Date on Master's Thesis/Doctoral Dissertation

8-2016

Document Type

Master's Thesis

Degree Name

M.S.

Department

Oral Biology

Degree Program

Oral Biology, MS

Committee Chair

Potempa, Jan

Committee Co-Chair (if applicable)

Demuth, Donald

Committee Member

Demuth, Donald

Committee Member

Scott, David

Committee Member

Lawrenz, Matthew

Author's Keywords

Porphyromonas gingivalis; Type 9 Secretion System; PG1604; C-terminal Domain; Por Z,; Secretory phenotype

Abstract

Background: The numerous virulence factors of Porphyromonas gingivalis are essential for the organism to invade host tissues while escaping immune responses [1, 2]. Some of these factors, like major proteases referred to as gingipains, are secreted by a unique secretion system called PerioGate or Type IX Secretion System (T9SS). T9SS consists of a number of membrane and periplasmic proteins, which provide transportation only for proteins that possess a conserved C-terminal domain (CTD). The CTD functions as a specific recognition signal for the T9SS. PG1604 of P. gingivalis W83 is a CTD-containing protein, which has been shown in our initial studies to be essential for secretion of CTD-proteins. However, unlike other CTD proteins, PorZ was also shown by proteomic studies to be present in the wild-type strain as the full-length protein containing the CTD domain (minus a signal peptide) [3, 4]. Objectives: Our objective was to probe structure-function relationship of the PG1604 protein by insertions of an oligohistidine motif or substitutions of six consecutive residues with hexahistidine within the C-terminal domain of PG1604. Effects of these mutations were monitored by various phenotypic tests and allowed to assess their influence on PG1604 maturation and function. Methods: 1) Oligohistidine insertions into the PG1604 protein by plasmid-based SLIM mutagenesis. 2) Generation of a P. gingivalis mutant strain expressing the modified PG1604 from the chromosome. 3) Phenotype analysis conducted on the whole set of the CTD altered PG1604, including the strain obtained in this study (AT001). Assays included: a) assessment of colony pigmentation on blood agar plates, b) the gingipains activity assay, c) western-blot analysis to detect the presence and subcellular localization of PG1604 (anti-PG1604 and anti-HIS tag antibodies) as well as the presence and processing of the Kgp and Rgp proteins. Results: 1) Some mutants showed the altered secretory phenotype detected by the strains failure to form pigmented colonies on the blood agar plate. 2) The lack of pigmentation correlated with severely decreased gingipains activities and with changes in proKgp and proRgp protein posttranslational processing visualized by western-blot. 4) The PG1604 protein was found to be localized mainly in the outer membrane. 5) HIS-tagged PG1604 may be purified from P. gingivalis culture for further analysis. Conclusions: 1) The CTD of the PG1604 protein is not cleaved off upon its transport through T9SS as full length ~80 kDa protein was shown by western-blot analysis with anti-PG1604 serum (migration at) and anti-HIS mAb for the PG1604 variant tagged with octahistidine at the C-terminus (P. gingivalis strain DM008). 2) The selective modifications of the PG1604 protein, hindered the proper maturation of secreted virulence factors (Kgp and Rgp) due to disrupted function of T9SS in P. gingivalis. Taken together based on our findings we concluded that PG1604 is a novel functional component of T9SS located on the bacterial surface in association with the outer membrane. The CTD of PG1604 is not only essential for translocation across the OM but also for this protein function in other CTD-proteins secretion and their posttranslational modifications (A-LPS attachment and proteolytic processing). Therefore, following the nomenclature of the T9SS components we designate the PG1604 protein as PorZ.

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