Cell division in Staphylococcus aureus : protein-protein interactions and super-resolution microscopy

Publication Type:
Thesis
Issue Date:
2014
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Bacterial cell division is an essential process and is increasingly recognized as an attractive drug target. Uncovering the proteins involved in cell division as well as their interactions will be the first step in designing compounds which could be used to treat infections caused by multi-drug resistant S. aureus strains. However, cell division remains relatively understudied in this organism due to its small size and difficult genetics. The aim of this study was to better understand the mechanism of cell division in S. aureus by identifying novel FtsZ-interacting components and confirming expected protein-protein interactions in vivo. The overall strategy involved tagging the essential cell division protein, FtsZ, with a green fluorescent protein (GFP) tag and using this fusion to isolate any FtsZ-interacting divisome components by GFP affinity purification. To circumvent the issue of lethality caused by non-functional FtsZ-GFP fusions, ftsZ-gfp was ectopically expressed from the plasmid-based protein localization system (pLOW/pGL485) which allows for regulated expression of gfp fusions in S. aureus. Using the FtsZ-GFP fusion for protein complex isolation, several cell division proteins that have been previously shown to interact with FtsZ in B. subtilis (SepF, EzrA, FtsA) were identified along with a chaperone protein called DnaK. Further analysis revealed that DnaK indeed interacts with FtsZ and EzrA but is particularly important for maintaining the stability of the curvature-sensing cell division protein, DivIVA. In B. subtilis, DivIVA appears to play a role in division site positioning but its role in S. aureus remains unknown. Genetic experiments conducted in this study with S. aureus divIVA mutants revealed a possible role for DivIVA in chromosome partitioning. It is therefore likely that one of the roles of DnaK is to maintain physiological levels of DivIVA to ensure efficient nucleoid partitioning in S. aureus. The same FtsZ-GFP fusion protein expressed from the plasmid pLOW/pGL485 system was used to examine the localization of FtsZ using high resolution microscopy to resolve a long standing question in the field: what is the architecture of the Z-ring in vivo? Super resolution 3D-SIM microscopy, which breaks the diffraction limit of conventional microscopes, revealed that the Z-ring in S. aureus was bead-like in appearance, heterogeneous in fluorescence intensity, highly dynamic and contained apparent gaps in the ring. This heterogeneous and dynamic Z-ring architecture is consistent with the iterative pinching model for Z-ring constriction where FtsZ undergo conformational changes from repeated cycles of polymerization-depolymerisation and is likely to provide the energy that drives constriction of the ring in vivo.
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