Porphyromonas species are closely associated with companion animal periodontitis which is one of the most common diseases in dogs and cats and leads to serious systemic diseases if left untreated. In this study, we evaluated the antimicrobial effects and mode of action of sodium tripolyphosphate (polyP3, Na5P3O10), a food additive with proven safety, using three pathogenic Porphyromonas species. The minimum inhibitory concentrations (MICs) of polyP3 against Porphyromonas gulae, Porphyromonas cansulci, and Porphyromonas cangingivalis were between 500 and 750 mg/L. PolyP3 significantly decreased viable planktonic cells as well as bacterial biofilm formation, even at sub-MIC concentrations. PolyP3 caused bacterial membrane disruption and this effect was most prominent in P. cangingivalis, which was demonstrated by measuring the amount of nucleotide leakage from the cells. To further investigate the mode of action of polyP3, high-throughput whole-transcriptome sequencing was performed using P. gulae. Approximately 30% of the total genes of P. gulae were differentially expressed by polyP3 (> 4-fold, adjusted p value < 0.01). PolyP3 influenced the expression of the P. gulae genes related to the biosynthesis of thiamine, ubiquinone, and peptidoglycan. Collectively, polyP3 has excellent antibacterial effects against pathogenic Porphyromonas species and can be a promising agent to control oral pathogenic bacteria in companion animals.
Animals ; Bacteria ; Biofilms ; Cats ; Dogs ; Food Additives ; Friends ; Humans ; Membranes ; Microbial Sensitivity Tests ; Peptidoglycan ; Periodontitis ; Pets ; Plankton ; Porphyromonas ; Sodium ; Thiamine ; Ubiquinone

BACKGROUND/AIMS: A Subset of patients with irritable bowel syndrome (IBS) may have mild inflammation due to immune activation. Toll-like receptors (TLRs) and cytokines may cause intestinal inflammation. We studied their expression in relation to gut microbiota. METHODS: Expression of TLRs and cytokines was assessed in 47 IBS patients (Rome III) and 25 controls using quantitative real-time polymerase chain reaction. Immunohistochemistry was further performed to confirm the expression of TLR-4 and TLR-5. RESULTS: Of 47 patients with IBS, 20 had constipation (IBS-C), 20 diarrhea (IBS-D), and 7 unclassified (IBS-U). The mRNA levels of TLR-4 and TLR-5 were up-regulated in IBS patients than controls (P = 0.013 and P < 0.001, respectively). Expression of TLR-4 and TLR-5 at protein level was 4.2-folds and 6.6-folds higher in IBS-D than controls. The mRNA levels of IL-6 (P = 0.003), C-X-C motif chemokine ligand 11 (CXCL-11) (P < 0.001) and C-X-C motif chemokine receptor 3 (CXCR-3) (P < 0.001) were higher among IBS patients than controls. Expression of IL-6 (P = 0.002), CXCL-11 (P < 0.001), and CXCR-3 (P < 0.001) were up-regulated and IL-10 (P = 0.012) was down-regulated in IBS-D patients than controls. Positive correlation was seen between TLR-4 and IL-6 (P = 0.043), CXCR-3, and CXCL-11 (P = 0.047), and IL-6 and CXCR-3 (P = 0.003). Stool frequency per week showed positive correlation with mRNA levels of TLR-4 (P = 0.016) and CXCR-3 (P = 0.005), but inversely correlated with IL-10 (P = 0.002). Copy number of Lactobacillus (P = 0.045) and Bifidobacterium (P = 0.011) showed correlation with IL-10 in IBS-C, while Gram-positive (P = 0.031) and Gram-negative bacteria (P = 0.010) showed correlation with CXCL-11 in IBS-D patients. CONCLUSIONS: Altered immune activation in response to dysbiotic microbiota may promote intestinal inflammation in a subset of patients with IBS.
Bifidobacterium ; Constipation ; Cytokines ; Diarrhea ; Gastrointestinal Microbiome ; Gram-Negative Bacteria ; Humans ; Immunohistochemistry ; Inflammation ; Interleukin-10 ; Interleukin-6 ; Irritable Bowel Syndrome ; Lactobacillus ; Microbiota ; Peptidoglycan ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Toll-Like Receptors

