Number Of Acyl Chains Research Articles

Essential role of Helicobacter pylori apolipoprotein N-acyltransferase (Lnt) in stomach colonization.

Bacterial lipoproteins are post-translationally modified with acyl chains, anchoring these proteins to bacterial membranes. In Gram-negative bacteria, three enzymes complete the modifications. Lgt (which adds two acyl chains) and LspA (which removes the signal peptide) are essential. Lnt (which adds a third acyl chain) is not essential in certain bacteria including Francisella tularensis, Neisseria gonorrhoeae, and Acinetobacter baumannii. Deleting lnt results in mild to severe physiologic changes. We previously showed lnt is not essential for Helicobacter pylori growth in vitro. Here, the physiologic consequences of deleting lnt in H. pylori and the role of Lnt in the host response to H. pylori were examined using in vitro and in vivo models. Comparing wild-type, Δlnt, and complemented mutant H. pylori, no changes in growth rates or sensitivity to acid or antibiotics were observed. Since deleting lnt changes the number of acyl chains on lipoproteins and the number of acyl chains on lipoproteins impacts the innate immune response through Toll-like receptor 2 (TLR2) signaling, primary human gastric epithelial cells were treated with a purified lipoprotein from wild-type or lnt mutant H. pylori. Differential gene expression analysis indicated that lipoprotein from the lnt mutant induced a more robust TLR2 response. In a complementary approach, we infected wild-type and Tlr2-/- mice and found that both the wild-type and complemented mutant strains successfully colonized the animals. However, the lnt mutant strain was unable to colonize either mouse strain. These results show that lnt is essential for H. pylori colonization and identifies lipoprotein synthesis as a target for therapeutic intervention.

Association between lipid-A-producing oral bacteria of different potency and fractional exhaled nitric oxide in a Norwegian population-based adult cohort

BackgroundLipid A is the primary immunostimulatory part of the lipopolysaccharide (LPS) molecule. The inflammatory response of LPS varies and depends upon the number of acyl chains and phosphate groups in lipid A which is specific for a bacterial species or strain. Traditional LPS quantification assays cannot distinguish between the acylation degree of lipid A molecules, and therefore little is known about how bacteria with different inflammation-inducing potencies affect fractional exhaled nitric oxide (FeNO). We aimed to explore the association between pro-inflammatory hexa- and less inflammatory penta-acylated LPS-producing oral bacteria and FeNO as a marker of airway inflammation.MethodsWe used data from a population-based adult cohort from Norway (n = 477), a study center of the RHINESSA multi-center generation study. We applied statistical methods on the bacterial community- (prediction with MiRKAT) and genus-level (differential abundance analysis with ANCOM-BC) to investigate the association between the oral microbiota composition and FeNO.ResultsWe found the overall composition to be significantly associated with increasing FeNO levels independent of covariate adjustment, and abundances of 27 bacterial genera to differ in individuals with high FeNO vs. low FeNO levels. Hexa- and penta-acylated LPS producers made up 2.4% and 40.8% of the oral bacterial genera, respectively. The Bray–Curtis dissimilarity within hexa- and penta-acylated LPS-producing oral bacteria was associated with increasing FeNO levels independent of covariate adjustment. A few single penta-acylated LPS producers were more abundant in individuals with low FeNO vs. high FeNO, while hexa-acylated LPS producers were found not to be enriched.ConclusionsIn a population-based adult cohort, FeNO was observed to be associated with the overall oral bacterial community composition. The effect of hexa- and penta-acylated LPS-producing oral bacteria was overall significant when focusing on Bray–Curtis dissimilarity within each of the two communities and FeNO levels, but only penta-acylated LPS producers appeared to be reduced or absent in individuals with high FeNO. It is likely that the pro-inflammatory effect of hexa-acylated LPS producers is counteracted by the dominance of the more abundant penta-acylated LPS producers in this population-based adult cohort involving mainly healthy individuals.

