Lipopolysaccharide Aggregates Research Articles

ВЗАИМОДЕЙСТВИЕ ЛИПОПОЛИСАХАРИД-СВЯЗЫВАЮЩИХ БЕЛКОВ С РАЗЛИЧНЫМИ ФОРМАМИ ЛИПОПОЛИСАХАРИДОВ

Lipopolysaccharide-binding proteins from two common jellyfish species Aurellia aurita and Ropelema asamushi were isolated and purified, and the interaction of lipopolysaccharides (LPS) of various structural types with LBP was studied. By inhibiting the interaction, it was found that both proteins specifically bind to the lipid and core fragments of the LPS molecule. There are two types of binding sites in LBP with Kd = 3,28 × 10-6 M and Kd = 0,13 × 10-6 M (for the protein from A. aurita) and Kd = 3,66 × 10-6 M and Kd = 0,27 × 10-6 M (for protein from R. asamushi). It has been shown by dynamic light scattering that the binding of LBP to R-LPS leads to the dissociation of LPS micelles. The sizes of LPS aggregates decrease from 200 nm to 25–30 nm in the composition of LPS–LSB complexes. The data of electrokinetic measurements indicate the neutralization of the negative charge of Rd-LPS (-42,2 mV) in the LPS-LSB-R. asamushi complex up to -4,4 mV. S-LPS micelles from E. coli do not disaggregate upon binding to LBP, which is apparently due to the shielding of lipid A by O-specific chains in the S-LPS molecule. The binding of LPS to LBP may affect their endotoxic properties.

Selective protein aggregation confines and inhibits endotoxins in wounds: Linking host defense to amyloid formation

Bacterial lipopolysaccharide (LPS) induces rapid protein aggregation in human wound fluid. We aimed to characterize these LPS-induced aggregates and their functional implications using a combination of mass spectrometry analyses, biochemical assays, biological imaging, cell experiments, and animal models. The wound-fluid aggregates encompass diverse protein classes, including sequences from coagulation factors, annexins, histones, antimicrobial proteins/peptides, and apolipoproteins. We identified proteins and peptides with a high aggregation propensity and verified selected components through Western blot analysis. Thioflavin T and Amytracker staining revealed amyloid-like aggregates formed after exposure to LPS invitro in human wound fluid and invivo in porcine wound models. Using NF-κB-reporter mice and IVIS bioimaging, we demonstrate that such wound-fluid LPS aggregates induce a significant reduction in local inflammation compared with LPS in plasma. The results show that protein/peptide aggregation is a mechanism for confining LPS and reducing inflammation, further emphasizing the connection between host defense and amyloidogenesis.

The influence of calcium ions (Ca2+) on the enzymatic hydrolysis of lipopolysaccharide aggregates to liberate free fatty acids (FFA) in aqueous solution.

The chemical environment in aqueous solutions greatly influences the ability of amphiphilic molecules such as lipopolysaccharides (LPS) to aggregate into different structural phases in aqueous solutions. Understanding the substrate's morphology and conditions of aqueous solution that favor both enzymatic activity and the disruption of LPS aggregates are crucial in developing agents that can counteract the new trend of multidrug resistance by gram-negative bacteria. In this study, we developed two LPS morphologies using LPS from Escherichia coli as a model to study the in vitro hydrolytic response when using a lipase treatment. The hydrolysis was performed using lipase b from Candida antarctica to understand the catalytic effect in removing fatty acids from its lipid A moiety on different LPS aggregates. Physical and chemical characterizations of the products included dynamic light scattering, small angle X-ray scattering, Fourier transform infrared spectroscopy, thin-layer chromatography, and gas chromatography. Our results suggest a trend of prominent hydrolytic response (72% enhancement) upon the addition of calcium ions to induce LPS aggregates into bilayer formations. Moreover, our results revealed the detection of myristic acid (C14:0) as the product of the hydrolysis when using RaLPS in its aggregate forms.

Topical application of Porphyromonas gingivalis into thegingival pocket in mice leads to chronic‑activeinfection, periodontitis and systemic inflammation.

