muramidase and Gram-Positive-Bacterial-Infections

Lactococcosis, caused by the Gram-positive bacterium Lactococcus garvieae, is a major concern in rainbow trout (Oncorhynchus mykiss) farms, which are regularly affected by outbreaks especially during the summer/fall months. In these farms, unvaccinated healthy and symptomatic fish can coexist with vaccinated fish. In the present study, innate (leukogram, serum lysozyme activity, peroxidase activity, antiprotease activity, bactericidal activity, total IgM and total proteins), and specific immune parameters (serum antibodies to L. garvieae) were assessed in unvaccinated adult rainbow trout naturally exposed to the pathogen, with or without evidence of clinical signs, or subjected to vaccination. Blood was drawn from all three groups, and blood smears were prepared. Bacteria were found in the blood smears of 70% of the symptomatic fish but not in any of the asymptomatic fish. Symptomatic fish showed lower blood lymphocytes and higher thrombocytes than asymptomatic fish (p ≤ .05). Serum lysozyme and bactericidal activity did not vary substantially among groups; however, serum antiprotease and peroxidase activity were significantly lower in the unvaccinated symptomatic group than in the unvaccinated and vaccinated asymptomatic groups (p ≤ .05). Serum total proteins and total immunoglobulin (IgM) levels in vaccinated asymptomatic rainbow trout were significantly higher than in unvaccinated asymptomatic and symptomatic groups (p ≤ .05). Similarly, vaccinated asymptomatic fish produced more specific IgM against L. garvieae than unvaccinated asymptomatic and symptomatic fish (p ≤ .05). This preliminary study provides basic knowledge on the immunological relationship occurring between the rainbow trout and L. garvieae, potentially predicting health outcomes. The approach we proposed could facilitate infield diagnostics, and several non-specific immunological markers could serve as reliable indicators of the trout's innate ability to fight infection.

Topics: Animals; Antibodies, Bacterial; Fish Diseases; Gram-Positive Bacterial Infections; Immunoglobulin M; Lactococcus; Muramidase; Oncorhynchus mykiss; Peroxidases

Nematode virulence factors are of interest for a variety of applications including biocontrol against insect pests and the alleviation of autoimmune diseases with nematode-derived factors. In silico "omics" techniques have generated a wealth of candidate factors that may be important in the establishment of nematode infections, although the challenge of characterizing these individual factors in vivo remains. Here we provide a fundamental characterization of a putative lysozyme and serine carboxypeptidase from the host-induced transcriptome of Heterorhabditis bacteriophora. Both factors accelerated the mortality rate following Drosophila melanogaster infections with Photorhabdus luminescens, and both factors suppressed phenoloxidase activity in D. melanogaster hemolymph. Furthermore, the serine carboxypeptidase was lethal to a subpopulation of flies and suppressed the upregulation of antimicrobial peptides as well as phagocytosis. Together, our findings suggest that this serine carboxypeptidase possess both toxic and immunomodulatory properties while the lysozyme is likely to confer immunomodulatory, but not toxic effects.

Topics: Animals; Carboxypeptidases; Drosophila melanogaster; Gram-Positive Bacterial Infections; Immunomodulation; Insect Proteins; Monophenol Monooxygenase; Muramidase; Nematoda; Nematode Infections; Photorhabdus; Virulence

Different evidences suggest that pericardial cells play an important role during the immune response against pathogens that invade the mosquito hemocoel. Previously, we identified two lysozyme genes in Anopheles albimanus heart transcriptome. The present study showed that one of these genes (ID

Topics: Animals; Anopheles; Cloning, Molecular; Computational Biology; Escherichia coli; Escherichia coli Proteins; Gram-Positive Bacterial Infections; Immunity, Innate; Insect Proteins; Micrococcus luteus; Muramidase; Pericardium; Phylogeny; Transcriptome; Up-Regulation

The sea urchin Lytechinus variegatus is considered a good candidate for aquaculture, but bacterial diseases are a major challenge in culture conditions. The innate immunological defenses of L. variegatus to bacterial challenges were assessed through hematology parameters, in vitro phagocytosis, lysozyme activity and total plasma protein concentrations in cell-free coelomic fluid. Adult sea urchins were inoculated with Microccocus lysodeikticus, Escherichia coli and Vibrio parahaemolyticus in the cavity coelomic. Filtrated and sterile seawater (FSW) injected and non-injected sea urchins were used as control groups. Righting time, external aspects and behavior of sea urchins were evaluated. Twenty-four hours post-inoculation, we found an increase in the population of colorless spherule cells (CLS), phagocytosis, and humoral responses in sea urchins challenged by bacterial inoculations. Righting time was not affected by the treatments and apparent external signs of disease were not observed at least during 96h post-inoculation. The immunological system of L. variegatus quickly eliminated pathogenic microorganisms. CLS and lysozyme activity cooperate in the immune defenses of L. variegatus, showing an extraordinary efficiency for adjusting the immune defenses under stress caused by microbes. We recommend that the cellular and humoral markers serve as routine tests to monitor health status in sea urchins.

