Recent bioinformatic analyses identified an operon encoding a potential Type I Secretion System (T1SS) in Treponema pallidum that we hypothesize functions to export key treponemal virulence factors that may contribute to the unique invasiveness and pathogenesis of this spirochete. The membrane fusion protein component (MFP) of T1SSs in other organisms has been shown to play a role in substrate recognition. Hence, the objective of this project is to use the putative MFP, Tp0965, of the potential T. pallidum T1SS to investigate protein-protein interactions with the T. pallidum virulence factor pallilysin (Tp0751) and assess the possibility of the latter being a T1SS substrate. Moreover, protein-protein interactions between Tp0965 and a Treponema phagedenis lysate are investigated with the goal of identifying putative T1SS substrates in this spirochete that could result in the discovery of novel T. pallidum virulence factors via amino acid sequence similarity.
Plate-based binding studies and pull-down assays showed a low level of interaction between recombinant Tp0965 and the previously characterized host-component-binding protease, pallilysin, suggesting that the export of this virulence factor could occur via the putative T1SS.
Additionally, bioinformatic analyses of the related but cultivable model spirochete T. phagedenis predicted the presence of a potential T1SS homologous to the putative T1SS in T. pallidum. Thus, a more global and unbiased pull-down assay using “bait” Tp0965 and a “prey” T. phagedenis lysate was carried out, followed by mass spectrometric analysis to identify putative novel T1SS substrates with potential homologs in T. pallidum. We successfully identified a T. phagedenis protein, TphBIg, that showed evidence of an interaction with Tp0965. TphBIg seems to possess characteristics of a T1SS substrate suggesting it may be secreted via this system in T. phagedenis. Upon bioinformatic analysis, it was found that TphBIg showed weak amino acid sequence similarity as well as some structural similarity to the T. pallidum protein, Tp0854.
Tp0854 is predicted to contain a sialidase and a phosphatase domain with an RTX motif, which is characteristic of some T1SS substrates. Thus, it was hypothesized that if Tp0854 had characteristics of a T1SS, it may interact with Tp0965. Therefore, the phosphatase domain containing the RTX motif was produced recombinantly and plate-based binding studies indeed suggested an interaction with Tp0965, confirming the in silico-predicted interaction.
Future experiments to characterize the potential T1SS and substrates in T. pallidum could comprise the functional and structural characterization of the novel putative T1SS substrate, Tp0854. This would include assays to investigate the putative sialidase and phosphatase activities of Tp0854, as well as the identification of Tp0854-Tp0965 interacting sites. Moreover, as a more definite test for T1SS substrate secretion, T. pallidum pallilysin and/or Tp0854 could be expressed heterologously in an E. coli strain harbouring an endogenous T1SS and test for secretion. Similarly, the reconstitution of the T. pallidum putative T1SS in liposomes could be used to further investigate the secretion of pallilysin and/or Tp0854 via this system.
Additionally, the optimized unbiased pull-down technique could be further applied to detect more protein-protein interactions within T. pallidum and potentially lead to the identification of more virulence factors that may be secreted via the T1SS.
These studies constitute the first investigation of a putative T1SS and substrates within T. pallidum. Thus, insight gained will lead to a better understanding of the mechanisms facilitating T. pallidum host invasion and may reveal new potential vaccine targets to prevent bacterial dissemination and chronic infection. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/7412 |
| Date | 20 July 2016 |
| Creators | Gaither, Claudia |
| Contributors | Cameron, Caroline E. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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