Antimicrobial activity of Geranyl acetate against cell wall synthesis proteins of P. aeruginosa and S. aureus using molecular docking and simulation.

Incidences of Methicillin-Resistant Staphylococcus aureus and Multi-Drug Resistant Pseudomonas aeruginosa causing skin and soft tissue infections are becoming more prevalent due to repeated mutations and changes in the environment. Coriandrum sativum, a well-known Indian herbal medicinal plant, is shown to have antioxidant, antibacterial, and anti-inflammatory activity. This comparative study focuses on the molecular docking (PyRx v0.9.8) of ligand binding domains of WbpE Aminotransferase involved in O-antigen assembly in Pseudomonas aeruginosa (3NU7) and Beta-Lactamase found in Staphylococcus aureus (1BLC) with selected phytocompounds of Coriandrum sativum along with a known binder and a clinical reference drug. This was followed by molecular dynamics simulation studies (GROMACS v2019.4) for the docked complexes (with Geranyl acetate) with the best binding affinities (-23.4304 kJ/mol with Beta-Lactamase and -28.4512 kJ/mol with WbpE Aminotransferase) and maximum hydrogen bonds. Molecular dynamics simulation studies for both the proteins demonstrated that the complex with Geranyl acetate showed stability comparable to the complex with reference drug observed via Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF) and H-bond analyses. Changes in the secondary structural elements indicated that Geranyl acetate could possibly cause improper functioning of WbpE Aminotransferase leading to disrupted cell wall formation. Further, MM/PBSA analyses showed significant binding affinity of Geranyl acetate with WbpE Aminotransferase and Beta-Lactamase. This study aims to provide rationale for further studies of Coriandrum sativum as an antimicrobial, and to contextualise the results in the current scenario of growing antimicrobial resistance. HIGHLIGHTSPhytoconstituents present in Coriandrum sativum show significant binding affinity to the proteins in Pseudomonas aeruginosa and Staphylococcus aureus.Geranyl acetate exhibited the highest binding affinity with WbpE Aminotransferase involved in O-antigen assembly in Pseudomonas aeruginosa (PDB ID:3NU7) and Beta-Lactamase found in Staphylococcus aureus (PDB ID: 1BLC)Molecular dynamics simulation analyses show that the phytoconstituent, Geranyl acetate has an effect similar to the clinical reference drug, thus exhibiting potential antibacterial activity.Communicated by Ramaswamy H. Sarma.

Shigella flexneri Adapts to Niche-Specific Stresses through Modifications in Cell Envelope Composition and Decoration.

Shigella flexneri is the primary causative agent of worldwide shigellosis. As the pathogen transverses the distinct niches of the gastrointestinal tract it necessitates dynamic adaptation strategies to mitigate host antimicrobials such as dietary fatty acids (FAs) and the bile salt, deoxycholate (DOC). This study investigates the dynamics of the S. flexneri cell envelope, by interrogating adaptations following FA or DOC exposure. We deciphered the effects of FAs and DOC on bacterial membrane fatty acid and lipopolysaccharide (LPS) compositions. We identified novel LPS-based strategies by the pathogen to support resistance to these host compounds. In particular, expression of S. flexneri very-long O antigen (VL-Oag) LPS was found to play a central role in stress mitigation, as VL-Oag protects against antimicrobial FAs, but its presence rendered S. flexneri susceptible to DOC stress. Collectively, this work underpins the importance for S. flexneri to maintain appropriate regulation of cell envelope constituents, in particular VL-Oag LPS, to adequately adapt to diverse stresses during infection.

Force spectroscopy of interactions between Yersinia pseudotuberculosis and Yersinia pestis cells and monoclonal antibodies using optical tweezers.

The interactions of a microbial cell with host cells and humoral factors play an important role in the development of infectious diseases. The study of these mechanisms contributes to the development of effective methods for the treatment of bacterial infections. One of the possible approaches to studying bacterial adhesion to host cells is based on the use of the optical trap method. The aim of this work was to assess the significance of lipopolysaccharide O-antigen on the adhesiveness of Yersinia pseudotuberculosis using a model system including a bacterial cell captured by a laser beam and monoclonal antibodies (mAbs) bound covalently to a glass substrate. Registered interaction forces between Y. pseudotuberculosis cells and complementary antibodies to the O-antigen of lipopolysaccharide (LPS) or the B antigen outer membrane protein were 5.9 ± 3.3 and 2.0 ± 1.8 pN, respectively. Interaction forces between O-antigen deficient Y. pestis cells and the mentioned mAbs were 4.2 ± 2.9 and 9.6 ± 4.9 pN. The results are qualitatively consistent with earlier data obtained by using a model system based on polymer beads sensitized with LPS from Y. pseudotuberculosis and Y. pestis and surfaces coated by the aforementioned antibodies. This indicates that the immunochemical activity of Y. pseudotuberculosis cells is mediated mainly by the lipopolysaccharide. The model described can be used in similar studies of physicochemical and immunochemical mechanisms of bacterial adhesiveness.

