Lipopolysaccharide Fraction Research Articles

Interferon lambda 4 is a gut antimicrobial protein

To withstand complex microbial challenges, the mammalian gut largely depends on the secretion of diverse antimicrobial proteins. Type III interferons (IFNλs) are ordinarily considered inducible antiviral cytokines involved in intestinal immunity. Unlike other IFNλs, we found that newly identified IFNλ4 is an intestinal antibacterial protein. Large amounts of natural IFNλ4 are present in the secretory layer of the intestinal tracts of healthy piglets, which suggests that IFNλ4 is in direct physiological contact with microbial pathogens. We also identified two biochemical functions of mammalian IFNλ4, the induction of bacterial agglutination and direct microbial killing, which are not functions of the other IFNλs. Further mechanistic investigations revealed that after binding to the carbohydrate fraction of lipopolysaccharide, mammalian IFNλ4 self-assembles into bacteria-surrounding nanoparticles that agglutinate bacteria, and that its unique cationic amphiphilic molecular structure facilitates the destruction of bacterial membranes. Our data reveal features of IFNλ4 distinct from those of previously reported IFNλs and suggest that noncanonical IFNλ4 is deeply involved in intestinal immunity, beyond simply cytokine signaling.

Apolipoprotein E-containing lipoproteins and their extracellular interactions with LRP1 affect LPS-induced inflammation.

The linkage between low-density lipoprotein receptor-related protein (LRP)1-mediated metabolism of apolipoprotein (apo) E-containing lipoproteins (apoE-LP) and the lipopolysaccharide (LPS)-induced inflammatory response contributes to the pathogenesis of sepsis; however, the underlying mechanisms are unclear. Therefore, in this study, the effects of apoE-LP and their constituents on the mRNA expression of interleukin (IL)-6 and LRP1 were evaluated using a culture system of human fibroblasts supplemented with LPS and apoE-containing emulsion particles (apoE-EP). The affinity of apoE-LP for LPS was examined using the interaction between fluorescence-labeled LPS and serum lipoprotein fractions. LPS-induced inflammation significantly upregulated the mRNA expression of IL-6 andLRP1. This upregulation was markedly suppressed by pre-incubation of LPS with apoE-EP or its constituents (apoE or EP). The suppressive effect of apoE-EP on IL-6 upregulation was attenuated in the presence of lactoferrin, an inhibitor of LRP1. The prepared apoE-EP and serum triglyceride-rich lipoproteins showed significant affinity for LPS. However, these affinities appeared to be lower than expected based on the extent to which IL-6 upregulation was suppressed by pre-incubation of LPS with apoE-EP. Overall, these results indicate that LPS-induced inflammation may be regulated by1) the LPS-neutralizing effect of apoE-LP, 2) anti-inflammatory effect of apoE, and 3) LRP1-mediated metabolic pathways.

Antibodies Recognizing Yersinia enterocolitica Lipopolysaccharides of Various Chemotypes in Synovial Fluids From Patients With Juvenile Idiopathic Arthritis.

Yersinia enterocolitica O:3 (YeO3) is considered to be associated with reactive arthritis (ReA), and its lipopolysaccharide (LPS) has been detected in synovial fluids from patients. Interestingly, YeO3 wild-type LPS was processed by host cells, resulting in truncated LPS molecules presenting the core region. Previously, we reported the immunogenicity but not adjuvanticity of YeO3 LPSs of wild (S) type, Ra, Rd, or Re chemotypes in mice. Here, we demonstrate the presence of YeO3 LPS chemotype-specific antibodies in all analyzed synovial fluids (SF) from patients with juvenile idiopathic arthritis (JIA). Interestingly, the high titer of antibodies specific for the Kdo-lipid A region was found in most tested SF. In contrast, only a few were positive for antibodies recognizing O-specific polysaccharides. Western blot analysis revealed the presence of antibodies reacting with fast-migrating LPS fractions and enterobacterial common antigen (ECA) in synovial fluid samples. Our data also suggest the importance of LPS-associated ECA for the antigenicity of endotoxin. Furthermore, we confirmed in vitro that Yersinia LPS processing leads to the exposure of its core region and enhanced potency of complement lectin pathway activation.

