AbstractThe Type VII/Ess protein secretion system (T7SS) is found in many Gram positive bacteria, including the opportunistic human and animal pathogen Staphylococcus aureus. Previous work has shown that the S. aureus T7SS machinery comprises six core components of which four, EsaA, EssA, EssB and EssC are integral membrane proteins. EssC, the largest T7 component, is a member of the FtsK/SpoIIIE family of membrane-bound ATPases that harness the energy of ATP hydrolysis with the movement of macromolecules. The aim of this thesis was to probe the organisation of the membrane-embedded components of the secretion system and to determine whether EssC plays a role in substrate recognition.
A range of detergents was tested for their ability to solubilise EsaA, EssB and EssC from the S. aureus inner membrane. The zwitterionic detergent Foscholine-12 was subsequently used for further study as it was the only detergent identified that was able to extract reasonable levels of each protein from the membrane. Blue-native PAGE analysis identified homomeric complexes of Foscholine-solubilised EsaA and EssB proteins. Crosslinking analysis in native membranes provided further evidence for EsaA and EssB homo-dimerisation, and also revealed the presence of high molecular weight multimers of EssC. Surprisingly, there was no evidence from crosslinking that any of the components interacted with each other, or with the EssA protein. Furthermore, EssC multimers detected in whole cells were not dependent upon the presence of any other Ess component or substrate protein for their assembly.
The EssC protein from S. aureus strains can be grouped into four variants (EssC1-EssC4) that have sequence variability in their C-terminal domains. These variants are associated with unique clusters of candidate substrate-encoding genes. Here it was shown that each EssC variant can interact with the remaining Ess components from strain RN6390 to facilitate secretion of EsxA but not the substrate protein EsxC. Thus, EssC appears to be a specificity determinant for T7 substrate secretion in S. aureus.
|Date of Award||2017|
|Supervisor||Tracy Palmer (Supervisor)|