Synthesis and supramolecular arrangement of new stearoyl acid-based phenalenone derivatives

Lipid-based photoantimicrobial supramolecular assemblies are promising structures for the development of new photodynamic treatments of wound bacterial infections. Phenalenone (1H-phenalene-1-one, PN) has been used to obtain four stearoyl acid-based phenalenone derivatives (PNC18), with different types of linkage and different number of acyl chains. After studying the interfacial properties of these compounds, nano-objects were formed with either pure PNC18 or mixtures of PNC18 with 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine. Morphologies of these nano-objects were analyzed by CryoTEM imaging. The distearoyl glycerol PN derivative appears promising for building photosensitive micelles or vesicles.

Aggregation of Lipid A Variants: A Hybrid Particle-Field Model

Lipid A is one of the three components of bacterial lipopolysaccharides constituting the outer membrane of Gram-negative bacteria, and is recognized to have an important biological role in the inflammatory response of mammalians. Its biological activity is modulated by the number of acyl-chains that are present in the lipid and by the dielectric medium, i.e., the type of counter-ions, through electrostatic interactions. In this paper, we report on a coarse-grained model of chemical variants of Lipid A based on the hybrid particle-field/molecular dynamics approach (hPF-MD). In particular, we investigate the stability of Lipid A bilayers for two different hexa- and tetra-acylated structures. Comparing particle density profiles along bilayer cross-sections, we find good agreement between the hPF-MD model and reference all-atom simulation for both chemical variants of Lipid A. hPF-MD models of constituted bilayers composed by hexa-acylated Lipid A in water are stable within the simulation time. We further validate our model by verifying that the phase behavior of Lipid A/counterion/water mixtures is correctly reproduced. In particular, hPF-MD simulations predict the correct self-assembly of different lamellar and micellar phases from an initially random distribution of Lipid A molecules with counterions in water. Finally, it is possible to observe the spontaneous formation and stability of Lipid A vesicles by fusion of micellar aggregates.

Recognition and protection of glycosphingolipids by synthetic nanoparticle receptors.

Nanoparticle receptors were synthesized through micellar imprinting to bind glycosphingolipids with 20-140 μM binding affinities, meanwhile distinguishing glycan composition, the number of acyl chains, and hydroxylation of acyl chains in the lipids. The strong binding enabled the receptors to protect their target glycolipids dispersed in lipid membranes from enzymatic degradation.

Development of double chain phosphatidylcholine functionalized polymeric monoliths for immobilized artificial membrane chromatography

This study described a simple synthetic methodology for preparing biomembrane mimicking monolithic column. The suggested approach not only simplifies the preparation procedure but also improves the stability of double chain phosphatidylcholine (PC) functionalized monolithic column. The physicochemical properties of the optimized monolithic column were characterized by scanning electron microscopy, energy-dispersive X-ray spectrometry, and nano-LC. Satisfactory column permeability, efficiency, stability and reproducibility were obtained on this double chain PC functionalized monolithic column. It is worth noting that the resulting polymeric monolith exhibits great potential as a useful alternative of commercial immobilized artificial membrane (IAM) columns for in vitro predication of drug-membrane interactions. Furthermore, the comparative study of both double chain and single chain PC functionalized monoliths indicates that the presence or absence of glycerol backbone and the number of acyl chains are not decisive for the predictive ability of IAM monoliths on drug-membrane interactions. This novel PC functionalized monolithic column also exhibited good selectivity for a protein mixture and a set of pharmaceutical compounds.