Biopolymer Skeleton Produced by Rhizobium radiobacter: Stoichiometric Alternation of Glycosidic and Amidic Bonds in the Lipopolysaccharide O-Antigen.

The lipopolysaccharide (LPS) O-antigen structure of the plant pathogen Rhizobium radiobacter strain TT9 and its possible role in a plant-microbe interaction was investigated. The analyses disclosed the presence of two O-antigens, named Poly1 and Poly2. The repetitive unit of Poly2 constitutes a 4-α-l-rhamnose linked to a 3-α-d-fucose residue. Surprisingly, Poly1 turned out to be a novel type of biopolymer in which the repeating unit is formed by a monosaccharide and an amino-acid derivative, so that the polymer has alternating glycosidic and amidic bonds joining the two units: 4-amino-4-deoxy-3-O-methyl-d-fucose and (2'R,3'R,4'S)-N-methyl-3',4'-dihydroxy-3'-methyl-5'-oxoproline). Differently from the O-antigens of LPSs from other pathogenic Gram-negative bacteria, these two O-antigens do not activate the oxidative burst, an early innate immune response in the model plant Arabidopsis thaliana, explaining at least in part the ability of this R. radiobacter strain to avoid host defenses during a plant infection process.

Structure, gene cluster of the O antigen and biological activity of the lipopolysaccharide from the rhizospheric bacterium Ochrobactrum cytisi IPA7.2.

Lipopolysaccharide (LPS) of Ochrobactrum cytisi IPA7.2, a bacterium isolated from the roots of Solanum tuberosum L., was extracted from dry bacterial cells and chemically characterized. The O-specific polysaccharide was obtained by mild acid hydrolysis of the LPS and studied by sugar analysis and 1H and 13C NMR spectroscopy, including 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY, 1H,13C HSQC, and 1H,13C HMBC experiments. The polysaccharide was linear and consisted of trisaccharide repeating units of the following structure: A putative O-antigen gene cluster of O. cytisi IPA7.2 was identified and found to be consistent with the O-specific polysaccharide structure. The LPS of Ochrobactrum cytisi IPA7.2 promoted the growth of potato microplants in vitro.

Glycoconjugate vaccine using a genetically modified O antigen induces protective antibodies to Francisella tularensis.

Francisella tularensis is the causative agent of tularemia, a category A bioterrorism agent. The lipopolysaccharide (LPS) O antigen (OAg) of F. tularensis has been considered for use in a glycoconjugate vaccine, but conjugate vaccines tested so far have failed to confer protection necessary against aerosolized pulmonary bacterial challenge. When F. tularensis OAg was purified under standard conditions, the antigen had a small molecular size [25 kDa, low molecular weight (LMW)]. Using milder extraction conditions, we found the native OAg had a larger molecular size [80 kDa, high molecular weight (HMW)], and in a mouse model of tularemia, a glycoconjugate vaccine made with the HMW polysaccharide coupled to tetanus toxoid (HMW-TT) conferred better protection against intranasal challenge than a conjugate made with the LMW polysaccharide (LMW-TT). To further investigate the role of OAg size in protection, we created an F. tularensis live vaccine strain (LVS) mutant with a significantly increased OAg size [220 kDa, very high molecular weight (VHMW)] by expressing in F. tularensis a heterologous chain-length regulator gene (wzz) from the related species Francisella novicida Immunization with VHMW-TT provided markedly increased protection over that obtained with TT glycoconjugates made using smaller OAgs. We found that protective antibodies recognize a length-dependent epitope better expressed on HMW and VHMW antigens, which bind with higher affinity to the organism.

Sulfated O-polysaccharide with anticancer activity from the marine bacterium Poseidonocella sedimentorum KMM 9023T.

