The rise in antibiotic resistance has prompted a renewed interest in bacteriophage research. A study of the molecular basis for recognition of the bacterial host has applications in detection, vaccine design and control of bacterial infections. Although the role of bacteriophage in bacterial evolution, virulence, clonal diversity is well recognized, the biochemical and structural basis for the interaction of bacteriophages with the cell envelope of Gram positive pathogens is poorly understood. This project will study the molecular mechanisms that mediate cell surface-associated processes such as phage adsorption and phage resistance primarily in Staphylococcus aureus but also in other Gram-positive bacteria, such as coagulase-negative staphylococci. We will use an integrated, multidisciplinary approach involving genetics, biochemistry, structural biology and proteomics.
Specific goals and objectives:
1) We will identify and characterize the staphylococcal cell wall receptors for phages WS2 by analyzing phage adsorption efficiency using a panel of isogenic cell wall mutants. The cell wall fragments will be isolated, purified and used for inhibition of phage adsorption in order to define the minimum cell wall structures served as phage receptor.
2) Identify the receptor binding proteins (Rbp) of phage WS2; serum and recombinant Rbps will be generated and immunogold electron microscopy used to localize the Rbp in the bacteriophage tail. The stoichiometry of the Rbps in WS2 will be determined using a quantitative proteomics approach. The interaction of phage Rbp and its receptor will be analyzed in detail using biochemical and biophysical methods (Pull down assay, Biacore, Fluorescence microscopy/FACS analysis ).
3) The 3D structures of Rbps will be determined by X-ray crystallography and their location in the bacteriophage mapped by cryo-electron microscopy/single particle analysis.
4) Recombinant Rbp will be used to develop agglutination test, a cell capture assay, and capture ELISA as detection methods for S. aureus.
A) Niche Skills
1. Microbiological, biochemical, biophysical assays
2. Analysis of proteomic data
3. Protein expression and characterisation
4. X-ray crystallography
5. Cryo-electron microscopy and tomography
B) Core and Generic Skills
The full range of skills normally associated with a practical PhD project in the Molecular Biosciences, particularly communication the development of the student as an individual scientist, and ability to design, execute and interpret experiments in a suitable manner
Any enquiries relating to the project and/or suitability should be directed to Dr Xia. http://www.manchester.ac.uk/research/guoqing.xia/
1) Winstel V, Liang C, Sanchez-Carballo P, Steglich M, Munar M, Penades JR, Nübel U, Holst O, Dandekar T, Peschel A, Xia G. Wall teichoic acid structure governs horizontal gene transfer between major bacterial pathogens. Nat. Commun, 2013; 4: Article number 2345.
2) Brown S, Xia G*, Luhachack LG, Campbella J, Meredith T, Chen C, Winstel V, Gekeler C, Irazoqui JE, Peschel A, Walker S * Methicillin resistance in Staphylococcus aureus requires glycosylated wall teichoic acids. Proc. Natl. Acad. Sci. USA, 2012 Nov 13;109:18909-14. ( * Joint corresponding author; Highlights, commentary in PNAS, recommended by Faculty of 1000)
3) Xia G, Wolz C. Phages of Staphylococcus aureus and their impact on host evolution. Infect Genet Evol. 2014 Jan;21: 593-601.
4) Karuppiah V, Collins RF, Thistlethwaite A, Gao Y, Derrick JP. Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis. Proc Natl Acad Sci U S A. 2013 Nov 26;110(48):E4638-47.
5) Berry JL, Phelan MM, Collins RF, Adomavicius T, Tønjum T, Frye SA, Bird L, Owens R, Ford RC, Lian LY, Derrick JP. Structure and assembly of a trans-periplasmic channel for type IV pili in Neisseria meningitidis. PLoS Pathog. 2012 Sep;8(9):e1002923.