muramidase and Helicobacter-Infections

Interleukin (IL)-8 is the key agent for initiating an inflammatory response to infection with Helicobacter pylori. Some strains of Lactobacillus spp. are known to colonize the stomach and suppress inflammation caused by H. pylori. In this study, we characterized two gastric-derived lactobacilli, Lactobacillus salivarius (LS) strains B37 and B60, capable of inhibiting H. pylori-induced IL-8 production by gastric epithelial cells.. Conditioned media from LS-B37 and LS-B60 suppressed H. pylori-induced IL-8 production and mRNA expression from AGS cells without inhibiting H. pylori growth. These conditioned media suppressed the activation of NF-κB but did not suppress c-Jun activation. IL-8 inhibitory substances in conditioned media of LS-B37 and LS-B60 are heat-stable and larger than 100 kDa in size. The inhibitory activity of LS-B37 was abolished when the conditioned medium was treated with α-amylase but still remained when treated with either proteinase K, trypsin, lipase or lysozyme. The activity of LS-B60 was abolished when the conditioned medium was treated with either amylase or proteinase K but still remained when treated with lysozyme. Treatment with lipase and trypsin also significantly affected the inhibitory activity of LS-B60 although the conditioned medium retained IL-8 suppression statistically different from media control.. These results suggest that L. salivarius strains B37 and B60 produce different immunomodulatory factors capable of suppressing H. pylori-induced IL-8 production from gastric epithelial cells. Our results suggest that the large, heat-stable immunomodulatory substance(s) present in the LCM of LS-B37 is a polysaccharide, while the one(s) of LS-B60 is either complex consisting of components of polysaccharide, lipid and protein or includes multiple components such as glycoprotein and lipoprotein.

Topics: alpha-Amylases; Anti-Inflammatory Agents; Cell Line; Culture Media, Conditioned; Endopeptidase K; Epithelial Cells; Gastric Mucosa; Gene Expression Regulation, Bacterial; Helicobacter Infections; Helicobacter pylori; Humans; Inflammation; Interleukin-8; Lactobacillus; Ligilactobacillus salivarius; Lipase; Muramidase; NF-kappa B; Probiotics; RNA, Messenger; Stomach; Trypsin

Two independent series of inhibitors of Helicobacter pylori glutamate racemase (MurI) were characterized for their kinetic mechanism, and one was used to generate resistant mutants in vitro. Mutant MurI enzymes from these strains were characterized by structural, genetic, kinetic and biophysical methods. Both inhibitor series, pyrazolopyrimidinediones and benzodiazepines, are uncompetitive with respect to the glutamate substrate, and the resistance mutations were found to act by reducing the affinity of MurI for substrate, thereby reducing the pool of enzyme-substrate complex available for binding inhibitor, while still allowing sufficient glutamate racemase activity for peptidoglycan construction. Uncompetitive inhibitors of a single-substrate, single-product enzyme are rare, and this work gives insight into an remarkable resistance mechanism. This article will discuss the projected clinical impact of H. pylori MurI resistance on these types of inhibitors.

Topics: Amino Acid Isomerases; Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Benzodiazepines; Binding Sites; Catalytic Domain; Cell Wall; Drug Resistance, Bacterial; Helicobacter Infections; Helicobacter pylori; Humans; Kinetics; Molecular Sequence Data; Muramidase; Mutation; Peptidoglycan; Protein Binding; Pyrimidinones; Sequence Alignment; Substrate Specificity

The prominent host muramidase lysozyme cleaves bacterial peptidoglycan (PG), and the enzyme is abundant in mucosal secretions. The lytic enzyme susceptibility of Gram-negative bacteria and mechanisms they use to thwart lytic enzyme activity are poorly studied. We previously characterized a Helicobacter pylori PG modification enzyme, an N-deacetylase (PgdA) involved in lysozyme resistance. In this study, another PG modification enzyme, a putative PG O-acetyltransferase (PatA), was identified. Mass spectral analysis of the purified PG demonstrated that a patA strain contained a greatly reduced amount of acetylated muropeptides, indicating a role for PatA in H. pylori PG O-acetylation. The PG modification mutant strains (pgdA, patA, or pgdA patA) were more susceptible to lysozyme killing than the parent, but this assay required high lysozyme levels (up to 50 mg/ml). However, addition of host lactoferrin conferred lysozyme sensitivity to H. pylori, at physiologically relevant concentrations of both host components (3 mg/ml lactoferrin plus 0.3 mg/ml lysozyme). The pgdA patA double mutant strain was far more susceptible to lysozyme/lactoferrin killing than the parent. Peptidoglycan purified from a pgdA patA mutant was five times more sensitive to lysozyme than PG from the parent strain, while PG from both single mutants displayed intermediate sensitivity. Both sensitivity assays for whole cells and for purified PGs indicated that the modifications mediated by PgdA and PatA have a synergistic effect, conferring lysozyme tolerance. In a mouse infection model, significant colonization deficiency was observed for the double mutant at 3 weeks postinoculation. The results show that PG modifications affect the survival of a Gram-negative pathogen.. Pathogenic bacteria evade host antibacterial enzymes by a variety of mechanisms, which include resisting lytic enzymes abundant in the host. Enzymatic modifications to peptidoglycan (PG, the site of action of lysozyme) are a known mechanism used by Gram-positive bacteria to protect against host lysozyme attack. However, Gram-negative bacteria contain a thin layer of PG and a recalcitrant outer membrane permeability barrier to resist lysis, so molecular modifications to cell wall structure in order to combat lysis remain largely unstudied. Here we show that two Helicobacter pylori PG modification enzymes (PgdA and PatA) confer a clear protective advantage to a Gram-negative bacterium. They protect the bacterium from lytic enzyme degradation, albeit via different PG modification activities. Many pathogens are Gram negative, so some would be expected to have a similar cell wall-modifying strategy. Understanding such strategies may be useful for combating pathogen growth.