Bilayer Properties of Lipid A from Various Gram-Negative Bacteria

Lipid A is the lipid anchor of a lipopolysaccharide in the outer leaflet of the outer membrane of Gram-negative bacteria. In general, lipid A consists of two phosphorylated N-acetyl glucosamine and several acyl chains that are directly linked to the two sugars. Depending on the bacterial species and environments, the acyl chain number and length vary, and lipid A can be chemically modified with phosphoethanolamine, aminoarabinose, or glycine residues, which are key to bacterial pathogenesis. In this work, homogeneous lipid bilayers of 21 distinct lipid A types from 12 bacterial species are modeled and simulated to investigate the differences and similarities of their membrane properties. In addition, different neutralizing ion types (Ca2+, K+, and Na+) are considered to examine the ion’s influence on the membrane properties. The trajectory analysis shows that (1) the area per lipid is mostly correlated to the acyl chain number, and the area per lipid increases as a function of the acyl chain number; (2) the hydrophobic thickness is mainly determined by the average acyl chain length with slight dependence on the acyl chain number, and the hydrophobic thickness generally increases with the average acyl chain length; (3) a good correlation is observed among the area per lipid, hydrophobic thickness, and acyl chain order; and (4) although the influence of neutralizing ion types on the area per lipid and hydrophobic thickness is minimal, Ca2+ stays longer on the membrane surface than K+ or Na+, consequently leading to lower lateral diffusion and a higher compressibility modulus, which agrees well with available experiments.

A molecular and biophysical comparison of macromolecular changes in imatinib-sensitive and imatinib-resistant K562 cells exposed to ponatinib.

Chronic myeloid leukemia (CML) is a type of hematological malignancy that is characterized by the generation of Philadelphia chromosome encoding BCR/ABL oncoprotein. Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib, and dasatinib, are used for the frontline therapy of CML. Development of resistance against these TKIs in the patients bearing T315I mutation is a major obstacle in CML therapy. Ponatinib, the third-generation TKI, is novel drug that is effective even in CML patients with T315I mutation. The exact mechanism of ponatinib in CML has been still unknown. In this study, we aimed to determine the potential mechanisms and structural metabolic changes activated by ponatinib treatment in imatinib-sensitive K562 human CML cell lines and 3μM-imatinib-resistant K562/IMA3 CML cell lines generated at our lab. Apoptotic and antiproliferative effects of ponatinib on imatinib-sensitive and 3μM-imatinib-resistant K562/IMA3 CML cells were determined by proliferation and apoptosis assays. Additionally, the effects of ponatinib on macromolecules and lipid profiles were also analyzed using Fourier transform infrared spectroscopy (FTIR). Our results revealed that ponatinib inhibited cell proliferation and induced apoptosis as determined by loss of mitochondrial membrane potential, increased caspase-3 enzyme activity, and transfer of phosphatidylserine to the plasma membrane in both K562 and K562/IMA-3 cells. Furthermore, cell cycle analyses revealed that ponatinib arrested K562 and K562/IMA-3 cells at G1 phase. Moreover, ponatinib treatment created a more ordered nucleic acid structure in the resistant cells. Although the lipid to protein ratio increased in imatinib-sensitive K562 cells with a little decrease in the K562/IMA-3 cells, ponatinib treatment indicated significant changes in the lipid composition such as a significant increase in the cellular cholesterol amounts much more in the K562/IMA-3 cells than the sensitive counterparts. Unsaturation in lipids was higher in the resistant cells; however, increases in lipids without phosphate and the number of acyl chains were much higher in the K562 cells. Taken together, all these results showed powerful antiproliferative and apoptotic effects of ponatinib in both imatinib-sensitive and imatinib-resistant CML cells in a dose-dependent manner, and hence, the use of ponatinib for the treatment of TKI-resistant CML patients may be an effective treatment approach in the clinic. More importantly, these results showed that FTIR spectroscopy can detect drug-induced physiological changes in cancer drug resistance.

Controlling carbon-nanotube-phospholipid solubility by curvature-dependent self-assembly.