The sulfated polysaccharides are of study interest due to their high structural diversity and broad spectrum of biological activity including antitumor properties. In this paper, we report on the structural analysis of sulfated O-specific polysaccharide (OPS) and in vitro anticancer activity of O-deacylated lipopolysaccharide (DPS) of the marine-derived bacterium Poseidonocella sedimentorum KMM 9023T achieved by a multidisciplinary approach (chemical analysis, NMR, MS, and bioassay). The OPS is shown to include two rare monosaccharide derivatives: 3-deoxy-9-O-methyl-d-glycero-d-galacto-non-2-ulosonic acid (Kdn9Me) and 3-O-acetyl-2-O-sulfate-d-glucuronic acid (D-GlcA2S3Ac). The structure of polysaccharide moiety of a previously unknown carbohydrate-containing biopolymer is established: →4)-α-Kdnp9Me-(2→4)-α-d-GlcpA2S3Ac-(1→. From a biological point of view, we demonstrate that DPS of the P. sedimentorum KMM 9023T has no cytotoxicity and inhibits colony formation of human HT-29, MCF-7 and SK-MEL-5 cells in a dose-dependent manner. The investigated polysaccharide is the second glycan isolated from the bacteria of the genus Poseidonocella: previously we studied the OPS of P. pacifica KMM 9010T (Kokoulin et al., 2017). Both polysaccharides are sulfated and contain rare residues of ulosonic acids. Thus, obtained findings provide a new knowledge about kinds and antitumor properties of sulfated polysaccharides and can be a starting point for further investigations of mechanisms of anticancer action of carbohydrate-containing biopolymers from marine Gram-negative bacteria.

Lipopolysaccharides of Pantoea agglomerans 7604 and 8674 with structurally related O-polysaccharide chains: Chemical identification and biological properties.

Structurally related O-specific polysaccharide (O-antigen) and lipid A components were obtained by mild acid degradation of the lipopolysaccharides (LPSs) of two strains of bacteria Pantoea agglomerans, 7604 and 8674. Studies by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy enabled elucidation of the following structures of the O-polysaccharides, which differ only in the linkage configuration of a side-chain glucose residue: R=α-d-Glcp in strain 7604 or ß-d-Glcp in strain 8674 Lipid A samples were studied by GC-MS and high-resolution ESI-MS and found to be represented by penta- and tetra-acyl species; lipid A of strain 8674 also included hexaacyl species. A peculiar feature of lipid A of both strains is the presence of the major cis-9-hexadecenoic (palmitoleic) acid, which has not been found in P. agglomerans strains studied earlier. The LPSs of both strains were pyrogenic, reduced the average adhesion and the index of adhesiveness and showed a relatively low level of lethal toxicity. O-antiserum against strain 7604 showed one-way cross-reactivity with the LPS of strain 8674, and O-antisera against both strains cross-reacted with LPSs of some other Р. agglomerans strains but more strains were serologically unrelated. These structural and serological data indicate immunochemical heterogeneity of Р. agglomerans strains and will find demand in classification of Р. agglomerans by O-antigens.

Structure and in vitro anticancer activity of sulfated O-polysaccharide from marine bacterium Poseidonocella pacifica KMM 9010T.

We presented the structure of the sulfated polysaccharide moiety and anticancer activity in vitro of the О-deacylated lipopolysaccharide (DPS) isolated from the marine bacterium Poseidonocella pacifica KMM 9010T. The structure of O-polysaccharide was investigated by chemical methods along with 1H and 13C NMR spectroscopy. The O-polysaccharide was built up of sulfated disaccharide repeating units consisted of d-rhamnose (d-Rhaр) and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdop): →7)-ß-Kdoр4Ac5S-(2→3)-ß-d-Rhaр2S-(1→. We demonstrated that the DPS from P. pacifica KMM 9010T non-toxic for normal mouse epidermal cells (JB6 Cl41 cell line) and inhibited colony formation of human colorectal carcinoma HT-29, breast adenocarcinoma MCF-7 and melanoma SK-MEL-5 cells in a dose-dependent manner.

