muramidase and lipoteichoic-acid

Topics: Acetylation; Acetylglucosamine; Bacterial Proteins; beta-Lactam Resistance; Cell Cycle; Cell Division; Cell Wall; Choline; Cytoskeletal Proteins; Humans; Lipopolysaccharides; Muramic Acids; Muramidase; Operon; Penicillin Resistance; Peptidoglycan; Phosphorylation; Polysaccharides; Streptococcus pneumoniae; Teichoic Acids

Streptococcus mutans is a representative oral pathogen that causes dental caries and pulpal inflammation. Its lipoteichoic acid (Sm.LTA) is known to be an important cell-wall virulence factor involved in bacterial adhesion and induction of inflammation. Since Sm.LTA-binding proteins (Sm.LTA-BPs) might play an important role in pathogenesis and host immunity, we identified the Sm.LTA-BPs in the saliva of caries-free and caries-positive human subjects using Sm.LTA-conjugated beads and LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Sm.LTA was conjugated to N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Sm.LTA-beads). Sm.LTA retained its biological properties during conjugation, as determined by the expression of nitric oxide and interferon-γ-inducible protein 10 in a murine macrophage cell line and activation of Toll-like receptor 2 (TLR2) in CHO/CD14/TLR2 cells. Sm.LTA-BPs were isolated from pooled saliva prepared from 10 caries-free or caries-positive human subjects each, electrophoresed to see their differential expression in each group, and further identified by high-resolution mass spectrometry. A total of 8 and 12 Sm.LTA-BPs were identified with statistical significance in the pooled saliva from the caries-free and caries-positive human subjects, respectively. Unique Sm.LTA-BPs found in caries-free saliva included histone H4, profilin-1 and neutrophil defensin-1, and those in caries-positive saliva included cystatin-C, cystatin-SN, cystatin-S, cystatin-D, lysozyme C, calmodulin-like protein 3 and β-actin. The Sm.LTA-BPs found in both groups were hemoglobin subunits α and β, prolactin-inducible protein, protein S100-A9, and SPLUNC2. Collectively, we identified Sm.LTA-BPs in the saliva of caries-free and caries-positive subjects, which exhibit differential protein profiles.

Topics: Actins; Animals; Bacterial Adhesion; Calmodulin; Cell Line; Chemokine CXCL10; CHO Cells; Cricetulus; Cystatin C; Cystatins; Defensins; Dental Caries; Histones; Humans; Lipopolysaccharide Receptors; Lipopolysaccharides; Macrophages; Mice; Muramidase; Nitric Oxide; Profilins; Salivary Cystatins; Salivary Proteins and Peptides; Streptococcus mutans; Teichoic Acids; Toll-Like Receptor 2; Virulence Factors

Amphioxus is a well-known model organism widely used for interspecies comparative genome study, developmental homology analysis and comparative immunological investigation. However, no study has been performed so far to evaluate the internal reference for quantitative RT-PCR (qRT-PCR) studies of gene expression in this important species. In this study, two software applications (geNorm and NormFinder) were used to evaluate the expression stability of 4 housekeeping genes (ACTB, GAPDH, 18S rRNA and EF1α) in 8 different normal tissues (whole body, gut, gut-free body, hepatic caecum, gill, hind-gut, notochord and muscle) and 2 tissues (gut and gut-free body) challenged with LPS and LTA in amphioxus Branchiostoma japonicum. Our results showed that in the normal tissues, the expression of 18S rRNA was most abundant, whereas the expression levels of the other three genes were close to each other, with the expression of ACTB being most unstable. Following challenge with LPS and LTA, all the four genes exhibited varied degrees of expression changes in the different tissues and the expression stabilities of the genes were also affected by the different experimental conditions. Yet, the overall ranking results produced by the two algorithms consistently indicated that the expression of EF1α showed the most least variation in the different tissues, suggesting that EF1α is a suitable internal control for qRT-PCR studies in amphioxus B. japonicum.

