The Lysozyme Inhibitor Thionine Acetate Is Also an Inhibitor of the Soluble Lytic Transglycosylase Slt35 from Escherichia coli.

Lytic transglycosylases such as Slt35 from E. coli are enzymes involved in bacterial cell wall remodelling and recycling, which represent potential targets for novel antibacterial agents. Here, we investigated a series of known glycosidase inhibitors for their ability to inhibit Slt35. While glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, thiamet G and miglitol had no effect, the phenothiazinium dye thionine acetate was found to be a weak inhibitor. IC50 values and binding constants for thionine acetate were similar for Slt35 and the hen egg white lysozyme. Molecular docking simulations suggest that thionine binds to the active site of both Slt35 and lysozyme, although it does not make direct interactions with the side-chain of the catalytic Asp and Glu residues as might be expected based on other inhibitors. Thionine acetate also increased the potency of the beta-lactam antibiotic ampicillin against a laboratory strain of E. coli.

Analysis of glucosyltransferase gene expression of clinical isolates of Streptococcus mutans obtained from dental plaques in response to sub-lethal doses of photoactivated disinfection.

BACKGROUND: Dental caries is a disease caused by a group of oral streptococcal microorganisms, primarily Strptococcus mutans. Photoactivated disinfection (PAD) has been proposed as a conservative method for treatment of carious lesions. Therefore, the aim of this study was to assess the effectiveness of indocyanine green (ICG)- and toluidine blue O (TBO)-PAD as supplementary tools in reducing the bacterial load and expression profiling of the gene associated with the biofilm formation in S. mutans. MATERIALS AND METHODS: S. mutans strains were isolated from dental plaque samples collected from 212 patients through conventional biochemical tests and molecular methods. After identification, S. mutans strains were photosensitized in vitro with TBO and ICG, which were excited at a specific light wavelength based on the photosensitizer. After evaluating doses of TBO- and ICG-PAD contributing to sub-significant reduction of CFU/mL, ROS levels were measured with DCFH-DA fluorescent probe. Eventually, TBO- and ICG-PAD effects on the gtfB gene expression were assessed using real-time quantitative reverse transcription polymerase chain reaction. RESULTS: S. mutans strains were isolated from 50 (23.5%) patients. In this study, maximum doses of TBO- and ICG-PAD contributing to sub-significant reduction of CFU/mL against S. mutans strains were 23.12 µM/mL TBO at fluence of 68.75 J/cm2 and 20.15 µM/mL ICG at fluence of 31.2 J/cm2. According to the results, 3.8- and 6.1-fold increases in the fluorescence were observed in ICG and TBO treated cells compared to the control, respectively. The expression of gtfB was down regulated to approximately 3.9- and 8.25-fold following treatment with TBO- and ICG-PAD, respectively. CONCLUSIONS: The gtfB gene expression profiling decreased in the bacterial cells, with ICG-PAD causing a greater reduction. PAD may be a promising therapy for dentinal carious lesions.

High-throughput identification of antibacterials against methicillin-resistant Staphylococcus aureus (MRSA) and the transglycosylase.

To identify new transglycosylase inhibitors with potent anti-methicillin-resistant Staphylococcus aureus (MRSA) activities, a high-throughput screening against Staphylococcus aureus was conducted to look for antibacterial cores in our 2M compound library that consists of natural products, proprietary collection, and synthetic molecules. About 3600 hits were identified from the primary screening and the subsequent confirmation resulted in a total of 252 compounds in 84 clusters which showed anti-MRSA activities with MIC values as low as 0.1 µg/ml. Subsequent screening targeting bacterial transglycosylase identified a salicylanilide-based core that inhibited the lipid II polymerization and the moenomycin-binding activities of transglycosylase. Among the collected analogues, potent inhibitors with the IC(50) values below 10 µM against transglycosylase were identified. The non-carbonhydrate scaffold reported in this study suggests a new direction for development of bacterial transglycosylase inhibitors.

Targeting bacterial secretion systems: benefits of disarmament in the microcosm.

Secretion systems are used by many bacterial pathogens for the delivery of virulence factors to the extracellular space or directly into host cells. They are attractive targets for the development of novel anti-virulence drugs as their inactivation would lead to pathogen attenuation or avirulence, followed by clearance of the bacteria by the immune system. This review will present the state of knowledge on the assembly and function of type II, type III and type IV secretion systems in Gram-negative bacteria focusing on insights provided by structural analyses of several key components. The suitability of transcription factors regulating the expression of secretion system components and of ATPases, lytic transglycosylases and protein assembly factors as drug targets will be discussed. Recent progress using innovative in vivo as well as in vitro screening strategies led to a first set of secretion system inhibitors with potential for further development as anti-infectives. The discovery of such inhibitors offers exciting and innovative opportunities to further develop these anti-virulence drugs into monotherapy or in combination with classical antibiotics. Bacterial growth per se would not be inhibited by such drugs so that the selection for mutations causing resistance could be reduced. Secretion system inhibitors may therefore avoid many of the problems associated with classical antibiotics and may constitute a valuable addition to our arsenal for the treatment of bacterial infections.

