Lipopolysaccharide Micelles Research Articles

NMR Structures and Interactions of Antimicrobial Peptides with Lipopolysaccharide: Connecting Structures to Functions.

Antimicrobial peptides (AMPs) establish the first line of host defense mechanism against invading microorganisms including bacteria, viruses, fungi and parasites. In recent years, emergence and spread of antibiotic resistance bacterial pathogens have dawn considerable interest in investigations of AMPs. The ability of AMPs to exert lethality against multiple drug-resistant (MDR) bacteria has incited promising avenues for antibiotic development. As a mode of action, most AMPs perturb the membrane organization of bacterial cells. The outer membrane lipopolysaccharide (LPS) of Gram-negative bacteria establishes a superior permeability barrier, in contrast to the peptidoglycan layer of Gram-positive bacteria. Due to LPS barrier, development of antibiotics for drug resistant Gram- negative bacteria are more complicated, with only fewer compounds in the pipeline. Recent studies have demonstrated that LPS actively regulate mode of action of AMPs on the lethality of Gram-negative bacteria. LPS, also known as endotoxin, is the primary agent for septic shock syndromes in intensive care unit killing over 120,000 people in the USA. Currently, anti-sepsis therapies are greatly lacking. Therefore, LPS has been considered as a target for the development of antimicrobial and antisepsis drugs. In recent and past few years, 3-D structures and interactions of a number of AMPs have been determined in complex with LPS micelles. These studies have generated molecular insights towards mode of action and synergistic activity of AMPs in the outer membrane. In this review, atomic resolution structures and interactions of potent AMPs with LPS are discussed providing novel insights of their mode of action.

New amphiphilic neamine derivatives active against resistant Pseudomonas aeruginosa and their interactions with lipopolysaccharides.

The development of novel antimicrobial agents is urgently required to curb the widespread emergence of multidrug-resistant bacteria like colistin-resistant Pseudomonas aeruginosa. We previously synthesized a series of amphiphilic neamine derivatives active against bacterial membranes, among which 3',6-di-O-[(2"-naphthyl)propyl]neamine (3',6-di2NP), 3',6-di-O-[(2"-naphthyl)butyl]neamine (3',6-di2NB), and 3',6-di-O-nonylneamine (3',6-diNn) showed high levels of activity and low levels of cytotoxicity (L. Zimmermann et al., J. Med. Chem. 56:7691-7705, 2013). We have now further characterized the activity of these derivatives against colistin-resistant P. aeruginosa and studied their mode of action; specifically, we characterized their ability to interact with lipopolysaccharide (LPS) and to alter the bacterial outer membrane (OM). The three amphiphilic neamine derivatives were active against clinical colistin-resistant strains (MICs, about 2 to 8 μg/ml), The most active one (3',6-diNn) was bactericidal at its MIC and inhibited biofilm formation at 2-fold its MIC. They cooperatively bound to LPSs, increasing the outer membrane permeability. Grafting long and linear alkyl chains (nonyl) optimized binding to LPS and outer membrane permeabilization. The effects of amphiphilic neamine derivatives on LPS micelles suggest changes in the cross-bridging of lipopolysaccharides and disordering in the hydrophobic core of the micelles. The molecular shape of the 3',6-dialkyl neamine derivatives induced by the nature of the grafted hydrophobic moieties (naphthylalkyl instead of alkyl) and the flexibility of the hydrophobic moiety are critical for their fluidifying effect and their ability to displace cations bridging LPS. Results from this work could be exploited for the development of new amphiphilic neamine derivatives active against colistin-resistant P. aeruginosa.

