利用的作用。burgdorfer。我们将在存在或不存在特定糖的情况下生长后通过Tn-seq筛选我们的转座子文库。基于对这些数据的分析，我们预计将确定参与特定碳源吸收和代谢的基因以及参与控制环境适应遗传程序的全球监管机构，我们将选择5个基因的子集进行测试，通过创建靶向缺失突变体和互补突变体。将在蜱和小鼠中以及在竞争试验中检测这些菌株的存活率。Tn-seq技术在B. Burgdorferi有可能大大加速我们对这种生物的发病机制及其适应宿主的机制的理解。