The Porphyromonas gingivalis O antigen is required for inhibition of apoptosis in gingival epithelial cells following bacterial infection.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis infection induces apoptosis inhibition in gingival epithelial cells; however, it is not fully understood which bacterial effectors are involved in this process. The aim of this study is to evaluate whether the P. gingivalis lipopolysaccharide (LPS), specifically the O-antigen region, affects adherence, invasion, viability and apoptosis of gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells (OKF6/TERT2 line) were infected by different freshly prepared P. gingivalis clinical isolates, obtained from subjects with chronic periodontitis (CP3 and CP4) and healthy individuals (H1 and H3). Periodontitis and healthy isolates show differences in O-antigen production, as healthy isolates lack the O-antigen region. In addition, cells were infected by a site-specific mutant lacking the O-antigen portion. After 24 h postinfection, cell proliferation, viability and apoptosis were evaluated by Trypan blue, MTS and annexin V assays, respectively. Bacterial invasion, adhesion and proliferation were measured by gentamicin/metronidazole protection assays. Finally, toll-like receptor (TLR)2 and TLR4 mRNA expression was evaluated by quantitative reverse transcription-polymerase chain reaction. Statistical analysis was performed using ANOVA, Tukey's or Dunnett's tests (p < 0.05). RESULTS: At 24 h postinfection, strains lacking the O-antigen region (healthy isolates and O-antigen ligase-deficient strain) were unable to increase proliferation and viability, or decrease apoptosis as compared with strains producing intact LPS (periodontitis isolates and reference strain). However, the presence of the O-antigen neither contributed to changes in the ability of the bacteria to adhere to or invade cells, nor to intracellular survival. The presence of O-antigen also increased the expression of TLR4 (nearly sixfold), which correlated with inhibition of apoptosis. CONCLUSION: The O-antigen region of P. gingivalis LPS is required to increase gingival epithelial cell viability upon infection by bacteria and this increase is attributable to a reduction in apoptosis. Moreover, although bacterial internalization is required, the effects observed are not due to alterations in P. gingivalis adherence, invasion or intracellular survival. Interestingly, inhibition of apoptosis correlates with increased TLR4 expression, suggesting a role for TLR4 in this process.

NMR study of the O-specific polysaccharide and the core oligosaccharide from the lipopolysaccharide produced by Plesiomonas shigelloides O24:H8 (strain CNCTC 92/89).

The structures of the O-specific polysacccharide and core oligosaccharide of the lipopolysaccharide from Plesiomonas shigelloides O24:H8, strain CNCTC 92/89, have been investigated by NMR spectroscopy and ESI mass spectrometry. The O-specific polysaccharide was found to be composed of a tetrasaccharide repeating unit consisting of [→3)-α-FucpNAc-(1→3)-α-GalpNAcA-(1→3)-α-QuipNAc-(1→] and of α-RhapNAc (1→4) linked to the GalpNAcA residue. An identical structure has been reported for the capsular polysaccharide of the clinical isolate of Vibrio vulnificus strain BO62316 [1]. The core oligosaccharide was composed of a decasaccharide which structure is identical with these in P. shigelloides serotype O54 [2] and serotype O37 [3].

Molecular and chemical analysis of the lipopolysaccharide from Aeromonas hydrophila strain AH-1 (Serotype O11).

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.

[SEROLOGICAL PROPERTIES AND BIOLOGICAL ACTIVITY OF PANTOEA AGGLOMERANS LIPOPOLYSACCHARIDES].

The serological and phytotoxic properties of lipopolysaccharide (LPS) of plant pathogens--Pantoea agglomerans were studied. It is known that the thin variations in the structure of the O-specific polysaccharides determining serological specificity of gram- negative bacteria and used as a molecular basis of serological classification schemes. For P. agglomerans still does not exist a classification scheme based on serology specificity of their LPS. The results of cross serological tests demonstrate immunochemical heterogeneity of species P agglomerans. Only three strains of the 8488, 8490 and 7969 according to the agglutination of O-antigens and direct hemagglutination and inhibition direct hemagglutination can be attributed to a single serogroup. Other strains--each separate group, although some have a relationship. Compared with control plants under the influence of seed treatment of LPS in plants may be reduced, and in some cases increased root length, height and weight sprout, depending on the strain from which the selected LPS. Dive seedlings of tomatoes in the solutions of the studied preparations FSC caused the loss, and after some time, restore turgor.

Synthesis and plant growth regulation activity of α-d-ManpNAc-(1→2)-[α-L-Rhap-(1→3)-]α-L-Rhap-(1→4)-ß-d-GlupNAc-(1→3)-α-L-Rhap, the repeating unit of O-antigen of Rhizobium trifolii 4s.

