Guy Gagnon

Tetracyclines and Chemically Modified Tetracycline-3 (CMT-3) Modulate Cytokine Secretion by Lipopolysaccharide-Stimulated Whole Blood

In addition to their bacteriostatic effect, tetracyclines, which are often used in the treatment of periodontitis, also present anti-inflammatory properties. In the present study, we investigated the effects of tetracycline (TC), doxycycline (doxy), and chemically modified tetracycline-3 (CMT-3) on the production of pro-inflammatory mediators and matrix metalloproteinases (MMPs) in an ex vivo human whole blood (WB) model stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). WB samples obtained from three periodontitis patients and six healthy subjects were stimulated with P. gingivalis LPS in the absence and presence of TC, doxy, or CMT-3. The secretion of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), MMP-8, and MMP-9 by the WB samples was determined using enzyme-linked immunosorbent assays. P. gingivalis LPS significantly increased the secretion of all cytokines and MMPs tested. While we observed inter-patient variations, TC, doxy, and CMT-3 caused reductions of LPS-induced cytokine secretion to various degrees. TC, doxy, and CMT-3 had no significant effect on MMP-8 and MMP-9 secretion by LPS-stimulated WB samples. In conclusion, we used a human WB model that takes into consideration relevant in vivo immune cell interactions in the presence of plasma proteins to show that TC, doxy, and CMT-3 can reduce the production of pro-inflammatory mediators. This property may contribute to the clinically proven benefits of these molecules in the treatment of periodontitis and other chronic inflammatory diseases.

Cloning, sequencing and expression in Escherichia coli of the ptsI gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system from Streptococcus salivarius

We present the cloning and sequencing of the ptsI gene, encoding enzyme I (EI) of the phosphoenolpyruvate (PEP): sugar phosphotransferase (PTS) transport system from Streptococcus salivarius. The ptsI gene corresponds to an open reading frame of 1731 nucleotides, which translates into a putative 577-amino acid (aa) protein with a M(r) of 62,948 and a pI of 4.49. The EI was produced in Escherichia coli under the control of its own promoter located immediately upstream of ptsI, a situation never previously reported for any other gene coding for an EI. The deduced aa sequence of the S. salivarius EI shows a high degree of similarity with the E. coli EI and the EI moiety of the multiphosphoryl transfer protein from Rhodobacter capsulatus. The S. salivarius EI also shares a highly conserved aa cluster with a non-PTS protein, the maize pyruvate:orthophosphate dikinase. The conserved cluster is located in a domain which is hypothesized to be the PEP-binding site.

Phosphotransferase system of Streptococcus salivarius: characterization of the ptsH gene and its product.

The Streptococcus salivarius ptsH gene encoding histidine-containing phosphocarrier protein (HPr) of the phosphotransferase system (PTS) has been cloned, sequenced, and found to be part of a ptsH, ptsI operon. Upstream from ptsH, putative -35 and -10 boxes and a Shine-Dalgarno sequence highly similar to the Escherichia coli consensus regulatory elements were identified. A second promoter, located in the ptsH coding sequence was also observed and is sufficient for the expression of the S. salivarius ptsI gene, encoding enzyme I of the PTS in E. coli [Gagnon et al., Gene 121 (1992) 71-78]. The amino acid sequence of S. salivarius HPr, inferred from the ptsH sequence, shared identity varying between 37 and 76% with known HPr from other bacteria. Moreover, the S. salivarius HPr shared 78% identity with an HPr-like protein of Aspergillus fumigatus, a eukaroytic mold that does not possess a functional PTS. Expression analysis of S. salivarius HPr in E. coli demonstrated that (i) S. salivarius ptsH is expressed in E. coli under the control of its own promoter, (ii) S. salivarius HPr synthesized by E. coli is completely processed by methionine aminopeptidase, and (iii) S. salivarius HPr is phosphorylated in vivo by E. coli enzyme I. It was also observed that, in E. coli, the copy number of pUC18 bearing S. salivarius ptsH was reduced more than 25-fold, as compared to pUC18 without an insertion.

Positive selection for resistance to 2-deoxyglucose gives rise, in Streptococcus salivarius, to seven classes of pleiotropic mutants, including ptsH and ptsI missense mutants.

We have used the toxic non‐metabolizabie glucose/ mannose analogue 2‐deoxygiucose to isolate a comprehensive collection of mutants of the phosphoenoipyruvate:sugar phosphotransferase system from Streptococcus salivarius. To increase the range of possible mutations, we isolated spontaneous mutants on different media containing 2‐deoxyglucose and various metabolizable sugars, either lactose, meli‐biose, galactose or fructose. We found that the frequency at which 2‐deoxygiucose‐resistant mutants Were isolated varied according to the growth substrate. The highest frequency was obtained with the combination galactose and 2‐deoxygiucose and was 15‐fold higher than the rate observed with the mixture melibiose and 2‐deoxygiucose, the combination that gave the lowest frequency. By combining results from: (i) Western biol analysis of IIIMan, a specific component of the phosphoenolpyruvate:mannose phosphotransferase system in S. salivarius; (ii) rocket immunoelectrophoresis of HPr and EI, the two general energy‐coupling proteins of the phosphotransferase system; and (iii) from gene sequencing, mutants could be assigned to seven classes. Class 1 was composed of strains devoid of IIIManL, a low‐molecular‐weight form of IIIManL (35200), class 2 was composed of strains exhibiting a reduced level of IIIManL, class 3 was composed of strains devoid of both forms of IIIMan (IIIManL as well as IIIManH, the high‐molecular‐weight form of IIIMan (38900)), class 4 was composed of mutants bearing a mutation in ptsH, the gene encoding HPr, class 5 was composed of mutants bearing a mutation in ptsl, the gene encoding EI, class 6 was composed of 2‐deoxygiucose‐resistant strains without any apparent defect in PTS components, and class 7 was composed of strains possessing both forms of IIIMan but abnormal levels of HPr and/or EI without any mutation in the ptsH and/or the ptsI genes. Preliminary characterization of representative strains of each class is reported.

