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1.
Chemical analyses, mass spectrometry, and NMR spectroscopy were applied to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas veronii strain Bs19, serotype O16. ESI-MS revealed that the most abundant LPS glycoforms have tetra-acylated or hexa-acylated lipid A species, consisting of a bisphosphorylated GlcN disaccharide with an AraN residue as a non-stoichiometric substituent, and a core oligosaccharide composed of Hep5Hex3HexN1Kdo1P1. Sugar and methylation analysis together with 1D and 2D 1H and 13C NMR spectroscopy were the main methods used, and revealed that the O-specific polysaccharide (OPS) of A. veronii Bs19 was built up of tetrasaccharide repeating units with the structure: →4)-α-d-Quip3NAc-(1→3)-α-l-Rhap-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→. This composition was confirmed by mass spectrometry. The charge-deconvoluted ESI FT-ICR MS recorded for the LPS preparations identified mass peaks of SR- and R-form LPS species, that differed by Δm = 698.27 u, a value corresponding to the calculated molecular mass of one OPS repeating unit (6dHexNAc6dHexHexHexNAc-H2O). Moreover, unspecific fragmentation spectra confirmed the sequence of the sugar residues in the OPS and allowed to assume that the elucidated structure also represented the biological repeating unit.  相似文献   

2.
Although human exposure to Gram-negative Vibrio vulnificus (V. vulnificus) lipopolysaccharide (LPS) has been reported to result in septic shock, its impact on the central nervous system’s innate immunity remains undetermined. The purpose of this study was to determine whether V. vulnificus MO6-24/O LPS might activate rat microglia in vitro and stimulate the release of superoxide anion (O2−), a reactive oxygen species known to cause oxidative stress and neuronal injury in vivo. Brain microglia were isolated from neonatal rats, and then treated with either V. vulnificus MO6-24/O LPS or Escherichia coli O26:B6 LPS for 17 hours in vitro. O2− was determined by cytochrome C reduction, and matrix metalloproteinase-2 (MMP-2) and MMP-9 by gelatinase zymography. Generation of cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 alpha (IL-1α), IL-6, and transforming growth factor-beta 1 (TGF-β1), chemokines macrophage inflammatory protein (MIP-1α)/chemokine (C-C motif) ligand 3 (CCL3), MIP-2/chemokine (C-X-C motif) ligand 2 (CXCL2), monocyte chemotactic protein-1 (MCP-1)/CCL2, and cytokine-induced neutrophil chemoattractant-2alpha/beta (CINC-2α/β)/CXCL3, and brain-derived neurotrophic factor (BDNF), were determined by specific immunoassays. Priming of rat microglia by V. vulnificus MO6-24/O LPS in vitro yielded a bell-shaped dose-response curve for PMA (phorbol 12-myristate 13-acetate)-stimulated O2− generation: (1) 0.1–1 ng/mL V. vulnificus LPS enhanced O2− generation significantly but with limited inflammatory mediator generation; (2) 10–100 ng/mL V. vulnificus LPS maximized O2− generation with concomitant release of thromboxane B2 (TXB2), matrix metalloproteinase-9 (MMP-9), and several cytokines and chemokines; (3) 1000–100,000 ng/mL V. vulnificus LPS, with the exception of TXB2, yielded both attenuated O2− production, and a progressive decrease in MMP-9, cytokines and chemokines investigated. Thus concentration-dependent treatment of neonatal brain microglia with V. vulnificus MO6-24/O LPS resulted in a significant rise in O2− production, followed by a progressive decrease in O2− release, with concomitant release of lactic dehydrogenase (LDH), and generation of TXB2, MMP-9, cytokines and chemokines. We hypothesize that the inflammatory mediators investigated may be cytotoxic to microglia in vitro, by an as yet undetermined autocrine mechanism. Although V. vulnificus LPS was less potent than E. coli LPS in vitro, inflammatory mediator release by the former was clearly more efficacious. Finally, we hypothesize that should V. vulnificus LPS gain entry into the CNS, it would be possible that microglia might become activated, resulting in high levels of O2− as well as neuroinflammatory TXB2, MMP-9, cytokines and chemokines.  相似文献   