Control of aqueous dispersion is central in the processing and usage of nanoscale hydrophobic objects. However, selecting dispersive agents based on the size and form of the hydrophobic object and the role of coating morphology in dispersion efficiency remain important open questions. Here, the effect of the substrate and the dispersing molecule curvature, as well as, the influence of dispersant concentration on the adsorption morphology are examined by molecular simulations of graphene and carbon nanotube (CNT) substrates with phospholipids of varying curvature as the dispersing agents. Lipid spontaneous curvature is increased from close to zero (effectively cylindrical lipid) to highly positive (effectively conical lipid) by studying double tailed dipalmitoylphosphadidylcholine (DPPC) and single tailed lysophosphadidylcholine (LPC) which differ in the number of acyl chains but have identical headgroup. We find that lipids are good dispersion agents for both planar and curved nanoparticles and induce a dispersive barrier nonsize selectively. Differences in dispersion efficiency arise from lipid headgroup density and their extension from the hydrophobic substrate in the adsorption morphology. We map the packing morphology contributing factors and report that the aggregate morphologies depend on the competition of interactions rising from (1) hydrophobicity driven maximization of lipid-substrate contacts and lipid self-adhesion, (2) tail bending energy cost, (3) preferential alignment along the graphitic substrate principal axes, and (4) lipid headgroup preferential packing. Curved substrates adjust the morphology by changing the balance between the interaction strengths. Jointly, the findings show substrate curvature and dimensions are a way to tune lipid adsorption to desired, self-assembling patterns. Besides engineering dispersion efficiency, the findings could bear significance in designing materials with defined molecular scale, molecular coatings for orientation specific CNT assembly or lipid-based molecular masks and patterning on graphene.

GC-MS and MALDI-TOF MS Profi ling of Sucrose Esters from Nicotiana tabacum and N. rustica

Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been applied for the first time to the analysis of the sucrose esters from the surface of Nicotiana L. leaves. The profiles obtained for the model plant N. tabacum were similar to those from the gas chromatography-flame ionization detector (GC-FID) analysis. The most reproducible results were obtained using a dihydroxybenzoic acid (DHB) matrix. The main advantage of this method is that crude plant extracts can be analysed without sample clean-up. GC-MS analysis of Aztec tobacco (N. rustica) extracts revealed the presence of three types of sucrose esters. All identified compounds had three C4-C8 acyl chains substituting the glucose moiety, while the fructose part of the molecule was substituted with 0, 1, or 2 acetyl groups. MALDI-TOF MS analysis of the sucrose ester fraction revealed the presence of compounds not eluting from a GC column. Combining the data from both GC-MS and MALDI-TOF MS experiments, we obtained a full sucrose ester profile, which is based on the molecular weight of the compounds and on the number of acyl chains in the molecule.

The Effect of Temperature, Cations, and Number of Acyl Chains on the Lamellar to Non-Lamellar Transition in Lipid-A Membranes: A Microscopic View

Lipopolysaccharides (LPS) are the main constituent of the outer bacterial membrane of Gram-negative bacteria. Lipid-A is the structural region of LPS that interacts with the innate immune system and induces inflammatory responses. It is formed by a phosphorylated β-d-glucosaminyl-(1→6)-α-N-glucosamine disaccharide backbone containing ester-linked and amide-linked long-chain fatty acids, which may vary in length and number depending on the bacterial strains and the environment. Phenotypical variation (i.e., number of acyl chains), cation type, and temperature influence the phase transition, aggregate structure, and endotoxic activity of Lipid-A. We have applied an extension of the GROMOS force field 45a4 carbohydrate parameter set to investigate the behavior of hexa- and pentaacylated Lipid-A of Pseudomonas aeruginosa at two temperatures (300 and 328 K) and in the presence of mono- and divalent cations (represented by Ca(2+) and Na(+), respectively) through molecular dynamics simulations. The distinct phase of Lipid-A aggregates was characterized by structural properties, deuterium order parameters, the molecular shape of the lipid units (conical versus cylindrical), and molecular packing. Our results show that Na(+) ions induce a transition from the lamellar to nonlamellar phase. In contrast, the bilayer integrity is maintained in the presence of Ca(2+) ions. Through these findings, we present microscopic insights on the influence of different cations on the molecular behavior of Lipid-A associated with the lamellar to nonlamellar transition.