Thermo-Nanoimprinted Biomimetic Probe for LPS and LTA Immunosensing

A complex prepolymerized film comprising monomers, cross-linkers, and initiator is usually used to create molecularly imprinted polymers. We herein exploit ready-to-use resist materials and link molecular surface imprinting with UV- and thermo-nanoimprinting techniques to create a sensor layer for the specific recognition of the bacterial surface markers lipopolysaccharide (LPS) and lipoteichoic acid (LTA). To account for the highly polar moieties of LPS and LTA, we evaluate different resist and stamp materials of distinct surface properties by AFM and molecularly imprinted sorbent assays. Thermo nanoimprinting of LPS and LTA micelles to Epon 1002F films exhibits excellent sensitivity of up to 13 times increased signals compared to those of the nonimprinted films and negligible cross-reaction with the tested nonspecific analyte. Additionally, the sensitivity and selectivity of the thermo nanoimprints is compared to conventional molecular surface imprints using a cocktail of acrylic monomers in QCM measurements.

NMR Structure of Temporin-1 Ta in Lipopolysaccharide Micelles: Mechanistic Insight into Inactivation by Outer Membrane

BackgroundAntimicrobial peptides (AMPs) play important roles in the innate defense mechanism. The broad spectrum of activity of AMPs requires an efficient permeabilization of the bacterial outer and inner membranes. The outer leaflet of the outer membrane of Gram negative bacteria is made of a specialized lipid called lipopolysaccharide (LPS). The LPS layer is an efficient permeability barrier against anti-bacterial agents including AMPs. As a mode of protection, LPS can induce self associations of AMPs rendering them inactive. Temporins are a group of short-sized AMPs isolated from frog skin, and many of them are inactive against Gram negative bacteria as a result of their self-association in the LPS-outer membrane.Principal FindingsUsing NMR spectroscopy, we have determined atomic resolution structure and characterized localization of temporin-1Ta or TA (FLPLIGRVLSGIL-amide) in LPS micelles. In LPS micelles, TA adopts helical conformation for residues L4-I12, while residues F1-L3 are found to be in extended conformations. The aromatic sidechain of residue F1 is involved in extensive packing interactions with the sidechains of residues P3, L4 and I5. Interestingly, a number of long-range NOE contacts have been detected between the N-terminal residues F1, P3 with the C-terminal residues S10, I12, L13 of TA in LPS micelles. Saturation transfer difference (STD) NMR studies demonstrate close proximity of residues including F1, L2, P3, R7, S10 and L13 with the LPS micelles. Notably, the LPS bound structure of TA shows differences with the structures of TA determined in DPC and SDS detergent micelles.SignificanceWe propose that TA, in LPS lipids, forms helical oligomeric structures employing N- and C-termini residues. Such oligomeric structures may not be translocated across the outer membrane; resulting in the inactivation of the AMP. Importantly, the results of our studies will be useful for the development of antimicrobial agents with a broader spectrum of activity.

Structure, activity and interactions of the cysteine deleted analog of tachyplesin-1 with lipopolysaccharide micelle: Mechanistic insights into outer-membrane permeabilization and endotoxin neutralization

Tachyplesin-1, a disulfide stabilized β-hairpin antimicrobial peptide, can be found at the hemocytes of horse shoe crab Tachypleus tridentatus. A cysteine deleted linear analog of tachyplesin-1 or CDT (KWFRVYRGIYRRR-NH2) contains a broad spectrum of bactericidal activity with a reduced hemolytic property. The bactericidal activity of CDT stems from selective interactions with the negatively charged lipids including LPS. In this work, CDT–LPS interactions were investigated using NMR spectroscopy, optical spectroscopy and functional assays. We found that CDT neutralized LPS and disrupted permeability barrier of the outer membrane. Zeta potential and ITC studies demonstrated charge compensation and hydrophobic interactions of CDT with the LPS-outer membrane, respectively. Secondary structure of the peptide was probed by CD and FT-IR experiments indicating β-strands and/or β-turn conformations in the LPS micelle. An ensemble of structures, determined in LPS micelle by NMR, revealed a β-hairpin like topology of the CDT peptide that was typified by an extended cationic surface and a relatively shorter segment of hydrophobic region. Interestingly, at the non-polar face, residue R11 was found to be in a close proximity to the indole ring of W2, suggesting a cation–π type interactions. Further, saturation transfer difference (STD) NMR studies established intimate contacts among the aromatic and cationic residues of CDT with the LPS micelle. Fluorescence and dynamic light scattering experiments demonstrated that CDT imparted structural destabilization to the aggregated states of LPS. Collectively, atomic resolution structure and interactions of CDT with the outer membrane-LPS could be exploited for developing potent broad spectrum antimicrobial and anti-sepsis agents.