Porphyromonas gingivalis (Pg), one of the 'red-complex' perio-pathogens known to play a critical role in the development of periodontitis, has been used in various animal models to mimic human bacteria-induced periodontitis. In order to achieve a more realistic animal model of human Pg infection, the present study investigated whether repeated small-volume topical applications of Pg directly into the gingival pocket can induce local infection, including periodontitis and systemic vascular inflammation in wild-type mice. Freshly cultured Pg was topically applied directly into the gingival pocket of the second molars for 5 weeks (3 times/week). After the final application, the mice were left in cages for 4 or 8 weeks and sacrificed. The status of Pg colony formation in the pocket, gingival inflammation, alveolar bone loss, the expression levels of pro-inflammatory cytokines in the serum and aorta, the presence of anti-Pg lipopolysaccharide (LPS) and gingipain (Kpg and RgpB) antibodies in the serum, as well as the accumulation of Pg LPS and gingipain aggregates in the gingiva and arterial wall were evaluated. The topical application of Pg into the gingival pocket induced the following local and systemic pathohistological changes in mice when examined at 4 or 8 weeks after the final topical Pg application: Pg colonization in the majority of gingival pockets; increased gingival pocket depths; gingival inflammation indicated by the increased expression of TNF-α, IL-6 and IL-1β; significant loss of alveolar bone at the sites of topical Pg application; and increased levels of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-17, IL-13, KC and IFN-γ in the serum in comparison to those from mice receiving PBS. In addition, the Pg application/colonization model induced anti-Pg LPS and gingipain antibodies in serum, as well as the accumulation of Pg LPS and gingipain aggregates in the gingivae and arterial walls. To the best of our knowledge, this mouse model represents the first example of creating a more sustained local infection in the gingival tissues of wild-type mice and may prove to be useful for the investigation of the more natural and complete pathogenesis of the bacteria in the development of local oral and systemic diseases, such as atherosclerosis. It may also be useful for the determination of a treatment/prevention/efficacy model associated with Pg-induced colonization periodontitis in mice.

A Novel Antimicrobial Peptide Derived from Bony Fish IFN1 Exerts Potent Antimicrobial and Anti-Inflammatory Activity in Mammals.

ABSTRACTType I interferons (IFN-Is) are critical antiviral cytokine in innate immunity but with limited direct defense ability against bacterial infections in mammals. In bony fish, despite all the IFN-Is (IFN1-4) act in antiviral immunity, studies demonstrate that IFN1 can remarkably contribute to host defense against bacterial infections. In this study, we found that IFN1 from grass carp (Ctenopharyngodon idella) contains an unusual cationic and amphipathic α-helical region (named as gcIFN-20, sequence: SYEKKINRHFKILKKNLKKK). The synthesized peptide gcIFN-20 could form α-helical structure in a membrane environment and exerts potent antimicrobial activity against multiple species of Gram-negative (G−) and Gram-positive (G+) bacteria with negligible toxicity. Mechanism studies showed gcIFN-20 kills G+ bacteria through membrane disruption and cytoplasm outflow while G− bacteria through membrane permeation and protein synthesis inhibition. In two mouse bacterial infection models, gcIFN-20 therapy could significantly reduce tissue bacterial loads and mortalities. In addition to the direct antibacterial activity, we also found that gcIFN-20 could significantly suppress the lipopolysaccharide (LPS)-induced pro-inflammatory cytokines in vitro and in vivo, obviously alleviated lung lesions in a mouse endotoxemia model. The mechanism is that gcIFN-20 interacts with LPS, causes LPS aggregation and neutralization. The antimicrobial and anti-inflammatory activities in vivo of gcIFN-20 in mammalian models suggested a promising agent for developing peptide-based antibacterial therapy.IMPORTANCE Type I interferons play crucial role in antiviral immunity in both vertebrates and invertebrates. The powerful antimicrobial activity is recently reported in nonmammalian vertebrates. The present study identified a novel antimicrobial peptide (gcIFN-20) derived from grass carp interferon 1, found gcIFN-20 exhibits forceful bactericidal and anti-inflammatory activity in mammals, and efficient therapeutic effect against two clinical severe extraintestinal pathogenic Escherichia coli and a mouse endotoxemia models. The antimicrobial mechanisms are membrane disruption and cytoplasm overflow for Gram-positive bacteria, while membrane permeation and protein synthesis inhibition for Gram-negative bacteria. The anti-inflammatory mechanisms can be aggregating and neutralizing lipopolysaccharide to attenuate the binding with receptors and facilitate phagocytosis. The results indicate that gcIFN-20 can be a promising novel therapeutic agent for bacterial diseases and inflammatory disorders, especially as a potential weapon for multidrug resistant strain infections.