Topics: Adjuvants, Immunologic; Animals; Chordata; Gene Expression Profiling; Gene Expression Regulation; Lipopolysaccharides; Muramidase; Peptide Elongation Factor 1; Reference Standards; Reverse Transcriptase Polymerase Chain Reaction; Teichoic Acids

The g-type lysozyme is a key protein of the innate immune system to fight bacterial infections. In this study we cloned and characterized the gene encoding for g-type lysozyme in Senegalese sole (Solea senegalensis). The deduced amino acid sequence comprised 195 residues containing the three conserved catalytic residues and two cysteines. A BAC analysis revealed that the gene is structured in 5 exons and 4 introns. Also, two polyadenylation signals that generate two cDNAs differing in 3'-UTR length were detected. Promoter analysis showed the presence of the main cis-acting elements involved in the transcriptional regulation of the gene. At genomic level, the g-type lysozyme was associated with mucolipin 1 and the peptidoglycan recognition protein 2 conforming a cluster of antidefensive genes with a well-conserved synteny across Percomorpha. FISH analysis using the BAC clone revealed a single hybridization signal located in an acrocentric chromosome pair. The phylogenetic analysis confirmed that the g-type lysozyme represents a complex group in fish that has been shaped by gene duplications and diversification with several positions under Darwinian selection. Expression analysis in juvenile tissues indicated that transcript levels were higher in gills, spleen and heart. During development, gene expression activated just at the beginning of metamorphosis, increasing progressively until climax. Hormonal treatments demonstrated that this gene was regulated positively by thyroid hormones during development and negatively by dexamethasone. In contrast, no response was observed after all-trans retinoic acid or 4-diethylaminobenzaldehyde treatments. Finally, treatments using lipopolysaccharide, lipoteichoic acid, peptidoglycan, zymosan and poly(I:C) activated gene expression in a time- and tissue-specific manner. Taken together, data indicate that g-type lysozyme is a high evolutionary conserved gene that diversified to adapt to changing environment and pathogen conditions. Gene expression can be activated by diverse pathogen stimuli and modulated by physiological factors with important consequences for the aquaculture of this species.

Topics: Age Factors; Amino Acid Sequence; Animals; Aquaculture; Base Sequence; Chromosomes, Artificial, Bacterial; Dexamethasone; DNA Primers; DNA, Complementary; Evolution, Molecular; Flatfishes; Gene Components; Gene Expression Profiling; Gene Expression Regulation, Developmental; In Situ Hybridization, Fluorescence; Lipopolysaccharides; Molecular Sequence Data; Muramidase; Peptidoglycan; Phylogeny; Promoter Regions, Genetic; Sequence Analysis, DNA; Teichoic Acids; Thyroid Hormones; Zymosan

The insertional inactivation of the dlt operon from Lactobacillus plantarum NCIMB8826 had a strong impact on lipoteichoic acid (LTA) composition, resulting in a major reduction in D-alanyl ester content. Unexpectedly, mutant LTA showed high levels of glucosylation and were threefold longer than wild-type LTA. The dlt mutation resulted in a reduced growth rate and increased cell lysis during the exponential and stationary growth phases. Microscopy analysis revealed increased cell length, damaged dividing cells, and perforations of the envelope in the septal region. The observed defects in the separation process, cell envelope perforation, and autolysis of the dlt mutant could be partially attributed to the L. plantarum Acm2 peptidoglycan hydrolase.

Topics: Alanine; Autolysis; Base Sequence; Cell Wall; DNA Primers; Esters; Kinetics; Lactobacillus plantarum; Lipopolysaccharides; Microscopy, Electron, Scanning; Muramidase; N-Acetylmuramoyl-L-alanine Amidase; Operon; Polymerase Chain Reaction; Restriction Mapping; Teichoic Acids

Mutations in the genes encoding enzymes responsible for the incorporation of D-Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase (alr) mutant is strictly dependent on an external supply of D-Ala to be able to synthesize peptidoglycan and to incorporate D-Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when D-Ala is removed at mid-exponential growth. AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent. To investigate the role of D-Ala on LTA in the increased cell lysis, a dltD mutant of L. lactis was investigated, since this mutant is only affected in the D-alanylation of LTA and not the synthesis of peptidoglycan. Mutation of dltD results in increased lysis, showing that D-alanylation of LTA also influences autolysis. Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In L. lactis, LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking D-Ala has been reported in other bacterial species to have an improved capacity for autolysin binding. Mutation of dltD in L. lactis, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion, D-Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L. lactis. Second, reduced amounts of D-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.

Topics: Alanine; Alanine Racemase; Bacteriolysis; Lactococcus lactis; Lipopolysaccharides; Muramidase; Peptidoglycan; Teichoic Acids

The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall of Enterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.