A Plausible Molecular Mechanism for the Synergistic Activity of Temporins at the Level of Lipopolysaccharide-Outer Membrane of Gram-Negative Bacteria

Gene-encoded antimicrobial peptides (AMPs) represent attractive candidates for the development of a new generation of anti-infective agents.There is compelling evidence that unlike conventional antibiotics, most AMPs do act by altering the membrane permeability of the target cell.However, before reaching it, they need to cross the microbial cell wall that, in Gram-negative bacteria, is surrounded by the lipopolysaccharide (LPS)-outer membrane, which forms an efficient barrier against a variety of molecules.In Nature, the frog skin temporins are among the shortest (10 to 14 residues) AMPs, with up to ten isoforms within the same specimen [1]. We have shown that some of them (temporin-1Tb, TB, and temporin-1Tl, TL) have a synergistic effect in killing Gram-negative bacteria [2]. This suggests an important strategy to overcome bacterial resistance due to the LPS layer. More precisely, we have found that LPS induces oligomerization of TB. This would prevent its translocation across the outer membrane making the peptide inactive on Gram-negative bacteria. Differently, TL is highly active on these bacteria, presumably due to the lack of such self-association. To investigate the interactions of TL and TB within LPS, we studied their structures and interactions in LPS micelles using NMR spectroscopy. Interestingly, TL assumes an anti-parallel dimeric helical structure stabilized by intimate packing between aromatic-aromatic and aromatic-aliphatic residues. In contrast, TB shows populations of helical and aggregated conformations. Note that the LPS-induced aggregated states of TB are largely destabilized in the presence of TL. Importantly, these results provide the first structural insights into the mechanism of action and synergism of AMPs at the level of the LPS-outer membrane.1 Mangoni ML (2006) CMLS 63: 1060-692 Mangoni ML et al. (2008) JBC 283:22907-17

NMR Structures and Interactions of Temporin-1Tl and Temporin-1Tb with Lipopolysaccharide Micelles: MECHANISTIC INSIGHTS INTO OUTER MEMBRANE PERMEABILIZATION AND SYNERGISTIC ACTIVITY

Temporins are a group of closely related short antimicrobial peptides from frog skin. Lipopolysaccharide (LPS), the major constituent of the outer membrane of gram-negative bacteria, plays important roles in the activity of temporins. Earlier studies have found that LPS induces oligomerization of temporin-1Tb (TB) thus preventing its translocation across the outer membrane and, as a result, reduces its activity on gram-negative bacteria. On the other hand, temporin-1Tl (TL) exhibits higher activity, presumably because of lack of such oligomerization. A synergistic mechanism was proposed, involving TL and TB in overcoming the LPS-mediated barrier. Here, to gain insights into interactions of TL and TB within LPS, we investigated the structures and interactions of TL, TB, and TL+TB in LPS micelles, using NMR and fluorescence spectroscopy. In the context of LPS, TL assumes a novel antiparallel dimeric helical structure sustained by intimate packing between aromatic-aromatic and aromatic-aliphatic residues. By contrast, independent TB has populations of helical and aggregated conformations in LPS. The LPS-induced aggregated states of TB are largely destabilized in the presence of TL. Saturation transfer difference NMR studies have delineated residues of TL and TB in close contact with LPS and enhanced interactions of these two peptides with LPS, when combined together. Fluorescence resonance energy transfer and (31)P NMR have pointed out the proximity of TL and TB in LPS and conformational changes of LPS, respectively. Importantly, these results provide the first structural insights into the mode of action and synergism of antimicrobial peptides at the level of the LPS-outer membrane.