Interaction of Lipopolysaccharide-Spiked Blood with Anti-Fouling Polymyxin B-Modified Glass.

Bacterial endotoxin, also known as lipopolysaccharide (LPS), plays a major role in the initiation of sepsis, a severe inflammatory condition. Removal of the toxin from blood is one accepted method of patient treatment. Polymyxin B (PMB)-modified columns have been employed successfully for this purpose via extra-corporeal blood-flow systems that incorporate a cartridge for toxin removal. Herein we demonstrate that PMB-modified glass beads are able to reduce the presence of LPS competitively with the equivalent fiber column used in a commercial cartridge. Analysis by gas chromatography-mass spectrometry and ELISA of released fatty acids from the toxin indicates that PMB does not physically capture or significantly remove LPS from the blood samples. In reality, interaction between the surface-bound PMB and the toxin may lead to disaggregation or monomerization of LPS aggregates. As aggregates are the bioactive form of LPS, it is possible that the monomerization of these entities may be the mechanism by which their toxicity is reduced. Moreover, this work indicates that LPS monomers are stabilized subsequent to disaggregation induced by PMB.

Cation-Driven Lipopolysaccharide Morphological Changes Impact Heterogeneous Reactions of Nitric Acid with Sea Spray Aerosol Particles.

Lipopolysaccharides (LPS) in sea spray aerosol (SSA) particles have recently been shown to undergo heterogeneous reactions with HNO3 in the atmosphere. Here, we integrate theory and experiment to further investigate how the most abundant sea salt cations, Na+, Mg2+, and Ca2+, impact HNO3 reactions with LPS-containing SSA particles. Aerosol reaction flow tube studies show that heterogeneous reactions of SSA particles with divalent cation (Mg2+ and Ca2+) and LPS signatures were less reactive with HNO3 than those dominated by monovalent cations (Na+). All-atom molecular dynamics simulations of model LPS aggregates suggest that divalent cations cross-link the oligosaccharide chains to increase molecular aggregation and rigidity, which changes the particle phase and morphology, decreases water diffusion, and consequently decreases the reactive uptake of HNO3. This study provides new insight into how complex chemical interactions between ocean-derived salts and biogenic organic species can impact the heterogeneous reactivity of SSA particles.

Human guanylate binding proteins: nanomachines orchestrating host defense.

Disease-causing microorganisms not only breach anatomical barriers and invade tissues but also frequently enter host cells, nutrient-enriched environments amenable to support parasitic microbial growth. Protection from many infectious diseases is therefore reliant on the ability of individual host cells to combat intracellular infections through the execution of cell-autonomous defense programs. Central players in human cell-autonomous immunity are members of the family of dynamin-related guanylate binding proteins (GBPs). The importance of these interferon-inducible GTPases in host defense to viral, bacterial, and protozoan pathogens has been established for some time; only recently, cell biological and biochemical studies that largely focused on the prenylated paralogs GBP1, GBP2, and GBP5 have provided us with robust molecular frameworks for GBP-mediated immunity. Specifically, the recent characterization of GBP1 as a bona fide pattern recognition receptor for bacterial lipopolysaccharide (LPS) disrupting the integrity of bacterial outer membranes through LPS aggregation, the discovery of a link between hydrolysis-induced GMP production by GBP1 and inflammasome activation, and the classification of GBP2 and GBP5 as inhibitors of viral envelope glycoprotein processing via suppression of the host endoprotease furin have paved the way for a vastly improved conceptual understanding of the molecular mechanisms by which GBP nanomachines execute cell-autonomous immunity. The herein discussed models incorporate our current knowledge of the antimicrobial, proinflammatory, and biochemical properties of human GBPs and thereby provide testable hypotheses that will guide future studies into the intricacies of GBP-controlled host defense and their role in human disease.

In vitro Analysis of O-Antigen-Specific Bacteriophage P22 Inactivation by Salmonella Outer Membrane Vesicles.

