Clinical Gastroenterology: The regulation of GDH expression in Clostridioides difficile

Clostridioides difficile infection (CDI), the most common cause of infective antibiotic-associated diarrhea in the western world, is diagnosed by a combination of tests that firstly detect the organism, then the toxins that it produces (Planche 2013). One such test that determines if the organism is present does so by detecting the extracellular enzyme glutamate dehydrogenase (GDH). GDH is normally cytoplasmic or membrane bound in most bacterial species, but in C. difficile appears to be both cytoplasmic and extracellular (Giriniathan 2014). GDH is produced during exponential growth, but regulation of this production has not been described (Giriniathan 2014). In clinical samples the amount of GDH detected in the sample appears to be related to bioload (Davies, eThesis), as higher amounts of GDH can be detected from samples with higher numbers of organisms, as measured by semi-quantitative culture. However, large amounts of GDH can also be detected from samples when no other test for C. difficile is positive. The reasons for this are unclear but could, in part, be related to the extremely large amounts of GDH excreted by the organism, a phenomenon also seen when testing samples from the Leeds in-vitro gut model (Davies, eThesis). The mechanisms that control GDH expression are currently unknown, nor do we know what the consequences of high GDH levels may be for pathogenesis, thus patient health. It is possible that some stimulants (e.g. antibiotics) are more effective than others at promoting GDH expression, depending on the extent of microbiome dysbiosis in the gut. In addition, GDH-positive/toxin-negative results are difficult for clinicians to interpret, potentially leading to misdiagnosis of the disease; elucidation of the regulation of C. difficile GDH excretion may therefore have clinical impact. 

Project: 

The production/ expression of GDH by C. difficile will be examined using single stage chemostats, using a quantitative GDH enzyme immunoassay and quantitative PCR assay. Growth and GDH expression will be compared between a wild type and two mutant strains (one lacking the GDH gene and one overexpressing the gene), in the presence of different stimuli/promoters of GDH production, such as amino acids and antibiotics. Findings would be extended using a clinically reflective in vitro triple-stage gut model, where the growth and expression of GDH can be monitored against a background of normal gut microbiota. Patient faecal samples will be used to validate if any of the GDH associations seen in model(s) are also found in vivo; e.g. if a certain antibiotic is associated with hyper-production of GDH in the model, are high GDH levels found in CDI patients who have received that antibiotic, compared with others.  

Techniques associated with project: 

Standard microbiology culture techniques for both aerobic and anaerobic bacteria, enzyme immunoassays and quantitative PCR. You will use molecular microbiology methodologies to genetically delete, and overexpress the GDH gene, gluD, in mutant strains. Set up and maintenance of chemostat models, both single stage and triple stage, of single and mixed bacterial cultures. Completion of paperwork for the appropriate regulatory approvals required for use of patient samples. Handling and processing of clinical samples.  

This project is available as part of the International PhD Academy: Medical Research

Eligibility:

You should hold a first degree equivalent to at least a UK upper second class honours degree in a relevant subject.

Candidates whose first language is not English must provide evidence that their English language is sufficient to meet the specific demands of their study. The Faculty of Medicine and Health minimum requirements are:

• British Council IELTS - score of 7.0 overall, with no element less than 6.5
• TOEFL iBT - overall score of 100 with the listening and reading element no less than 22, writing element no less than 23 and the speaking element no less than 24.

How to apply:

Applications can be made at any time. To apply for this project applicants should complete an online application form and attach the following documentation to support their application. 

• a full academic CV
• degree certificate and transcripts of marks
• Evidence that you meet the University's minimum English language requirements (if applicable)

To help us identify that you are applying for this project please ensure you provide the following information on your application form;

• Select PhD in Medicine, Health and Human Disease as your programme of study
• Give the full project title and name the supervisors listed in this advert

Any queries regarding the application process should be directed to fmhpgradmissions@leeds.ac.uk