Systemic inflammation elevates cytosolic prolyl oligopeptidase protein expression but not peptidase activity in the cerebral cortices of familial Alzheimer`s disease modeling mice

Changes in brain prolyl oligopeptidase (PREP) expression and activity have been associated with neuroinflammation and Alzheimer`s disease (AD). The role of PREP in AD, which onset can be contributed by peripheral infection-induced inflammation, is unknown. The aim of the study was to elucidate further the association of PREP with AD pathology and inflammation. Here, we quantitated PREP protein expression by liquid chromatography-tandem mass spectrometry-based quantitative targeted absolute proteomics and determined PREP peptidase activity in the cerebral cortices of familial AD (APdE9) and lipopolysaccharide (LPS)-induced inflammation mouse models. PREP activity was investigated using the fluorogenic substrate Suc-Gly-Pro-AMC. PREP expression was significantly increased (by 2-fold) in the brain cytosolic fraction of LPS treated APdE9 mice, while the peptidase activity remained unaltered. In the cortical crude membrane fraction, the PREP expression and activity were decreased by 35–40% in the LPS treated APdE9 mice. In conclusion, cortical cytosolic and membrane-bound PREP expression levels and enzyme activities were altered due to LPS-induced inflammation in the AD mouse model. Since the cytosolic protein expression increased without any concomitant increase in the peptidase activity, it is likely that PREP activity is affected by other factors than protein expression alone.

Extracts of Gluconacetobacter hansenii GK-1 induce Foxp3+T cells in food-allergic mice by an IL-4-dependent or IL-4-independent mechanism.

The biological activities of acetic acid bacteria (AAB) as Gram-negative bacteria have attracted our interests, especially in their inhibitory effects on allergic responses. To clarify the underlying mechanism that improves allergic symptoms by ingestion of the AAB Gluconacetobacter hansenii, we examined whether different extracts of heat-killed G. hansenii GK-1 could reduce the interleukin (IL)-4 production of immune cells from food-allergic model of OVA23-3, transgenic mice with ovalbumin (OVA)-specific T-cell-receptor genes. A hot-water extract fraction (FII) of G. hansenii GK-1 significantly decreased the in vitro IL-4 production of spleen cells of OVA23-3 mice compared with those stimulated with OVA alone. The IL-4 inhibitory effect was also observed for FIV (purified lipopolysaccharide (LPS) fraction), but the activity was lower than for FII or LPS from Escherichia coli. Unlike LPS from Escherichia coli, FIV significantly inhibited the LPS-induced IL-6 production of the spleen cells. The addition of FII or FIV to a Foxp3+T cell-inducing culture showed that FII significantly promoted the rate of Foxp3+CD4+T cells of OVA-stimulated mesenteric lymph node cells from recombination-activating-gene (RAG)-2-deficient food-allergic inflammatory OVA23-3 (R23-3) mice with suppression of IL-4 production, while FIV induced Foxp3+T cells from RAG-2-deficient DO11.10 non-inflammatory mice. Structure analysis showed a lack of O-antigen in FIV, which seemed to lead to the weak biological activities of FIV observed. The present study suggests that extracts of G. hansenii GK-1 to inhibit IL-4 production of immune cells and/or promote regulatory T cell differentiation synergistically play important roles in improving allergic symptoms safely as well as normal condition.

Lipopolysaccharide Is a 4-Aminoarabinose Donor to Exogenous Polyisoprenyl Phosphates through the Reverse Reaction of the Enzyme ArnT.

Modification of the lipid A portion of LPS with cationic monosaccharides provides resistance to polymyxins, which are often employed as a last resort to treat multidrug-resistant bacterial infections. Here, we describe the use of fluorescent polyisoprenoids, liquid chromatography-mass spectrometry, and bacterial genetics to probe the activity of membrane-localized proteins that utilize the 55-carbon lipid carrier bactoprenyl phosphate (BP). We have discovered that a substantial background reaction occurs when B-strain E. coli cell membrane fractions are supplemented with exogenous BP. This reaction involves proteins associated with the arn operon, which is necessary for the covalent modification of lipid A with the cationic 4-aminoarabinose (Ara4N). Using a series of arn operon gene deletion mutants, we identified that the modification was dependent on ArnC, which is responsible for forming BP-linked Ara4N, or ArnT, which transfers Ara4N to lipid A. Surprisingly, we found that the majority of the Ara4N-modified isoprenoid was due to the reverse reaction catalyzed by ArnT and demonstrate this using heat-inactivated membrane fractions, isolated lipopolysaccharide fractions, and analyses of a purified ArnT. This work provides methods that will facilitate thorough and rapid investigation of bacterial outer membrane remodeling and the evaluation of polyisoprenoid precursors required for covalent glycan modifications.