Bacteriophages use a large number of different bacterial cell envelope structures as receptors for surface attachment. As a consequence, bacterial surfaces represent a major control point for the defense against phage attack. One strategy for phage population control is the production of outer membrane vesicles (OMVs). In Gram-negative host bacteria, O-antigen-specific bacteriophages address lipopolysaccharide (LPS) to initiate infection, thus relying on an essential outer membrane glycan building block as receptor that is constantly present also in OMVs. In this work, we have analyzed interactions of Salmonella (S.) bacteriophage P22 with OMVs. For this, we isolated OMVs that were formed in large amounts during mechanical cell lysis of the P22 S. Typhimurium host. In vitro, these OMVs could efficiently reduce the number of infective phage particles. Fluorescence spectroscopy showed that upon interaction with OMVs, bacteriophage P22 released its DNA into the vesicle lumen. However, only about one third of the phage P22 particles actively ejected their genome. For the larger part, no genome release was observed, albeit the majority of phages in the system had lost infectivity towards their host. With OMVs, P22 ejected its DNA more rapidly and could release more DNA against elevated osmotic pressures compared to DNA release triggered with protein-free LPS aggregates. This emphasizes that OMV composition is a key feature for the regulation of infective bacteriophage particles in the system.

Polysaccharide Chain Length of Lipopolysaccharides From Salmonella Minnesota Is a Determinant of Aggregate Stability, Plasma Residence Time and Proinflammatory Propensity in vivo.

Lipopolysaccharides (LPS) originate from the outer membrane of Gram-negative bacteria and trigger an inflammatory response via the innate immune system. LPS consist of a lipid A moiety directly responsible for the stimulation of the proinflammatory cascade and a polysaccharide chain of variable length. LPS form aggregates of variable size and structure in aqueous media, and the aggregation/disaggregation propensity of LPS is known as a key determinant of their biological activity. The aim of the present study was to determine to which extent the length of the polysaccharide chain can affect the nature of LPS structures, their pharmacokinetics, and eventually their proinflammatory properties in vivo. LPS variants of Salmonella Minnesota with identical lipid A but with different polysaccharide moieties were used. The physical properties of LPS aggregates were analyzed by zetametry, dynamic light scattering, and microscopy. The stability of LPS aggregates was tested in the presence of plasma, whole blood, and cultured cell lines. LPS pharmacokinetics was performed in wild-type mice. The accumulation in plasma of rough LPS (R-LPS) with a short polysaccharidic chain was lower, and its hepatic uptake was faster as compared to smooth LPS (S-LPS) with a long polysaccharidic chain. The inflammatory response was weaker with R-LPS than with S-LPS. As compared to S-LPS, R-LPS formed larger aggregates, with a higher hydrophobicity index, a more negative zeta potential, and a higher critical aggregation concentration. The lower stability of R-LPS aggregates could be illustrated in vitro by a higher extent of association of LPS to plasma lipoproteins, faster binding to blood cells, and increased uptake by macrophages and hepatocytes, compared to S-LPS. Our data indicate that a long polysaccharide chain is associated with the formation of more stable aggregates with extended residence time in plasma and higher inflammatory potential. These results show that polysaccharide chain length, and overall aggregability of LPS might be helpful to predict the proinflammatory effect that can be expected in experimental settings using LPS preparations. In addition, better knowledge and control of LPS aggregation and disaggregation might lead to new strategies to enhance LPS detoxification in septic patients.

Understanding lipopolysaccharide aggregation and its influence on activation of Factor C

The quantification of lipopolysaccharide (LPS) shed by bacteria within aqueous samples is typically performed by binding LPS to a protein called Factor C within a lysate prepared from the blood of horseshoe crabs (Limulus amebocyte lysate (LAL)). How the state of aggregation of LPS impacts Factor C activation, however, is not understood, particularly in the presence of select salts and non-ionic surfactants that are commonly incorporated into pharmaceutical formulations. To address this open question, herein we report on the aggregation status of LPS in aqueous solution, characterized using angle-dependent static and dynamic light scattering with and without chelating salts and polysorbate surfactants, and its correlation with activation of Factor C. Because the aggregation status of LPS is kinetically controlled, care was taken to compare LPS aggregation and activity using identically prepared samples. By plotting LPS activity versus the LPS aggregate size distribution over varied solution conditions, we found a positive correlation between LPS aggregate sizes between 30 and 50 nm and LAL activity. Overall, our results support the hypothesis that activation of Factor C is dependent of LPS aggregate size, and that the modulating effects of salts and surfactants on activation of Factor C is associated with changes in the LPS aggregation.

