Context: Although oral infectious diseases have been attributed to bacteria, drug treatments remain ineffective because bacteria and their products exist as biofilms. Cationic liposomes have been suggested to electrostatically interact with the negative charge on the bacterial surface, thereby improving the effects of conventional drug therapies. However, the electrostatic interaction between oral bacteria and cationic liposomes has not yet been examined in detail.Objective: The aim of the present study was to examine the behavior of cationic liposomes and Streptococcus mutans in planktonic cells and biofilms.Materials and methods: Liposomes with or without cationic lipid were prepared using a reverse-phase evaporation method. The zeta potentials of conventional liposomes (without cationic lipid) and cationic liposomes were −13 and 8 mV, respectively, and both had a mean particle size of approximately 180 nm. We first assessed the interaction between liposomes and planktonic bacterial cells with a flow cytometer. We then used a surface plasmon resonance method to examine the binding of liposomes to biofilms. We confirmed the binding behavior of liposomes with biofilms using confocal laser scanning microscopy.Results: The interactions between cationic liposomes and S. mutans cells and biofilms were stronger than those of conventional liposomes. Microscopic observations revealed that many cationic liposomes interacted with the bacterial mass and penetrated the deep layers of biofilms.Discussion and conclusion: In this study, we demonstrated that cationic liposomes had higher affinity not only to oral bacterial cells, but also biofilms than conventional liposomes. This electrostatic interaction may be useful as a potential drug delivery system to biofilms.
The aim of this study was to examine the ability of a poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butylmethacrylate-co-2-methacryloyloxyethyloxy-p-azidobenzoate) (PMBPAz) coating on polymethyl methacrylate (PMMA)-based dental resin to inhibit bacterial plaque formation, as well as the polymer's durability against water soaking and chemical exposure.Successful application of PMBPAz on PMMA surfaces was confirmed by x-ray photoelectron spectroscopy (XPS) and measuring the static air contact angle in water. The anti-adhesive effects to bacterial plaque were evaluated using Streptococcus mutans biofilm formation assay. The mechanical and chemical durabilities of the PMBPAz coating on the PMMA surfaces were examined using soaking and immersion tests, respectively.XPS signals for phosphorus and nitrogen atoms and hydrophilic status on PMMA surfaces treated with PMBPAz were observed, indicating the presence of the polymer on the substrates. The treated PMMA surfaces showed significant inhibition of S mutans biofilm formation compared to untreated surfaces. The PMBPAz coating was preserved after water soaking and chemical exposure. In addition, water soaking did not decrease the ability of treated PMMA to inhibit biofilm formation compared to treated PMMA specimens not subjected to water soaking.This study suggests that PMBPAz coating may represent a useful modification to PMMA surfaces for inhibiting denture plaque accumulation.
Food allergy is a life-threatening response to specific foods, and microbiota imbalance (dysbiosis) in gut is considered a cause of this disease. Meanwhile, the host immune response also plays an important role in the disease. Notably, interleukin 33 (IL-33) released from damaged or necrotic intestinal epithelial cells facilitates IL-2-producing CD4 helper T (Th2) responses. However, causal relationships between the gut and oral dysbiosis and food allergy remain unknown. In this study, we analyzed effects of gut and oral dysbiosis on development of food allergy. A murine model of food allergy was established via ovalbumin (OVA) injection in BALB/c mice. Viable fecal bacteria were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). il33 expression in colon-26 mouse colon cells stimulated by isolated fecal bacteria was quantified by real-time PCR. Intestinal T cells from the mice were analyzed by flow cytometry. Salivary IgA levels were quantified by enzyme-linked immunosorbent assay (ELISA), and IgA-bound oral bacteria were detected by flow cytometry. Among fecal bacteria, the abundance of Citrobacter sp. increased in the feces of allergic mice and induced il33 expression in colon-26 cells. Orally administered Citrobacter koseri JCM1658 exacerbated systemic allergic symptoms and reduced intestinal Th17 cells. Salivary IgA and IgA-bound oral bacteria increased in the allergic mice. Based on the results described above, food allergy induced both gut and oral dysbiosis. Citrobacter sp. aggravated allergy symptoms by inducing IL-33 release from intestinal epithelial cells.
Oral streptococci, including cariogenic bacterium Streptococcus mutans, comprise a large percentage of human supragingival plaque, which contacts both tooth surfaces and gingiva. Eukaryotic cells are able to take up macromolecules and particles, including bacteria, by endocytosis. Increasing evidence indicates endocytosis may be used as an entry process by bacteria. We hypothesized that some endocytosed bacteria might survive and obtain nutrients, such as amino acids, until they are killed. To verify this hypothesis, we focused on bacterial utilization of branched-chain amino acids (BCAAs; isoleucine, leucine and valine) in host cells. A branched-chain aminotransferase, IlvE (EC 2.6.1.42), has been suggested to play an important role in internal synthesis of BCAAs in S. mutans UA159. Therefore, we constructed an ilvE-deficient S. mutans 109c strain and confirmed that it had similar growth behavior as reported previously. 14C radioactive leucine uptake assays showed that ilvE-deficient S. mutans took up more leucine both inside and outside of host cells. We further clarified that a relative decrease of BCAAs in host cells caused enhanced endocytic and autophagic activity. In conclusion, S. mutans is endocytosed by host cells and may survive and obtain nutrients, such as BCAAs, inside the cells, which might affect cellular functions of host cells.
Summary Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps ( NET s). These are extracellular net‐like fibers comprising DNA and antimicrobial components such as histones, LL ‐37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NET s to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram‐negative periodontopathogenic bacteria, Porphyromonas gingivalis , Prevotella intermedia , Fusobacterium nucleatum , and Aggregatibacter actinomycetemcomitans . We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD , putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA ‐ and nucD ‐encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA , which required both Mg 2+ and Ca 2+ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NET s.
Dextranase (Dex) is an enzyme that hydrolyzes glucan, a polymer of glucose synthesized from sucrose by glucosyltransferases (GTFs). By comparing amino acid sequences of Dexs and GTFs, we found that the Dex enzymes of Streptococcus mutans , Streptococcus sobrinus , Streptococcus downei and Streptococcus salivarius had similar amino acid sequences to those of the catalytic sites of GTFs of mutans streptococci. We therefore examined the amino acid essential in Dex catalysis by molecular genetic approaches in this study. Site‐directed mutagenesis was used to convert the Asp‐385 of the Dex molecule of S. mutans Ingbritt to Glu, Asn, Thr or Val. Replacement of Asp‐385 with any of the amino acids resulted in complete disappearance of Dex activity. However, replacement of other Asp residues did not affect the enzyme activity. The inactive enzymes still retained dextran‐binding ability. These results suggest that Asp‐385 of the Dex of S. mutans Ingbritt was essential for enzyme activity and the catalytic and substrate‐binding sites were located at different sites within the Dex molecule.
