Biological role of an arginine residue present in a histidine-rich peptide which inhibits hemagglutination of Porphyromonas gingivalis

Inhibitory effects of synthetic fragments in histatin 8, having the sequence Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, on hemagglutination by Porphyromonas gingivalis 381 were examined. The hemagglutinating activity was reduced much more by the peptide Lys-His-His-Ser-His-Arg-Gly-Tyr than by the peptides Lys-His-His-Ser-His and/or Lys-Phe-His-Glu-Lys. These results suggest that the arginine residue may have an important role in the inhibition of hemagglutination by P. gingivalis.     

Regulation of protease‐activated receptor‐2 expression in gingival fibroblasts and Jurkat T cells by Porphyromonas gingivalis

Periodontal disease destroys the tooth‐supporting tissues as a result of chronic inflammation elicited by bacterial accumulation on tooth surfaces. Porphyromonas gingivalis is a major periodontal pathogen, with a significant capacity to perturb connective tissue homeostasis and immune responses in the periodontium, attributed to its virulence factors, including a group of secreted cysteine proteases (gingipains). PAR‐2 (protease‐activated receptor‐2) is a G‐protein‐coupled receptor activated upon proteolytic cleavage, mediating intracellular signalling events related to infection and inflammation, such as cytokine production. GF (gingival fibroblasts) and T cells have central roles in periodontal inflammation, which can potentially be mediated by PAR‐2. The aims of this study were to investigate the effects of P. gingivalis on PAR‐2 gene expression in human GF and Jurkat T cells, using quantitative real‐time PCR, and to evaluate the involvement of gingipains. After 6 h of challenge with ascending concentrations of P. gingivalis, PAR‐2 expression was up‐regulated in both cell types by approximately 5‐fold, compared with the control. The P. gingivalis concentration required for maximal PAR‐2 induction was 4‐fold greater in GF than Jurkat T cells. Heat inactivation or chemical inhibition of cysteine proteases abolished the capacity of P. gingivalis to induce PAR‐2 expression in Jurkat T cells. In conclusion, P. gingivalis can induce PAR‐2 expression in GF and Jurkat T cells, potentially attributed to its gingipains. These findings denote that P. gingivalis may perturb the host immune and inflammatory responses by enhancing PAR‐2 expression, thus contributing to the pathogenesis of periodontal disease.     

A universal stress protein of Porphyromonas gingivalis is involved in stress responses and biofilm formation

Porphyromonas gingivalis is recognized as one of the major periodontal pathogens in subgingival plaque, which is implicated in the progression of chronic periodontal disease. We analyzed the role of upsA in P. gingivalis 381 and its uspA-deficient mutant CW301 under various stress conditions. In general, the uspA mutant was less tolerant to a variety of environmental stresses relative to the parental strain. In addition, gene expression of uspA is upregulated during biofilm formation. Biofilm formation of the uspA mutant was also less than that of strain 381. In conclusion, the uspA gene affecting the stress responses of P. gingivalis is required for optimal biofilm formation.     

Prevention of biofilm formation on titanium surfaces modified with conjugated molecules comprised of antimicrobial and titanium-binding peptides

Specific binding of antimicrobial peptides to titanium (Ti) surfaces may serve to prevent biofilm formation, leading to a reduction in peri-implantitis. This study evaluated the binding behavior of conjugated molecules consisting of antimicrobial and hexapeptidic Ti-binding peptides (minTBP-1) using the quartz crystal microbalance (QCM-D) technique, and investigated the effect of modification of Ti surfaces with these peptides on the bioactivity of Porphyromonas gingivalis. Four kinds of peptide were prepared: histatin 5 (DSHAKRHHGYKRKFHEKHHSHRGY), minTBP-1 + histatin 5 (RKLPDAPDSHAKRHHGYKRKFHEKHHSHRGY), lactoferricin (FQWQRNMRKVR), and minTBP-1 + lactoferricin (RKLPDAPGGFQWQRNMRKVR). The QCM-D analysis demonstrated that significantly larger increases in peptide adsorption were observed in the conjugated peptides than in antimicrobial peptides alone. In addition, ATP activity in P. gingivalis in peptide-modified specimens significantly decreased compared to that in the Ti control. These results indicate that surface modification with conjugated molecules consisting of antimicrobial and Ti-binding peptides is a promising method for reduction of biofilm formation on Ti surfaces.     

Porphyromonas gingivalis exacerbates the progression of fatty liver disease via CD36-PPARγ pathway

Periodontal diseases have been reported to have a multidirectional association with metabolic disorders. We sought to investigate the correlation between periodontitis and diabetes or fatty liver disease using HFD-fed obese mice inoculated with P. gingivalis. Body weight, alveolar bone loss, serological biochemistry, and glucose level were determined to evaluate the pathophysiology of periodontitis and diabetes. For the evaluation of fatty liver disease, hepatic nonalcoholic steatohepatitis (NASH) was assessed by scoring steatosis, inflammation, hepatocyte ballooning and the crucial signaling pathways involved in liver metabolism were analyzed. The C-reactive protein (CRP) level and NASH score in P. gingivalis-infected obese mice were significantly elevated. Particularly, the extensive lobular inflammation was observed in the liver of obese mice infected with P. gingivalis. Moreover, the expression of metabolic regulatory factors, including peroxisome proliferator-activated receptor γ (Pparγ) and the fatty acid transporter Cd36, was up-regulated in the liver of P. gingivalis-infected obese mice. However, inoculation of P. gingivalis had no significant influence on glucose homeostasis, insulin resistance, and hepatic mTOR/AMPK signaling. In conclusion, our results indicate that P. gingivalis can induce the progression of fatty liver disease in HFD-fed mice through the upregulation of CD36-PPARγ axis.     