Topics: Animals; Escherichia coli; Escherichia coli Infections; Gram-Positive Bacterial Infections; Immunity, Innate; Lytechinus; Micrococcus; Muramidase; Phagocytosis; Vibrio Infections; Vibrio parahaemolyticus

Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires a complex network of transcriptional regulators and several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa variable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promote E. faecalis infections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis.

Topics: Animals; Antigens, Surface; Antimicrobial Cationic Peptides; Bacterial Proteins; Enterococcus faecalis; Gram-Positive Bacterial Infections; Muramidase; Mutagenesis; Mutation; Polysaccharides; Virulence; Zebrafish

Lysozyme is a key component of the innate immune system and plays an important role in antibacterial infection. In this study, we analyzed the expression and activity of a chicken-type (c-type) lysozyme (named SmLysC) from turbot (Scophthalmus maximus). SmLysC is composed of 143 residues and shares 67-90% overall sequence identities with the c-type lysozymes of a number of teleost fish. SmLysC possesses a typical c-type lysozyme domain, which contains the conserved residues E50 and D67 that form the putative catalytic site. SmLysC expression was detected, in increasing order, in head kidney, gill, heart, muscle, brain, spleen, blood, and liver. Bacterial infection caused significant inductions of SmLysC expression in head kidney, spleen, and liver in a time-dependent manner. Immunoblot analysis indicated that SmLysC has a subcellular localization in the extracellular milieu. Recombinant SmLysC (rSmLysC) was able to bind to bacterial cells and inhibit bacterial growth. Enzyme assay showed that the optimal temperature and pH of rSmLysC were 37 °C and pH 6.0 respectively. In contrast to rSmLysC, the mutant protein rSmLysCM1, which bears alanine substitutions at E50 and D67, displayed drastically reduced bacteriolytic activity. rSmLysC was able to inhibit the growth of several fish bacterial pathogens in a manner that depended on the dose of the protein; however, Gram-positive bacteria were in general more sensitive to rSmLysC than Gram-negative bacteria. Together these results indicate that SmLysC is a functional lysozyme that is likely to participate in innate immune defense against extracellular bacterial pathogens, in particular those of Gram-positive nature.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Fish Proteins; Flatfishes; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Gram-Positive Bacteria; Gram-Positive Bacterial Infections; Immunoblotting; Muramidase; Organ Specificity; Plasmids; Polymerase Chain Reaction; Recombinant Proteins; Sequence Alignment; Sequence Analysis, DNA; Species Specificity

The prevalence of infections with enterococci is increasing worldwide. However, little is known about the mechanisms which enable these opportunistic pathogens to cause infections of their host. Here we demonstrate that Enterococcus faecium in the presence of lysozyme induces necrosis in human and mouse cells after 4 h indicated by disrupted cellular membranes of epithelial (HeLa), myeloid (U937, J774A.1) and lymphoid (Jurkat J16, thymocytes), but not intestinal epithelial cells (CaCo-2, CMT-93). Using an appropriate mutant strain it was shown that the enterococcal surface-protein SgrA is involved in cell death induction in mouse cells (J774A.1, thymocytes). Microscopic analyses of epithelial cells 30 min post infection revealed that lysozyme increases adhesion of E. faecium to HeLa, but not CaCo-2 cells. At that time the phalloidin-FITC-stained cytoskeleton of infected cells was still intact, whereas 2 h post infection the F-actin network of HeLa, but not CaCo-2 cells was disrupted. Hence, the early, lysozyme-mediated increase of bacterial adherence plays an important role for cell death induction by E. faecium in HeLa cells. Moreover, bacterial extracellular hydrogen peroxide might contribute to necrosis induction, since the rate of propidium iodide-positive HeLa and J774A.1 cells was lowered after infection with a ROS-deficient E. faecium mutant.

Topics: Animals; Bacterial Adhesion; Bacterial Proteins; Caco-2 Cells; Cell Death; Cell Line; Enterococcus faecium; Epithelial Cells; Gram-Positive Bacterial Infections; HeLa Cells; Humans; Mice; Muramidase; Necrosis; Reactive Oxygen Species; U937 Cells

Lysozyme is a widely distributed antimicrobial protein having specificity for cleaving the beta-(1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (GlcNAc) of peptidoglycan of the bacterial cell walls and thus efficiently contributes to protection against infections caused mainly by Gram-positive bacteria. In the present study, we assembled a full-length cDNA of a novel invertebrate-type lysozyme from Eisenia andrei earthworm (EALys) by RT-PCR and RACE system. The primary structure of EALys shares high homology with other invertebrate lysozymes; however the highest, 72% identity, was shown for the destabilase I isolated from medicinal leech. Recombinant EALys expressed in Escherichia coli exhibited the lysozyme and isopeptidase activity. Moreover, real-time PCR revealed increased levels of lysozyme mRNA in coelomocytes of E. andrei after the challenge with both Gram-positive and Gram-negative bacteria.

