Investigation of the potential regulator proteins associated with the expression of major surface protein and dentilisin in Treponema denticola

Abstract

Objective Treponema denticola is involved in 'chronic' periodontitis pathogenesis. The mechanism underlying the regulation of the expression of its virulence factors, such as major surface protein (Msp) and prolyl-phenylalanine specific protease (dentilisin) is yet to be clarified. We determined the gene expression profiles of Msp- and dentilisin-deficient mutants of T. denticola to identify the regulation network of gene expression concomitant with the inactivation of these virulence genes. Methods Gene expression profiles of T. denticola ATCC 35405 (wild type), dentilisin-deficient mutant K1, and msp-deficient mutant DMSP3 were determined using DNA microarray analysis and quantitative real-time reverse transcription PCR (qRT-PCR). Msp and dentilisin protein levels were determined by immunoblotting and proteolytic activity assays. Results In addition to several differentially expressed genes, dentilisin expression was reduced in DMSP3; msp expression was significantly reduced in K1 (p < 0.05), both at the gene and protein levels. To identify the regulatory system involved, the expression levels of the potential regulators whose expression showed changes in the mutants were evaluated using qRT-PCR. Transcriptional regulators TDE_0127 and TDE_0814 were upregulated in K1, and the potential repressor, TDE_0344, was elevated in DMSP3. Conclusions Dentilisin and Msp expression were interrelated, and gene expression regulators, such as TDE_0127, may be involved in their regulation.

Keywords: DNA-binding protein; Treponema denticola; dentilisin; gene expression; major surface protein; repressor.

Figure 1.

Expression of msp and prtP determined using qRT-PCR. (a) Expression of msp. (b) Expression of prtP (dentilisin); 35405: T. denticola ATCC 35405 (wild type), K1: dentilisin mutant, DMSP3: msp mutant. The expression of each gene is presented as fold-change over that in the wild type strain. Data are presented as means ± SD (n = 6), and statistically significant differences are indicated using asterisks (one-way ANOVA with Dunnett’s multiple comparison test; *p < 0.05 compared to the wild type).

Figure 2.

SDS-PAGE and immunoblot analysis of sonicates of T. denticola ATCC 35405, DMSP3, and K1. SDS-PAGE analysis (a) and immunoblot analysis (b).

Figure 3.

Dentilisin activity of wild type (35405) and msp-deficient mutant (DMSP3). Data are presented as means ± SD, and statistically significant differences are indicated by asterisks (student t-test; *p < 0.05 compared to the wild type). 35405: T. denticola ATCC 35405, DMSP3: T. denticola DMSP3, cell: cells of T. denticola, sup: culture supernatant of T. denticola, log: log phase, stationary: stationary phase.

Figure 4.

Gene expression of the potential regulator proteins in wild type (35405), msp-deficient mutant (DMSP3), and prtP-deficient mutant (K1). Gene expression levels were evaluated using qRT-PCR with a Taqman probe. The expression of TDE_0127 (a), TDE_0344 (b), and TDE_0814 (c) are presented as fold-change with respect to that of the wild type strain. Data are presented as means ± SD (n = 6), and statistically significant differences are indicated by asterisks (one-way ANOVA with Dunnett’s multiple comparison test; *p < 0.05 compared to wild type).