Mapping residue-specific contacts of polymyxin B with lipopolysaccharide by saturation transfer difference NMR: Insights into outer-membrane disruption and endotoxin neutralization

High-resolution interactions studies of molecules with lipopolysaccharide (LPS) or endotoxin are important for health, diseases and environment. LPS is the major constituent of the outer layer of the outer membrane of the gram-negative bacteria. LPS provides an efficient barrier against permeation of variety of compounds including antibacterial agents and antimicrobial peptides. In the intensive care units, LPS is known for the fatal septic shock syndromes. Because of LPS toxicity, high affinity LPS sensors are sought-after for the assessment of the quality of water and pharmaceutical products. Therefore, elucidation of binding epitopes of LPS interacting molecules would be vital for the development of antimicrobial, antiendotoxic molecules. Polymyxin B (PMB), an antibacterial cyclic lipo-peptide, is well known for its LPS sequestering and neutralizing activities. Here, we have used saturation transfer difference (STD) NMR methods for characterizing interactions of PMB with LPS from E. coli 0111:B4 and P. aeruginosa. The dissociation constants of the LPS-PMB complexes were obtained from concentration dependent STD studies. Further a detailed epitope mapping of PMB has been carried out in E. coli 0111:B4 LPS micelles. Experiments including one-dimensional 1H STD, two-dimensional 1H-1H STD-TOCSY and naturalabundance 13C-1H STD-HSQC, are performed to determine the site(s) of interactions of PMB with endotoxin at atomic resolution. Our studies reveal that the hydrophobic sidechains of PMB including a part of the N-terminus lipidic tail demonstrate close contacts with LPS. In contrast, cyclic backbone structure of PMB has the lowest STD effects suggesting a rather loose association with endotoxin.

Structure, Interactions, and Antibacterial Activities of MSI-594 Derived Mutant Peptide MSI-594F5A in Lipopolysaccharide Micelles: Role of the Helical Hairpin Conformation in Outer-Membrane Permeabilization

Lipopolysaccharide (LPS) provides a well-organized permeability barrier at the outer membrane of Gram-negative bacteria. Host defense cationic antimicrobial peptides (AMPs) need to disrupt the outer membrane before gaining access to the inner cytoplasmic membrane or intracellular targets. Several AMPs are largely inactive against Gram-negative pathogens due to the restricted permeation through the LPS layer of the outer membrane. MSI-594 (GIGKFLKKAKKGIGAVLKVLTTG) is a highly active AMP with a broad-spectrum of activities against bacteria, fungi, and virus. In the context of LPS, MSI-594 assumes a hairpin helical structure dictated by packing interactions between two helical segments. Residue Phe5 of MSI-594 has been found to be engaged in important interhelical interactions. In order to understand plausible structural and functional inter-relationship of the helical hairpin structure of MSI-594 with outer membrane permeabilization, a mutant peptide, termed MSI-594F5A, containing a replacement of Phe5 with Ala has been prepared. We have compared antibacterial activities, outer and inner membrane permeabilizations, LPS binding affinity, perturbation of LPS micelles structures by MSI-594 and MSI-594F5A peptides. Our results demonstrated that the MSI-594F5A has lower activities against Gram-negative bacteria, due to limited permeabilization through the LPS layer, however, retains Gram-positive activity, akin to MSI-594. The atomic-resolution structure of MSI-594F5A has been determined in LPS micelles by NMR spectroscopy showing an amphipathic curved helix without any packing interactions. The 3D structures, interactions, and activities of MSI-594 and its mutant MSI-594F5A in LPS provide important mechanistic insights toward the requirements of LPS specific conformations and outer membrane permeabilization by broad-spectrum antimicrobial peptides.

NMR Structure of Pardaxin, a Pore-forming Antimicrobial Peptide, in Lipopolysaccharide Micelles: MECHANISM OF OUTER MEMBRANE PERMEABILIZATION

