Expression of the tpr Protease Gene of Porphyromonas gingivalis Is Regulated by Peptide Nutrients

Abstract

The Tpr protease of Porphyromonas gingivalis W83 is a membrane-associated enzyme capable of hydrolyzing chromogenic substrates for trypsin and bacterial collagenases. A previous study by us indicated that Tpr expression was increased under conditions of nutrient limitation. In the present study, we further characterized expression of the tpr gene using atpr::lacZ reporter gene construct under a range of nutrient conditions. In P. gingivalis, transcription of tpr was initiated 215 bp upstream of the coding region and regulation of tpr expression was at the level of transcription. Deletion mutations in the tprupstream region identified the promoter region immediately upstream of the transcription start site, determined by primer extension analysis. Three identical 17-bp direct repeats identified within the 5′ end oftpr mRNA were involved in tpr regulation. In anEscherichia coli background, tpr transcription was initiated after an AT-rich region upstream of tpr but not at the P. gingivalis start site. Tpr expression inP. gingivalis was suppressed by the addition of peptide and protein nutrients to a peptide-limited growth medium but was only slightly affected by addition of free amino acids. Low-molecular-weight fractions of brain heart infusion rich in phenylalanine, proline, and alanine had the greatest inhibitory effects on expression of thetpr::lacZ construct. Addition of the dipeptide phenylalanyl-phenylalanine to the growth medium resulted in a 10-fold decrease in tpr expression. This suggests that specific phenylalanine-containing peptides are a major factor controlling Tpr expression. Neither hemin starvation, heat shock, nor pH change had significant effects on Tpr expression.

Role of bacterial proteinases in matrix destruction and modulation of host responses

“…This effect of iron limitation, however, was probably due to a decrease in the culture growth rate since a similar change in gene expression was observed in a P. gingivalis 381 culture grown either slowly on diluted medium (351) or in chemostatcontrolled conditions (219). A second very well-designed study which concentrated on the regulation of tpr gene expression has yielded significant seminal information (208). It was found that Tpr proteinase production is negatively controlled by the increased availability of peptides, especially those containing phenylalanine, but not by free amino acids.…”

“…Peptides and cleaved collagen fibers generated at this step would then become substrates for periodontain and the Tpr proteinase. Significantly, as described above, the Tpr proteinase would initially be strongly upregulated by a deficiency of peptide nutrients (208) providing P. gingivalis with more proteolytic power in periods of starvation. Peptides and partially degraded proteins produced at this stage would also become perfect targets for periodontain, which cleaves very efficiently only peptides and denatured proteins, and without any discrete specificity for selected amino acid residues.…”

“…M84471, AF020499), which is homologous to members of the papain or calpain families of cysteine proteinases. Its expression is upregulated in P. gingivalis cultures starved with regard to the availability of peptides (35,208).…”

A consensus Porphyromonas gingivalis promoter sequence

“…These factors may have a bearing on the observed poor expression of P. gingivalis genes in E. coli from endogenous promoters. The mRNAs of two genes which are expressed in E. coli, tpr and ¢mA, have each been shown to be initiated from a site other than that used in P. gingivalis [10,12].…”

“…Recently, the complete genome of P. gingivalis strain W83 has been determined (www.tigr.org) and although amino acid sequences can be deduced using this approach, little information can be gathered regarding the regulatory elements of these genes, and how protein expression is controlled. Transcription start points (tsp) have been reported for the P. gingivalis genes, prtT, which encodes a cysteine proteinase [7], hemR, a haemin-regulated gene [8], the groESL operon [9], the proteinase tpr [10], sodA the superoxide dismutase gene [11] and the ¢mbrillin gene ¢mA [12]. Promoter-like sequences were subsequently designated for prtT, groESL and tpr using the Escherichia coli sigma-70 (c 70 ) consensus binding sequence, although E. coli is only distantly related to the Porphyromonas genus [13].…”