Lipopolysaccharide of Legionella pneumophila Serogroup 1 Facilitates Interaction with Host Cells.

Legionella pneumophila is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the L. pneumophila strain Heysham-1, lacking the O-acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with Acanthamoeba castellanii cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to A. castellanii, suggesting that the O-acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose O-acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of L. pneumophila sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection.

Heterogenicity within the LPS Structure in Relation to the Chosen Genomic and Physiological Features of the Plant Pathogen Pectobacterium parmentieri.

Pectobacterium parmentieri is a pectinolytic plant pathogenic bacterium causing high economic losses of cultivated plants. The highly devastating potential of this phytopathogen results from the efficient production of plant cell wall-degrading enzymes, i.e., pectinases, cellulases and proteases, in addition to the impact of accessory virulence factors such as motility, siderophores, biofilm and lipopolysaccharide (LPS). LPS belongs to pathogen-associated molecular patterns (PAMPs) and plays an important role in plant colonization and interaction with the defense systems of the host. Therefore, we decided to investigate the heterogeneity of O-polysaccharides (OPS) of LPS of different strains of P. parmentieri, in search of an association between the selected genomic and phenotypic features of the strains that share an identical structure of the OPS molecule. In the current study, OPS were isolated from the LPS of two P. parmentieri strains obtained either in Finland in the 1980s (SCC3193) or in Poland in 2013 (IFB5432). The purified polysaccharides were analyzed by utilizing 1D and 2D NMR spectroscopy (1H, DQF-COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY and HMBC) in addition to chemical methods. Sugar and methylation analyses of native polysaccharides, absolute configuration assignment of constituent monosaccharides and NMR spectroscopy data revealed that these two P. parmentieri strains isolated in different countries possess the same structure of OPS with a very rare residue of 5,7-diamino-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (pseudaminic acid) substituted in the position C-8: →3)-ß-d-Galf-(1→3)-α-d-Galp-(1→8)-ß-Pse4Ac5Ac7Ac-(2→6)-α-d-Glcp-(1→6)-ß-d-Glcp-(1→. The previous study indicated that three other P. parmentieri strains, namely IFB5427, IFB5408 and IFB5443, exhibit a different OPS molecule than SCC3193 and IFB5432. The conducted biodiversity-oriented assays revealed that the P. parmentieri IFB5427 and IFB5408 strains possessing the same OPS structure yielded the highest genome-wide similarity, according to average nucleotide identity analyses, in addition to the greatest ability to macerate chicory tissue among the studied P. parmentieri strains. The current research demonstrated a novel OPS structure, characteristic of at least two P. parmentieri strains (SCC3193 and IFB5432), and discussed the observed heterogenicity in the OPS of P. parmentieri in a broad genomic and phenotype-related context.

Potential of Silver Nanoparticles in Overcoming the Intrinsic Resistance of Pseudomonas aeruginosa to Secondary Metabolites from Carnivorous Plants.

Carnivorous plants are exemplary natural sources of secondary metabolites with biological activity. However, the therapeutic antimicrobial potential of these compounds is limited due to intrinsic resistance of selected bacterial pathogens, among which Pseudomonas aeruginosa represents an extreme example. The objective of the study was to overcome the intrinsic resistance of P. aeruginosa by combining silver nanoparticles (AgNPs) with secondary metabolites from selected carnivorous plant species. We employed the broth microdilution method, the checkerboard titration technique and comprehensive phytochemical analyses to define interactions between nanoparticles and active compounds from carnivorous plants. It has been confirmed that P. aeruginosa is resistant to a broad range of secondary metabolites from carnivorous plants, i.e., naphthoquinones, flavonoids, phenolic acids (MBC = 512 µg mL-1) and only weakly sensitive to their mixtures, i.e., extracts and extracts' fractions. However, it was shown that the antimicrobial activity of extracts and fractions with a significant level of naphthoquinone (plumbagin) was significantly enhanced by AgNPs. Our studies clearly demonstrated a crucial role of naphthoquinones in AgNPs and extract interaction, as well as depicted the potential of AgNPs to restore the bactericidal activity of naphthoquinones towards P. aeruginosa. Our findings indicate the significant potential of nanoparticles to modulate the activity of selected secondary metabolites and revisit their antimicrobial potential towards human pathogenic bacteria.

