Background: Dipeptidyl peptidases (DPPs) are required for protein metabolism in Porphyromonas gingivalis. Results: Asp/Glu-specific novel DPP (DPP11) was discovered and characterized. Conclusion: DPP11 ensures efficient degradation of oligopeptide substrates in Gram-negative anaerobic rods. Significance: This observation suggests further variation of substrate specificity in the DPP members.
Background: Dipeptidyl-peptidases (DPPs) are key factors for amino acid metabolism and bacterial growth of asaccharolytic Porphyromonas gingivalis. Results: DPP5, which is specific for Ala and hydrophobic residues, is expressed in the periplasmic space of P. gingivalis. Conclusion: DPP5 was discovered in prokaryotes for the first time. Significance: The discovery of DPP5 expands understanding of amino acid and energy metabolism in prokaryotes.
BackgroundThe effects of tongue cleaning on reconstruction of bacterial flora in dental plaque and tongue coating itself are obscure. We assessed changes in the amounts of total bacteria as well as Fusobacterium nucleatum in tongue coating and dental plaque specimens obtained with and without tongue cleaning.MethodsWe conducted a randomized examiner-blind crossover study using 30 volunteers (average 23.7 ± 3.2 years old) without periodontitis. After dividing randomly into 2 groups, 1 group was instructed to clean the tongue, while the other did not. On days 1 (baseline), 3, and 10, tongue coating and dental plaque samples were collected after recording tongue coating score (Winkel tongue coating index: WTCI). After a washout period of 3 weeks, the same examinations were performed with the subjects allocated to the alternate group. Genomic DNA was purified from the samples and applied to SYBR® Green-based real-time PCR to quantify the amounts of total bacteria and F. nucleatum.ResultsAfter 3 days, the WTCI score recovered to baseline, though the amount of total bacteria in tongue coating was significantly lower as compared to the baseline. In plaque samples, the bacterial amounts on day 3 and 10 were significantly lower than the baseline with and without tongue cleaning. Principal component analysis showed that variations of bacterial amounts in the tongue coating and dental plaque samples were independent from each other. Furthermore, we found a strong association between amounts of total bacteria and F. nucleatum in specimens both.ConclusionsTongue cleaning reduced the amount of bacteria in tongue coating. However, the cleaning had no obvious contribution to inhibit dental plaque formation. Furthermore, recovery of the total bacterial amount induced an increase in F. nucleatum in both tongue coating and dental plaque. Thus, it is recommended that tongue cleaning and tooth brushing should both be performed for promoting oral health.
V8 protease, a member of the glutamyl endopeptidase I family, of Staphylococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coli expression system for GluV8, as well as its homologue from Staphylococcus epidermidis (GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. C‐terminal His6‐tagged proGluSE was successfully expressed from the full‐length sequence as a soluble form. By contrast, GluV8 was poorly expressed by the system as a result of autodegradation; however, it was efficiently obtained by swapping its preprosegment with that of GluSE, or by the substitution of four residues in the GluV8 prosequence with those of GluSE. The purified proGluV8 was converted to the mature form in vitro by thermolysin treatment. The prosegment was essential for the suppression of proteolytic activity, as well as for the correct folding of GluV8, indicating its role as an intramolecular chaperone. Furthermore, the four amino acid residues at the C‐terminus of the prosegment were sufficient for both of these roles. In vitro mutagenesis revealed that Ser237 was essential for proteolytic activity, and that Val69 was indispensable for the precise cleavage by thermolysin and was involved in the proteolytic reaction itself. This is the first study to express quantitatively GluV8 in E. coli, and to demonstrate explicitly the intramolecular chaperone activity of the prosegment of glutamyl endopeptidase I.
Severe periodontitis is known to aggravate diabetes mellitus, though molecular events related to that link have not been fully elucidated. Porphyromonas gingivalis, a major pathogen of periodontitis, expresses dipeptidyl peptidase 4 (DPP4), which is involved in regulation of blood glucose levels by cleaving incretins in humans. We examined the enzymatic characteristics of DPP4 from P. gingivalis as well as two other periodontopathic bacteria, Tannerella forsythia and Prevotella intermedia, and determined whether it is capable of regulating blood glucose levels. Cellassociated DPP4 activity was found in those microorganisms, which was effectively suppressed by inhibitors of human DPP4, and molecules sized 73 kDa in P. gingivalis, and 71 kDa in T. forsythia and P. intermedia were immunologically detected. The k cat /K m values of recombinant DPP4s ranged from 721 Ϯ 55 to 1,283 Ϯ 23 M Ϫ1 s Ϫ1 toward Gly-Pro-4-methylcoumaryl-7-amide (MCA), while those were much lower for His-Ala-MCA. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis showed His/Tyr-Ala dipeptide release from the N termini of incretins, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide, respectively, with the action of microbial DPP4. Moreover, intravenous injection of DPP4 into mice decreased plasma active GLP-1 and insulin levels, accompanied by a substantial elevation in blood glucose over the control after oral glucose administration. These results are the first to show that periodontopathic bacterial DPP4 is capable of modulating blood glucose levels the same as mammalian DPP4; thus, the incidence of periodontopathic bacteremia may exacerbate diabetes mellitus via molecular events of bacterial DPP4 activities.
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