Dependence on size and shape of non-nature amino acids in the enhancement of lipopolysaccharide (LPS) neutralizing activities of antimicrobial peptides

HypothesisRelease of lipopolysaccharides (LPS) from bacteria into bloodstream may cause serious unwanted stimulation of the host immune system. P-113 is a clinically active histidine-rich antimicrobial peptide. Nal-P-113, a β-naphthylalanine-substituted P-113, is salt-resistant but has limited LPS neutralizing activity. We suspected the size and shape of the non-natural bulky amino acid may affect its LPS neutralizing activity. Herein, antimicrobial, LPS neutralizing, and antiproteolytic effects of phenylalanine- (Phe-P-113), β-naphthylalanine- (Nal-P-113), β-diphenylalanine- (Dip-P-113), and β-(4,4′-biphenyl)alanine- (Bip-P-113) substituted P-113 were studied. ExperimentsStructure-activity relationships of P-113, Phe-P-113, Nal-P-113, Dip-P-113, and Bip-P-113 were evaluated using antimicrobial activity assays, serum proteolytic assays, peptide-induced permeabilization of large unilamellar vesicles, zeta potential measurements, dynamic light scattering measurement of LPS aggregation, and Limulus amebocyte lysate assays for measuring LPS neutralization. In vitro and in vivo LPS neutralizing activities were further confirmed by LPS-induced inflammation inhibition in an endotoxemia mouse model. FindingsBip-P-113 and Dip-P-113 had the longest and widest non-nature amino acids, respectively. Bip-P-113 enhanced salt resistance, serum proteolytic stability, peptide-induced permeabilization, zeta potential measurements, LPS aggregation, and in vitro and in vivo LPS neutralizing activities. These results could help design novel antimicrobial peptides that have enhanced stability in vivo and that can have potential therapeutic applications.

A novel cecropin B-derived peptide with antibacterial and potential anti-inflammatory properties.

Cecropins, originally found in insects, are a group of cationic antimicrobial peptides. Most cecropins have an amphipathic N-terminal segment and a largely hydrophobic C-terminal segment, and normally form a helix-hinge-helix structure. In this study, we developed the novel 32-residue cecropin-like peptide cecropin DH by deleting the hinge region (Alanine-Glycine-Proline) of cecropin B isolated from Chinese oak silk moth, Antheraea pernyi. Cecropin DH possesses effective antibacterial activity, particularly against Gram-negative bacteria, with very low cytotoxicity against mammalian cells. Interactions between cecropin DH and the highly anionic lipopolysaccharide (LPS) component of the Gram-negative bacterial outer membrane indicate that it is capable of dissociating LPS micelles and disrupting LPS aggregates into smaller assemblies, which may play a vital role in its antimicrobial activity. Using LPS-stimulated mouse macrophage RAW264.7 cells, we found that cecropin DH exerted higher potential anti-inflammatory activity than cecropin B, as demonstrated by the inhibition of pro-inflammatory cytokines nitric oxide production and secretion of tumor necrosis factor-α. In conclusion, cecropin DH has potential as a therapeutic agent for both antibacterial and anti-inflammatory applications.

An MD2-derived peptide promotes LPS aggregation, facilitates its internalization in THP-1 cells, and inhibits LPS-induced pro-inflammatory responses.

MD2, a 160-residue accessory glycoprotein, is responsible for the recognition and binding of Gram-negative bacterial membrane component, lipopolysaccharide (LPS). Internalization of pathogen inside the mononuclear phagocytes has also been attributed to MD2 which leads to the clearance of pathogens from the host. However, not much is known about the segments in MD2 that are responsible for LPS interaction or internalization of pathogen inside the defense cells. A 16-residue stretch (MD54) from MD2 protein has been identified that possesses a short heptad repeat sequence and four cationic residues enabling it to participate in both hydrophobic and electrostatic interactions with LPS. An MD54 analog of the same size was also designed in which a leucine residue at a heptadic position was replaced with an alanine residue. MD54 but not its analog, MMD54 induced aggregation of LPS and aided in its internalization within THP-1 monocytes. Furthermore, MD54 inhibited LPS-induced nuclear translocation of NF-κB in PMA-treated THP-1 and TLR4/MD2/CD14-transfected HEK-293Tcells and the production of pro-inflammatory cytokines. In addition, in in vivo experiments, MD54 showed marked protection and survival of mice against LPS-induced inflammation and death. Overall, we have identified a short peptide with heptad repeat sequence from MD2 that can cause aggregation of LPS and abet in its internalization within THP-1 cells, resulting in attenuation of LPS-induced pro-inflammatory responses in vitro and in vivo.