3.
A group of virulent Aeromonas hydrophila, A. sobria, and A. veronii biovar sobria strains isolated from humans and fish have been described; these strains classified to serotype O11 are serologically related by their lipopolysaccharide (LPS) O-antigen (O-polysaccharide), and the presence of an S-layer consisting of multiple copies of a crystalline surface array protein with a molecular weight of 52 kDa in the form of a crystalline surface array which lies peripheral to the cell wall. A. hydrophila strain AH-1 is one of them. We isolated the LPS from this strain and determined the structure of the O-polysaccharide, which was similar to that previously described for another strain of serotype O11. The genetics of the O11-antigen showed the genes (wbO11 cluster) in two sections separated by genes involved in biosynthesis and assembly of the S-layer. The O11-antigen LPS is an example of an ABC-2-transporter-dependent pathway for O-antigen heteropolysaccharide (disaccharide) assembly. The genes involved in the biosynthesis of the LPS core (waaO11 cluster) were also identified in three different chromosome regions being nearly identical to the ones described for A. hydrophila AH-3 (serotype O34). The genetic data and preliminary chemical analysis indicated that the LPS core for strain AH-1 is identical to the one for strain AH-3.  相似文献   

4.
Three different fucoidan fractions were isolated and purified from the brown alga, Sargassum mcclurei. The SmF1 and SmF2 fucoidans are sulfated heteropolysaccharides that contain fucose, galactose, mannose, xylose and glucose. The SmF3 fucoidan is highly sulfated (35%) galactofucan, and the main chain of the polysaccharide contains a →3)-α-l-Fucp(2,4SO3−)-(1→3)-α-l-Fucp(2,4SO3−)-(1→ motif with 1,4-linked 3-sulfated α-l-Fucp inserts and 6-linked galactose on reducing end. Possible branching points include the 1,2,6- or 1,3,6-linked galactose and/or 1,3,4-linked fucose residues that could be glycosylated with terminal β-d-Galp residues or chains of alternating sulfated 1,3-linked α-l-Fucp and 1,4-linked β-d-Galp residues, which have been identified in galactofucans for the first time. Both α-l-Fucp and β-d-Galp residues are sulfated at C-2 and/or C-4 (and some C-6 of β-d-Galp) and potentially the C-3 of terminal β-d-Galp, 1,4-linked β-d-Galp and 1,4-linked α-l-Fucp residues. All fucoidans fractions were less cytotoxic and displayed colony formation inhibition in colon cancer DLD-1 cells. Therefore, these fucoidan fractions are potential antitumor agents.  相似文献   

5.
A new chrysolaminarin, named CL2, with a molecular mass of 7.75 kDa, was purified from the marine diatom, Odontella aurita, using DEAE-52 cellulose anion-exchange chromatography and Sephadex G-200 gel-filtration chromatography. The monosaccharide and structural analysis revealed that CL2 was a glucan mainly composed of glucose, which was linked by the β-d-(1→3) (main chain) and β-d-(1→6) (side chain) glycosidic bond, demonstrated by infrared spectroscopy (IR) and nuclear magnetic resonance (NMR). The antioxidant activity tests revealed that the CL2 presented stronger hydroxyl radical scavenging activity with increasing concentrations, but less was effective on reducing power analysis and scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The influences of nitrogen concentration and light intensity on chrysolaminarin production of O. aurita were further investigated in a glass column photobioreactor, and a record high chrysolaminarin productivity of 306 mg L−1 day−1 was achieved. In conclusion, the chrysolaminarin CL2 from O. aurita may be explored as a natural antioxidant agent for application in aquaculture, food and pharmaceutical areas.  相似文献   

6.
Two new indole-diterpenoids (1 and 2) and a new isocoumarin (3), along with the known β-aflatrem (4), paspalinine (5), leporin B (6), α-cyclopiazonic acid (7), iso-α-cyclopiazonic acid (8), ditryptophenaline (9), aflatoxin B1 (10), 7-O-acetylkojic acid (11) and kojic acid (12), were isolated from the fermentation broth of the marine-derived fungus, Aspergillus flavus OUCMDZ-2205. The structures of Compounds 1–12 were elucidated by spectroscopic analyses, quantum ECD calculations and the chemical method. New Compound 1 exhibited antibacterial activity against Staphylococcus aureus with a MIC value of 20.5 μM. Both new Compounds 1 and 2 could arrest the A549 cell cycle in the S phase at a concentration of 10 μM. Compound 1 showed PKC-beta inhibition with an IC50 value of 15.6 μM. In addition, the absolute configurations of the known compounds, 4–6 and leporin A (6a), were also determined for the first time.  相似文献   

