The bacterial type six secretion system (T6SS) is an injectisome responsible for the translocation of effector molecules directly into host cells or competing bacteria. The system is widely distributed among proteobacteria and is found in both clinically relevant strains as well as environmental stains and represents an important system for the study of both microbial ecology and virulence. The apparatus itself is believed to have arisen from a combination of genes from bacteria and bacteriophage due to seqeuence and structural identity between T6SS components and structural bacteriophage proteins. The current model of the T6SS apparatus consists essentially of an inverted phage body that is attached to the donor cell membrane complex. The phage-like structure can contract and force a sharp needle point complex along with effector proteins into the target cell. The phage derived components have received a considerable amount of attention and the mode of assembly is relatively well understood. However, little detailed information on the assembly and function of the membrane embedded complex is available. The first major goal of this thesis was to structurally characterize the proteins of the membrane embedded complex of the type six secretion system. The structures of IglE and TssL from Francisella sp. were solved and represent a platform for further characterization of the T6SS assembly and function. The periplasmic domain of a TssL homologue from P. aeruginosa was also solved and this structure represents a subset of evolved TssL proteins that bind peptidoglycan through an unknown mechanism. Biochemical and structural analysis probed this system but came short of a definitive model for peptidoglycan binding. However, the data collected from this study will further the field of peptidoglycan binding modules and help to characterise differences among T6SSs.
The translocated proteins of the T6SS are often bactericidal and attack the peptidoglycan, lipid bilayer or DNA of the target cell. However, one secreted substrate, Tse2 from Pseudomonas aeruginosa is targeted to other neighbouring cells of the same species. This toxin shares no sequence identity with any known protein but has been shown to be toxic to not only bacteria but also yeast and mammalian cells. The structure of the complex between Tse2 and its immunity protein was solved and led to two interrelated discoveries. The first was that the molecular details behind the immunity protein inhibiting Tse2 where it binds directly to the active site. The second was that based on structural identity with ADP-ribosylating toxins, the active site of Tse2 was identified. These results carry the study of this protein forward significantly although the precise function of Tse2 remains unknown. This structure is the first co-structure of a cytotoxic T6SS substrate and has significant implications for the cell in terms of handling the toxin for delivery rather than self intoxication. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/6043 |
| Date | 28 April 2015 |
| Creators | Robb, Craig |
| Contributors | Boraston, Alisdair Bennett |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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