Lipid A from Oligotropha carboxidovorans Lipopolysaccharide That Contains Two Galacturonic Acid Residues in the Backbone and Malic Acid A Tertiary Acyl Substituent.

The free-living Gram-negative bacterium Oligotropha carboxidovorans (formerly: Pseudomonas carboxydovorans), isolated from wastewater, is able to live in aerobic and, facultatively, in autotrophic conditions, utilizing carbon monoxide or hydrogen as a source of energy. The structure of O. carboxidovorans lipid A, a hydrophobic part of lipopolysaccharide, was studied using NMR spectroscopy and high-resolution mass spectrometry (MALDI-ToF MS) techniques. It was demonstrated that the lipid A backbone is composed of two d-GlcpN3N residues connected by a ß-(1→6) glycosidic linkage, substituted by galacturonic acids (d-GalpA) at C-1 and C-4' positions. Both diaminosugars are symmetrically substituted by 3-hydroxy fatty acids (12:0(3-OH) and 18:0(3-OH)). Ester-linked secondary acyl residues (i.e., 18:0, and 26:0(25-OH) and a small amount of 28:0(27-OH)) are located in the distal part of lipid A. These very long-chain hydroxylated fatty acids (VLCFAs) were found to be almost totally esterified at the (ω-1)-OH position with malic acid. Similarities between the lipid A of O. carboxidovorans and Mesorhizobium loti, Rhizobium leguminosarum, Caulobacter crescentus as well as Aquifex pyrophylus were observed and discussed from the perspective of the genomic context of these bacteria.

Definitive structural assessment of enterococcal diheteroglycan.

Enterococcus faecalis is one of most important nosocomial and often multi-antibiotic resistant pathogens responsible for infections that are difficult to treat. Previously, a cell-wall polysaccharide termed diheteroglycan (DHG) was isolated and characterized as a promising vaccine candidate. However, the configuration of its lactic acid (LA) residue attached to the galactofuranoside unit was not assessed, although it influences conformation of DHG chain in terms of biological recognition and immune evasion. This study proves the R configuration of the LA residue by means of chemical analysis, investigation of intramolecular NMR nuclear Overhauser effects and molecular dynamics simulations of native DHG and corresponding R and S models, which were obtained by using pyranoside-into-furanoside rearrangement. As alternative treatment and prevention strategies for E. faecalis are desperately needed, this discovery may offer the prospect of a synthetic vaccine to actively immunize patients at risk.

The structure of O-polysaccharide isolated from the type strain of Pectobacterium versatile CFBP6051T containing an erwiniose - higher branched monosaccharide.

Utilizing sugar, methylation, and absolute configurations analyses as well as NMR spectroscopy, the chemical repeating unit of the O-specific polysaccharide of Pectobacteriumversatile CFBP6051T was identified as: The polymer contains residues of an unusual, higher-branched monosaccharide, named erwiniose (3,6,8-trideoxy-4-C-(R-1-hydroxyethyl)-d-gulo-octose). Comparison of the P. versatile CFBP6051T O-polysaccharide with those isolated from strains of other Pectobacterium species indicated high differentiation in their structures within this genus.

Structure of the lipopolysaccharide O-antigen of Serratia spp. strains 10.1WK and 1XS plant endophytes isolated from O. biennis and L. corniculatus.