7.
The A. salmonicida A450 LPS O-antigen, encoded by the wbsalmo gene cluster, is exported through an ABC-2 transporter-dependent pathway. It represents the first example of an O-antigen LPS polysaccharide with three different monosaccharides in their repeating unit assembled by this pathway. Until now, only repeating units with one or two different monosaccharides have been described. Functional genomic analysis of this wbsalmo region is mostly in agreement with the LPS O-antigen structure of acetylated l-rhamnose (Rha), d-glucose (Glc), and 2-amino-2-deoxy-d-mannose (ManN). Between genes of the wbsalmo we found the genes responsible for the biosynthesis and assembly of the S-layer (named A-layer in these strains). Through comparative genomic analysis and in-frame deletions of some of the genes, we concluded that all the A. salmonicida typical and atypical strains, other than A. salmonicida subsp. pectinolytica strains, shared the same wbsalmo and presence of A-layer. A. salmonicida subsp. pectinolytica strains lack wbsalmo and A-layer, two major virulence factors, and this could be the reason they are the only ones not found as fish pathogens.  相似文献   

8.
The production, characterization, and antioxidant capacity of the carotenoid fucoxanthin from the marine diatom Odontella aurita were investigated. The results showed that low light and nitrogen-replete culture medium enhanced the biosynthesis of fucoxanthin. The maximum biomass concentration of 6.36 g L−1 and maximum fucoxanthin concentration of 18.47 mg g−1 were obtained in cultures grown in a bubble column photobioreactor (Ø 3.0 cm inner diameter), resulting in a fucoxanthin volumetric productivity of 7.96 mg L−1 day−1. A slight reduction in biomass production was observed in the scaling up of O. aurita culture in a flat plate photobioreactor, yet yielded a comparable fucoxanthin volumetric productivity. A rapid method was developed for extraction and purification of fucoxanthin. The purified fucoxanthin was identified as all-trans-fucoxanthin, which exhibited strong antioxidant properties, with the effective concentration for 50% scavenging (EC50) of 1,1-dihpenyl-2-picrylhydrazyl (DPPH) radical and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical being 0.14 and 0.03 mg mL−1, respectively. Our results suggested that O. aurita can be a natural source of fucoxanthin for human health and nutrition.  相似文献   

9.
Bullacta exarata is one of the most economically important aquatic species in China, noted for not only its delicious taste and nutritional value, but also for its pharmacological activities. In order to explore its potential in medical applications, a mannoglucan designated as BEPS-IB was isolated and purified from the foot muscle of B. exarata after papain digestion. Chemical composition analysis indicated BEPS-IB contained mainly d-glucose and d-mannose in a molar ratio of 1:0.52, with an average molecular weight of about 94 kDa. The linkage information was determined by methylation analysis, and the anomeric configuration and chain linkage were confirmed by IR and 2D NMR. The results indicated BEPS-IB was composed of Glcp6Manp heptasaccharide repeating unit in the backbone, with occasional branch chains of mannose residues (14%) occurring in the backbone mannose. Further antioxidant assay indicated BEPS-IB exhibited positive antioxidant activity in scavenging superoxide radicals and reducing power. This is the first report on the structure and bioactivity of the mannoglucan from the B. exarata.  相似文献   

10.
Algae contain a number of anti-inflammatory bioactive compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA) and chlorophyll a, hence as dietary ingredients, their extracts may be effective in chronic inflammation-linked metabolic diseases such as cardiovascular disease. In this study, anti-inflammatory potential of lipid extracts from three red seaweeds (Porphyra dioica, Palmaria palmata and Chondrus crispus) and one microalga (Pavlova lutheri) were assessed in lipopolysaccharide (LPS)-stimulated human THP-1 macrophages. Extracts contained 34%–42% total fatty acids as n-3 PUFA and 5%–7% crude extract as pigments, including chlorophyll a, β-carotene and fucoxanthin. Pretreatment of the THP-1 cells with lipid extract from P. palmata inhibited production of the pro-inflammatory cytokines interleukin (IL)-6 (p < 0.05) and IL-8 (p < 0.05) while that of P. lutheri inhibited IL-6 (p < 0.01) production. Quantitative gene expression analysis of a panel of 92 genes linked to inflammatory signaling pathway revealed down-regulation of the expression of 14 pro-inflammatory genes (TLR1, TLR2, TLR4, TLR8, TRAF5, TRAF6, TNFSF18, IL6R, IL23, CCR1, CCR4, CCL17, STAT3, MAP3K1) by the lipid extracts. The lipid extracts effectively inhibited the LPS-induced pro-inflammatory signaling pathways mediated via toll-like receptors, chemokines and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling molecules. These results suggest that lipid extracts from P. lutheri, P. palmata, P. dioica and C. crispus can inhibit LPS-induced inflammatory pathways in human macrophages. Therefore, algal lipid extracts should be further explored as anti-inflammatory ingredients for chronic inflammation-linked metabolic diseases.  相似文献   

