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

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.