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is an important element against permeability of bactericidal agents, including antimicrobial peptides. However, structural determinants of antimicrobial peptides for LPS recognition are not clearly understood. Pardaxins (Pa1, Pa2, Pa3, and Pa4) are a group of pore-forming bactericidal peptides found in the mucous glands of sole fishes. Despite having a low net positive charge, pardaxins contain a broad spectrum of antibacterial activities. To elucidate the structural basis of LPS interactions of pardaxins, herein, we report the first three-dimensional structure of Pa4 bound to LPS micelles. The binding kinetics of Pa4 with LPS is estimated using [(15)N-Leu-19] relaxation dispersion NMR experiments. LPS/Pa4 interactions are further characterized by a number of biophysical methods, including isothermal titration calorimetry, (31)P NMR, saturation transfer difference NMR, dynamic light scattering, and IR spectroscopy. In the LPS-Pa4 complex, Pa4 adopts a unique helix-turn-helix conformation resembling a "horseshoe." Interestingly, the LPS-bound structure of Pa4 shows striking differences with the structures determined in lipid micelles or organic solvents. Saturation transfer difference NMR identifies residues of Pa4 that are intimately associated with LPS micelles. Collectively, our results provide mechanistic insights into the outer membrane permeabilization by pardaxin.

Designed β-Boomerang Antiendotoxic and Antimicrobial Peptides: STRUCTURES AND ACTIVITIES IN LIPOPOLYSACCHARIDE

Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like beta-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nM concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the beta-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate beta-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane.

Helical Hairpin Structure of a Potent Antimicrobial Peptide MSI‐594 in Lipopolysaccharide Micelles by NMR Spectroscopy

Essential understanding: Elucidation of structural requirements and interactions of antimicrobial peptides with lipopolysaccharide (LPS) are essential to understand the mechanism of action of antimicrobial peptides. The highly active antimicrobial peptide MSI-594 (see figure for electrostatic potential surface) acquires a novel helical hairpin structure in complex with LPS. The structure and interactions of MSI-594 with LPS presented here provide important insights into the mechanism of outer membrane permeabilization by antimicrobial peptides.

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant ( K a) of 2.85 × 10 6 M − 1 and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (Δ G 0), − 8.8 kcal mol − 1 , obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.

Perturbation of Lipopolysaccharide (LPS) Micelles by Sushi 3 (S3) Antimicrobial Peptide: THE IMPORTANCE OF AN INTERMOLECULAR DISULFIDE BOND IN S3 DIMER FOR BINDING, DISRUPTION, AND NEUTRALIZATION OF LPS

S3 peptide, derived from the Sushi 3 domain of Factor C, which is the lipopolysaccharide (LPS)-sensitive serine protease of the horseshoe crab coagulation cascade, was shown previously to harbor antimicrobial activity against Gram-negative bacteria. However, the mechanism of action remains poorly understood at the molecular level. Here we demonstrate that the intermolecular disulfide bonding of S3 resulting in S3 dimers is indispensable for its interaction with LPS. The binding properties of the S3 monomer and dimer to LPS were analyzed by several approaches including enzyme-linked immunosorbent assay (ELISA)-based assay, surface plasmon resonance, and fluorescence correlation spectroscopy (FCS). It is evident that the S3 dimer exhibits stronger binding to LPS, demonstrating 50% LPS-neutralizing capability at a concentration of 1 mum. Circular dichroism spectrometry revealed that the S3 peptide undergoes conformational change in the presence of a disulfide bridge, transitioning from a random coil to beta-sheet structure. Using a fluorescence correlation spectroscopy monitoring system, we describe a novel approach for examining the mechanism of peptide interaction with LPS in the native environment. The strategy shows that intermolecular disulfide bonding of S3 into dimers plays a critical role in its propensity to disrupt LPS micelles and consequently neutralize LPS activity. S3 dimers display detergent-like properties in disrupting LPS micelles. Considering intermolecular disulfide bonds as an important parameter in the structure-activity relationship, this insight provides clues for the future design of improved LPS-binding and -neutralizing peptides.