The synthesis of a pentasaccharide 2 containing acetamido-2-deoxy-d-glucose and acetamido-2-deoxy-d-mannose related to the cell wall polysaccharide of Rhizobium trifolii 4s has been achieved by a [2+3] approach from commercially available l-rhamnose, d-glucose, and d-glucosamine as the starting materials. The target molecule was equipped with a p-methoxylphenyl handle at the reducing terminus to allow for further glycoconjugate formation via selective cleavage of this group. The bioassay suggested that the synthetic pentasaccharide 2 can stimulate the growth of wheat coleoptile similarly to indole-3-acetic acid (IAA), and promote the wheat seedling development before winter by seed treatment at a concentration of 20mg/L.

Different effects of P. gingivalis LPS and E. coli LPS on the expression of interleukin-6 in human gingival fibroblasts.

OBJECTIVE: Gingival fibroblasts (GFs) produce pro-inflammatory cytokines in response to stimulation with lipopolysaccharide (LPS) of Porphyromonas gingivalis, which is thought to be mediated by activation of toll-like receptors (TLR)2 and TLR4. The present study investigated the expression of interleukin (IL)-6, TLR2, and TLR4 in GFs of seven different donors upon stimulation with P. gingivalis LPS. The effects of P. gingivalis LPS were compared with those of TLR4 agonist Escherichia coli LPS and TLR2 agonist Pam3CSK4. MATERIALS AND METHODS: GFs were stimulated with P. gingivalis LPS, E. coli LPS or Pam3CSK4 and the expression of IL-6, TLR2 and TLR4 was measured by qPCR. The surface expression of TLR2 and TLR4 was measured by flow cytometry. RESULTS: In GFs from three donors, P. gingivalis LPS and Pam3CSK4 induced a markedly lower increase in IL-6 expression than E. coli LPS. This was accompanied by significant down-regulation of the TLR2 and TLR4 expression. In GFs from another four donors, an increase in IL-6 expression upon stimulation with P. gingivalis LPS and Pam3CSK4 was similar or even higher than that induced by E. coli LPS. In GFs of these donors, all stimuli induced an up-regulation of both mRNA and protein expression of TLR2 and did not influence that of TLR4. CONCLUSIONS: This study suggests that P. gingivalis LPS and E. coli LPS differently regulate cytokine production in human gingival fibroblasts. Regulation of the expression level of TLR2 and TLR4 by periodontal pathogens might be an important factor controlling the inflammatory response in GFs.

Similarities and differences of innate immune responses elicited by smooth and rough LPS.

The lipopolysaccharide is the major component of Gram-negative bacteria outer membrane. LPS comprises three covalently linked regions: the lipid A, the rough core oligosaccharide, and the O-antigenic side chain determining serotype specificity. Wild-type LPS (sLPS) contains the O-antigenic side chain and is referred to as smooth. Rough LPS (rLPS) does not contain the O-side chain. Most wt bacteria and especially wt Enterobacteriaceae express prevalently the sLPS form although some truncated rLPS molecules always reach the external membrane. The two sLPS and rLPS forms are used almost indistinctly to study the effects on innate immune cells. Nevertheless, there is evidence that their mechanism of action may be different. For instance, while sLPS requires CD14 for the initiation of both MyD88-dependent and independent signal transduction pathways at least at low doses, rLPS leads to MyD88-dependent responses in the absence of CD14 even at low doses. Here we have identified additional differences in the signaling capacity of the two LPS species in the mouse. We have found that rLPS, diversely from sLPS, is capable of activating in dendritic cells (DCs) the Ca(2+)/calcineurin and NFAT pathway in a CD14-independent manner, moreover it is also capable per se of activating the inflammasome and eliciting IL-1ß secretion independent of the presence of additional stimuli required instead for sLPS. The ability of rLPS of activating the inflammasome in vitro has as a direct consequence a higher efficiency of rLPS-exposed DCs in activating natural killer (NK) cells compared to sLPS-exposed DCs. However, diversely from possible predictions, we found that the different efficiencies of the two LPS species in eliciting innate responses are almost nullified in vivo. Therefore, sLPS and rLPS induce nearly similar in vivo innate responses but with different mechanisms of signaling.

Vibrio cholerae O1 Ogawa detoxified lipopolysaccharide structures as inducers of cytokines and oxidative species in macrophages.

