Impact of biofilm matrix components on interaction of commensal Escherichia coli with the gastrointestinal cell line HT-29.

Commensal Escherichia coli form biofilms at body temperature by expressing the extracellular matrix components curli fimbriae and cellulose. The role of curli fimbriae and cellulose in the interaction of commensal E. coli with the intestinal epithelial cell line HT-29 was investigated. Expression of curli fimbriae by the typical commensal isolate E. coli TOB1 caused adherence and internalization of the bacteria and triggered IL-8 production in HT-29 cells. In particular, induction of IL-8 production was complex and involved curli-bound flagellin. While cellulose alone had no effect on the interaction of TOB1 with HT-29 cells, co-expression of cellulose with curli fimbriae decreased adherence to, internalization and IL-8 induction of HT-29 cells. Investigation of a panel of commensal isolates showed a partial correlation between expression of curli fimbriae and enhanced internalization and IL-8 production. In addition, a high immunostimulatory flagellin was identified. Thus, the consequences of expression of extracellular matrix components on commensal bacterial-host interactions are complex.

Formation and analysis of mannosylerythritol lipids secreted by Pseudozyma aphidis.

Pseudozyma aphidis DSM 70725 was found to be a novel producer of mannosylerythritol lipids (MELs). The MELs were quantified by HPLC. Glucose as carbon source for precultivation supported growth well. By contrast, at concentrations >30 g l(-1) in preculture, subsequent MEL formation in the main culture with soybean oil as sole carbon source was reduced. The type of substrate supply considerably influenced MEL formation. High concentrations of soybean oil (80 ml l(-1)) at init favored the production process when compared to a stepwise (20 ml l(-1)) addition. Mannose or erythritol were suitable second carbon sources that enhanced the MEL yield with soybean oil as preferred primary substrate. After 10 days, a maximum yield of 75 g l(-1) was attained during shake-flask cultivation. Biofuel (rapeseed oil methyl ester) also resulted in high yields of MEL, but glucose reduced the MEL yield. Analysis by GC-MS showed that all fatty acids contained in MEL and derived from soybean oil or related methyl ester were degraded by C2-units to differing extents. The surface (water/air) and interfacial (water/hexadecane) tension of the MELs produced from different carbon sources were reduced to a minimum of 26.2 mN m(-1) and 1 mN m(-1), respectively.

Diglucosyl-glycerolipids from the marine sponge-associated Bacillus pumilus strain AAS3: their production, enzymatic modification and properties.

The marine strain Bacillus pumilus strain AAS3, isolated from the Mediterranean sponge Acanthella acuta, produced a diglucosyl-glycerolipid, 1,2-O-diacyl-3-[beta-glucopyranosyl-(1-6)-beta-glucopyranosyl)]glycerol, with 14-methylhexadecanoic acid and 12-methyltetradecanoic acid as the main fatty acid moieties (GGL11). On a 30 l scale, using artificial seawater supplemented with glucose (20 g/l), yeast extract (10 g/l), and suitable nitrogen/phosphate sources, growth-associated glycoglycerolipid production reached its maximum yield of 90 mg/l after 11 h. Lipase-catalyzed modification of the native substance led to the deacylated parent compound (GG11), which could be reacylated using the same enzyme system to afford a new dipentenoyl-diglucosylglycerol (GGL12) as the major product upon addition of 4-pentenoic acid to the medium. GGL11 decreased the surface tension of water from 72 mN/m to 29 mN/m and the interfacial tension of the water/ n-hexadecane system from 44 to 5 mN/m. Anti-tumor-promoting studies on this class of diglucosyl glycerol products showed that the carbohydrate/glycerol backbone (GG11) has a more potent inhibitory activity than the acylated compounds. The diglucosyl-glycerol GG11 strongly inhibited growth of the tumor cell lines HM02 and Hep G2 (50% inhibition at approximately 1 microg/ml), while the glycerolipids GGL11 and GGL12 were less active or had no effect.

Proteome reference map of Pseudomonas putida strain KT2440 for genome expression profiling: distinct responses of KT2440 and Pseudomonas aeruginosa strain PAO1 to iron deprivation and a new form of superoxide dismutase.