Apoptosis of human breast carcinoma cells in the presence of cis-platin and L-/D-PPMP: IV. Modulation of replication complexes and glycolipid: Glycosyltransferases.

Apoptosis of human breast carcinoma cells (SKBR-3, MCF-7, and MDA-468) has been observed after treatment of these cells with anti-cancer drug cis-platin and glycosphingolipid biosynthesis inhibitor L- and D-PPMP, respectively. These drugs initiated apoptosis in a dose-dependent manner as measured by phenotypic morphological changes, by binding of a fluorescent phophatidyl serine-specific dye (PSS-380) onto the outer leaflet of the cell membranes, and by activation of caspases, -3, -8, and -9. It was observed that in two hours very little apoptotic process had started but predominant biochemical changes occurred after 6 h. DNA degradation started after 24 hours of drug treatment. However, very little is known about the stability of the ';Replication Complexes'' during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during its replication, repair, and recombination processes. Previously, DNA helicase-III was characterized as a component of the replication complexes isolated from embryonic chicken brains as well as breast and colon carcinoma cells. Helicase activities were measured by a novel method (ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of the nicked ACT-DNA, by determining values obtained from +/- aphidicolin-treated incubation mixtures. In all three breast carcinoma cell lines, a common trend was observed: a decrease of activities of DNA polymerase-alpha and Helicase III. A sharp decrease of activities of the glycolipid sialyltransferases: SAT-2 (CMP-NeuAc; GD3 alpha2-8 sialyltransferase) and SAT-4 (CMP-NeuAc: GM1a alpha2-3 sialyltransferase) was observed in the apoptotic carcinoma cells treated with L-PPMP compared with cis-platin.

Successive glycosyltransfer activity and enzymatic characterization of pectic polygalacturonate 4-alpha-galacturonosyltransferase solubilized from pollen tubes of Petunia axillaris using pyridylaminated oligogalacturonates as substrates.

Polygalacturonate 4-alpha-galacturonosyltransferase (pectin synthase) was solubilized from pollen tubes of Petunia axillaris and characterized. To accomplish this, an assay method using fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as acceptor substrates was developed. When the pollen tube enzyme was solubilized with 0.5% (v/v) Triton X-100 and was incubated with PA-OGA and UDP-galacturonic acid (UDP-GalUA), successive transfer activity of more than 10 GalUAs from UDP-GalUA to the nonreducing end of PA-OGA was observed by diethylaminoethyl high-performance liquid chromatography. This activity was time- and enzyme concentration-dependent. The optimum enzyme activity was observed at pH 7.0 and 30 degrees C. Among the PA-OGAs investigated, those with a degree of polymerization of more than 10 were preferred as substrates. The crude pollen tube enzyme had an apparent K(m) value of 13 microM for the PA-OGA with a degree of polymerization 11 and 170 microM for UDP-GalUA. The characteristics of the P. axillaris pollen tube enzyme and the usefulness of fluorogenic PA-OGAs for the assay of this enzyme are discussed.

Enzymatic synthesis of lipopolysaccharide in Escherichia coli. Purification and properties of heptosyltransferase i.

Heptosyltransferase I, encoded by the rfaC(waaC) gene of Escherichia coli, is thought to add L-glycero-D-manno-heptose to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide core. Lipopolysaccharide isolated from mutants defective in rfaC lack heptose and all other sugars distal to heptose. The putative donor, ADP-L-glycero-D-manno-heptose, has never been fully characterized and is not readily available. In cell extracts, the analog ADP-mannose can serve as an alternative donor for RfaC-catalyzed glycosylation of the acceptor, Kdo2-lipid IVA. Using a T7 promoter construct that overexpresses RfaC approximately 15,000-fold, the enzyme has been purified to near homogeneity. NH2-terminal sequencing confirms that the purified enzyme is the rfaC gene product. The subunit molecular mass is 36 kDa. Enzymatic activity is dependent upon the presence of Triton X-100 and is maximal at pH 7.5. The apparent Km (determined at near saturating concentrations of the second substrate) is 1.5 mM for ADP-mannose and 4.5 microM for Kdo2-lipid IVA. Chemical hydrolysis of the RfaC reaction product at 100 degrees C in the presence of sodium acetate and 1% sodium dodecyl sulfate generates fragments consistent with the inner Kdo residue of Kdo2-lipid IVA as the site of mannosylation. The analog, Kdo-lipid IVA, functions as an acceptor, but is mannosylated at less than 1% the rate of Kdo2-lipid IVA. The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose. Mannosylation of Kdo2-lipid IVA catalyzed by RfaC proceeds in high yield and may be useful for the synthesis of lipopolysaccharide analogs. Pure RfaC can also be used together with Kdo2-[4'-32P]lipid IVA to assay for the physiological donor (presumably ADP-L-glycero-D-manno-heptose) in a crude, low molecular weight fraction isolated from wild type cells.