Multidrug resistance in several strains of Vibrio cholerae has encouraged anti-cholera vaccine developmental attempts using various subcellular moieties. In order to examine the immunological efficacy of detoxified LPS (dLPS)-derived saccharide immunogens, ex vivo activation of mouse peritoneal macrophages (MPhis) was investigated. The immunomodulatory effect was evaluated via induction of the pro-inflammatory cytokines tumour necrosis factor-alpha, interleukin (IL)-1 alpha and IL-6 and acceleration of nitric oxide (NO) and reactive oxygen species (ROS). Immunologically active structures triggered mouse peritoneal MPhis to secrete cytokines and release NO/ROS, even at concentrations as low as 12.5 microg ml(-1). It was found that the O-specific polysaccharide moiety was more immunologically efficient than the glycolipid one, probably due to the position of 3-deoxy-D-manno-octulosonic acid. The results revealed effective structure-immunomodulating relationships of dLPS-derived moieties that are desirable in subcellular anti-cholera vaccine design.

[Biological activity of native and modified lipopolysaccharides of Pragia fontium].

Modified lipopolysaccharides (LPS) of Pragia fontium were obtained with germanium complexes (IV) of 2-aminobenzoylhydrazon of salicylic aldehyde (2-NH2-H2Bs), 2-hydroxybenzoylhydrazon salicylic aldehyde (2-OH-H2Bs) and nicotinoylhydrazon of salicylic aldehyde (H2Ns). The modification of LPS was confirmed by IR spectroscopy. Comparative investigations of pyrogenic activity of native and modified LPS showed, that only P. fontium 20125 LPS, modified by germanium complexes with 2-hydroxybenzoylhydrazon of salicylic aldehyde (2-OH-H2Bs) has lost the pyrogenic activity. In the homological reactions of double immunodiffusion in agar it was shown that all modified LPS unlike the native ones lose completely antigenic activity.

Pulmonary collectins selectively permeabilize model bacterial membranes containing rough lipopolysaccharide.

We have reported that Gram-negative organisms decorated with rough lipopolysaccharide (LPS) are particularly susceptible to the direct antimicrobial actions of the pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D). In this study, we examined the lipid and LPS components required for the permeabilizing effects of the collectins on model bacterial membranes. Liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), with or without rough Escherichia coli LPS (J5), smooth E. coli LPS (B5), or cholesterol, were loaded with self-quenching probes and exposed to native or oxidatively modified SP-A. Fluorescence that resulted from permeabilization of liposomes and diffusion of dyes was assessed by microscopy or fluorimetry. Human SP-A and melittin increased the permeability of J5 LPS/POPE liposomes, but not B5 LPS/POPE liposomes or control (POPE only) liposomes. At a human SP-A concentration of 100 microg/mL, the permeability of the J5 LPS/POPE membranes increased 4.4-fold (p < 0.02) compared to the control with no added SP-A. Rat SP-A and SP-D also permeabilized the J5-containing liposomes. Incorporation of cholesterol into J5 LPS/POPE liposomes at a POPE:cholesterol molar ratio of 1:0.15 blocked human SP-A or melittin-induced permeability (p < 0.05) compared to cholesterol-free liposomes. Exposure of human SP-A to surfactant lipid peroxidation blocked the permeabilizing activity of the protein. We conclude that SP-A permeabilizes phospholipid membranes in an LPS-dependent and rough LPS-specific manner, that the effect is neither SP-A- nor species-specific, and that oxidative damage to SP-A abolishes its membrane destabilizing properties. Incorporation of cholesterol into the membrane enhances resistance to permeabilization by SP-A, most likely by increasing the packing density and membrane rigidity.

Structure-dependent modulation of a pathogen response in plants by synthetic O-antigen polysaccharides.

Many phytopathogenic bacteria display lipopolysaccharides (LPS) with the O-chain repeating unit [alpha-l-Rha-(1-->3)-alpha-l-Rha-(1-->3)-alpha-l-Rha-(1-->2)](n)(). This trisaccharide unit was synthesized and oligomerized to obtain hexa- and nonasaccharides. The deprotected rhamnans were effective in suppressing the hypersensitive response (HR) and in inducing PR-1 gene expression in Arabidopsis thaliana. Conformational analysis of the oligorhamnans by NMR spectroscopy and molecular dynamics calculations revealed that a coiled structure develops with increasing chain length of the oligosaccharide. This is associated with increasing efficacy in HR suppression and PR-1 gene expression. We therefore infer that the coiled structure of phytopathogenic bacteria is a plant-recognizable pathogen-associated molecular pattern (PAMP).