Nanocochleates containing N-Octylglicoside extracted Vibrio cholerae antigens elicited high vibriocidal antibodies titers after intragastric immunization in a mice model

Biological detergents are used in research laboratories, to extract or solubilize proteins from cell membranes. In order to evaluate the ability to extract antigens from the bacterial cell surface of the wild Vibrio cholerae strain C7258 and study their immunogenic potential by forming proteoliposomes and cochleate and preserving their immunogenicity, the non-ionic detergent, n-Octylglucoside (n-OG), and the Zwitterionic detergent (3-cholamidopropyl dimethylammonio 1-propanesulfonate; CHAPS) were tested in concentrations between 5 and 15%. The anionic detergent sodium deoxycholate (DOC) was used as a reference. Electrophoretic, immunochemical and electron microscopy techniques have characterized the extracts and their chromatographic fractions. With CHAPS and n-OG detergents in concentrations between 5 and 15%, a higher yield was obtained in the extraction of proteins and lipopolysaccharides (LPS) and other components from the bacterial surface compared to 10% DOC. When using 10% DOC, 15% CHAPS and n-OG between 5 and 15%, stable proteoliposomes were formed, of average size between 82 and 93 nm in diameter, with known proportions of proteins, LPS and other components. In some of the concentrations, liposomes were formed with almost pure proteins. Some cholera outer membrane proteins like the 17 kDa protein, which corresponds to the mannose-sensitive hemagglutinin (MSHA), which mediates the adhesion to the brush border of the small intestine and the outer membrane protein U (OMPU) were identified with monoclonal antibodies (mAbs) and purified. The fundamental components of liposomes, proteins and LPS, retained their molecular weights, when compared with known standards and by processing programs of electrophoretic profiles and their antigenicity, without alterations due to the extraction procedure, as could be verified by immune identification techniques with monoclonal antibodies in the case of LPS, significant antigens in this pathogen. The main purpose of the present work was to show that a new anticholera vaccine formulation based on cochleates, containing selected protein and LPS fraction extracted by detergents, is able to elicit protective high titers of bactericidal antibodies after intragastric immunization in the mice model. The objective was achieved.

Morphometric characteristics of effects induced by <i>Ensifer meliloti</i> lipopolysaccharide fractions on parenchymatous organs in laboratory rats with secondary immunodeficiency

Introduction. Gram-negative bacteria-derived lipopolysaccharides (LPS) are better known as bacterial endotoxins. However, an increasing body of evidence has been accumulated regarding a whole range of LPS-bound physiological effects also observed in normal settings. In particular, LPS derived from some bacterial species was shown to exhibit an immunomodulating activity.Study objective — to characterize physiological effects of Ensifer meliloti lipopolysaccharides in modelled rat induced immunodeficiency.Materials and methods. Biological activity of intraperitoneally administered E. meliloti LPS fractions was studied for 21 days in 60 outbred male rats after induction of a minimal immunodeficiency state 24 hours later after inoculating cytostatic agent cyclophosphamide (CF). Animals were euthanized on day 22 followed by conducting an autopsy and morphometric study of internal organs. Later, paraffin-embedded sections of parenchymal organs were stained with hematoxylin-eosin and examined histologically by light microscopy.Results. It was found that at the end of the experiment cyclophosphamide applied to laboratory animals insignificantly decreased weight of liver and kidney, but not that of heart and spleen (compared to intact animals). In contrast, lung weight was solely significantly increased in immunodeficient rats compared to control. Intraperitoneally administered LPS fractions during secondary immunodeficiency affected weight parameters in the liver and kidney as the most intensively blood supplied organs suggesting its systemic effects. Quantity of follicles with large germinal centers as well as secondary follicles and lymphatic sheath formation in splenic stroma was increased that features activated immune response. Moreover, hepatic lymphoid infiltration in the portal tracts and reversal to normal vascular pattern were found as well. In contrast, LPS and Licopid administered to rats resulted in marked lung hyperplasia of lymphoid tissue containing large germinal centers.Conclusion. The data obtained indicate that E. meliloti-derived LPS fractions administered to rats with secondary immunodeficiency positively affected immunoreactivity.

Bacillus Calmette-Guerin polysaccharide nucleic acid vs Bacillus Calmette-Guerin vaccine in the treatment of warts: A comparative, double-blind, controlled study.

Bacillus Calmette-Guerin polysaccharide nucleic acid (BCG-PSN) is a relatively new generation, bacteria lipopolysaccharide fraction extracted from BCG vaccine. It has a strong immunomodulatory effect with higher efficacy and fewer side effects. To assess the clinical efficacy and safety of BCG-PSN vs the viable BCG vaccine in treatment of multiple warts. One hundred twenty patients with warts were allocated into three groups each containing 40 patients. Each group received intralesionally 0.1 mL of BCG-PSN, BCG vaccine, saline in group A, B, and C, respectively, for a maximum of five sessions, 3 weeks apart. Complete response occurred in 77.5% of group A vs 63.8% in group B. Partial response detected in 7.9% of control group (P < .0001). The distant warts showed 72.5% complete clearance in group A compared to 58.3% in group B (P = .19). Group A showed fewer number of sessions (P = .03) and earlier response than group B (P = .0003). A significant increase in the frequency of adverse effects was found among group B than group A (P < .0001). No recurrence was seen in both groups. BCG-PSN immunotherapy has a higher therapeutic efficacy with almost no serious side effects compared to the BCG vaccine in warts therapy.