The genome sequence of Pseudomonas putida strain KT2440, a nutritionally versatile, saprophytic and plant root-colonizing Gram-negative soil bacterium, was recently determined by K. E. Nelson et al. (2002, Environ Microbiol 4: 799-808). Here, we present a two-dimensional gel protein reference map of KT2440 cells grown in mineral salts medium with glucose as carbon source. Proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis, in conjunction with an in-house database developed from the genome sequence of KT2440, and approximately 200 two-dimensional gel spots were assigned. The map was used to assess the genomic response of KT2440 to iron limitation stress and to compare this response with that of the closely related facultative human pathogen Pseudomonas aeruginosa strain PAO1. The synthesis of about 25 proteins was affected in both strains, including four prominent upregulated ferric uptake regulator (Fur) protein-dependent proteins, but there were also striking differences in their proteome responses, for example in the expression of superoxide dismutases (Sod), which may indicate important roles of iron-responsive functions in the adaptation of these two bacteria to different lifestyles. The Sod enzyme of KT2440 was shown to be a novel heterodimer of the SodA and SodB polypeptides.

Structural and functional cellular changes induced by Burkholderia pseudomallei rhamnolipid.

In this study we report that extracellular Burkholderia pseudomallei rhamnolipid induced cytopathic changes characterized by retraction, rounding up, and, finally, detachment in phagocytic and nonphagocytic cell lines. These changes were due to a progressive reorganization of the F-actin network resulting in impaired cell cycle progression and a reduced phagocytic function of macrophages.

Structural analysis and antibody response to the extracellular glutathione S-transferases from Onchocerca volvulus.

Onchocerca volvulus is a human pathogenic filarial parasite which, like other parasitic nematodes, is capable of surviving in an immunologically competent host by employing a variety of immune evasion strategies and defense mechanisms including the detoxification and repair mechanisms of the glutathione S-transferases (GSTs). In this study we analyzed the glycosylation pattern and the immunological properties of extracellular O. volvulus GST1a and -1b (OvGST1a and -1b). The enzymes differ in only 10 amino acids, and both are glycoproteins that have cleavable signal peptides and unusual N-terminal extensions. These characteristics have not been described for other GSTs so far. Mass spectrometry analyses indicate that both enzymes carry high-mannose type oligosaccharides on at least four glycosylation sites. Glycosylation sites 1 to 3 of OvGST1a (OvGST1b sites 2 to 4) are occupied by truncated N-glycans (Man(2)GlcNAc2 to Man(5)GlcNAc(2)), and N glycosylation site 4 of OvGST1a (OvGST1b site 5) carries Man(5)GlcNAc2 to Man(9)GlcNAc(2). To analyze the capacity of these secretory GSTs to stimulate host immune responses, we studied the antibody responses of onchocerciasis patients against the native affinity-purified OvGST1a and -1b. By enzyme-linked immunosorbent assay we showed that OvGST1a and -1b are immunodominant antigens, with less than 7% nonresponder patients. A direct comparison of the antibody responses to the glycosylated and deglycosylated forms demonstrates the high immunogenicity of the N-glycans. Analyses of the antibody responses to the unusual N-terminal extension show an enhanced recognition of this portion by patients as opposed to recognition of the recombinant protein without extension.

Production and molecular characterization of clinical phase i anti-melanoma mouse IgG3 monoclonal antibody R24.

R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglioside GD3 expressed by cells of neuroectodermal origin. The anti-tumor activity of R24 has been demonstrated in initial phase I and pilot trials in patients suffering from metastatic melanoma. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important antibody. Growth, metabolism, and IgG production of R24 secreting hybridoma cells were analyzed on 1 L bioreactor bench scale using repeated-batch mode. The amount of 57 mg of pure mab was obtained from 1.6 L crude supernatant by protein A chromatography. Western blot binding assays with sugar-specific lectins revealed glycosylation of the heavy chains, whereas no carbohydrates were detectable on the light chains. Because glycosylation is essential for antibody effector functions in vivo (such as complement fixation or binding to macrophage Fc receptors), mab R24 was subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Released glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Six major biantennary chains of the complex glycosylation phenotype were found with variations in galactosylation and core fucosylation. The predominant N-linked structure, indicating the high degree of agalactosyl glycoforms, was the agalacto biantennary chain with a relative percentage of 57% (51% core-fucosylated, 6% nonfucosylated). The second most abundant oligosaccharide was the monogalacto biantennary chain amounting to 30% (26% core- and 4% nonfucosylated). The antibody contained 0.46 microg sialic acid per mg protein, which splits into 0.243 microg Neu5Gc and 0.217 microg Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06. Furthermore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interactions were measured with an optical biosensor (BIAcore). From the structural data obtained in this study it is concluded that glycosylation of the antibody may be important in the clinical outcome of targeted anti-cancer immunotherapy.

Rocaglamides, glycosides, and putrescine bisamides from Aglaia dasyclada.