O-specific polysaccharides (O-PSs) isolated from lipopolysaccharides of Serratia spp., strains 10.1WK and 1XS, which are endophytic bacteria of Oenothera biennis (common evening-primrose) and Lotus corniculatus (bird's-foot trefoil), plants growing on a petroleum hydrocarbon polluted site in the Silesia region, were investigated. The high-molecular-weight O-PS fractions liberated from lipopolysaccharides by mild acid hydrolysis were studied using chemical methods, MALDI-TOF mass spectrometry, and a set of 1D and 2D NMR spectroscopy techniques. It was found that both O-specific polysaccharides were built of an identical trisaccharide repeating unit composed of d-Rhap and d-Manp residues. The following structure of the O-PSs of Serratia spp. strains 10.1WK and 1XS was established: →4)-α-d-Rhap-(1 â†’ 3)-ß-d-Manp-(1 â†’ 4)-ß-d-Rhap-(1→.

Spectroscopic studies on the supramolecular interactions of methyl benzoate derivatives with p-sulfocalix[6]arene macrocycles.

This paper is a continuation of our previous research and aims to further investigate and elucidate the nature and mechanisms of noncovalent supramolecular interactions between four methyl benzoate derivatives (I-IV), which are capable of exhibiting Twisted Intramolecular Charge Transfer (TICT) and/or Excited State Intramolecular Proton Transfer (ESIPT)-type behavior, and chemical and biological nanocavities. Photophysical and photochemical properties of molecules I-IV in aqueous solution in the presence of well-recognized macrocyclic host p-sulfocalix[6]arenes (SCA[6]) have been studied using steady-state, time-resolved and 1H NMR spectroscopic techniques. The changes in the ground- and excited-state spectroscopic characteristics (absorption and fluorescence spectra, time-resolved fluorescence spectra, fluorescence decay times and 1H NMR spectra) undergo significant modifications upon encapsulation of the investigated methyl benzoate derivative in the macromolecular cavity. For the two compounds (I and II), the interactions with the macrocycles with a hydrophobic SCA[6] cavity lead to the formation of stable inclusion complexes with 1:1 stoichiometry, both in the ground and excited state, while the stoichiometry of the III-SCA[6] and IV-SCA[6] complexes in the ground and excited states is 1:2. The values of the equilibrium constants have been determined from the spectroscopic data using Benesi-Hildebrand and nonlinear regression procedures. The location of the organic molecule inside the SCA[6] has been investigated by 1H NMR experiments. The changes in macrocyclic compound-induced NMR chemical shifts clearly indicate that the chemical structure of inclusion complexes is very different for methyl benzoate derivative-SCA[6] and methyl benzoate derivative-CB[7] systems. Finally, we have shown, using time-dependent fluorescence Stokes shift, that very fast solvation dynamics of pure water is markedly different from that of the confined water molecule in SCA[6] system.

Host-adaptive traits in the plant-colonizing Pseudomonas donghuensis P482 revealed by transcriptomic responses to exudates of tomato and maize.

Pseudomonads are metabolically flexible and can thrive on different plant hosts. However, the metabolic adaptations required for host promiscuity are unknown. Here, we addressed this knowledge gap by employing RNAseq and comparing transcriptomic responses of Pseudomonas donghuensis P482 to root exudates of two plant hosts: tomato and maize. Our main goal was to identify the differences and the common points between these two responses. Pathways upregulated only by tomato exudates included nitric oxide detoxification, repair of iron-sulfur clusters, respiration through the cyanide-insensitive cytochrome bd, and catabolism of amino and/or fatty acids. The first two indicate the presence of NO donors in the exudates of the test plants. Maize specifically induced the activity of MexE RND-type efflux pump and copper tolerance. Genes associated with motility were induced by maize but repressed by tomato. The shared response to exudates seemed to be affected both by compounds originating from the plants and those from their growth environment: arsenic resistance and bacterioferritin synthesis were upregulated, while sulfur assimilation, sensing of ferric citrate and/or other iron carriers, heme acquisition, and transport of polar amino acids were downregulated. Our results provide directions to explore mechanisms of host adaptation in plant-associated microorganisms.

Structure of the lipopolysaccharide O-antigen of endophytic Pseudomonas sp. strain L1.

