Date on Master's Thesis/Doctoral Dissertation
Oral Biology, MS
Committee Co-Chair (if applicable)
T9SS; proteins; gingivalis; secretion; cargo; CTD
Background: Porphyromonas gingivalis, a keystone-pathogen associated with periodontitis, utilizes the Type IX Secretion System (T9SS) to secrete proteins bearing the conserved C-terminal domain (CTD), many of which are essential virulence factors. These include, besides gingipains and PPAD, 29 other proteins. During or after the outer membrane translocation the CTD is cleaved off and a glycan moiety is covalently attached anchoring the secreted proteins at the bacterial cell surface. Objective: Most of the CTD bearing proteins have only hypothetical functions assigned. It is plausible that these CTD bearing proteins may also play a crucial role in P. gingivalis pathogenesis. Therefore, it would be of interest to determine their precise role, which could be accomplished through protein purification and subsequent characterization. Three CTD proteins were chosen as first targets: C25 peptidase (PG_RS01820), Carboxypeptidase D (PG_RS01060; Cpg70) and Periodontain (PG_RS06260; PrtT related). Methods: It was shown previously that the insertion of a hexahistidine affinity tag upstream of the CTD in RgpB and PPAD results in secretion of soluble, non-glycosylated, mature forms of these enzymes into the culture media, this facilitates their purification by nickel-chelate affinity chromatography. This same approach was applied in the present study. Results: Isogenic mutant strains of P. gingivalis with the insertion of a hexahistidine affinity tag upstream of the CTD were generated via homologous recombination. The presence of soluble proteins bearing the hexahistidine tag in growth media were confirmed for carboxypeptidase D (CPG70) and periodontain (PrtT related) expressing strains. These two proteins were then purified to homogeneity by Ni-Sepharose affinity chromatography. Conclusion: Purification of two CTD bearing proteins out of three chosen targets, confirmed that the applied method can be successfully used for purification of other proteins from this family. In the case of C25 peptidase the failure to find the His-tagged protein can be due to very low expression level.
Goel, Apoorv, "Modification of T9SS cargo proteins of Porphyromonas gingivalis for their secretion in soluble form and purification by affinity chromatography." (2016). Electronic Theses and Dissertations. Paper 2415.