Interplay of glycosylation & cell surface display of T9SS cargo proteins in oral pathogens – New avenues for treatment of periodontitis?

Background.

Periodontitis is a widespread oral bio-film disease for which no targeted therapies currently exist. It is associated with the bacteria Porphyromonas gingivalis (Pg) and Tannerella forsythia (Tf), both of which rely on a sophisticated Type IX protein secretion system (T9SS) to transport virulence factors across the OM and anchor them to the cell surface. The T9SS exports cargo proteins contain a structurally conserved C-terminal signal domain (CTD), and many of them are glycosylated [1,2]. Within the T9SS machinery, the OM shuttle protein PorV binds to glycoproteins as they are secreted, while the signal peptidase/sortase PorU cleaves the CTD and anchors the cargo to the cell surface via an LPS-derived sugar [3,4]. This process is facilitated by PorZ that delivers LPS to PorU. While the T9SS has been extensively studied in Pg, our research has shown that in Tf, O-glycosylation occurs at a conserved amino acid motif [5] in the periplasm prior to CTD cleavage and secretion [6]. Glycosylation in Pg remains less well understood [2].
This project aims to unravel the interplay between protein O-glycosylation and the T9SS, as well as the specificity of cell surface display for T9SS cargo glycoproteins.

Hypotheses.

1. Glycosylation of T9SS cargo proteins influences their secretion and subsequent surface attachment. 2. The interaction between the CTD of cargo (glyco)proteins and LPS exhibits species-specificity. 3. Proteins can be engineered to function as T9SS cargo proteins.

Methods.

The study investigates the glycosylated Tf S-layer proteins TfsA and TfsB as model cargo for the T9SS, focusing on glycosylation's role in export and interaction, using also components of the T9SS from Pg. The S-layer proteins, glycosylated at 15 surface-exposed sites, are hypothesized to interact with the PorV shuttle's inner surface. i) Protein export and glycosylation: In vivo studies in Tf cells will use glycosylation variants of TfsA/B to analyze export rates and surface display, complemented by struc-tural modeling of S-layer glycoprotein-PorV interactions. ii) LPS and PorZ interactions: The interaction of Tf LPS with Tf and Pg PorZ, and truncated S-layer proteins, will be analyzed using biophysical methods to determine the specificity of LPS anchoring and the role of O-glycosylation. iii) Chimeric protein export: Chimeric proteins combining the CTD of TfsA/B with the Tf fucosyltransferase GtfE will be tested for T9SS-mediated export. iv) Surface display and glycosylation: TfsA/B, with native or swapped Tf/Pg CTDs, will be expressed in Tf, Pg, and Pg engineered to produce Tf LPS linkages. Surface display will be assessed via SDS-PAGE and TEM, with glycosylation patterns analyzed by glycoproteomics This study has the potential to uncover novel molecular targets for therapies against periodontitis.

REFERENCES
1. Gorasia, D.G., Hanssen, E., Veith, P.D., Reynolds, E.C. (2021) Structural characterization of the Type IX Secre-tion System in Porphyromonas gingivalis. Meth Mol Biol 2210, 113-121. doi:10.1007/978-1-0716-0939-2_11
2. Veith, P.D., Shoji, M., Scott, N.E., Reynolds, E.C. (2022) Characterization of the O-glycoproteome of Porphyromonas gingivalis. Microbiol Spectr 10, e0150221. doi:10.1128/spectrum.01502-21
3. Veith, P.D., Shoji, M., O'Hair, R.A.J., Leeming, M.G., Nie, S., Glew, M.D., Reid, G.E., Nakayama, K., Reynolds, E.C. (2020) Type IX secretion system cargo proteins are glycosylated at the C-terminus with a novel linking sugar of the Wbp/Vim pathway. mBio 11doi:10.1128/mBio.01497-20
4. Lauber, F., Deme, J.C., Liu, X., Kjær, A., Miller, H.L., Alcock, F., Lea, S.M., Berks, B.C. (2024) Structural insights into the mechanism of protein transport by the Type 9 Secretion System translocon. Nat Microbiol 9, 1089-1102. doi:10.1038/s41564-024-01644-7
5. Tomek, M.B., Maresch, D., Windwarder, M., Friedrich, V., Janesch, B., Fuchs, K., Neumann, L., Nimeth, I., Zwickl, N.F., Dohm, J.C., Everest-Dass, A., Kolarich, D., Himmelbauer, H., Altmann, F., Schäffer, C. (2018) A general protein O-glycosylation gene cluster encodes the species-specific glycan of the oral pathogen Tannerella forsythia: O-glycan biosynthesis and immunological implications. Front Microbiol 9, 2008. doi:10.3389/fmicb.2018.02008
6. Tomek, M.B., Neumann, L., Nimeth, I., Koerdt, A., Andesner, P., Messner, P., Mach, L., Potempa, J.S., Schäffer, C. (2014) The S-layer proteins of Tannerella forsythia are secreted via a type IX secretion system that is decoupled from protein O-glycosylation. Mol Oral Microbiol 29, 307-320. doi:10.1111/omi.12062