Tualang Honey and its Methanolic Fraction Improve LPS-induced Learning and Memory Impairment in Male Rats: Comparison with Memantine

Background: Tualang honey (TH) has been shown to exert beneficial effects on learning and memory function in various animal models. However, its learning and memory effects in lipopolysaccharide (LPS) rat model have not been elucidated. Objective: The present study aimed to investigate the cognitive-enhancing effects of TH and its methanolic fraction in comparison to the clinically approved N-methyl-D-aspartate (NMDA) receptor antagonist (memantine) using LPS rat model. Methods: A total of ninety male Sprague Dawley rats were divided into 5 groups: (i) control, (ii) untreated LPS (iii) LPS treated with 200 mg/kg TH, (iv) LPS treated with 150 mg/kg methanol fraction of TH (MTH) and (v) LPS treated with 10 mg/kg memantine. All treatments were administered intraperitoneally once daily for 14 days. Morris water maze (MWM) and novel object recognition (NOR) tests were performed to assess spatial and recognition memory function. Results: The present study confirmed that LPS significantly impairs spatial and recognition memory and alone treatment with TH or MTH improved spatial and recognition memory comparable to memantine. Conclusion: Both TH and its methanolic fraction improved spatial and recognition memory of LPS rat model comparable to memantine. Thus, TH and its methanolic fraction have potential preventivetherapeutic effects for neurodegenerative diseases involving neuroinflammation.

The Role of Legionella pneumophila Serogroup 1 Lipopolysaccharide in Host-Pathogen Interaction.

The Legionella pneumophila TF3/1 mutant of the Corby strain, which possesses a point mutation in the active site of the O-acetyltransferase, synthesized the polysaccharide chain with a reduced degree of substitution with O-acetyl groups. The mutant did not produce a high-molecular-weight lipopolysaccharide (LPS) fraction above 12 kDa. The disturbances in LPS synthesis have an effect on the composition of other macromolecules (lipids and proteins), as indicated by differences in the infrared absorption spectra between the L. pneumophila Corby strain and its TF3/1 mutant. The wild-type strain contained less N+–CH3 and C-N groups as well as more CH3 groups than the mutant. The fatty acid composition showed that the wild type strain synthesized more branched acyl residues (a15:0, i16:0, and a17:0), a less unsaturated acid (16:1), and a straight-chain acid (18:0) than the mutant. The mutant synthesized approximately twice more a long-chain fatty acid (20:0) than the wild type. The main differences in the phospholipids between both strains were found in the classes of phosphatidylcholines and phosphatidylglycerols (PG). Substantial differences in the cell surface topography of these bacteria and their nanomechanical properties were shown by atomic force microscopy (AFM). The wild type strain had no undulated surface and produced numerous vesicles. In the case of the mutant type, the vesicles were not numerous, but there were grooves on the cell surface. The average roughness of the cell surface of the mutant was approximately twofold higher than in the wild-type strain. In turn, the wild-type strain exhibited much better adhesive properties than the mutant. The kinetic study of the interaction between the L. pneumophila strains and Acanthamoeba castellanii monitored by Förster resonance energy transfer revealed a pronounced difference, i.e., almost instantaneous and highly efficient binding of the L. pneumophila Corby strain to the amoeba surface, followed by penetration into the amoeba cells. This process was clearly not as efficient in the case of the mutant. The results point to LPS and, in particular, to the length of the polysaccharide fraction as an important L. pneumophila determinant involved in the process of adhesion to the host cell.

Infrared spectroscopy with multivariate analysis to interrogate the interaction of whole cells and secreted soluble exopolimeric substances of Pseudomonas veronii 2E with Cd(II), Cu(II) and Zn(II).

Extracellular polymeric substances (EPS) are bacterial products associated to cell wall or secreted to the liquid media that form the framework of microbial mats. These EPS contain functional groups as carboxyl, amino, hydroxyl, phosphate and sulfhydryl, able to interact with cations. Thus, EPS may be considered natural detoxifying compounds of metal polluted waters and wastewaters. In this work Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) in combination with multivariate analysis (Principal Component Analysis-PCA-) were used to study the interaction of Cd(II), Cu(II) and Zn(II) and Pseudomonas veronii 2E cells, including bound EPS and cell wall, and its different soluble EPS fractions, previously characterized as Cd(II) ligands of moderate strength. Amino groups present in exopolysaccharide fraction were responsible for Zn(II) and Cu(II) complexation, while carboxylates chelated Cd(II). In lipopolysaccharide fraction, phosphoryl and carboxyl sites were involved in Cd(II) and Cu(II) binding, while Zn(II) interacted with amino groups. Similar results were obtained from cells. These studies confirmed that FTIR-PCA is a rapid analytical tool to provide valuable information regarding the functional groups in biomolecules related to metal interaction. Moreover, a discrimination and identification of functional groups present in both EPS and cells that interacted with Cd(II), Zn(II) and Cu(II) was demonstrated.