Topics: Acetyltransferases; Animals; Disease Models, Animal; Helicobacter Infections; Helicobacter pylori; Host-Pathogen Interactions; Mass Spectrometry; Mice; Microbial Viability; Muramidase; Peptidoglycan; Virulence

Backgroud: Helicobacter pylori (Hp) rarely proliferates in patients with fundic gland polyps (FGPs). We recently found that FGPs express lysozyme, one of the natural defence substances against infection. We aimed to assess the degree of lysozyme expression in a cohort of consecutive FGPs.. A total of 153 gastric biopsies were investigated: 93 with FGPs, 30 with normal mucosa (Nm), 15 with Hp-induced chronic gastritis (Hp-gastritis) and 15 with chronic gastritis without Hp infection (non-Hp-gastritis). Sections were stained with anti-lysozyme (muramidase).. Lysozyme was slightly to moderately expressed in the surface and foveolar pits, being markedly expressed in the neck glands in Nm, in non-Hp and Hp-gastritis. The ratio of lysozyme neck glands-foveoli was higher in non-Hp than in Nm and even higher in Hp-gastritis. In FGPs, lysozyme was markedly expressed in the surface, the foveolar pits and the cells that partly or entirely covered the microcysts.. While the moderate expansion of the lysozyme-producing cells of the neck glands in Hp-gastritis might be insufficient to eradicate these bacteria, the overproduction of lysozyme in the epithelium covering FGP could be an explanation for the lack of Hp proliferation in these patients.

Topics: Biopsy; Chronic Disease; Gastric Fundus; Gastric Mucosa; Gastritis; Helicobacter Infections; Helicobacter pylori; Humans; Muramidase; Polyps; Stomach Diseases

Helicobacter pylori has frequently been isolated from human dental plaque, and oral spread via saliva is thought to be one of its principal modes of transmission. Among other innate defence systems human saliva contains peroxidase enzymes and lysozyme. The sensitivity of H. pylori to physiological concentrations of lactoperoxidase and its salivary substrate thiocyanate, and different amounts of hydrogen peroxide (H(2)O(2)) was investigated in buffer and in human whole saliva. The effect of lysozyme was also studied in saliva. All tested H. pylori strains, ATCC 43504(T) and five clinical isolates, were efficiently inhibited by the peroxidase system with high concentrations of H(2)O(2) in buffer. The inhibition was stronger at lower pH. However, in human saliva these high concentrations of H(2)O(2) generated less hypothiocyanite, the antibacterial product of the peroxidase system and the effects of the peroxidase system were weaker. Physiological concentration of lysozyme was not bacteriocidal against H. pylori, nor did it enhance the effect of the peroxidase system in saliva. Thus, further studies are needed to enhance the efficacy of peroxidase systems in human saliva to make it more beneficial not only against dental but also against gastric pathogens.