A phytochemical analysis of the leaves of Aglaia dasyclada collected in Yunnan Province (People's Republic of China) yielded five cyclopentabenzofurans (1-5) of the rocaglamide family that are common secondary metabolites of Aglaia species as well as four biogenetically related compounds of the aglain (7), aglaforbesin (8) and forbaglin (9, 10) types. In addition, the cinnamic acid amide dasyclamide (6), which is a putative biogenetic precursor of these compounds (7-10), was isolated. The structures of the new compounds (6-10) were assigned unambiguously from the combined use of 1D and 2D NMR spectroscopy and mass spectrometry.

Application of MS and NMR to the structure elucidation of complex sugar moieties of natural products: exemplified by the steroidal saponin from Yucca filamentosa L.

An approach, using well characterized procedures, is presented that should be of general applicability for the structural elucidation of complex sugar moieties of natural products. The methods used are exemplified by the structure elucidation of a new gitogenin-based steroidal saponin that has a strong leishmanicidal activity similar to preparations used in clinical practice and has been isolated by bioactivity-guided fractionation of the ethanolic extract of Yucca filamentosa L. leaves. The saponin has been characterized as 3-O-((beta-D-glucopyranosyl-(1-->3)- beta-D-glucopyranosy-(1-->2))(alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->3))-beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranosyl)-25R,5alpha-spirostan-2alpha,3beta-diol.

The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix.

Production of cellulose has been thought to be restricted to a few bacterial species such as the model organism Acetobacter xylinus. We show by enzymatic analysis and mass spectrometry that, besides thin aggregative fimbriae, the second component of the extracellular matrix of the multicellular morphotype (rdar) of Salmonella typhimurium and Escherichia coli is cellulose. The bcsA, bcsB, bcsZ and bcsC genes responsible for cellulose biosynthesis are not regulated by AgfD, the positive transcriptional regulator of the rdar morphotype. Transcription of the bcs genes was not co-expressed with the rdar morphotype under any of the environmental conditions examined. However, cellulose biosynthesis was turned on by the sole expression of adrA, a gene encoding a putative transmembrane protein regulated by agfD, indicating a novel pathway for the activation of cellulose synthesis. The co-expression of cellulose and thin aggregative fimbriae leads to the formation of a highly hydrophobic network with tightly packed cells aligned in parallel in a rigid matrix. As the production of cellulose would now appear to be a property widely distributed among bacteria, the function of the cellulose polymer in bacteria will have to be considered in a new light.

Monodesmosidic saponins from Herniaria hirsuta.

Two new monodesmosidic saponins, herniaria saponins E and F, were isolated from the aerial parts of Herniaria hirsuta. On the basis of chemical and spectral evidence, their structures were established to be 2-O-acetyl medicagenic acid 28-O-beta-D-xylopyranosyl- (1-->4)-alpha-L-rhamnopyranosyl(1-->2)-[beta-D-glucopyranosyl(1-->6)]-be ta- D-glucopyranoside (herniaria saponin E, 1) and medicagenic acid 28-O-beta-D-xylopyranosyl(1-->4)-alpha-L- rhamnopyranosyl(1-->2)-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D- glucopyranosyl-(1-->6)]-beta-D-glucopyranoside (herniaria saponin F, compound 2).

Characterization of the carbohydrate chains of the secreted form of the human epidermal growth factor receptor.

The human epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein having 11 potential N-glycosylation sites in its extracellular domain. N-Glycosylation is needed for proper membrane insertion, EGF binding and receptor functioning. The human epidermoid carcinoma A431 cell line secretes a soluble 105 kDa glycoprotein (sEGFR) that represents the extracellular domain of the membrane-bound form, and its glycosylation pattern has been investigated. After liberation of the oligosaccharides from sEGFR with PNGase F, the glycans were fractionated along different routes, including Concanavalin A affinity chromatography, anion-exchange chromatography, HPLC and high-pH anion-exchange chromatography. The oligosaccharide fractions were characterized by 500- and 600-MHz 1H-NMR spectroscopy and mass spectrometry (FAB, ESI, and MALDI-TOF). The oligomannose-type glycans range from Man5GlcNAc2 to Man8GlcNAc2 and account for 17% of the total carbohydrate moiety. Furthermore, di-, tri'- and tetraantennary complex-type structures are present, both neutral and (alpha2-3)-sialylated (up to tetrasialo), comprising 24 and 59%, respectively, of the total carbohydrate moiety. In this study, 32 new complex-type glycans are characterized containing the Le(x), Le(Y), and sialyl-Le(x) determinants, the bloodgroup A and H antigens, as well as the ALe(Y) determinant. This first comprehensive glycosylation study on a human nonrecombinant receptor shows the immense heterogeneity of the glycosylation of sEGFR.