Липополисахарид Sinorhizobium meliloti стимулирует гемопоэз при вторичном иммунодефиците в эксперименте

Липолисахариды (ЛПС, эндотоксины) грамотрицательных бактерий обладают выраженной биологической активностью, в том числе терапевтической, однако для S. meliloti таких данных нет. Цель работы - экспериментальное изучение гемопоэтической активности 4 фракций липополисахаридов, выделенных из S. meliloti, при индуцированном иммунодефиците у мышей. Методика. Сформировано 7 групп лабораторных мышей (по 10 особей в каждой): 1-я группа - интактные (контроль 1), 2-я - 7-я группа - мыши с иммунодефицитным состоянием, индуцированным однократным внутрибрюшинным введением циклофосфамида. Через 1 сут после моделирования иммунодефицита в течение 21 сут ежедневно мышам 3-й группы вводили препарат сравнения Ликопид® (химическое название: [4-O-(2-ацетиламино-2-дезокси-β-D-глюкопиранозил)-N-ацетилмурамил]-L-аланил-D-α-глутамиламид - синтетический аналог бактериальных гликопептидов из группы иммуностимулирующих средств). Мышам 4-7-й групп - вводили исследуемые фракции липолисахаридов - ЛПС-1, ЛПС-2, ЛПС-3 и ЛПС-4 соответственно. Для ликопида разовая доза составляла 0,1 мл (0,05 мг/мл), для исследуемых фракций ЛПС S. meliloti - 0,2 мл (10 пг/мл). Иммунодефицитным мышам 2-й группы фракции липополисахаридов и препарат сравнения Ликопид® не вводили Через 21 сут мышей выводили из эксперимента. Изучали весовые характеристики органов подопытных животных и лейкоцитарную формулу. Результаты. Введение мышам на фоне вторичного экспериментального иммунодефицита ликопида сопровождалось снижением количества палочкоядерных нейтрофилов и моноцитопенией; при введении фракции ЛПС-1 возрастало количество сегментоядерных нейтрофилов; ЛПС-2 - имели место снижение содержания палочкоядерных нейтрофилов и лимфоцитоз; ЛПС-3 - наблюдали снижение содержания палочкоядерных нейтрофилов и лимфоцитоз на фоне значимого увеличения количества сегментоядерных нейтрофилов; ЛПС-4 - констатировалось увеличение числа базофилов, снижение содержания палочкоядерных нейтрофилов и лимфоцитоз на фоне значимого увеличения количества сегментоядерных нейтрофилов. Заключение. Фракции ЛПС Sinorhizobium meliloti проявляют модулирующие эффекты, схожие с механизмами «экстренного миелопоэза» при физиологичном варианте течения бактериальных инфекций. Lipolysaccharides (LPS, endotoxins) of gram-negative bacteria have a pronounced biological activity, including therapeutic activity; however, there is no such data for S. meliloti. Aim. To conduct an experimental study of hematopoietic activity of four lipopolysaccharide fractions isolated from S. meliloti under induced immunodeficiency in mice. Methods. 7 groups of 10 laboratory mice each were formed: group 1, intact mice (control 1); groups 2-7, mice with immunodeficiency induced by a single intraperitoneal injection of cyclophosphamide. Mice of group 3 were daily injected with a comparison agent, Licopid® (Chemical name: [4-O- (2-acetylamino-2-deoxy-β-D-glucopyranosyl) -N-acetylmuramyl] -L-alanyl-D-α-glutamyl amide; single dose, 0.1 ml (0.05 mg/ml)) for 21 days starting one day after the induction of immunodeficiency. Mice of groups 3-7 were injected with the studied S. meliloti LPS fractions, LPS-1, LPS-2, LPS-3, and LPS-4, respectively (single dose, 0.2 ml (10 pg/ml)). Immunodeficient mice of group 2 received neither the comparison agent, Licopid® nor LPS fractions. The mice were euthanized at 21 days. Weight characteristics of animal organs and white blood count were studied. Results. Administration of Licopid® to mice with secondary experimental immunodeficiency was associated with decreased count of stab neutrophils and monocytopenia; LPS-1 fraction increased the count of segmented neutrophils; LPS-2 decreased the count of stab neutrophils and induced lymphocytosis; LPS-3 decreased the count of stab neutrophils and induced lymphocytosis associated with a significant increase in the count of segmented neutrophils; LPS-4 induced basophilia, decreased count of stab neutrophils, and lymphocytosis associated with a significant increase in the count of segmented neutrophils. Conclusion. Sinorhizobium meliloti LPS fractions exerted modulating effects similar to the mechanisms of “emergency myelopoiesis” in the physiological course of bacterial infections.