11.
A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(d-Pro-d-Phe) (1), cyclo(d-Tyr-d-Pro) (2), phenethyl 5-oxo-l-prolinate (3), cyclo(l-Ile-l-Pro) (4), cyclo(l-Leu-l-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1–6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.  相似文献   

12.
In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11). The monosaccharide is non-enzymatically converted to 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH), then reduced to 2-keto-3-deoxy-d-gluconate (KDG) by a specific reductase, and metabolized through the Entner–Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%–88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.  相似文献   

13.
Two new secondary metabolites, namely, pinodiketopiperazine A (1) and 6,7-dihydroxy-3-methoxy-3-methylphthalide (2), along with alternariol 2,4-dimethyl ether (3) and l-5-oxoproline methyl ester (4), which were isolated from a natural source for the first time but have been previously synthesized, were characterized from the marine sediment-derived fungus Penicillium pinophilum SD-272. In addition, six known metabolites (5–10) were also identified. Their structures were elucidated by analysis of the NMR and mass spectroscopic data. The absolute configuration of compound 1 was determined by experimental and calculated ECD spectra. Compound 2 displayed potent brine shrimp (Artemia salina) lethality with LD50 11.2 μM.  相似文献   

14.
Intracellular fucoidanase was isolated from the marine bacterium, Formosa algae strain KMM 3553. The first appearance of fucoidan enzymatic hydrolysis products in a cell-free extract was detected after 4 h of bacterial growth, and maximal fucoidanase activity was observed after 12 h of growth. The fucoidanase displayed maximal activity in a wide range of pH values, from 6.5 to 9.1. The presence of Mg2+, Ca2+ and Ba2+ cations strongly activated the enzyme; however, Cu2+ and Zn2+ cations had inhibitory effects on the enzymatic activity. The enzymatic activity of fucoidanase was considerably reduced after prolonged (about 60 min) incubation of the enzyme solution at 45 °C. The fucoidanase catalyzed the hydrolysis of fucoidans from Fucus evanescens and Fucus vesiculosus, but not from Saccharina cichorioides. The fucoidanase also did not hydrolyze carrageenan. Desulfated fucoidan from F. evanescens was hydrolysed very weakly in contrast to deacetylated fucoidan, which was hydrolysed more actively compared to the native fucoidan from F. evanescens. Analysis of the structure of the enzymatic products showed that the marine bacteria, F. algae, synthesized an α-l-fucanase with an endo-type action that is specific for 1→4-bonds in a polysaccharide molecule built up of alternating three- and four-linked α-l-fucopyranose residues sulfated mainly at position 2.  相似文献   

15.
Pure compound screening has previously identified the indolglyoxylamidospermidine ascidian metabolites didemnidine A and B (2 and 3) to be weak growth inhibitors of Trypanosoma brucei rhodesiense (IC50 59 and 44 μM, respectively) and Plasmodium falciparum (K1 dual drug resistant strain) (IC50 41 and 15 μM, respectively), but lacking in selectivity (L6 rat myoblast, IC50 24 μM and 25 μM, respectively). To expand the structure–activity relationship of this compound class towards both parasites, we have prepared and biologically tested a library of analogues that includes indoleglyoxyl and indoleacetic “capping acids”, and polyamines including spermine (PA3-4-3) and extended analogues PA3-8-3 and PA3-12-3. 7-Methoxy substituted indoleglyoxylamides were typically found to exhibit the most potent antimalarial activity (IC50 10–92 nM) but with varying degrees of selectivity versus the L6 rat myoblast cell line. A 6-methoxyindolglyoxylamide analogue was the most potent growth inhibitor of T. brucei (IC50 0.18 μM) identified in the study: it, however, also exhibited poor selectivity (L6 IC50 6.0 μM). There was no apparent correlation between antimalarial and anti-T. brucei activity in the series. In vivo evaluation of one analogue against Plasmodium berghei was undertaken, demonstrating a modest 20.9% reduction in parasitaemia.  相似文献   

