Annotation:
In 2017, the World Health Organization declared Staphylococcus aureus to be an antibiotic-resistant pathogen for which new therapeutics are urgently needed. Upon infection, S. aureus forms biofilms that can only be treated by the long-term application of several antibiotics in high doses or the surgical removal of the infected tissues. An alternative approach, phage therapy, has not been approved for clinical use, because the effects of phage infection on a biofilm are not sufficiently characterized. The student will study the dynamics of the propagation of a phage in a S. aureus biofilm and molecular details of its replication in a cell. He/she will determine how sub-populations of metabolically dormant or phage-resistant cells in a biofilm provide herd immunity against phage infection. In addition, he/she will use focused ion beam milling together with cryo-electron microscopy and tomography to determine high-resolution structures of previously uncharacterized phage replication and assembly intermediates in S. aureus cells.
Recommended literature:
Rigort et al., Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc Natl Acad Sci U S A 109, 4449-4454 (2012).
N. Cerca, R. Oliveira, J. Azeredo, Susceptibility of Staphylococcus epidermidis planktonic cells and biofilms to the lytic action of staphylococcus bacteriophage K. Lett Appl Microbiol 45, 313-317 (2007).
M. Jemielita, M. J. Taormina, A. Delaurier, C. B. Kimmel, R. Parthasarathy, Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques. J Biophotonics 6, 920-928 (2013).
J. Girstmair et al., Light-sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development. BMC Dev Biol 16, 22 (2016).
J. Novacek et al., Structure and genome release of Twort-like Myoviridae phage with a double-layered baseplate. Proc Natl Acad Sci U S A 113, 9351-9356 (2016).
Requirements on candidates:
Ideal candidate should have background in one of the following: molecular biology, biochemistry, physical chemistry, structural biology or informatics.
Keywords:
Virus, phage, structure, cryo-EM, cryo-ET, LSFM, light sheet fluorescence microscopy, biofilm, staphylococcus, phage therapy
Annotation:
Diseases caused by enteroviruses include upper and lower respiratory tract infections, gastroenteritis, hand-foot-and-mouth-disease, and life-threatening meningo-encephalitis. Rhinoviruses are responsible for 40% of common cold cases, which result in a yearly cost of tens of billions of US$ in treatments and lost working hours worldwide. The student will study the replication cycle of enteroviruses in situ by a combination of focused ion beam milling (FIBM) and cryo-electron tomography (cryo-ET). He/she will investigate the changes in intra-cellular organization induced by enterovirus infection, and the assembly of progeny virions. To facilitate the structural studies he/she will utilize patterned grids for electron microscopy to enable the automation of the FIBM of lamellas from tissue culture cells and the preparation of columnal sample geometry to increase the completeness of recordable cryo-ET data beyond the limit imposed by commonly used slab-shaped samples.
Recommended literature:
Palmenberg AC, et al. (2009) Sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution. Science 324(5923):55-59.
Fendrick AM, Monto AS, Nightengale B, & Sarnes M (2003) The economic burden of non-influenza- related viral respiratory tract infection in the United States. Archives of internal medicine 163(4):487-494.
Tuthill TJ, Groppelli E, Hogle JM, & Rowlands DJ (2010) Picornaviruses. Curr Top Microbiol Immunol 343:43-89.
Schaffer M, et al. (2017) Optimized cryo-focused ion beam sample preparation aimed at in situ
structural studies of membrane proteins. J Struct Biol 197(2):73-82.
Ren J, et al. (2013) Picornavirus uncoating intermediate captured in atomic detail. Nature communications 4:1929.
Requirements on candidates:
Ideal candidate should have background in one of the following: molecular biology, biochemistry, physical chemistry, structural biology or informatics.
Keywords:
Virus, structure, cryo-EM, cryo-ET, replication, assembly, capsid, replication factory, membrane, replicase
