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A proteomic map of host cell rewiring by pathogens


Project Description

We aim to develop advanced methods in mass spectrometry-based proteomics and apply them to the question of how host cellular defences are manipulated by bacterial pathogens.

Bacterial pathogens residing within host cells must subvert cellular defences in order to avoid destruction and progress infection. The mechanisms by which bacteria can ’rewire’ host cell functions are poorly understood. The delineation of mechanistic details of how host defence systems are manipulated by pathogens is a key goal in contemporary infection biology research and success in this area may lead to the identification of pharmacological intervention targets in the host cell1. This host-directed strategy is attractive from the perspective of antimicrobial resistance (AMR) as it sidesteps the possibility of pathogens evolving resistance by targeting host components of bacterial rewiring.

The principal targets for pathogenic host cell manipulation are the primary functional units of the cell, namely proteins. The functional state of proteins depends on their cellular abundance, post translation modification (PTM) status, and assembly into protein complexes. To assess the state of the proteome we require quantitative measurement technologies. We have recently jointly developed diaPASEF2,3, a mass spectrometry method for sensitive protein quantification from minute quantities of protein (10-200ng) that can be applied to at high throughput (up to 50 samples per day). In this project, we will extend/optimize diaPASEF to assess the state of the proteome during the course of cellular infection with bacterial pathogens. Specifically, our aims are:

(1) Extend the diaPASEF method by:
(i) Optimize the diaPASEF parameter space to maximize ion utilization and selectivity enabling deep proteome coverage
(ii) Adapt diaPASEF for samples enriched for PTMs with relevance in host cell infection biology (phosphorylation, ubiquitination, ISGylation, sumoylation, neddylation)
(iii) Extend diaPASEF for use with our previously developed methods for targeted and global assessment of protein complex assembly state4,5

(2) Apply the methods developed to measure protein abundance, PTM state, and protein complex assembly state, in a host-centric approach to delineating bacterial rewiring in 2 clinically relevant pathogens:
(i) Klebsiella pneumonia is a global concern due to the increasing isolation of multidrug resistant strains. Klebsiella targets PTMs (SUMOylation, Neddylation and ISGlyation) to promote infection, however there is a lack of understanding of which are the critical PTMs modified proteins counteracting the infection. (collaboration with Bengoechea lab)
(ii) Legionella pneumophila, is an emerging respiratory pathogen and the cause of Legionnaires’ disease. L. pneumophila injects more than 300 effector proteins into macrophages and epithelial cells. These actively change the host proteome and signalling, through modulation of translation, cellular stress responses and the ubiquitin system, ultimately establishing an intracellular niche for replication. The function of the majority of effectors and a global view of the changes occurring remain elusive. (collaboration with Schroeder lab).

These pathogens utilize diverse virulence mechanisms and, as such, will demonstrate the broad applicability of our method. The results will provide a broad, dynamic, and integrated map of the host systems targeted by each pathogen. We will assess the functional consequences of removing selected key host cell proteins (by knockdown/out, chemical inhibition) using standard infection assays. Overall, the outcome will be a broadly applicable approach to determine mechanisms of host-cell rewiring demonstrated in 2 clinically relevant bacterial pathogens.

Start Date: 1 October 2020
Duration: 3 years

The project will be supervised by Dr Ben Collins (Queen’s University School of Biological Sciences/Institute for Global Food Security), Professor Jose Bengoechea and Dr Gunnar Neels Schroeder (Queen’s University School of Medicine, Dentistry and Biomedical Sciences), and Dr Stephanie Kaspar Schoenfeld (Bruker Daltonics).

Skills required by applicants:

The student should have a background or interest in learning skills in the following areas:
- Mass spectrometry based proteomics
- Cellular microbiology and host-pathogen interactions
- Bioinformatics and statistics
However, the student will be trained in advanced mass spectrometry methodology and computational methods (Collins lab and Bruker Daltonics) and cellular microbiology (Bengoechea and Schroeder labs). The student will be expected to make a number of research visits to Bruker Daltonics (Bremen, Germany) and budget for research visits and additional training courses has been reserved.

Funding Notes

The studentship will be funded by the Department for the Economy (DfE) and Bruker Daltonics (industrial partner).

Only UK/EU students are eligible to apply. Please read the full information on eligibility criteria: View Website

Not all applicants may be eligible to receive a full studentship - please note the Residency and Citizenship requirements in the document linked to above.

References

1Zumla, A. et al. Lancet Infect. Dis. 16, e47–e63 (2016).

2Meier, F. et al. bioRxiv 656207 (2019).

3diaPASEF: data independent acquisition – parallel accumulation serial fragmentation

4Heusel, M. et al. Mol. Syst. Biol. 15, e8438 (2019).

5 Collins, BC et al. Nature Methods 10, 12 (2013): 1246–53.

How good is research at Queen’s University Belfast in Public Health, Health Services and Primary Care?

FTE Category A staff submitted: 29.60

Research output data provided by the Research Excellence Framework (REF)

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