Topics: Animals; Bacillus subtilis; Bacterial Adhesion; Carbon-Nitrogen Lyases; Chitinases; Cloning, Molecular; Echinodermata; Endopeptidases; Escherichia coli; Escherichia coli Infections; Glucosamine; Gram-Positive Bacterial Infections; Hirudo medicinalis; Host-Pathogen Interactions; Hydrolysis; Muramic Acids; Muramidase; Oligochaeta; Sequence Homology; Virulence

The systemic immune response of Trichoplusia ni after Bacillus thuringiensis (Bt) exposure was evaluated by comparing the expression of genes encoding antimicrobial peptides (AMPs) in Bt-susceptible and -resistant T. ni strains that were either exposed or not to XenTari (Bt-XT). AMP genes were detected by RT-PCR using primers for attacin, gloverin, lebocin, lysozyme, and peptidoglycan recognition peptide (PGRP). In general, AMP genes were detected more frequently in Mexican field strains previously exposed to Bt (SALX and GTOX) than in a Mexican laboratory strain (NL), but expression was similar to the AMP expression in USA laboratory strains (US and USX). Among the AMPs, transcripts for lebocin were the least detected (11.7%) and those for lysozyme were the most detected (84.8%) in all samples. Lebocin was detected only in 2nd instar and pupa. All untreated controls expressed attacin. Attacin and gloverin were not detected in any midgut sample, and their highest detection was in pupa. Lysozyme was rarely detected in 2nd instar larvae from any strain or treatment but was detected in almost all midgut and hemolymph samples. Overall, AMPs were found more in T. ni strains previously exposed to Bt-XT, especially lebocin and globerin (1.8-fold increase) and PGRP (3.8-fold increase). The data suggest that the expression of AMPs in T. ni correlates to previous Bt exposure.

Topics: Animals; Bacillus thuringiensis; Carrier Proteins; Gene Expression; Gram-Positive Bacterial Infections; Insect Proteins; Intercellular Signaling Peptides and Proteins; Lepidoptera; Mexico; Muramidase; Proteins; Reverse Transcriptase Polymerase Chain Reaction

Listeria monocytogenes is a human intracellular pathogen that is able to survive in the gastrointestinal environment and replicate in macrophages, thus bypassing the early innate immune defenses. Peptidoglycan (PG) is an essential component of the bacterial cell wall readily exposed to the host and, thus, an important target for the innate immune system. Characterization of the PG from L. monocytogenes demonstrated deacetylation of N-acetylglucosamine residues. We identified a PG N-deacetylase gene, pgdA, in L. monocytogenes genome sequence. Inactivation of pgdA revealed the key role of this PG modification in bacterial virulence because the mutant was extremely sensitive to the bacteriolytic activity of lysozyme, and growth was severely impaired after oral and i.v. inoculations. Within macrophage vacuoles, the mutant was rapidly destroyed and induced a massive IFN-beta response in a TLR2 and Nod1-dependent manner. Together, these results reveal that PG N-deacetylation is a highly efficient mechanism used by Listeria to evade innate host defenses. The presence of deacetylase genes in other pathogenic bacteria indicates that PG N-deacetylation could be a general mechanism used by bacteria to evade the host innate immune system.

Topics: Acetylation; Amidohydrolases; Animals; Cell Line; Cell Survival; Gram-Positive Bacterial Infections; Humans; Immune System; Immunity, Innate; Interleukin-6; Listeria; Macrophages; Mice; Muramidase; Mutation; Peptidoglycan; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Two almost-identical strains of Eubacterium aerofaciens isolated from the normal human gut flora were used. The cell wall (CW) of one strain with a peptidoglycan (PG) type A4alpha induces chronic arthritis in the rat after a single intraperitoneal injection, whereas CW of the other with PG type A4beta induces only a transient acute arthritis. The CW of the arthritogenic E. aerofaciens was a twofold-more-potent stimulator of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein 1 (MCP-1) than the nonarthritogenic CW. After degradation with mutanolysin, the capacity of the arthritogenic PG to stimulate production of TNF-alpha and MCP-1 was significantly increased, whereas that of the nonarthritogenic PG was significantly decreased. In other words, after enzyme degradation the arthritogenic PG had a four- to fivefold-stronger stimulatory capacity than that of the enzyme-treated nonarthritogenic PG. These findings indicate that the arthritogenicity of CW or a PG is not dependent on the enzyme resistance alone but also on how the PG fragments released by enzyme degradation stimulate the production of proinflammatory cytokines.

Topics: Animals; Arthritis, Infectious; Arthritis, Rheumatoid; Carbohydrate Sequence; Cell Wall; Chemokine CCL2; Digestive System; Endopeptidases; Eubacterium; Gram-Positive Bacterial Infections; Liver; Macrophages, Peritoneal; Molecular Sequence Data; Muramic Acids; Muramidase; Peptidoglycan; Rats; Species Specificity; Spleen; Synovial Membrane; Tumor Necrosis Factor-alpha