Secondary products in mycorrhizal roots of tobacco and tomato.

Colonization of the roots of various tobacco species and cultivars (Nicotiana glauca Grah., N. longiflora Cav., N. rustica L., N. tabacum L., N. tabacum L. cv. Samsun NN, N. sanderae hort. Sander ex Wats.) as well as tomato plants (Lycopersicon esculentum L. cv. Moneymaker) by the arbuscular mycorrhizal fungus Glomus intraradices Schenck and Smith resulted in the accumulation of several glycosylated C13 cyclohexenone derivatives. Eight derivatives were isolated from the mycorrhizal roots by preparative high performance liquid chromatography (HPLC) and spectroscopically identified (MS and NMR) as mono-, di- and triglucosides of 6-(9-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one and monoglucosides of 6-(9-hydroxybutyl)-1,5-dimethyl-4-cyclohexen-3-one-1-carboxylic acid and 6-(9-hydroxybutyl)-1,1-dimethyl-4-cyclohexen-3-one-5-carboxylic acid. In contrast to the induced cyclohexenone derivatives, accumulation of the coumarins scopoletin and its glucoside (scopolin) in roots of N. glauca Grah. and N. tabacum L. cv. Samsun NN, was markedly suppressed.

Betalains from Christmas cactus.

The presence of 14 betalain pigments have been detected by their characteristic spectral properties in flower petals of Christmas cactus (Schlumbergera x buckleyi). Along with the known vulgaxanthin I, betalamic acid, betanin and phyllocactin (6'-O-malonylbetanin), the structure of a new phyllocactin-derived betacyanin was elucidated by various spectroscopic techniques and carbohydrate analyses as betanidin 5-O-(2'-O-beta-D-apiofuranosyl-6'-O-malonyl)-beta-D-glucopyranosid e. Among the more complex betacyanins occurring in trace amounts, the presence of a new diacylated betacyanin ¿betanidin 5-O-[(5"-O-E-feruloyl)-2'-O-beta-D- apiofuranosyl-6'-O-malonyl)]-beta-D-glucopyranoside¿ has been ascertained. Furthermore, the accumulation of betalains during flower development and their pattern in different organs of the flower has been examined.

Production and structure elucidation of glycoglycerolipids from a marine sponge-associated microbacterium species.

The bacterium Microbacterium sp., isolated from the sponge Halichondria panicea, produced four unusual cell-associated glycoglycerolipids and one diphosphatidylglycerol when grown on marine broth and on artificial seawater media. The lipids were isolated by chromatography on silica columns and their structures elucidated using a combination of multidimensional NMR and MS techniques. The main compound was 1-O-acyl-3-[alpha-glucopyranosyl-(1-3)-(6-O-acyl-alpha-mannopyranosyl )]glycerol (GGL.2) with 14-methyl-hexadecanoic acid and 12-methyl-tetradecanoic acid positioned at C-6 of the mannose unit and at the glycerol moiety. Glycolipid production was correlated with growth and reached a maximum value of 200 mg/L when grown on artificial seawater medium with 20 g/L glucose. The main compound decreased the surface tension of water down to 33 mN/m and the interfacial tension of the water/n-hexadecane system down to 5 mN/m. In addition to this good surface-active behavior, the main glycoglycerolipid showed antitumor activities.

Metabolic shifts do not influence the glycosylation patterns of a recombinant fusion protein expressed in BHK cells.

BHK-21 cells expressing a human IgG-IL2 fusion protein, with potential application in tumor-targeted therapy, were grown under different nutrient conditions in a continuous system for a time period of 80 days. At very low-glucose (< 0.5 mM) or glutamine (< 0. 2 mM) concentrations, a shift toward an energetically more efficient metabolism was observed. Cell-specific productivity was maintained under metabolically shifted growth conditions and at the same time an almost identical intracellular ATP content, obtained by in vivo (31)P NMR experiments, was observed. No significant differences in the oligosaccharide structures were detected from the IgG-IL2 fusion protein preparations obtained by growing cells under the different metabolic states. By using oligosaccharide mapping and MALDI/TOF-MS, only neutral diantennary oligosaccharides with or without core alpha1-6-linked fucose were detected that carried no, one or two beta1-4-linked galactose. Although the O-linked oligosaccharide structures that are present in the IL2 moiety of the protein were studied with less detail, the data obtained from the hydrazinolysis procedure point to the presence of the classical NeuAcalpha2-3Galbeta1-3GalNAc structure. Here, it is shown that under different defined cellular metabolic states, the quality of a recombinant product in terms of O- and N-linked oligosaccharides is stable, even after a prolonged cultivation period. Moreover, unaffected intracellular ATP levels under the different metabolic states were observed.