Lipopolysaccharide-Dependent Membrane Permeation and Lipid Clustering Caused by Cyclic Lipopeptide Colistin.

Polyanionic lipopolysaccharides (LPS) play an important role in regulating the permeability of the outer membrane (OM) of Gram-negative bacteria. Impairment of the LPS-enriched OM is essential in initiating the bactericidal activity of polymyxins. We are interested in how colistin (polymyxin E) affects the membrane permeability of LPS/phospholipid bilayers. Our vesicle leakage experiment showed that colistin binding enhanced bilayer permeability; the maximum increase in the bilayer permeability was positively correlated with the LPS fraction. Addition of magnesium ions abolished the effect of LPS in enhancing bilayer permeabilization. To describe the vesicle leakage behavior from a structural perspective, we performed liquid atomic force microscopy (AFM) measurements on planar lipid bilayers. We found that colistin caused the formation of nano- and macroclusters that protruded from the bilayer by ∼2 nm. Moreover, cluster development was promoted by increasing the fraction of LPS or colistin concentration but inhibited by magnesium ions. To explain our experimental data, we proposed a lipid clustering model where colistin binds to LPS to form large-scale complexes segregated from zwitterionic phospholipids. The discontinuity (and thickness mismatch) at the edge of LPS–colistin clusters will create a passage that allows solutes to permeate through. The proposed model is consistent with all data obtained from our leakage and AFM experiments. Our results of LPS-dependent membrane restructuring provided useful insights into the mechanism that could be used by polymyxins in impairing the permeability barrier of the OM of Gram-negative bacteria.

Sensing of Escherichia coli and LPS by mammary epithelial cells is modulated by O-antigen chain and CD14.

Escherichia coli is one of the major pathogens causing mastitis in dairy cattle. Yet, the factors which mediate the ability for E. coli to develop in the bovine mammary gland remain poorly elucidated. In a mouse model, infections induced by the reference mastitis E. coli P4 showed a strong colonisation of the mammary gland, while this strain had a low stimulating power on cells of the PS bovine mammary epithelial cell line. In order to understand if such a reduced response contributes to the severity of infection, a library of random mutants of P4 strain was screened to identify mutants inducing stronger response of PS cells. Among hyper-stimulating mutants, six were altered in genes involved in biosynthesis of lipopolysaccharide (LPS) and had lost their O-polysaccharide region, suggesting that the presence of O-antigen impairs the response of PS cells to LPS. Using purified smooth (S) and rough (R) fractions of LPS, we showed that the R-LPS fraction induced a stronger response from PS cells than the smooth LPS fraction. Biological activity of the S-LPS fraction could be restored by the addition of recombinant bovine CD14 (rbCD14), indicating a crucial role of CD14 in the recognition of S-LPS by Mammary Epithelial Cells (MEC). When S-LPS and R-LPS were injected in udder quarters of healthy lactating cows, an inflammation developed in all infused quarters, but the S-LPS induced a more intense pro-inflammatory response, possibly in relation to sizeable concentrations of CD14 in milk. Altogether, our results demonstrate that the O-antigen modulates the pro-inflammatory response of MEC to LPS, that S-LPS and R-LPS trigger different responses of MEC and that these responses depend on the presence of CD14.

RAW264.7 대식세포에서 LPS 매개 iNOS/NO 생성에 대한 protopanaxadiol saponin 및 protopanaxatriol saponin의 억제효과