Aggregation of serum proteins with lipopolysaccharide (LPS): characterization of the precipitable LPS-protein complex

We have employed an in vitro system to study the interactions between rat serum proteins and lipopolysaccharide (LPS) under conditions similar to those commonly employed in endotoxemia studies. Immediately after mixing, a small fraction of the LPS micelles form a readily precipitable aggregate with serum proteins. Aggregate formation is measurable at LPS concentrations equivalent to maximal serum loads at the lethal threshold in endotoxic shock studies. The amount of aggregate increases with increasing LPS concentration. While the protein content of this aggregate remains stable over the 60 min studied, the LPS content decreases. Only three serum protein species comprise 85-90% of the protein bound in the aggregate: albumin, complement factor C3b and iC3b, and immunoglobulins G and M. Much larger amounts of serum protein-LPS aggregates are formed using serum from animals pre-exposed to sublethal levels of LPS. The results help better characterize the physical-chemical properties of LPS as it is employed in endotoxic shock models in normal and pre-exposed animals and may help explain the altered clearance profiles experienced in these models.

Soluble CD14 acts as a shuttle in the neutralization of lipopolysaccharide (LPS) by LPS-binding protein and reconstituted high density lipoprotein.

We have recently shown that lipopolysaccharide (LPS)-binding protein (LBP) is a lipid transfer protein that catalyzes two distinct reactions: movement of bacterial LPS (endotoxin) from LPS micelles to soluble CD14 (sCD14) and movement of LPS from micelles to reconstituted high density lipoprotein (R-HDL) particles. Here we show that LBP facilitates a third lipid transfer reaction: movement of LPS from LPS-sCD14 complexes to R-HDL particles. This action of LBP is catalytic, with one molecule of LBP enabling the movement of multiple LPS molecules into R-HDL. LBP-catalyzed movement of LPS from LPS-sCD14 complexes to R-HDL neutralizes the capacity of LPS to stimulate polymorphonuclear leukocytes. Our findings show that LPS may be transferred to R-HDL either by the direct action of LBP or by a two-step reaction in which LPS is first transferred to sCD14 and subsequently to R-HDL. We have observed that the two-step pathway of LPS transfer to R-HDL is strongly favored over direct transfer. Neutralization of LPS by LBP and R-HDL was accelerated more than 30-fold by addition of sCD14. Several observations suggest that sCD14 accelerates this reaction by serving as a shuttle for LPS: addition of LBP and sCD14 to LPS micelles resulted in LPS-sCD14 complexes that could diffuse through a 100-kD cutoff filter; LPS-sCD14 complexes appeared transiently during movement of LPS to R-HDL facilitated by purified LBP; and sCD14 could facilitate transfer of LPS to R-HDL without becoming part of the final LPS-R-HDL complex. Complexes of LPS and sCD14 were formed transiently when LPS was incubated in plasma, suggesting that these complexes may play a role as intermediates in the neutralization of LPS under physiological conditions. These findings detail a new activity for sCD14 and suggest a novel mechanism for lipid transfer by LBP.

Radioiodination of S-type Lipopolysaccharide. Possibilities and limitations of labelling of the carbohydrate chain by periodate oxidation

S-type Lipopolysaccharide (LPS) from E. coli O 139:K 82 (B) was radiolabelled by coupling 125I-tyramine to the carbohydrate chain of LPS after oxidation of sugar residues carrying vicinal hydroxy groups, with sodium metaperiodate. The specific activity amounted to 1.3 kBq/microgram LPS. As LPS is a mixture of molecules with different carbohydrate chain lengths that are associated to large micelles of high kinetic stability, the preparation and the properties of the tracer exhibit some peculiarities. Most important, partial cleavage of the carbohydrate chains of LPS is caused on radioiodination by the procedure described. As a consequence, the tracer differs markedly from the starting LPS with respect to carbohydrate chain length distribution. Another feature of special interest is the variation of the specific activity (radioactivity/unit mass) for LPS molecules of different carbohydrate chain lengths. Because of the kinetic stability of LPS micelles, the equilibration of radioiodinated and non-radioactive LPS aggregates requires their solubilization by a detergent and its subsequent removal by dialysis. It could be demonstrated that short-chain LPS molecules predominantly associate to larger micelles than long-chain molecules. Regardless of these restrictions, the 125I-LPS proved to be useful for certain analytical purposes.

Uptake and subcellular distribution of Escherichia coli lipopolysaccharide by isolated rat type II pneumocytes.