Biochemical and functional characterization of the Tn-specific lectin from Salvia sclarea seeds.

SSL, the lectin isolated from Salvia sclarea seeds, recognizes the Tn antigen (GalNAcalpha-O-Ser/Thr), a specific marker of many human carcinomas. Two-dimensional electrophoresis, amino-acid and amino-sugar analysis, and MALDI-TOF MS showed that SSL is an acidic (pI 5.5), 60-61-kDa dimeric glycoprotein composed of apparently identical subunits linked by a single disulfide bond. The apparent molecular mass of SSL in solution determined by equilibrium sedimentation analytical ultracentrifugation was 59 +/- 9 kDa. This value did not change in the pH range 2.5-8.5, indicating that SSL does not associate into higher order structures. Tandem mass spectrometry and methylation analysis of N-glycans released from SSL by hydrazinolysis indicated that SSL possesses 2-3 glycosylation sites occupied with the typical plant glycans Manalpha1-6[(Manalpha1-3)(Xylbeta1-2)]Manbeta1-4 -GlcNAcbeta1-4(Fucalp ha1-3)GlcNAc and [(Manalpha1-3/6)(Xylbeta1-2)]Manbeta1-4-GlcNAcbeta1 -4(Fucalpha1-3)Glc NAc. The influence of adjacent Tn structures on the binding of two Tn-specific lectins (SSL and the isolectin B4 from Vicia villosa) and an anti-Tn monoclonal antibody (mAb 83D4) was evaluated using synthetic Tn glycopeptides. The binding of both lectins to the synthetic Tn glycopeptides was independent of the density of Tn structures. On the other hand, mAb 83D4 only reacted with glycopeptides displaying two or three consecutive Tn structures.

Fucosylated hybrid-type N-glycans on the secreted human epidermal growth factor receptor from swainsonine-treated A431 cells.

N-Glycans linked to the human secreted form of epidermal growth factor receptor were isolated from A431 cells after swainsonine treatment. Analysis of the oligosaccharides by (1)H NMR spectroscopy and mass spectrometry shows the presence of oligomannose- and (alpha2-3)-sialylated hybrid-type glycans. The major hybrid-type oligosaccharide chains are fucosylated at the Asn-bound GlcNAc residue. Smaller amounts of the hybrid-type structures are also fucosylated at peripheral GlcNAc residues, constituting the sialyl-Le(x) antigen. No complex-type glycans are found, suggesting the absence of alpha-mannosidase III. An assay for alpha-mannosidase III on the A431 cells in the absence and presence of 6 microM swainsonine shows that Man(5)GlcNAc(2) is not converted into Man(3)GlcNAc(2), thereby confirming that these cells do not contain alpha-mannosidase III activity.

Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells.

The analysis of many natural glycoproteins and their recombinant counterparts from mammalian hosts has revealed that the basic oligosaccharide structures and the site occupancy of glycosylated polypeptides are primarily dictated by the protein conformation. The equipment of many frequently used host cells (e.g. BHK-21 and CHO-cells) with glycosyltransferases, nucleotide-sugar synthases and transporters appears to be sufficient to guarantee complex-type glycosylation of recombinant proteins with a high degree of terminal alpha2-3 sialylation even under high expression conditions. Some human tissue-specific terminal carbohydrate motifs are not synthesized by these cells since they lack the proper sugar-transferring enzymes (e.g. alpha1-3/4 fucosyltransferases, alpha2-6 sialyltransferases). Glycosylation engineering of these hosts by stable transfection with genes encoding terminal human glycosyltransferases allows to obtain products with tailored (human tissue-specific) glycosylation in high yields. Using site-directed mutagenesis, unglycosylated polypeptides can be successfully converted in N- and/or O-glycoproteins by transferring glycosylation domains (consisting of 7-17 amino acids) from donor glycoproteins to different loop regions of acceptor proteins. The genetic engineering of glycoproteins and of host cell lines are considered to provide a versatile tool to obtain therapeutic glyco-products with novel/improved in-vivo properties, e.g. by introduction of specific tissue-targeting signals by a rational design of terminal glycosylation motifs.