Proteomics-based analysis of a counter-oxidative stress system in Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative anaerobic pathogen associated with chronic periodontitis. Although anaerobic, P. gingivalis exhibits a high degree of aerotolerance, which enables it to survive within periodontal pockets. The aim of the present study was to examine the effect of oxidative stress on protein expression in P. gingivalis to obtain a better understanding of the mechanism underlying its aerotolerance. To accomplish this, P. gingivalis cells were grown under conditions of hemin limitation (0.01 microg/mL) to avoid the oxygen protective effect of hemin on oxidative stress. The proteins were then extracted from cultures either left untreated or subjected to oxidative stress and separated by 2-DE. The resultant protein expression profiles were examined by image scanning, and those found to differ depending on the presence or absence of aeration were subjected to MALDI-MS and then analyzed using the ORF database of P. gingivalis W83 from The Institute of Genomic Research. Oxidative stress was found to affect the expression of numerous proteins in P. gingivalis cells. In particular, the levels of HtpG, GroEL, DnaK, AhpC, TPR domain protein, and trigger factor were substantially increased.     

Kinetic analysis of PPi-dependent phosphofructokinase from Porphyromonas gingivalis

We have previously cloned the gene encoding a pyrophosphate-dependent phosphofructokinase (PFK), designated PgPFK, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease. In this study, recombinant PgPFK was purified to homogeneity, and biochemically characterized. The apparent K(m) value for fructose 6-phosphate was 2.2 mM, which was approximately 20 times higher than that for fructose 1,6-bisphosphate. The value was significantly greater than any other described PFKs, except for Amycolatopsis methanolica PFK which is proposed to function as a fructose 1,6 bisphosphatase (FBPase). The PgPFK appears to serves as FBPase in this organism. We postulate that this may lead to the gluconeogenic pathways to synthesize the lipopolysaccharides and/or glycoconjugates essential for cell viability.     

PG27 is a novel membrane protein essential for a Porphyromonas gingivalis protease secretion system

Porphyromonas gingivalis secretes endopeptidase gingipains, which are important virulence factors of this bacterium. Gingipains are transported across the inner membrane via the Sec system, followed by transport across the outer membrane via an unidentified pathway. The latter transport step is suggested to be mediated via a novel protein secretion pathway. In the present study, we report a novel candidate as an essential factor for the latter transport step. The PG0027 gene of P. gingivalis W83 encodes novel protein PG27. In a PG0027 deletion mutant (83K10), the activities of Arg-gingipain and Lys-gingipain were severely reduced, while the activities of secreted exopeptidases DPPIV, DPP-7, and PTP-A were unaffected. Protein localization was investigated by cell-surface biotinylation, subcellular fractionation, and immunoblot analysis. In the wild-type W83, Arg-gingipains in membrane fraction were detected as cell surface proteins. In contrast, in 83K10, Arg-gingipains were trapped in the periplasm and hardly secreted into an extracellular milieu. PG27 was suggested to be exposed to the cell surface by a cell surface biotinylation experiment; however, PG27 was detected in both inner and outer membrane fractions by subcellular fractionation experiments. Taken together, we suggest that PG27 is a unique membrane protein essential for a novel secretion pathway.     

对氨基苯甲酸对牙龄卟啉单胞菌胰酶样蛋白酶活性的影响

目的：测定不同浓度PABA对牙龄卟啉单胞菌胰酶样蛋白酶（TLP）活性的影响,以探讨血链球菌在龈下菌微生态平衡中的作用。方法：在1/2浓度的BH培养基中加入不同浓度的PABA和一定浊度的P.gingivalis,行常规培养。测定培养物上清液和菌细胞中TLP活性及蛋白质含量,计算TLP的比活。结果：PABA对P.gingivalis TLP的比活产生促进作用。结论：PABA影响P.gingivalis TLP的活性提示血链球菌可能对龈下菌斑的微生态平衡具调节作用。     

Structural and functional analyses of the Porphyromonas gingivalis type IX secretion system PorN protein

Porphyromonas gingivalis, the major human pathogen bacterium associated with periodontal diseases, secretes virulence factors through the Bacteroidetes-specific type IX secretion system (T9SS). Effector proteins of the T9SS are recognized by the complex via their conserved C-terminal domains (CTDs). Among the 18 proteins essential for T9SS function in P. gingivalis, PorN is a periplasmic protein that forms large ring-shaped structures in association with the PorK outer membrane lipoprotein. PorN also mediates contacts with the PorM subunit of the PorLM energetic module, and with the effector’s CTD. However, no information is available on the PorN structure and on the implication of PorN domains for T9SS assembly and effector recognition. Here we present the crystal structure of PorN at 2.0-Å resolution, which represents a novel fold with no significant similarity to any known structure. In agreement with in silico analyses, we also found that the N- and C-terminal regions of PorN are intrinsically disordered. Our functional studies showed that the N-terminal disordered region is involved in PorN dimerization while the C-terminal disordered region is involved in the interaction with PorK. Finally, we determined that the folded PorN central domain is involved in the interaction with PorM, as well as with the effector’s CTD. Altogether, these results lay the foundations for a more comprehensive model of T9SS architecture and effector transport.