Polymorphic Phase Behavior of Cardiolipin Derivatives Studied by Coarse-Grained Molecular Dynamics

Cardiolipin (CL) is a negatively charged four acyl chain lipid, associated with energy production in bacterial and mitochondrial membranes. Due to the shape of CL, negative curvatures of aggregates are favorable if the charges in the head group can be reduced. The phase polymorphism of CL, and of associated derivatives with 2, 3, 4, or 5 chains, has been determined previously and offers a model system in which micellar, lamellar, and inverse hexagonal phases can be observed. We present an extension to a previously established coarse-grained molecular dynamics model with the aim of reproducing the different CL phases with two adjustable parameters: the number of acyl chains and the effective head group charge. With molecular dynamics simulations of large lipid systems, we observed transitions between different phases on the nanosecond to microsecond time scale. Charge screening by high salt or low pH was successfully modeled by a reduction of phosphate charge, which led to the adoption of aggregates with more negative curvature. Although specific ion binding at the interface and other atomistic details are sacrificed in the coarse-grained model, we found that it captures general phase features over a large range of aggregate geometries.

Structural characterization of lipid A from nontypeable and type f Haemophilus influenzae: Variability of fatty acid substitution

Lipid A isolated by mild acid hydrolysis from lipopolysaccharides of 22 nontypeable and 2 type f Haemophilus influenzae strains was investigated using electrospray ionization coupled to quadrupole ion trap mass spectrometry. The lengths, positions, and number of acyl chains in the lipid A molecule were determined using multiple-step tandem mass spectrometry (MS n ). All of the analyzed strains showed a major lipid A molecule comprising β-2-amino-2-deoxy- d-glucopyranose-(1 → 6)-α-2-amino-2-deoxy- d-glucopyranose phosphorylated at the C4′ and C1 positions. The C2/C2′ and C3/C3′ positions were substituted by amide-linked and ester-linked 3-hydroxytetradecanoic acid chains, respectively. The fatty acid chains on C3′ and C2′ were further esterified by tetradecanoic acid chains. In all strains, minor amounts of lipid A molecules with different acylation patterns were identified. Thus, structures comprising the hexaacylated lipid A with the C2 or C3 position being substituted by 3-hydroxydecanoic acid, and hexaacylated lipid A with the C3 and C3′ positions being substituted by 3-hydroxydodecanoic or dodecanoyloxytetradecanoic acid, respectively, were found. In addition, lipid A with an acetyl group attached to the 3-hydroxytetradecanoic acid groups attached to the C2 or C3 position was detected in two nontypeable H. influenzae strains.

Implications of modifying cardiolipin acyl composition by diet. 1. Cardiolipin acyl chain is an important determinant in the binding to antiphospholipid antibodies in SLE sera

Antiphospholipid antibodies (aPL), prevalent in sera of patients with systemic lupus erythematosus (SLE), have been linked to thrombosis, thrombocytopenia, recurrent miscarriages, neurological disorders and ischemic heart disease. Most evidence suggests that phosphodiester-linked phosphate groups are the reactive epitope of cardiolipin (CL) in binding to aPL. Little attention has been given to the acyl moiety. To address this problem we have evaluated the ELISA binding of 12 highly positive IgG anticardiolipin antibody-positive SLE sera to: bovine CL (86.1% 18:2n-6), monolyso CL (MLCL; bovine CL minus 1 fatty acid), dilyso CL (DLCL; minus 2 fatty acids), tetraoleoyl CL (TOCL), myristoyl CL (MCL) and E. coli CL. The reductions in binding of the IgG aPL antibodies relative to bovine CL were as follows: DLCL 83%; MLCL 70.7%; MCL 58%; and TOCL 14% ( P<0.05). These data suggest that the number of acyl chains and the unsaturation of the acyl chain of CL may be important determinants in the binding to aPL present in SLE sera. To investigate the nutritional relevance of this finding, we examined the incorporation of several dietary fatty acid classes into the CL pool of mice. Mice were fed diets containing n-6 (safflower oil), n-9 (olive oil) or n-3 fatty acids as either 18:3n-3 (linseed oil) or 20:5n-3/22:6n-3 (fish oil) for a 5 month period. The feeding of fish oil and olive oil resulted in replacement of a substantial portion of 18:2n-6 with 22:6n-3 or 18:1n-9, respectively. These results suggest that there may be therapeutic value in modifying the CL acyl composition by nutritional means with respect to binding to pathogenic aPL.