Nucleotide-binding domain 1 (Nod1) is a cytosolic receptor that is responsible for the recognition of a bacterial peptidoglycan motif containing meso-diaminophimelic acid. In this study, we sought to identify the role of Nod1 in host defense in vivo against pulmonary infection by multidrug resistant Acinetobacter baumannii. Wildtype (WT) and Nod1-deficient mice were intranasally infected with 3×107 CFU of A. baumannii and sacrificed at 1 and 3 days post-infection (dpi). Bacterial CFUs, cytokines production, histopathology, and mouse β-defensins (mBD) in the lungs of infected mice were evaluated. The production of cytokines in response to A. baumannii was also measured in WT and Nod1-deficient macrophages. The bacterial clearance in the lungs was not affected by Nod1 deficiency. Levels of IL-6, TNF-α, and IL-1β in the lung homogenates were comparable at days 1 and 3 between WT and Nod1-deficient mice, except the TNF-α level at day 3, which was higher in Nod1-deficient mice. There was no significant difference in lung pathology and expression of mBDs (mBD1, 2, 3, and 4) between WT and Nod1-deficient mice infected with A. baumannii. The production of IL-6, TNF-α, and NO by macrophages in response to A. baumannii was also comparable in WT and Nod1-deficient mice. Our results indicated that Nod1 does not play an important role in host immune responses against A. baumannii infection.
Acinetobacter baumannii* ; Acinetobacter* ; Animals ; Cytokines ; Cytosol ; Interleukin-6 ; Lung ; Macrophages ; Mice* ; Pathology ; Peptidoglycan

Antibiotic resistance is a major global concern that primarily affects public health. Texiobactin is a newly discovered antibiotic produces by soil microbes isolated from natural environment. Drug is active against Gram-positive bacteria as it inhibits biosynthesis of peptidoglycan. Infection of methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae in mice elicits a good response reduce bacterial load. Although extensive efforts have been made to discover new antibiotics but results are still not satisfactory to meet the demands of public health. Recently it has been shown that the discovery of texiobactin by iChip will be a great stone mile to discover more antibiotics.
Animals ; Anti-Bacterial Agents ; Bacterial Load ; Drug Resistance, Microbial ; Gram-Positive Bacteria ; Methicillin-Resistant Staphylococcus aureus ; Mice ; Peptidoglycan ; Public Health ; Soil ; Streptococcus pneumoniae

Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca2+, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 microg/ml BG, 100 microg/ml peptidoglycan (PGN), or 10 microM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca2+ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca2+ uniporter has an important regulatory role in BG-induced mast cell degranulation.
Animals ; Calcimycin ; Calcium* ; Cytosol ; Exocytosis ; Inflammation ; Ion Transport* ; Mast Cells* ; Membrane Potentials ; Mice* ; Mitochondria ; Peptidoglycan ; Ruthenium Red ; Shock

Peptidoglycan (PG), the gram positive bacterial pathogen-associated molecular patterns (PAMP), is detected in a high proportion in macrophage-rich atheromatous regions, and expression of chemokine CXCL8, which triggers monocyte arrest on early atherosclerotic endothelium, is elevated in monocytes/macrophages in human atherosclerotic lesion. The aim of this study was to investigate whether PG induced CXCL8 expression in the cell type and to determine cellular signaling pathways involved in that process. Exposure of THP-1 cell, human monocyte/macrophage cell line, to PG not only enhanced CXCL8 release but also profoundly induced il8 gene transcription. PG-induced release of CXCL8 and induction of il8 gene transcription were blocked by OxPAPC, an inhibitor of TLR-2/4 and TLR4, but not by polymyxin B, an inhibitor of LPS. PG-mediated CXCL8 release was significantly attenuated by inhibitors of PI3K-Akt-mTOR pathways. PKC inhibitors, MAPK inhibitors, and ROS quenchers also significantly attenuated expression of CXCL8. The present study proposes that PG contributes to inflammatory reaction and progression of atherosclerosis by inducing CXCL8 expression in monocytes/macrophages, and that TLR-2, PI3K-Akt-mTOR, PKC, ROS, and MAPK are actively involved in the process.
Atherosclerosis ; Cell Line ; Endothelium ; Humans ; Interleukin-8 ; Monocytes ; Peptidoglycan* ; Polymyxin B

The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of gram-positive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells.
Bacteria ; Bacterial Toxins ; Calcium ; Calcium Signaling* ; Calcium-Transporting ATPases ; Cytokines ; Epithelial Cells ; Epithelium* ; Gram-Positive Bacteria ; Humans ; Inflammation ; Inositol 1,4,5-Trisphosphate Receptors ; Interleukin-8* ; Peptidoglycan* ; Periodontal Diseases ; Phospholipases ; Reticulum ; Thapsigargin ; Toll-Like Receptor 2 ; Toll-Like Receptors ; Type C Phospholipases

