Principal Supervisor: Dr Meera Unnikrishnan - WMS
Co-supervisor: Dr Chrystala Constantinidou - WMS
PhD project title: Elucidating mechanisms of host cell manipulation by Staphylococcus aureus
University of Registration: University of Warwick
Background: Staphylococcus aureus, a highly versatile bacterial pathogen, is a leading cause of infections in animals and humans. It causes mastitis, a severe infection of the mammary glands in dairy cows. S. aureus is responsible for 10%-20% of the clinical and subclinical mastitis cases studied. Clinical mastitis significantly impacts the health and welfare of dairy animals, and can cause death in worst cases. Decreased milk production and increased expenditure due to mastitis have resulted in huge financial losses in the dairy industry worldwide. Losses of up to €300 per cow per year have been attributed to staphylococcal mastitis. Subclinical mastitis is a chronic condition common in dairy herds with no visible clinical symptoms. Persistent sub clinical infections are very hard to eradicate.
Persistent infections by S. aureus have been attributed to its ability survive intracellularly. Although primarily an extracellular pathogen, S. aureus has now been recognized to have an intracellular lifestyle. Intracellular residence in immune and non-immune cells in the udder can provide a niche for the bacterium to evade antibiotics and immune responses in subclinical mastitis. Presence of intracellular bacteria within phagocytic and epithelial cells has been reported from experimental bovine udder infections and from recurrent human staphylococcal infections like tonsillitis and rhinosinusitis. In spite of the importance of intracellular niches in chronic infection, we do not have a good understanding of the staphylococcal factors that mediate intracellular survival or the precise mechanisms by which intracellular bacteria cause persistent infection.
Transposon (Tn) mutant libraries have been extensively used to study global gene requirements for survival in a given condition, such as infection, intracellular survival etc., for many bacterial pathogens, including S. aureus, using conventional and other approaches based on DNA microarray hybridizations. Recently, a novel method which combines high-throughput sequencing technology and high-density transposon-mutant libraries-Transposon Directed Insertion Site Sequencing (TraDIS, also known as Tn-seq) was described, where thousands of mutants were sequenced in parallel. Major advantages of TraDis include comprehensive identification of gene requirements under a selected condition in a quantitative fashion. This method has been employed in the identification of genes essential for bacterial growth in conditions relevant to pathogenesis such as biofilms and in different animal infection models.
The broad aim of this proposal is to apply TraDIS to identify comprehensively S. aureus genes required for the bacterium to survive intracellularly during infection. The specific aims of this project are:
- TraDis analysis of genes required for invasion and survival of S. aureus within host cells
- Characterisation of proteins that control S. aureus intracellular infection
BBSRC Strategic Research Priority: Food Security
Techniques that will be undertaken during the project:
The student will be trained in molecular, cellular microbiology techniques and advanced bioinformatic methods. Specific techniques will include cDNA library preparations, genome sequencing and analysis, bacterial cloning, transfections, siRNA studies, qRT-PCR. The student will receive training in mammalian cell culture, immunofluorescence staining and advanced microscopy techniques.
Contact: Dr Meera Unnikrishnan, University of Warwick