Topics: Bacterial Proteins; Carrier Proteins; Cell Wall; Chromatography, High Pressure Liquid; Enterococcus faecalis; Hexosyltransferases; Lipopolysaccharides; Muramidase; Muramoylpentapeptide Carboxypeptidase; Oligopeptides; Penicillin-Binding Proteins; Peptidoglycan; Peptidyl Transferases; Teichoic Acids

The cationic tear proteins lactoferrin and lysozyme exhibit co-operative antistaphylococcal properties. The purpose of this study was to determine the mechanism of action of this co-operation on Staphylococcus epidermidis. Following blocking of lipoteichoic acid (LTA) binding sites, the effects on binding of lactoferrin and susceptibility to lactoferrin and lysozyme were determined. The effect of lactoferrin on autolysis and LTA release was also examined. Maximal susceptibility occurred on addition of lactoferrin first followed by lysozyme. Blocking the LTA binding sites both reduced lactoferrin binding and decreased susceptibility. Autolytic activity decreased and LTA release increased in the presence of lactoferrin. These results suggest that binding of lactoferrin to LTA is important in its synergy with lysozyme and interferes with the autolysins present on the LTA. It is proposed that, on binding to the anionic LTA of S. epidermidis, the cationic protein lactoferrin decreases the negative charge, allowing greater accessibility of lysozyme to the underlying peptidoglycan.

Topics: Anti-Bacterial Agents; Bacteriolysis; Culture Media; Drug Synergism; Lactoferrin; Lipopolysaccharides; Microbial Sensitivity Tests; Muramidase; Ophthalmic Solutions; Staphylococcus epidermidis; Teichoic Acids

The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined. Autolysis was optimal at pH 6.3 and 55 degrees C in 0.1 M-sodium acetate/phosphate buffer. The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth. Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations. The autolysin of C. acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase. The enzyme was identical to the extracellular muramidase in terms of M(r), isoelectric point and NH2-terminal amino acid sequence. The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol. A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed.

Topics: Amino Acid Sequence; Amino Acids; Amino Alcohols; Amino Sugars; Autolysis; Cell Division; Cell Wall; Chelating Agents; Clostridium; Hot Temperature; Hydrogen-Ion Concentration; Ions; Lipopolysaccharides; Molecular Sequence Data; Mucoproteins; Muramidase; Phospholipids; Sequence Homology, Nucleic Acid; Subcellular Fractions; Teichoic Acids

The antibacterial properties of lysozyme for Streptococcus mutans BHT may be a function of its binding to cell components other than to peptidoglycan. Inhibitors of muramidase activity, including histamine and N-acetyl-D-glucosamine, only partially blocked the bacteriostatic effects on this strain. Greater than 20 mM histamine alone inhibited growth suggesting a bacteriostatic potential. An autoclaved saline extract was then prepared from stationary phase cultures in a chemically-defined medium. As little as 31.25 micrograms of the extract significantly blocked the effect of 50 micrograms lysozyme and complete enzyme inhibition was achieved with 62.5 micrograms. The extract was fractionated and location of potential binding components determined by a precipitin method consisting of diffusing the samples into 1.2 per cent agarose containing lysozyme. Binding components eluted in the first peak of a Sephacryl S-300 column, bound to DEAE-cellulose, but desorbed with gradient elution (0.1-1.0 M tris-HCl buffer, pH 8.0). The eluted material was then applied to an affinity column containing purified lysozyme coupled to epoxy-activated Sepharose 6B. Non-absorbed anionic material precipitated only with protamine. Lysozyme-binding fractions eluted in a sharp peak with 1.0 M tris-HCl buffer (pH 8.0), did not bind wheat-germ agglutinin, contained less than 50 micrograms protein, 95 micrograms sugar, 66.7 micrograms phosphorus, less than 0.25 mequiv lipid and no detectable nucleic acids. The peak material reacted with antiserum directed against polyglycerol phosphate, indicating that it contained acylated or, possibly, deacylated lipoteichoic acid. The findings suggest that the antibacterial properties of lysozyme for Strep. mutans BHT may, in part, be modified (or possibly regulated) by binding to molecules such as lipoteichoic acid.

Topics: Glycerophosphates; Lipopolysaccharides; Muramidase; Phosphatidic Acids; Polysaccharides, Bacterial; Protein Binding; Streptococcus mutans; Teichoic Acids

Topics: Bacterial Proteins; Bacteriolysis; Cell Wall; Lipopolysaccharides; Muramidase; Phosphatidic Acids; Streptococcus pneumoniae; Teichoic Acids; Trypsin