PURPOSE: The present study aimed to determine the role played by beta-defensin 124 (DEFB124) in the innate immunity of prostate epithelial RWPE-1 cells during bacterial infection. MATERIALS AND METHODS: The expression of DEFB124 was examined by quantitative real-time polymerase chain reaction (PCR), Western blotting, and immunocytochemistry. Enzyme-linked immunosorbent assays and quantitative real-time PCR were performed to determine the production of cytokines and chemokines. Western blotting and chromatin immunoprecipitation studies were performed to assess the interaction between DEFB124 and nuclear factor-kappa B (NF-kappaB) in peptidoglycan (PGN)-stimulated RWPE-1 cells. By chemotaxis assay, we assessed the effect of DEFB124 on the migration of monocytes. RESULTS: Exposure to PGN induced DEFB124 upregulation and NF-kappaB activation through IkappaBalpha phosphorylation and IkappaBalpha degradation. Bay11-7082, an NF-kappaB inhibitor, blocked PGN-induced DEFB124 production. Also, NF-kappaB was shown to be a direct regulator and to directly bind to the -3.14 kb site of the DEFB124 promoter in PGN-treated human prostate epithelial RWPE-1 cells. When DEFB124 was overexpressed in RWPE-1 cells, interestingly, the production of cytokines (interleukin [IL] 6 and IL-12) and chemokines (CCL5, CCL22, and CXCL8) was significantly increased. These DEFB124-upregulated RWPE-1 cells markedly induced chemotactic activity for THP-1 monocytes. CONCLUSIONS: Taken together, these results provide strong evidence for the first time that increased DEFB124 expression via NF-kappaB activation in PGN-exposed RWPE-1 cells enhances the production of cytokines and chemokines, which may contribute to an efficient innate immune defense.
Bacterial Infections ; Blotting, Western ; Chemokines ; Chemotaxis ; Chromatin Immunoprecipitation ; Cytokines ; Defensins ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunity, Innate* ; Immunohistochemistry ; Monocytes ; NF-kappa B ; Peptidoglycan ; Phosphorylation ; Prostate* ; Real-Time Polymerase Chain Reaction ; Up-Regulation

Streptococcus mutans is a common Gram-positive bacterium and plays a significant role in dental caries. Tobacco and/or nicotine have documented effects on S. mutans growth and colonization. Sortase A is used by many Gram-positive bacteria, including S. mutans, to facilitate the insertion of certain cell surface proteins, containing an LPXTGX motif such as antigen I/II. This study examined the effect of nicotine on the function of sortase A to control the physiology and growth of S. mutans using wild-type S. mutans NG8, and its isogenic sortase-defective and -complemented strains. Briefly, the strains were treated with increasing amounts of nicotine in planktonic growth, biofilm metabolism, and sucrose-induced and saliva-induced antigen I/II-dependent biofilm formation assays. The strains exhibited no significant differences with different concentrations of nicotine in planktonic growth assays. However, they had significantly increased (P≤0.05) biofilm metabolic activity (2- to 3-fold increase) as the concentration of nicotine increased. Furthermore, the sortase-defective strain was more sensitive metabolically to nicotine than the wild-type or sortase-complemented strains. All strains had significantly increased sucrose-induced biofilm formation (2- to 3-fold increase) as a result of increasing concentrations of nicotine. However, the sortase-defective strain was not able to make as much sucrose- and saliva-induced biofilm as the wild-type NG8 did with increasing nicotine concentrations. These results indicated that nicotine increased metabolic activity and sucrose-induced biofilm formation. The saliva-induced biofilm formation assay and qPCR data suggested that antigen I/II was upregulated with nicotine but biofilm was not able to be formed as much as wild-type NG8 without functional sortase A.
Amino Acid Motifs ; Aminoacyltransferases ; drug effects ; genetics ; Antigens, Bacterial ; drug effects ; Bacterial Adhesion ; drug effects ; Bacterial Proteins ; drug effects ; genetics ; Biofilms ; drug effects ; Cysteine Endopeptidases ; drug effects ; genetics ; Dose-Response Relationship, Drug ; Humans ; Mutation ; genetics ; Nicotine ; administration & dosage ; pharmacology ; Peptidoglycan ; drug effects ; genetics ; Saliva ; physiology ; Streptococcus mutans ; drug effects ; enzymology ; growth & development ; Sucrose ; pharmacology

Helicobacter pylori (H. pylori) is one of the most common human infection world-wide. However, only a limited proportion of the infected population developed gastrointestinal diseases such as peptic ulcer disease, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. These various outcomes of H. pylori infection may result from bacterial virulence factors, host factors such as genetic diversities, and environmental influences. Bacterial factors such as cagA PAI, vacA, adhesin and outer membrane proteins, and peptidoglycans are known to be associated with specific gastrointestinal diseases such gastric adenocarcinoma. Various cytokines including interleukin (IL)-1beta, IL-10, and tumor necrosis factor-alpha, and host immune reaction to the bacteria are closely related to specific diseases such as gastric adenocarcinoma and duodenal ulcer. In this article, we reviewed each factors and their relevance to the disease outcome.
Adenocarcinoma ; Bacteria ; Cytokines ; Duodenal Ulcer ; Gastrointestinal Diseases ; Genetic Variation ; Helicobacter ; Helicobacter pylori ; Humans ; Interleukin-10 ; Interleukins ; Lymphoma, B-Cell, Marginal Zone ; Membrane Proteins ; Peptic Ulcer ; Peptidoglycan ; Stomach Diseases ; Tumor Necrosis Factor-alpha ; Virulence Factors