Activation of beef-heart cytochrome c oxidase by cardiolipin and analogues of cardiolipin

Beef-heart cytochrome c oxidase lacking endogenous lipids can be prepared by cholate-mediated exchange with dimyristoylphosphatidylcholine (Powell, G.L., Knowles, P.F. and Marsh, D. (1985) Biochim. Biophys. Acta 816, 191–194). These preparations retained practically no endogenous cardiolipin (less than 0.19 mol cardiolipin per mol of oxidase) but in Tween 80 they retained unaltered electron transport activity. Resupplementation of the dimyristoylphos-phatidylcholine-substituted cytochrome oxidase with cardiolipin and cardiolipin analogues with different numbers of acyl chains or with a methylated headgroup enhanced the activity of the reconstituted enzyme to an extent dependent on the structure of the cardiolipin derivative. The Eadie-Hofstee plot showed biphasic kinetic behavior for all reconstituted preparations, even those completely lacking cardiolipin. This biphasic substrate dependence of the kinetics was simulated using the model of Brzezinski, P. and Malmström, B.G. (Proc. Natl. Acad. Sci. USA 83 (1986) 4282–4286), which implicates two interconverting enzyme conformations in the proton transport step. The activation of cytochrome c oxidase by the cardiolipin analogues could be explained in terms of an electrostatic enhancement of the surface concentrations of both cytochrome c and protons, and a facilitated interconversion between the two enzyme conformations.

Spin label studies on the selectivity of lipid-protein interaction of cardiolipin analogues with the Na 2+/K 2+-ATPase

The selectivity of lipid-protein interaction for various spin-labelled cardiolipin analogoues in Na +/K +- ATPase membranes from Squalus acanthias has been investigated by ESR spectroscopy. Cardiolipin derivatives with different numbers of acyl chains, or in which the headgroup charge has been removed by methylation of the phosphate groups, all show a pronounced selectivity relative to phosphatidylcholine. Maximally three times more of the cardiolipin analogue is associated with the protein, than is phosphatidylcholine. The selectivity pattern in the absence of salt is in the order: cardiolipin ≈ monolysocardiolipin ≥ acylcardiolipin > dimethylcardiolipin ⪢ phosphatidylcholine, where acylcardiolipin has the spin label chain attached to the centre-OH group of the headgroup. The degree of association of the negatively charged cardiolipinds with the protein is reduced by salt, corresponding to the lower selectivity for dimethylcardiolipin. It is concluded that the selectivity of the Na +/K +-ATPase for cardiolipin is not solely of electrostatic origin, nor is it likely the originate in the larger number of fatty acid chains relative to diacyl phospholipids.

Leukocyte chemoattraction by 1,2-diacylglycerol.

Previous reports have demonstrated the hydrolysis of inositol phospholipids in polymorphonuclear leukocytes (PMN) in response to chemoattractants and in lymphocytes in response to the mitogen phytohemagglutinin. We investigated the role of 1,2-diacylglycerol, one of the products of receptor-linked phosphatidylinositol hydrolysis, in mediating the migratory response of leukocytes. In an under-agarose migration system, we found 1,2-dioctanoylglycerol to be a strong chemoattractant for human PMN, 6C3HED (a mouse thymic lymphoma), and Jurkat (a human T-cell leukemia). By using a modified Boyden chamber assay, the migratory response of PMN to 1,2-dioctanoylglycerol was determined to be primarily chemotactic. Analysis of structural analogs indicated that both the position and number of acyl chains are important in determining chemoattractant activity. These studies demonstrate that exogenous 1,2-diacylglycerol can stimulate the directed migration of leukocytes. They further suggest that the formation of 1,2-diacylglycerol following receptor-mediated stimulation may represent a common step in the migratory responses of myeloid and lymphoid cells.