The O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide of Pseudomonas sp. Strain L1, the endophytic bacteria of Lolium perenne (ryegrass) plants growing in soil in an industrial area in the Silesia region (Zabrze, Southern Poland). The high-molecular-weight O-PS fraction liberated from Pseudomonas sp. L1 lipopolysaccharide by mild acid hydrolysis was studied using chemical methods, MALDI-TOF mass spectrometry, and 1D and 2D NMR spectroscopy techniques. It was found that the O-specific polysaccharide was built of tetrasaccharide repeating units composed of d-FucpN, d-Fucp4N, and two d-QuipN residues. The following structure of the O-PS of Pseudomonas sp. Strain L1 was established: [Formula: see text].

The chemical structure of the O-antigen and enterobacterial common antigen of Pectobacterium brasiliense NCPPB 4609TS.

Pectobacterium brasiliense is a widely distributed phytopathogenic bacterium that causes diseases such as soft rot and blackleg, leading to significant yield losses in potatoes as well as other vegetables and ornamental plants. Lipopolysaccharide (LPS) is an important virulence factor that plays an essential role in colonisation of plant tissues and overcoming the host defence mechanisms. The O-polysaccharide from the LPS of P. brasiliense strain NCPPB 4609TS (=CFBP 6617TS = LMG 21371TS = IFB5390) was structurally characterised using spectroscopic techniques and chemical methods. The analyses revealed that the polysaccharide repeating unit consists of Gal, GlcN and an unusual 3-amino-3,6-dideoxyglucose decorated with (R)-3-hydroxybutyric acid according to the structure shown below: In addition, another polysaccharide was isolated from bacterial cells, analysis of which led to the identification of an enterobacterial common antigen, containing N-acetyl-d-glucosamine, N-acetyl-d-mannosaminouronic acid, and 4-acetamido-4,6-dideoxy-d-galactose.

Novel polysaccharide and polysaccharide-peptide conjugate from Rosa rugosa Thunb. pseudofruit - Structural characterisation and nutraceutical potential.

A novel bioactive polysaccharopeptide (C1) and polysaccharide (C2) with an average molecular weight of 180 kDa and 70 kDa were isolated from R. rugosa pseudofruit. The composition of the macromolecules was established using 1H NMR, FT-IR, GC-MS, SDS-PAGE coupled with enzymatic cleavage, and proteomic analyses (LC-MS). C1 was found to contain 60.56 ± 1.82 % of sugars and 21.17 ± 0.47 % of uronic acids. Its main neutral monosaccharides were arabinose, rhamnose, galactose, glucose, fucose, and mannose. C1 was found to be a polysaccharopeptide containing pectinesterase-like protein. C2 was composed of 32.85 ± 0.97 % of sugars and 48.77 ± 1.15 % of uronic acids. Its main neutral monosaccharides were galactose, glucose, rhamnose, arabinose, and mannose. A promising nutraceutical value of the polysaccharides was revealed. Assays showed strong α-glucosidase inhibitory activity of both macromolecules and considerable antiradical potential and moderate lipoxygenase inhibitory activity of the crude polysaccharide. Moreover, antiproliferative activity of C2 was observed.

The chemical structure of the O-polysaccharide isolated from the lipopolysaccharide of Pectobacterium brasiliense IFB5527, a phytopathogenic bacterium of high economic importance.

Pectobacterium brasiliense is a widespread plant pathogenic bacterium classified to the Pectobacteriaceae family, which causes significant economic losses because of the developed soft rot and blackleg symptoms on potatoes and a wide spectrum of crops, vegetables, and ornamentals. One of the key virulence factors is a lipopolysaccharide due to its involvement in efficient colonisation of plant tissues and overcoming the host defence mechanisms. Thus, we structurally characterised the O-polysaccharide from the LPS of P. brasiliense strain IFB5527 (HAFL05) using chemical methods followed by GLC and GLC-MS as well as 1D and 2D NMR spectroscopy. The analyses revealed that the polysaccharide repeating unit consists of Fuc, Glc, GlcN and an unusual N-formylated 6-deoxy amino sugar, Qui3NFo, and has the structure shown below.