16.
This study investigates the occurrence of diarrhetic shellfish toxins (DSTs) and their producing phytoplankton species in southern Brazil, as well as the potential for toxin accumulation in co-occurring mussels (Perna perna) and octopuses (Octopus vulgaris). During the spring in 2012 and 2013, cells of Dinophysis acuminata complex were always present, sometimes at relatively high abundances (max. 1143 cells L−1), likely the main source of okadaic acid (OA) in the plankton (max. 34 ng L−1). Dinophysis caudata occurred at lower cell densities in 2013 when the lipophilic toxins pectenotoxin-2 (PTX-2) and PTX-2 seco acid were detected in plankton and mussel samples. Here, we report for the first time the accumulation of DSTs in octopuses, probably linked to the consumption of contaminated bivalves. Perna perna mussels were consistently contaminated with different DSTs (max. 42 µg kg−1), and all octopuses analyzed (n = 5) accumulated OA in different organs/tissues: digestive glands (DGs) > arms > gills > kidneys > stomach + intestine. Additionally, similar concentrations of 7-O-palmytoyl OA and 7-O-palmytoly dinophysistoxin-1 (DTX-1) were frequently detected in the hepatopancreas of P. perna and DGs of O. vulgaris. Therefore, octopuses can be considered a potential vector of DSTs to both humans and top predators such as marine mammals.  相似文献   

17.
Dual choice bioassays were used to evaluate the antifeedant property of essential oil and methanolic extract of Alpinia galanga (L.) (locally known as lengkuas) against two species of termites, Coptotermes gestroi (Wasmann) and Coptotermes curvignathus (Holmgren) (Isoptera: Rhinotermitidae). A 4-cm-diameter paper disc treated with A. galanga essential oil and another treated with either methanol or hexane as control were placed in a petri dish with 10 termites. Mean consumption of paper discs (miligram) treated with 2,000 ppm of essential oil by C. gestroi was 3.30 ± 0.24 mg and by C. curvignathus was 3.32 ± 0.24 mg. A. galanga essential oil showed significant difference in antifeedant effect, 2,000 ppm of A. galanga essential oil was considered to be the optimum concentration that gave maximum antifeedant effect. The essential oil composition was determined using gas chromatography-mass spectrometry. The major component of the essential oil was 1,8-cineol (61.9%). Antifeedant bioassay using 500 ppm of 1,8-cineol showed significant reduction in paper consumption by both termite species. Thus, the bioactive agent in A. galangal essential oil causing antifeeding activity was identified as 1,8-cineol. Repellent activity shows that 250 ppm of 1,8-cineol caused 50.00 ± 4.47% repellency for C. gestroi, whereas for C. curvignathus 750 ppm of 1,8-cineol was needed to cause similar repellent activity (56.67 ± 3.33%). C. curvignathus is more susceptible compare to C. gestroi in Contact Toxicity study, the lethal dose (LD50) of C. curvignathus was 945 mg/kg, whereas LD50 value for C. gestroi was 1,102 mg/kg. Hence 1,8-cineol may be developed as an alternative control against termite in sustainable agriculture practices.  相似文献   

18.
Two new peptides, chujamides A (1) and B (2), were isolated from the marine sponge Suberites waedoensis, which was collected from Korean waters. Based upon the results of the combined spectroscopic analyses, the structures of these compounds were determined to be proline-riched and cyclic cystine bridged dodeca- and undecapeptides. The absolute configurations of all amino acid residues were determined to be l by advanced Marfey’s analysis. The new compounds exhibited weak cytotoxicities against A549 and K562 cell-lines, and compound 2 also demonstrated moderate inhibitory activity against Na+/K+-ATPase.  相似文献   

19.
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20.
3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)2-lipid A is the conserved structure domain of lipopolysaccharide found in most Gram-negative bacteria, and it is believed to stimulate the innate immune system through the TLR4/MD2 complex. Therefore, Kdo2-lipid A is an important stimulator for studying the mechanism of the innate immune system and for developing bacterial vaccine adjuvants. Kdo2-lipid A has not been chemically synthesized to date and could only be isolated from an Escherichia coli mutant strain, WBB06. WBB06 cells grow slowly and have to grow in the presence of tetracycline. In this study, a novel E. coli mutant strain, WJW00, that could synthesize Kdo2-lipid A was constructed by deleting the rfaD gene from the genome of E. coli W3110. The rfaD gene encodes ADP-l-glycero-d-manno-heptose-6-epimerase RfaD. Based on the analysis by SDS-PAGE, thin layer chromatography (TLC) and electrospray ionization mass spectrometry (ESI/MS), WJW00 could produce similar levels of Kdo2-lipid A to WBB06. WJW00 cells grow much better than WBB06 cells and do not need to add any antibiotics during growth. Compared with the wild-type strain, W3110, WJW00 showed increased hydrophobicity, higher cell permeability, greater autoaggregation and decreased biofilm-forming ability. Therefore, WJW00 could be a more suitable strain than WBB06 for producing Kdo2-lipid A and a good base strain for developing lipid A adjuvants.  相似文献   

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