Dentilisin Expression by Treponema denticola in Biofilms Under Peptide Limitation

Treponema denticola (Td) is a proteolytic bacterium implicated in periodontitis. The outer sheath protease dentilisin (PrtP) is considered an important virulence factor of Td, as it degrades proteins in the gingiva and crevicular fluid, generating peptides that aid the bacterium's survival. Objective: to determine the relative transcription rate of prtP and dentilisin activity in biofilm and planktonic Td cells incubated under the nutritional stress of peptide limitation. Methods: Td 35405 cells were grown on fibronectin coated plates in spirochete medium (SM) for 4 days. Biofilm and planktonic cells were collected separately and exposed to SM with serially diluted trypticase peptide for 3-h. Total RNA was extracted from the bacterial cell pools, DNAse treated and reverse transcribed to cDNA. Quantitative real-time PCR was conducted to analyze prtP differential gene expression. The 16S rRNA gene was used as an internal standard to normalize prtP cycle threshold values (Ct). Dentilisin activity was measured colorimetricaly using SAAPFNA as substrate. Results: After 4-d growth, the Ct values were lower in biofilm than planktonic Td cells. Upon peptide limitation, prtP expression in biofilm cells remained constant. In contrast, planktonic cells showed decreased expression of prtP especially in SM with 1/64 the concentration of trypticase (1/64SM). At this peptide concentration, levels of prtP expression increased by 2-fold (P < 0.001) in biofilm versus planktonic cells. At 1/64SM, the activity of the expressed dentilisin showed an increase of 27% (P < 0.001) and 20% (P < 0.001) in biofilm and planktonic cells, respectively. Conclusions: Expression of prtP may be constitutive in biofilm cells regardless of short-term peptide limitation. However, planktonic cells significantly decreased expression at the most diluted trypticase concentration tested. Td cells in biofilm and planktonic phases differentially regulate dentilisin expression in response to peptide limitation. Supported by CIHR MGP-5619.