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The photorhabdus virulence cassette: the role of payload tags in loading bacterial syringes
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Evans, Rhys David (2023) The photorhabdus virulence cassette: the role of payload tags in loading bacterial syringes. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3985154~S1
Abstract
The Photorhabdus virulence cassette (PVC) is an elegant, multi-protein, contractile nanostructure which injects bioactive polypeptide ‘payloads’ across eukaryotic cell membranes. N-terminal leader sequences (LSs) from native payloads are necessary for loading and can also associate heterologous proteins into the hollow tube of the PVC ready for injection. Previous works identified an accessory protein encoded in the PVC operon, Pvc15, to be responsible for payload loading via an interaction with the LS. This thesis builds on these findings and concludes that Pvc15 also uses the LS to stabilise payloads and that the Pvc15 hexamer’s ATPase activity is necessary for association of the LS-tagged payload to the PVC chassis. Bioinformatic analyses in this work found that Pvc15 encodes an elusive N-domain and two tandem AAA domains: D1 and D2. E. coli carrying plasmids encoding the PVC operon and the LS-tagged payload were used to investigate the effects of mutations in either the LS or Pvc15 using quantitative western blot. Domain D2 was found to stabilise the Pnf payload via the LS. Given that only mutation of D2 was necessary to abolish the ATPase, D2 was identified as the sole functional ATPase domain. In addition, truncations to the LS of less than 50 amino acids conferred payload stability and abolished its capacity to load into the PVC. Overall, this thesis provides insight into the interactions between Pvc15 and the LS. Elucidating the nature of PVC loading may enable the development of PVCs as a therapeutic synthetic biology protein delivery toolkit in the near future.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QR Microbiology | ||||
Library of Congress Subject Headings (LCSH): | Bacterial protein -- Research, Proteins -- Physiological transport, Proteins -- Structure, Cell membranes, Heterorhabditidae, Pathogenic bacteria | ||||
Official Date: | August 2023 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Waterfield, Nicholas R., James, John R. | ||||
Sponsors: | Biotechnology and Biological Sciences Research Council (Great Britain) ; Midlands Integrative Biosciences Training Partnership | ||||
Extent: | xx, 249 pages : illustrations | ||||
Language: | eng |
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