Regulation of glycolipid synthesis in HL-60 cells by antisense oligodeoxynucleotides to glycosyltransferase sequences: effect on cellular differentiation.

Treatment of the human promyelocytic leukemia cell line HL-60 with antisense oligodeoxynucleotides to UDP-N-acetylgalactosamine:beta-1,4-N-acetylgalactosaminyl-transferase (GM2-synthase; EC 2.4.1.92) and CMP-sialic acid:alpha-2,8-sialyltransferase (GD3-synthase; EC 2.4.99.8) sequences effectively down-regulated the synthesis of more complex gangliosides in the ganglioside synthetic pathways after GM3, resulting in a remarkable increase in endogenous GM3 with concomitant decreases in more complex gangliosides. The treated cells underwent monocytic differentiation as judged by morphological changes, adherent ability, and nitroblue tetrazolium staining. These data provide evidence that the increased endogenous ganglioside GM3 may play an important role in regulating cellular differentiation and that the antisense DNA technique proves to be a powerful tool in manipulating glycolipid synthesis in the cell.

Inhibition of UDP-GlcNAc:Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase from acute myeloid leukaemia cells by photoreactive nitrophenyl substrate derivatives.

Cells from patients with acute myeloid leukaemia (AML) contain an abnormally high UDP-GlcNAc: Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (core 2 beta 6-Gn-T) activity. Upon UV irradiation at 350 nm, the substrate Gal beta 1-3GalNAc alpha-p-nitrophenyl acted as an effective inhibitor for this enzyme but not for several other transferases. Preincubation with Gal beta 1-3GalNAc alpha-benzyl but not GalNAc alpha-benzyl protected core 2 beta 6-Gn-T from inhibition indicating that the inhibitor is specific for the substrate binding site of core 2 beta 6-Gn-T. A number of other nitrophenyl-sugar derivatives similarly acted as inhibitors for core 2 beta 6-Gn-T. GalNAc alpha-pnp at higher concentrations also inactivated UDP-Gal: GalNAc-R beta 3-galactosyltransferase from rat liver and AML cells and inhibition could be reduced by substrate protection. These results suggest that pnp-sugar derivatives may prove useful as specific inhibitors of glycosyltransferases and as affinity labels.

Folimycin (concanamycin A), a specific inhibitor of V-ATPase, blocks intracellular translocation of the glycoprotein of vesicular stomatitis virus before arrival to the Golgi apparatus.

Folimycin (concanamycin A) specifically inhibited vacuolar-type ATPase as far as examined. Folimycin blocked excretion of the glycoprotein (G protein) of vesicular stomatitis virus into the medium and, instead, G protein was accumulated intracellularly. The intracellularly accumulated G protein electrophoresed a little faster than mature one. The N-glycan of the G protein was endoglycosidase H-sensitive, and terminal galactose and N-acetylglucosamine were not detected essentially on sequential digestion with exoglycosidases, indicating that processings known to occur in the Golgi apparatus do not take place in the presence of folimycin. The oligosaccharide chain of the G protein was determined to have a composition of Man8GlcNAc2 as analyzed by Bio-Gel P-4 column chromatography and high-performance liquid chromatography following digestion with alpha- and then with beta-mannosidase. Activities of mannosidase I and glycosyltransferases prepared from baby hamster kidney cells were not inhibited as far as examined, indicating that the incompleteness of the N-glycosidic chain in folimycin-treated cells is not caused by inhibition of processing enzymes. Taken together these observations suggest that folimycin blocks the intracellular translocation of G protein before the step of trimming by mannosidase I which is confined to the cis compartment of the Golgi. The intracellular localization of G protein as revealed by fluorescence microscopy was in good accordance with this assumption.

Effect of non-steroidal anti-inflammatory drugs (NSAIDS) on glycosyltransferase activity from human osteoarthritic cartilage.

The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the activity of glycosyltransferases required for the synthesis of the polysaccharide chains of proteoglycans, was studied in human osteoarthritic cartilage in vitro. Using exogenous acceptors, salicylate and indomethacin suppressed the activity of glucuronyl- and xylosyltransferases in a concentration-dependent manner, but had little effect on N-acetylgalactosaminyl- and galactosyltransferases. When used at a concentration derived from the values found in the synovial fluid, salicylate, indomethacin and chloroquine significantly suppressed the activity of glucuronyl- and xylosyltransferases, while tiaprofenic acid, paracetamol (acetaminophen), floctafenine, ketoprofen, ibuprofen and tenoxicam had no effect on the enzymes. An alteration of some glycosyltransferases could explain the reported suppressive effect of some NSAIDs on cartilage proteoglycan synthesis.