The structure of an abequose - containing O-polysaccharide isolated from Pectobacterium aquaticum IFB5637.

Soft rot and blackleg diseases, caused by pectinolytic bacteria from the numerous species of Dickeya and Pectobacterium, pose a serious threat to the world potato production. Besides, infections triggered by these pectinolytic bacteria lead to huge economic losses in the cultivation of other crops, vegetables, and ornamentals. Strains belonging to the genus Pectobacterium tend to be isolated from various environments such as rotten or asymptomatic plants, weeds, soil or water. The main virulence factors of these phytopathogenic bacteria involve plant cell wall degrading enzymes (PCWDEs) i.e. pectinases, cellulases and proteases. Among accessory virulence factors, there is often lipopolysaccharide (LPS) listed. This constituent of the external part of bacterial cell wall contains lipid A, inner and outer core in addition to O-polysaccharide (OPS). LPS plays an important role in plant-microbe interactions, in particular during the first step of pathogen recognition. In this study we present the chemical structure of OPS of the first Pectobacterium aquaticum strain (IFB5637) isolated from water in Poland. The OPS consists of two common hexoses, such as mannose and glucose, as well as an abequose (3,6-dideoxy-d-xylo-hexose), the first 3,6-dideoxyhexose identified among the Pectobacteriaceae family: According to our best knowledge this is the first determined structure of the OPS of P. aquaticum.

The First Polish Isolate of a Novel Species Pectobacterium aquaticum Originates from a Pomeranian Lake.

Pectinolytic bacteria from the genus Pectobacterium cause high economic losses in various crops, vegetables, and ornamentals including potato. Thus far, these strains have been isolated from distinct environments such as rotten or asymptomatic plants, soil, and waterways. The prevalence of soft rot Pectobacteriaceae in different depths of Pomeranian lakes was performed by a qualified scuba diver over 2 years of monitoring. It allowed for the isolation and broad characterization of a strain from the newly established species Pectobacterium aquaticum. Phylogenetic analysis on the sequences of dnaX and recA genes revealed the highest similarity of this strain to P. aquaticum CFBP 8637T. In addition to the determination of analytical profile index (API 20E), we discovered that this strain possesses a smooth form of a lipopolysaccharide with O-polysaccharide consisting of mannose, glucose, and abequose. Moreover, the characterized strain, described as P. aquaticum IFB5637, produced plant-cell-wall-degrading enzymes, such as pectinases, cellulases, proteases, and was capable of macerating potato and chicory tissues under laboratory conditions. In view of more frequent irrigation of seed potato fields resulting from the ongoing climate warming, it is important to monitor the occurrence of potential disease-causing agents in natural waterways.

Promising Potential of Crude Polysaccharides from Sparassis crispa against Colon Cancer: An In Vitro Study.

The aim of the present study was to evaluate in vitro the beneficial potential of crude polysaccharides from S. crispa (CPS) in one of the most common cancer types-colon cancer. The determination of the chemical composition of CPS has revealed that it contains mostly carbohydrates, while proteins or phenolics are present only in trace amounts. 1H NMR and GC-MS methods were used for the structural analysis of CPS. Biological activity including anticancer, anti-inflammatory and antioxidant properties of CPS was investigated. CPS was found to be non-toxic to normal human colon epithelial CCD841 CoN cells. Simultaneously, they destroyed membrane integrity as well as inhibited the proliferation of human colon cancer cell lines: Caco-2, LS180 and HT-29. Antioxidant activity was determined by various methods and revealed the moderate potential of CPS. The enzymatic assays revealed no influence of CPS on xanthine oxidase and the inhibition of catalase activity. Moreover, pro-inflammatory enzymes such as cyclooxygenase-2 or lipooxygenase were inhibited by CPS. Therefore, it may be suggested that S. crispa is a valuable part of the regular human diet, which may contribute to a reduction in the risk of colon cancer, and possess promising activities encouraging further studies regarding its potential use as chemopreventive and therapeutic agent in more invasive stages of this type of cancer.