본 연구는 RAW264.7 세포에서 lipopolysaccharide (LPS) 처리에 의한 염증매개인자의 유도에 대한 고려인삼 사포닌 분획인 20(S)-protopanaxadiol saponins (PDS)과 20(S)-protopanaxatriol saponins (PTS)의 조절효능을 탐구하였다. 이를 위해 RAW264.7 세포에 PDS 또는 PTS를 <TEX>$150{\mu}g/ml$</TEX>의 농도로 LPS (<TEX>$10{\mu}g/ml$</TEX> 처리 이전이나 처리 이후 또는 LPS와 동시에 처리하였으며, 처리된 세포에서 nitric oxide (NO)의 방출량, 유도성 nitric oxide synthase (iNOS) 및 cyclooxygenase-2 (COX-2)의 발현 량을 분석하였다. PDS에 비하여 PTS는 RAW264.7 세포에 LPS와 동시에 처리하여 24시간 동안 배양했을 때 LPS 처리에 의해 유도된 NO의 생성을 강하게 감소시켰다. RAW264.7 세포에 LPS (<TEX>$10{\mu}g/ml$</TEX>를 2시간 동안 처리한 후에 PDS 또는 PTS를 <TEX>$150{\mu}g/ml$</TEX> 농도로 24시간 동안 처리하면 두 인삼 사포닌 성분 모두 NO의 생성을 강하게 감소시켰다. RAW264.7 세포에 PDS 또는 PTS를 <TEX>$150{\mu}g/ml$</TEX> 농도로 2시간 동안 처리한 후에 LPS (<TEX>$10{\mu}g/ml$</TEX>를 24시간 동안 처리했을 경우에도 두 인삼 사포닌 성분 모두 LPS 처리에 의해 유도된 NO 생성을 강하게 감소시켰다. LPS 처리에 의한 NO 생성을 저해하는 효과는 PDS에 비하여 PTS가 더 강하게 나타났다. PDS와 PTS 모두 <TEX>$150{\mu}g/ml$</TEX> 처리농도에서 LPS (<TEX>$10{\mu}g/ml$</TEX>처리에 의해 유도된 iNOS와 COX-2의 발현 역시 상당히 감소시켰다. 따라서 본 연구의 결과는 RAW264.7 대식세포에서 PDS와 PTS 두 인삼 사포닌 성분은 LPS 처리에 의한 염증활성화에 강한 억제효과를 가지고 있음을 의미하며, 전염증성 효소인 iNOS와 COX-2 발현의 감소조절을 통하여 NO의 생성을 억제함으로써 항 염증효과가 나타남을 제시한다. This study was performed to investigate the modulatory effects of two prototypes of Panax ginseng saponin fractions, 20(S)-protopanaxadiol saponins (PDS) and 20(S)-protopanaxatriol saponins (PTS), on the induction of inflammatory mediators in lipopolysaccharide (LPS)-treated RAW264.7 murine macrophage cells. For this purpose, RAW264.7 cells were treated with LPS (<TEX>$10{\mu}g/ml$</TEX>) before, after, or simultaneously with PDS or PTS (<TEX>$150{\mu}g/ml$</TEX>), and the released level of nitric oxide (NO) and expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were evaluated. When RAW264.7 cells were treated with LPS and ginseng saponin fractions simultaneously for 24 hr, PTS, compared to PDS, more strongly attenuated the NO production induced by LPS treatment. When the cells were pretreated with LPS for 2 hr followed by PDS or PTS treatment for 24 hr, both ginseng saponins strongly reduced NO release. The pretreatment of RAW264.7 cells with PDS or PTS for 2 hr followed by LPS treatment for 24 hr significantly attenuated the LPS-induced production of NO. PTS showed stronger inhibitory potency to NO generation than PDS. Our western blot experiment showed that both PDS and PTS (<TEX>$150{\mu}g/ml$</TEX>) also significantly down-regulated the expressions of iNOS and COX-2 induced by LPS treatment. Our results suggest that both PDS and PTS possess strong protective effects against LPS-stimulated inflammation and that their protective effects are mediated by the suppression of NO synthesis via down-regulation of pro-inflammatory enzymes, iNOS, and COX-2 in the RAW264.7 cells.

Chemical characterization of Pseudomonas veronii 2E soluble exopolymer as Cd(II) ligand for the biotreatment of electroplating wastes

The production, isolation, purification and characterization of soluble Extracellular Polymeric Substances (EPS) secreted by Pseudomonas veronii 2E were studied. Optimal production was obtained in medium M9-14 g l−1 glycerol, 25 °C. Spectrophotometric and potentiometric titration studies showed that EPS consisted on extracellular DNA, proteins and polysaccharides as the main components. DNase I, Proteinase K and further sodium deoxycholate treatments yielded similar proportions of lipopolysaccharides (LPSs) and exopolysaccharides (ExPs). ExP was subjected to total acid hydrolysis and then analyzed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD), showing the presence of fucose, galactosamine, glucosamine, galactose, glucose, mannose and glucuronic acid. Further treatment with oxalic acid confirmed the presence of a pyruvilated sugar. The ability of the secreted products to interact with Cd(II) was studied by anodic stripping voltammetry (ASV). LPS fraction contained the highest ligand concentration (279 ± 10 μmoles Cd(II) binding sites g LPS−1). This information is a valuable tool for the application of EPS in industrial wastewater treatment.