Influence of glycemic control on Epstein-Bar and Cytomegalovirus infection in periodontal pocket of type 2 diabetic subjects

OBJECTIVE The aim of the present study was to evaluate the influence of glycemic control on the frequency of Epstein-Bar (EBV) and Cytomegalovirus (CMV) in periodontal pockets of type 2 diabetic subjects with chronic periodontitis. DESIGN Forty-six subjects presenting generalized chronic periodontitis and type 2 diabetes mellitus (DM) were selected for this study. Polymerase chain reaction (PCR) was used to determine the presence of EBV and CMV in shallow [Probing Depth (PD)≤3mm], moderate (PD=4-6mm) and deep (PD>7mm) pockets. HbA1c levels ≤7%, >7 to <10%, and ≥10% defined good, moderate and poor glycemic control, respectively. RESULTS Higher frequency of EBV was found in the shallow pockets of the subjects with poor glycemic control (p<0.05; chi-square test). Moreover, EBV-free subjects presented moderate or good glycemic control. Glycemic control did not influence the frequency of CMV in all pocket categories. CONCLUSION Poor glycemic control in type 2 diabetic subjects can increase the occurrence of EBV in shallow periodontal pockets.

Periodontal Disease is Not Associated With Preeclampsia in Canadian Pregnant Women

BACKGROUND The findings from the studies on the relationship between periodontal disease and preeclampsia are inconsistent. The objective of this study is to examine the relationship between periodontal disease and preeclampsia. METHODS A multicenter case-control study was conducted in Quebec, Canada. Preeclampsia was defined as blood pressure ≥140/90 mm Hg and ≥1+ proteinuria after 20 weeks of gestation. Periodontitis was defined as the presence of ≥4 sites with a probing depth ≥5 mm and a clinical attachment loss ≥3 mm at the same sites. RESULTS A total of 92 preeclamptic women and 245 controls were analyzed. The percentage of periodontal disease was 18.5% in preeclamptic women and 19.2% in normotensive women (crude odds ratio [OR] = 0.96, 95% confidence interval [CI] = 0.52 to 1.77). After adjusting for confounding variables, periodontitis remained not associated with preeclampsia (adjusted OR = 1.13, 95% CI = 0.59 to 2.17). CONCLUSION This study does not support the hypothesis of an association between periodontal disease and preeclampsia.

Regulation of ptsH and ptsl gene expression in Streptococcus salivarius ATCC 25975

The transcriptional regulation of the Streptococcus salivarius ptsH and ptsl genes coding for the general energy‐coupling proteins HPr and enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system were investigated. These genes form an operon with the gene order ptsH–ptsl. Three distinct mRNA species were detected: a 0.5 kb transcript specific for ptsH, and two long transcripts (2.2 arid 2.4 kb) covering the whole pts operon. Transcription of all these mRNAs initiated at the same nucleotide located 9 bp downstream from a promoter located immediately upstream from the ptsH gene. The presence of a high‐energy stem–loop structure (T0) located at the begining of ptsl was responsible for the premature transcrrption termination generating the 0.5 kb ptsH‐specific transcript. The long transcripts ended in the poly(U) region of two rho‐independent‐like terminators (T1 and T2) at the 3′ end of ptsl. Studies with a 2‐deoxyglucose‐resistant spontaneous mutant of S. salivarius (L26) that produces an HPr–El fusion protein suggest that the regulation of HPr and El expression involves transcriptional as well as translational mechanisms.

Response of periodontitis and healthy patients in a Porphyromonas gingivalis-stimulated whole-blood model

AIM   To investigate the inflammatory responses of periodontitis patients and healthy patients in a whole-blood model stimulated with Porphyromonas gingivalis. METHODS   Whole blood collected from 17 periodontitis patients and six healthy patients was stimulated with Porphyromonas gingivalis cells. The secretion of cytokines and matrix metalloproteinases was quantified by enzyme-linked immunosorbent assay. An analysis of covariance with the ancova model was used to evaluate the significance of differences in secreted host molecules by whole blood from the periodontitis and healthy groups. RESULTS   Porphyromonas gingivalis induced the secretion of interleukin-1β, interleukin-6, interleukin-8, tumor necrosis factor-α, monocyte chemoattractant protein-1, interferon inducible protein-10 by whole blood from patients in the periodontitis and healthy groups. Matrix metalloproteinase-8 and -9 levels secreted by whole blood also increased following stimulation. No significant differences (P < 0.05) in the amounts of secreted host molecules were observed between periodontitis and healthy patients. CONCLUSION   This study suggests that Porphyromonas gingivalis can provoke an inflammatory response and promote the progression of periodontitis by inducing the secretion of high levels of cytokines and matrix metalloproteinases by a mixed leukocyte population. However, the whole-blood model did not reveal any significant differences in the inflammatory response between periodontitis patients (n = 17) and periodontally-healthy patients (n = 6).