Application of spectroscopic methods for structural analysis of chitin and chitosan.

Chitin, the second most important natural polymer in the world, and its N-deacetylated derivative chitosan, have been identified as versatile biopolymers for a broad range of applications in medicine, agriculture and the food industry. Two of the main reasons for this are firstly the unique chemical, physicochemical and biological properties of chitin and chitosan, and secondly the unlimited supply of raw materials for their production. These polymers exhibit widely differing physicochemical properties depending on the chitin source and the conditions of chitosan production. The presence of reactive functional groups as well as the polysaccharide nature of these biopolymers enables them to undergo diverse chemical modifications. A complete chemical and physicochemical characterization of chitin, chitosan and their derivatives is not possible without using spectroscopic techniques. This review focuses on the application of spectroscopic methods for the structural analysis of these compounds.

The chemical structure of polysaccharides isolated from the Ochrobactrum rhizosphaerae PR17T.

The bacteria from Ochrobactrum genus are commonly found in the soil and association with the roots of plants. The O-polysaccharide and the glucan were isolated from the Ochrobactrum rhizosphaerae PR17T strain. Purified polysaccharides were analysed using chemical methods and NMR spectroscopy. Sugar and absolute configuration assignment combined with NMR data revealed the chemical structure of the repeating unit of the O.

The structure of the O-polysaccharide isolated from pectinolytic gram-negative bacterium Dickeya aquatica IFB0154 is different from the O-polysaccharides of other Dickeya species.

The species Dickeya aquatica was established in 2014 after the genomic characterization of the pectinolytic bacteria isolated from water. It was demonstrated that D. aquatica was able to cause symptoms of soft rot on the fruit of tomato and cucumber. According to earlier works, lipopolysaccharides are regarded as an important virulence factor of Pectobacteriaceae. An O-specific polysaccharide containing d-Fuc and l-Rha was obtained by mild acid hydrolysis of the lipopolysaccharide of D. aquatica IFB0154 (strain Dw044 isolated in Finland). By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as a linear disaccharide of the structure shown below. The rhamnose residue was partially acetylated at O-2 or O-3. OAc (~40%) ↓2 →3)-α-d-Fucp-(1 â†’ 4)-α-l-Rhap-(1→ ↑3 OAc (~30%) The O-polysaccharides isolated from Dickeya dianthicola IFB0485 and Dickeya zeae IPO946 have a different structure, identical to that previously described for several strains of Dickeya solani and Dickeya dadantii 3937.

Decreased Triacylglycerol Content and Elevated Contents of Cell Membrane Lipids in Colorectal Cancer Tissue: A Lipidomic Study.

Recent evidence suggests that lipid composition in cancer tissues may undergo multiple alterations. However, no comprehensive analysis of various lipid groups in colorectal cancer (CRC) tissue has been conducted thus far. To address the problem in question, we determined the contents of triacylglycerols (TG), an energetic substrate, various lipids necessary for cell membrane formation, among them phospholipids (phosphatidylcholine, phosphatidylethanolamine), sphingolipids (sphingomyelin) and cholesterol (free, esterified and total), and fatty acids included in complex lipids. 1H-nuclear magnetic resonance (1H-NMR) and gas chromatography-mass spectrometry (GC-MS) were used to analyze the lipid composition of colon cancer tissue and normal large intestinal mucosa from 25 patients. Compared with normal tissue, cancer tissues had significantly lower TG content, along with elevated levels of phospholipids, sphingomyelin, and cholesterol. Moreover, the content of oleic acid, the main component of TG, was decreased in cancer tissues, whereas the levels of saturated fatty acids and polyunsaturated fatty acids (PUFAs), which are principal components of polar lipids, were elevated. These lipidome rearrangements were associated with the overexpression of genes associated with fatty acid oxidation, and the synthesis of phospholipids and cholesterol. These findings suggest that reprogramming of lipid metabolism might occur in CRC tissue, with a shift towards increased utilization of TG for energy production and enhanced synthesis of membrane lipids, necessary for the rapid proliferation of cancer cells.