Deacylated lipopolysaccharides inhibit biofilm formation by Gram-negative bacteria

The extracellular polysaccharides of Vibrio vulnificus play different roles during biofilm development. Among them, the effect of lipopolysaccharide (LPS), which is crucial for bacterial adherence to surfaces during the initial stage of biofilm formation, on the formation process was examined using various types of LPS extracts. Exogenously added LPS strongly inhibited biofilm formation in a dose-dependent manner. In addition, the exogenous addition of a deacylated form of LPS (dLPS) also inhibited biofilm formation. However, an LPS fraction extracted from a mutant not able to produce O-antigen polysaccharides (O-Ag) did not have an inhibitory effect. Furthermore, biofilm formation by several Gram-negative bacteria was inhibited by dLPS addition. In contrast, biofilm formation by Gram-positive bacteria was not influenced by dLPS but was affected by lipoteichoic acid. Therefore, this study demonstrates that O-Ag in LPS is important for inhibiting biofilm formation and may serve an efficient anti-biofilm agent specific for Gram-negative bacteria.

The influence of a modified lipopolysaccharide O-antigen on the biosynthesis of xanthan in Xanthomonas campestris pv. campestris B100.

BackgroundThe exopolysaccharide xanthan is a natural product which is extensively used in industry. It is a thickening agent in many fields, from oil recovery to the food sector. Xanthan is produced by the Gram negative bacterium Xanthomonas campestris pv. campestris (Xcc). We analyzed the lipopolysaccharide (LPS) of three mutant strains of the Xcc wild type B100 to distinguish if the xanthan production can be increased when LPS biosynthesis is affected.ResultsThe Xcc B100 O-antigen (OA) is composed of a linear main chain of rhamnose residues with N-acetylfucosamine (FucNAc) side branches at every second rhamnose. It is the major LPS constituent. The O-antigen was missing completely in the mutant strain H21012 (deficient in wxcB), since neither rhamnose nor FucNAc could be detected as part of the LPS by MALDI-TOF-MS, and only a slight amount of rhamnose and no FucNAc was found by GC analysis. The LPS of two other mutants was analyzed, Xcc H28110 (deficient in wxcK) and H20110 (wxcN). In both of them no FucNAc could be detected in the LPS fraction, while the rhamnose moieties were more abundant than in wild type LPS. The measurements were carried out by GC and confirmed by MALDI-TOF-MS analyses that indicated an altered OA in which the branches are missing, while the rhamnan main chain seemed longer than in the wild type. Quantification of xanthan confirmed our hypothesis that a missing OA can lead to an increased production of the extracellular polysaccharide. About 6.3 g xanthan per g biomass were produced by the Xcc mutant H21012 (wxcB), as compared to the wild type production of approximately 5 g xanthan per g biomass. In the two mutant strains with modified OA however, Xcc H28110 (wxcK) and Xcc H20110 (wxcN), the xanthan production of 5.5 g and 5.3 g, respectively, was not significantly increased.ConclusionsMutations affecting LPS biosynthesis can be beneficial for the production of the extracellular polysaccharide xanthan. However, only complete inhibition of the OA resulted in increased xanthan production. The inhibition of the FucNAc side branches did not lead to increased production, but provoked a novel LPS phenotype. The data suggests an elongation of the linear rhamnan main chain of the LPS OA in both the Xcc H28110 (wxcK) and Xcc H20110 (wxcN) mutant strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0710-y) contains supplementary material, which is available to authorized users.

Supramolecular structure of enterobacterial wild-type lipopolysaccharides (LPS), fractions thereof, and their neutralization by Pep19-2.5.

Lipopolysaccharides (LPS) belong to the strongest immune-modulating compounds known in nature, and are often described as pathogen-associated molecular patterns (PAMPs). In particular, at higher concentrations they are responsible for sepsis and the septic shock syndrome associated with high lethality. Since most data are indicative that LPS aggregates are the bioactive units, their supramolecular structures are considered to be of outmost relevance for deciphering the molecular mechanisms of its bioactivity. So far, however, most of the data available addressing this issue, were published only for the lipid part (lipid A) and the core-oligosaccharide containing rough LPS, representing the bioactive unit. By contrast, it is well known that most of the LPS specimen identified in natural habitats contain the smooth-form (S-form) LPS, which carry additionally a high-molecular polysaccharide (O-chain). To fill this lacuna and going into a more natural system, here various wild-type (smooth form) LPS including also some LPS fractions were investigated by small-angle X-ray scattering with synchrotron radiation to analyze their aggregate structure. Furthermore, the influence of a recently designed synthetic anti-LPS peptide (SALP) Pep19-2.5 on the aggregate structure, on the binding thermodynamics, and on the cytokine-inducing activity of LPS were characterized, showing defined aggregate changes, high affinity binding and inhibition of cytokine secretion. The data obtained are suitable to refine our view on the preferences of LPS for non-lamellar structures, representing the highest bioactive forms which can be significantly influenced by the binding with neutralizing peptides such as Pep19-2.5.