Topics: Buffers; Helicobacter Infections; Helicobacter pylori; Humans; Hydrogen Peroxide; Lactoperoxidase; Muramidase; Saliva; Thiocyanates

The increase in the transcellular passage of intact antigens across the digestive epithelium infected with Helicobacter pylori may interfere with the regulation of mucosal immune responses. The aim of this work was to study the capacity of Helicobacter infection to inhibit the development of oral tolerance or to promote allergic sensitization and the capacity of a gastro-protective agent, rebamipide, to interfere with these processes in mice. Oral tolerance to ovalbumin (OVA) was studied in 48 C3H/He 4-week-old mice divided into four groups: (i) OVA-sensitized mice; (ii) OVA-"tolerized" mice (that is, mice that were rendered immunologically tolerant); (iii) H. felis-infected, OVA-tolerized mice; (iv) and H. felis-infected, OVA-tolerized, rebamipide-treated mice. Oral sensitization to hen egg lysozyme (HEL) was studied in 48 mice divided into four groups: (i) controls; (ii) HEL-sensitized mice; (iii) H. felis-infected, HEL-sensitized mice; and (iv) H. felis-infected, HEL-sensitized, rebamipide-treated mice. Specific anti-OVA or anti-HEL immunoglobulin E (IgE) and IgG1/IgG2a serum titers were measured by enzyme-linked immunosorbent assay. Additionally, the capacity of rebamipide to interfere with antigen presentation and T-cell activation in vitro, as well as absorption of rebamipide across the epithelial monolayer, was tested. H. felis infection led to the inhibition of oral tolerance to OVA, but rebamipide prevented this inhibitive effect of H. felis. H. felis infection did not enhance the sensitization to HEL, but rebamipide inhibited the development of this sensitization. Moreover, rebamipide inhibited in a dose-dependent manner antigen presentation and T-cell activation in vitro and was shown to be able to cross the epithelium at a concentration capable of inducing this inhibitory effect. We conclude that H. felis can inhibit the development of oral tolerance to OVA in mice and that this inhibition is prevented by rebamipide.

Topics: Administration, Oral; Alanine; Anaphylaxis; Animals; Antigen Presentation; Antigens; Chickens; Female; Gastritis; Helicobacter Infections; Immune Tolerance; Immunity, Mucosal; Immunoglobulin E; Immunoglobulin G; In Vitro Techniques; Intestines; Mice; Mice, Inbred C3H; Muramidase; Ovalbumin; Quinolones; T-Lymphocytes

Helicobacter pylori is an important pathogen of the gastric system. The clinical outcome of infection is thought to be correlated with some genetic features of the bacterium. However, due to the extreme genetic variability of this organism, it is hard to draw definitive conclusions concerning its virulence factors. Here we describe a novel H. pylori gene which expresses an autolytic enzyme that is also capable of degrading the cell walls of both gram-positive and gram-negative bacteria. We designated this gene lys. We found this gene and observed its expression in a number of unrelated clinical strains, a fact that suggests that it is well conserved in the species. A comparison of the nucleotide sequences of lys and the hypothetical gene HP0339 from H. pylori strain ATCC 26695 revealed almost total identity, except for the presence of an insertion consisting of 24 nucleotides in the lys sequence. The coding sequences of lys and HP0339 show a high degree of homology with the coding sequence of bacteriophage T4 lysozyme. Because of this similarity, it was possible to model the three-dimensional structures of both the lys and HP0339 products.

Topics: Amino Acid Sequence; Bacteria; Bacterial Proteins; Bacteriolysis; Bacteriophage T4; Cell Wall; Cloning, Molecular; Helicobacter Infections; Helicobacter pylori; Humans; Models, Molecular; Molecular Sequence Data; Muramidase; Sequence Alignment; Sequence Analysis, DNA

Granulocyte infiltration was studied in 88 biopsies of antrum mucosa from patients with B-gastritis. Evidence of IgA-, IgG- and IgM-antibodies as well as of lysozyme in the mucosa was demonstrated by immunohistochemical methods. Helicobacter pylori (Hp) is coated by antibodies and a significant correlation between extent of opsonisation and number of plasma cells in the connective tissue of the lamina propria could be stated. Thus, the infiltration of plasma cells is a specific immune response against Hp. In the depths of gastric pits the antibody-coating of bacteria is faint. Instead, lysozyme and lactoferrin are produced there. By means of a Cross-sectional study a model is developed which characterizes B-gastritis as a dynamic process. Lagging behind, the inflammation follows the motile bacteria resulting in a patchy distribution of inflamed areas in the mucosa. At the peak of these local inflammation-waves the production of antibodies and lysozyme is intensified. Coating the bacteria with IgG and IgM results in complement activation liberating chemotaxin C5a. Consequently, there is a massive granulocyte infiltration leading to local reduction or eradication of Hp.

Topics: Antibodies, Bacterial; Antibody Specificity; Biopsy; Gastric Mucosa; Gastritis; Helicobacter Infections; Helicobacter pylori; Humans; Immunoenzyme Techniques; Immunoglobulins; Muramidase

Topics: Antibody Formation; Biopsy; Gastric Mucosa; Gastritis; Helicobacter Infections; Helicobacter pylori; Humans; Immunoglobulins; Lactoferrin; Lymphatic System; Muramidase; Plasma Cells