The interaction between Clostridium difficile, the intestinal microbiome and disease outcome

艰难梭菌、肠道微生物组和疾病结果之间的相互作用

• 批准号: 1923156
• 金额: --
• 依托单位: London School of Hygiene & Tropical Medicine
• 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-1923156
• 关键词: interaction; between; Clostridium; difficile; intestinal

The purpose of this project is to investigate interactions between Clostridium difficile and the gut microbiome. C. difficile infection (CDI) occurs when the gut microbiome of patients is disturbed, usually by broad spectrum antibiotics, resulting in species that usually out-compete C. difficile and prevent infection not being present. Research has shown C. difficile produces high levels of an antibacterial compound; para-cresol. It is thought this contributes to CDI relapse by maintenance of dysbiosis of the gut microbiome. P-cresol is produced by degradation of tyrosine via the intermediate para-hydroxyphenylacetate (pHPA). The pathway by which pHPA is produced from tyrosine is not known however it has been demonstrated p-cresol is produced from pHPA via the actions of pHPA decarboxylase encoded by the hpdBCA operon.The first aim of this project is to investigate the triggers of p-cresol production in C. difficile through measurement of transcription and translation of the hpdBCA operon, making use of a number of gene reporter. I will attempt to elucidate the pathway by which tyrosine is degraded to pHPA using nuclear magnetic resonance (NMR) and RNA sequencing to produce metabolite and transcriptome profiles respectively.The second aim of the project is to determine levels of p-cresol in vivo and what effects p-cresol has on the microbiome. I will use in vitro and in vivo methods making use of techniques such as NMR to determine metabolite profiles and 16S rRNA sequencing to investigate microbial diversity. These techniques will be applied to samples from murine models of infection and human faecal and urine samples from both healthy controls and patients with infection including time course samples from sufferers of relapse and from patients who died of infection. Through comparison of metabolite profiles between healthy and infected patients, and using the disease severity scores from health records associated with these samples it may be possible to use metabolite profiles as a diagnostic for infection and disease severity.The third aim is to investigate the effect of p-cresol on the microbiome using in vitro and in vivo techniques. I will compare the effects of wild type C. difficile strains alongside mutants unable to produce p-cresol. I will use culture techniques to determine the effect p-cresol has on individual bacterial species before using samples from a murine model, healthy human samples and an in vitro "gut soup" model of human infection to determine p-cresol's wider effect on microbial diversity. To investigate microbial diversity I will employ 16S rRNA sequencing and culture techniques.The final aim is to determine the mechanism by which p-cresol inhibits bacterial growth. Using an assay for quantification of inorganic phosphate release used as a marker of cell membrane integrity I will determine the effect of p-cresol has on membrane integrity. I will use fluorescent microscopy techniques to analyse what effect p-cresol has on membrane integrity, this technique allows monitoring of the movement of molecules through the cell membrane.This impact this research could have is by elucidating p-cresol's production pathway and impact on the microbiome, it may be possible to identify novel therapeutic targets which it is hoped could be targeted and reduce infection relapse as a result.This work fits in with all three of the MRC's skill priorities. For quantitative skills, I will be developing skills in bioinformatics and statistics and applying them to transcriptomics, metabolomics and 16S rRNA sequencing. I will gain skills in interdisciplinary research by using health records combined with data generated in this project. Finally, I will be working with samples from in vivo murine models providing me with skills in these methods.

该项目的目的是研究艰难梭菌和肠道微生物组之间的相互作用。C.艰难梭菌感染（CDI）发生在患者的肠道微生物组受到干扰时，通常是由广谱抗生素引起的，导致通常竞争C.艰难，并防止感染不存在。研究表明，C。艰难梭菌产生高水平的抗菌化合物;对甲酚。据认为，这通过维持肠道微生物组的生态失调而导致CDI复发。对甲酚是通过中间体对羟基苯乙酸酯（pHPA）降解酪氨酸而产生的。pHPA由酪氨酸生成的途径尚不清楚，但已证实pHPA通过hpdBCA操纵子编码的pHPA脱羧酶的作用生成对甲酚。difficile通过测量hpdBCA操纵子的转录和翻译，利用许多基因报告基因。我将尝试阐明酪氨酸降解为pHPA的途径，使用核磁共振（NMR）和RNA测序分别产生代谢产物和转录组profiles.The项目的第二个目的是确定体内对甲酚的水平和对甲酚对微生物组的影响。我将使用体外和体内方法，利用NMR等技术确定代谢产物谱和16 S rRNA测序来研究微生物多样性。这些技术将应用于来自感染的鼠模型的样品和来自健康对照和感染患者的人粪便和尿液样品，包括来自复发患者和死于感染的患者的时间过程样品。通过比较健康和感染患者之间的代谢产物谱，并使用与这些样本相关的健康记录中的疾病严重程度评分，可以使用代谢产物谱作为感染和疾病严重程度的诊断。第三个目的是使用体外和体内技术研究对甲酚对微生物组的影响。我将比较野生型C的效果。艰难菌株以及不能产生对甲酚的突变体。我将使用培养技术来确定对甲酚对单个细菌物种的影响，然后使用来自小鼠模型，健康人体样本和体外“肠道汤”人类感染模型的样本来确定对甲酚对微生物多样性的更广泛影响。为了研究微生物多样性，我将采用16 S rRNA测序和培养技术，最终目的是确定对甲酚抑制细菌生长的机制。使用定量无机磷酸盐释放的试验（用作细胞膜完整性的标志物），我将确定对甲酚对膜完整性的影响。我将使用荧光显微镜技术来分析对甲酚对细胞膜完整性的影响，这项技术可以监测分子通过细胞膜的运动。这项研究可能产生的影响是通过阐明对甲酚的生产途径和对微生物组的影响，有可能确定新的治疗靶点，希望能够靶向这些靶点，从而减少感染复发。MRC的所有三个技能优先级。对于定量技能，我将培养生物信息学和统计学方面的技能，并将其应用于转录组学，代谢组学和16 S rRNA测序。我将通过使用健康记录与本项目中生成的数据相结合，获得跨学科研究的技能。最后，我将使用来自体内小鼠模型的样本，为我提供这些方法的技能。