PhD Scholarship project 1

SBBS Research Scholarships 2022: Project 1

The impact of motility on Streptococcus gallolyticus pathogenesis in colorectal cancer patients

PI: Dr Jennifer Mitchell

Project Description:

Introduction:

The pathophysiology of the gut bacterium Streptococcus gallolyticus subsp. gallolyticus (Sgg) remains largely unknown (1,2). It is an opportunistic pathogen that has been shown to specifically colonise colorectal cancer tissues via adherence to collagen and, following colonisation, translocation occurs through the intestinal barrier into the bloodstream resulting in bacteremia, sepsis and Infective Endocarditis.

Sgg were previously believed to be non-motile and their mechanism of gut wall translocation was unknown, however, our work has shown that these bacteria exhibit twitching motility. We hypothesise that Type IV pili are responsible for this novel phenomenon (3). In Sgg isolates, we have found that the only operon encoding a Type IV pilus is the com operon that is involved in competence or DNA uptake by the bacteria (4). We have also determined that exposure of the Sgg clinical strains to glucose and other sugars supresses this motility and consequently prevents invasion of colorectal cancer cells (5).

This Project:

We are currently making isogenic mutants of sgg by allele replacement mutagenesis. We are targeting the com genes that encode the Type IV pilus and the ccpA gene that is the master regulator for catabolite repression in Sgg. The ccpA gene switches off responsive genes in the presence of excess sugar. These mutant strains will help to determine the effect of their absence on motility and colonic cell invasion.

Two other closely related strains of bovis group streptococci, Streptococcus pasteurianus and Streptococcus infantarius can also cause bloodstream infections but are not associated with causing infections in patients with cancers but rather neonatal infections. We are interested in also testing the motility of these strains, characterizing their response to sugars and other gut metabolites and in doing so determine why they are less likely to cause infections in cancer patients.

All three species encode the com pilus, and we aim to characterize the motility, expression and function of the operon in all three strains in response to gut metabolites. Of interest to us is the potential for lysed or necrotic cells of the human gut to provide a released and tissue bound DNA anchor point for these pili that allows them to enter human colonic epithelial cells and translocate the gut wall. We will be characterizing the DNA binding activity of the com proteins in order to investigate this fully.

Another important aspect of the observation of motility in Sgg is that the com operon is prevalent in many medically important strains of streptococci. It has been observed in Streptococcus pneumoniae that the com pilus enables binding to DNA, but no one has tested is ability to confer motility. As part of this project, we will test whether S. pneumoniae and other oral streptococci are motile, whether the com operon contributes to this and whether metabolites present in their natural niches impact upon motility.

Finally, the impact of these observations needs to be tested in a model of infection. We will be collaborating with cancer researchers in Ireland and the US to test whether the isogenic com mutants that we generate have a reduction in pathogenicity in these models and whether growth of the bacteria in tumor conditioned media influences motility and com gene expression. We will also test whether drugs that have been previously observed to inhibit competence can be repurposed to prevent pathogenesis of these organisms in vivo and potentially be used as treatments for severe bloodstream infections and pneumonia.

Aims and timeline:

1. Characterize the motility of S. gallolyticus, S. pasteurianus, S infantarius in response to sugars, temperature, amino acids, nucleic acids, bile metabolites and fatty acids. (Months 1-12)
2. Assess the motility of these strains along/towards DNA gradients: self DNA, synthesized DNA and host DNA (necrotic DNA and apoptotic DNA) (Months 13-24)
3. Determine the DNA binding specificity of com pilin proteins in these organisms. (Months 25-36)
4. Assess the potential motility of related streptococci with competence pili especially pneumococci (Months 13-24)
5. To assess the impact of Type IV pilus gene deletions in in-vivo and ex-vivo models of infection and the impact of Tumor conditioned media on pilus gene expression. (Months 30-44)
6. Thesis Write up. (Months 45-48)

Training:

The student will be enrolled as part of the Infection Biology PhD Program and complete Infection Biology Modules and the Engage and postgrad teaching module as well as Conway modules so that they are up to date in both infection biology and cancer research and achieve their module quota to graduate.

The student will be supported to attend one local and one international conference per year to disseminate their work and publish the results of their research each year. The student will also be supported to become a member and apply for funding from the Microbiology Society and apply for any funding from UCD and other sources to fund their work as all my previous students have done. In addition, we will collaborate with Irish and international research groups to facilitate any expertise or training required to complete the project that is not already present in the lab or provided by core facilities in the Conway.

The student will also be supported to present their research at lab meeting each week and encouraged to critique their lab colleagues research as part of their training and team building. They will also be encouraged to take an active role in the UCD Micro Soc in order to meet other postgrads in SBBS and beyond and gain opportunities to network and present their work to a local peer audience.

Minimum Requirements: Applicants should have a BSc (Hons) (minimum grade 2.1 or above) or Master’s degree in biological sciences/microbiology/molecular biology The successful candidate should be enthusiastic, self-motivated and should be able to interact well within a larger research team.  EU applicants only.  

Conditions:

1. September/October 2022 start date.
2. Full EU fees + €18,000 per annum stipend over four years.
3. The student will be enrolled in a Structured PhD programme, associated with the School.
4. Each student is required to demonstrate in appropriate laboratory practicals as part of their funded scholarship(with no additional payment made).  Demonstrating hours and lab practicals are detailed and assigned by the SBBS Demonstrating Committee (maximum hours: 288 per annum) .
5. The student and supervisor are required to submit an application for an IRC Postgraduate Scholarshipin the first year of their SBBS-funded research scholarship.

Enquiries and applications (to include cover letter and CV) to Dr Jennifer Mitchell. Email: jennifer.mitchell@ucd.ie

Closing date: Friday 29th July, 2022 .