Treatment of isolated rat Type II pneumocytes with Escherichia coli lipopolysaccharide (LPS) induces a number of ultra-structural changes which become evident after 60 min of incubation. By using post-embedding immunolabeling methods and electron microscopy, we have followed the fate of LPS after different times of incubation. After an initial period of accumulation in the pneumocyte microvilli, the LPS molecules enter the cytoplasm, forming discrete patches which are dispersed in some areas. After longer incubation times, LPS localize in condensed chromatin-free areas inside the nuclei. LPS micelles were visualized after freeze-fracture and compared with the LPS-labeled membrane areas, showing that LPS micelles aggregate in particular membrane zones. The sugar-specific staining in microvilli areas, where Maclura pomifera agglutinin (MPA)-gold particles bind, indicates the presence of galactose derivatives in these membrane structures. Pre-treatment of pneumocytes with LPS inhibited the MPA-gold labeling, suggesting a relation between the MPA receptor and a possible LPS receptor. Finally, double immunolabeling experiments indicated an apparent LPS-tubulin association in some particular membrane regions, which could not be observed when LPS and actin were co-localized.

Demonstration of an Interaction between Transferrin and Lipopolysaccharide – An in vitro Study

Transferrin is reported to be a major lipopolysaccharide binding protein of human plasma, at least in vitro. By use of the limulus-amebocyte-lysate test the influence of transferrin on endotoxicity was studied. In the absence of any other protein human iron-free transferrin was able to strongly enhance endotoxicity in a concentration-dependent manner. Similar results were obtained when transferrin was added to primarily heat-inactivated plasma. Even in this assay the endotoxin recovery increased when transferrin was exogenously added. On the other hand, transferrin inhibited endotoxicity when inactivation of the plasma samples was performed after the addition of endotoxin and transferrin. These results lead to the conclusion that transferrin in fact interacts with lipopolysaccharide in a biologically important manner. In order to achieve neutralization of endotoxin, however, other plasma constituents are needed. The hypothetical function of transferrin is possibly a disaggregation of lipopolysaccharide micelles, following the interaction between the two molecules. The present data should justify further studies in order to clarify a possible benefit of the substitution of transferrin during gram-negative sepsis.

Ultrastructural and Immunocytochemical Study of the Uptake and Distribution of Bacterial Lipopolysaccharide in Human Monocytes

Interaction of bacterial lipopolysaccharide (LPS) with monocytes stimulates production of a variety of mediators that are involved in the pathogenesis of septic shock and wound repair. We report here the mechanisms of LPS uptake and intracellular distribution of LPS in human monocytes. Ficoll-Hypaque-purified peripheral mononuclear cells (PBMC) were exposed to LPS from rough Escherichia coli (J5) or to biotin-conjugated LPS (biotin-LPS) from smooth E. coli (0111:B4), or to fluorescein isothiocyanate-conjugated LPS of E. coli (055:B5) at 37 degrees C for various times and processed for electron microscopy, immunocytochemistry, and flow cytometry. Monocytes were identified by the presence of numerous cytoplasmic peroxidase-positive granules or by monoclonal antibodies against monocyte. LPS micelles were identified by their specific bilayer structure, staining of horseradish peroxidase reaction product, or colloidal gold using biotin-LPS or a monoclonal antibody to LPS. Binding of LPS to cell surface was observed 5 min after incubation with LPS. Intracellular localization of LPS micelles was found 30 min following exposure to LPS. Prolonged incubation with LPS increased intracellular LPS. Intracellularly, LPS micelles were found in large membrane-bound vacuoles, in small vesicles, and in the cytoplasm and nucleus. They were also observed in association with the cytomembrane of various organelles. The overall results indicate that LPS may be taken up by monocytes by direct passive diffusion through ruptures of plasma membrane, pinocytosis, and phagocytosis, involving specific and/or nonspecific binding, and suggest that peripheral blood monocytes play an important role in clearance of LPS; that LPS may have broad effects on cell functions; and that the nonspecific binding to various cytomembranes may be destructive to cell organelles and cells in general.