Understanding the dynamic interactions between Salmonella and the intestinal microbiota

了解沙门氏菌和肠道微生物群之间的动态相互作用

• 批准号: 1959827
• 金额: --
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1959827
• 关键词: Understanding; dynamic; interactions; between; Salmonella

Background: Salmonella is a dangerous bacterium that causes unacceptable levels of food poisoning, and more hospital admissions in the UK than any other food-borne pathogen (about 2,500 each year). This project addresses the BBSRC Strategic research priority 1 "Agriculture and food security".Hypothesis: The competitive interactions between Salmonella and the intestinal microbiota involve intra-bacterial cell-to-cell contact and chemical signaling.Research Plan: The foodborne pathogen Salmonella faces fierce competition from the resident intestinal microbiota. However Salmonella Typhimurium (Salmonella) has developed strategies enabling them to undergo a rapid growth-burst resulting in disease. The studentship will characterize the Salmonella-microbiota interactions, identifying molecular mechanisms mediating growth-bursts and pathogenesis. We will focus on Bacteroides initially as a representative component of the intestinal microbiota. 1. The in vitro growth conditions will be refined to mimic the gastro-intestinal environment including varying media components. We will use mono- and co-cultures of Salmonella with Bacteroides, and observe any changes to growth kinetics during co-culture. 2. Salmonella expresses three distinct secretion systems required for virulence: SPI-1 T3SS, SPI-2 T3SS, and SPI-6 T6SS. Using a panel of mutants, reporter strains, and antibodies, we will examine the importance of the three secretion systems to direct cell-to-cell interactions using mono- and co-culture growth kinetics, and examining changes in secreted protein profiles. 3. Important signalling molecules and/or metabolites generated during co-culture of Bacteroides and Salmonella will be identified. Using a variety of bioassays/reporters we will screen culture supernatants for known bacterial cell-cell signalling molecules, and for effects on the expression of a panel of key Salmonella virulence genes. LC MS/MS-based metabolomics will be used to identify differences in the extracellular small molecule/metabolite profiles of supernatants (Paul Williams, Nottingham). Putative signal molecules will be purified allowing examination of their activity in growth stimulation/reporter bioassays. 4. We will globally identify genes regulated during co-culture of Bacteroides-Salmonella using deep RNA sequencing (Jay Hinton, Liverpool). These transcriptional profiles will generate a set of candidate genes regulated by direct interactions between Salmonella and Bacteroides. These will form the basis of testable hypotheses to investigate further using gene knockout mutants and phenotypic screens.5. Depending on progress, we will investigate in vivo colonization dynamics using mono-associated gnotobiotic mice colonized by Bacteroides, and orally challenged with wild-type Salmonella and selected gene knock-out mutants (Lindsay Hall, Quadram Institute, Norwich). Bacteria will be enumerated in the intestine, mesenteric lymph nodes, and organs using standard methods.

背景资料：沙门氏菌是一种危险的细菌，会导致不可接受的食物中毒，在英国住院人数比任何其他食源性病原体都多（每年约2，500人）。本研究课题为BBSRC战略研究重点1“农业与食品安全”。假设：沙门氏菌与肠道菌群之间的竞争性相互作用涉及细菌内细胞间的接触和化学信号传导。研究计划：食源性病原体沙门氏菌面临来自肠道菌群的激烈竞争。然而，鼠伤寒沙门氏菌（沙门氏菌）已经开发出策略，使他们能够经历一个快速的生长爆发，导致疾病。该研究将表征沙门氏菌-微生物群相互作用，确定介导生长爆发和发病机制的分子机制。我们首先将关注拟杆菌作为肠道微生物群的代表性组成部分。1.将优化体外生长条件，以模拟胃肠道环境，包括不同的培养基组分。我们将使用沙门氏菌与拟杆菌的单培养物和共培养物，并观察共培养期间生长动力学的任何变化。2.沙门氏菌表达毒力所需的三种不同分泌系统：SPI-1 T3 SS、SPI-2 T3 SS和SPI-6 T6 SS。使用一组突变体，报告菌株和抗体，我们将研究的重要性，三个分泌系统直接细胞间的相互作用，使用单和共培养生长动力学，并检查分泌蛋白质的变化。3.将鉴定拟杆菌和沙门氏菌共培养期间产生的重要信号分子和/或代谢物。使用各种生物测定/报告，我们将筛选已知的细菌细胞-细胞信号分子的培养上清液，并对一组关键沙门氏菌毒力基因的表达的影响。将使用基于LC MS/MS的代谢组学鉴别上清液的细胞外小分子/代谢物谱的差异（Paul威廉姆斯，诺丁汉）。将纯化推定的信号分子，以检查其在生长刺激/报告基因生物测定中的活性。4.我们将使用深度RNA测序（Jay欣顿，利物浦）全面鉴定类杆菌-沙门氏菌共培养期间调控的基因。这些转录谱将产生一组由沙门氏菌和拟杆菌之间的直接相互作用调节的候选基因。这些将构成可测试假设的基础，以使用基因敲除突变体和表型筛选进行进一步研究。5.根据进展情况，我们将使用类杆菌定殖的单一相关的无菌小鼠研究体内定殖动力学，并用野生型沙门氏菌和选定的基因敲除突变体进行口服攻击（林赛霍尔，Quadram研究所，诺维奇）。将使用标准方法对肠道、肠系膜淋巴结和器官中的细菌进行计数。