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

本项目的目的是研究艰难梭菌与肠道微生物群之间的相互作用。艰难梭菌感染（CDI）发生在患者肠道微生物群受到干扰时，通常是被广谱抗生素干扰，导致通常与艰难梭菌竞争并预防感染的物种不存在。研究表明艰难梭菌产生高水平的抗菌化合物；para-cresol。据认为，这有助于CDI复发通过维持肠道微生物群的生态失调。对甲酚是通过中间体对羟基苯基乙酸酯（pHPA）降解酪氨酸而产生的。酪氨酸产生pHPA的途径尚不清楚，但已证明对甲酚是通过hpdBCA操纵子编码的pHPA脱羧酶的作用由pHPA产生的。本项目的第一个目的是利用一些基因报告基因，通过测量hpdBCA操纵子的转录和翻译，研究艰难梭菌对甲酚产生的触发因素。我将尝试通过核磁共振（NMR）和RNA测序分别产生代谢物和转录组图谱来阐明酪氨酸降解为pHPA的途径。该项目的第二个目标是确定体内对甲酚的水平以及对甲酚对微生物组的影响。我将使用体外和体内方法，利用核磁共振等技术来确定代谢物谱和16S rRNA测序来研究微生物多样性。这些技术将应用于感染小鼠模型的样本和健康对照者和感染患者的人类粪便和尿液样本，包括复发患者和死于感染的患者的时间过程样本。通过比较健康和感染患者之间的代谢物谱，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，有可能将代谢物谱用作感染和疾病严重程度的诊断。第三个目的是利用体外和体内技术研究对甲酚对微生物组的影响。我将比较野生型艰难梭菌菌株和不能产生对甲酚的突变菌株的影响。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、健康人类样本和体外人类感染“肠道汤”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16S 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。用一种测定无机磷酸盐释放量的方法作为细胞膜完整性的标志，我将确定对甲酚对细胞膜完整性的影响。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，这种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，希望能够靶向并减少感染复发。这项工作符合MRC的所有三个技能优先级。在定量技能方面，我将培养生物信息学和统计学的技能，并将其应用于转录组学、代谢组学和16S rRNA测序。通过使用健康记录结合这个项目产生的数据，我将获得跨学科研究的技能。最后，我将与来自活体小鼠模型的样品一起工作，为我提供这些方法的技能。

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.