Inflammasome Activation by Bacterial Outer Membrane Vesicles Requires Guanylate Binding Proteins.

ABSTRACTThe Gram-negative bacterial cell wall component lipopolysaccharide (LPS) is recognized by the noncanonical inflammasome protein caspase-11 in the cytosol of infected host cells and thereby prompts an inflammatory immune response linked to sepsis. Host guanylate binding proteins (GBPs) promote infection-induced caspase-11 activation in tissue culture models, and yet their in vivo role in LPS-mediated sepsis has remained unexplored. LPS can be released from lysed bacteria as “free” LPS aggregates or actively secreted by live bacteria as a component of outer membrane vesicles (OMVs). Here, we report that GBPs control inflammation and sepsis in mice injected with either free LPS or purified OMVs derived from Gram-negative Escherichia coli. In agreement with our observations from in vivo experiments, we demonstrate that macrophages lacking GBP2 expression fail to induce pyroptotic cell death and proinflammatory interleukin-1β (IL-1β) and IL-18 secretion when exposed to OMVs. We propose that in order to activate caspase-11 in vivo, GBPs control the processing of bacterium-derived OMVs by macrophages as well as the processing of circulating free LPS by as-yet-undetermined cell types.

Aggregation of thrombin-derived C-terminal fragments as a previously undisclosed host defense mechanism

Effective control of endotoxins and bacteria is crucial for normal wound healing. During injury, the key enzyme thrombin is formed, leading to generation of fibrin. Here, we show that human neutrophil elastase cleaves thrombin, generating 11-kDa thrombin-derived C-terminal peptides (TCPs), which bind to and form amorphous amyloid-like aggregates with both bacterial lipopolysaccharide (LPS) and gram-negative bacteria. In silico molecular modeling using atomic resolution and coarse-grained simulations corroborates our experimental observations, altogether indicating increased aggregation through LPS-mediated intermolecular contacts between clusters of TCP molecules. Upon bacterial aggregation, recombinantly produced TCPs induce permeabilization of Escherichia coli and phagocytic uptake. TCPs of about 11 kDa are present in acute wound fluids as well as in fibrin sloughs from patients with infected wounds. We noted aggregation and colocalization of LPS with TCPs in such fibrin material, which indicates the presence of TCP-LPS aggregates under physiological conditions. Apart from identifying a function of proteolyzed thrombin and its fragments, our findings provide an interesting link between the coagulation system, innate immunity, LPS scavenging, and protein aggregation/amyloid formation.

The antimicrobial effects of the alginate oligomer OligoG CF-5/20 are independent of direct bacterial cell membrane disruption

Concerns about acquisition of antibiotic resistance have led to increasing demand for new antimicrobial therapies. OligoG CF-5/20 is an alginate oligosaccharide previously shown to have antimicrobial and antibiotic potentiating activity. We investigated the structural modification of the bacterial cell wall by OligoG CF-5/20 and its effect on membrane permeability. Binding of OligoG CF-5/20 to the bacterial cell surface was demonstrated in Gram-negative bacteria. Permeability assays revealed that OligoG CF-5/20 had virtually no membrane-perturbing effects. Lipopolysaccharide (LPS) surface charge and aggregation were unaltered in the presence of OligoG CF-5/20. Small angle neutron scattering and circular dichroism spectroscopy showed no substantial change to the structure of LPS in the presence of OligoG CF-5/20, however, isothermal titration calorimetry demonstrated a weak calcium-mediated interaction. Metabolomic analysis confirmed no change in cellular metabolic response to a range of osmolytes when treated with OligoG CF-5/20. This data shows that, although weak interactions occur between LPS and OligoG CF-5/20 in the presence of calcium, the antimicrobial effects of OligoG CF-5/20 are not related to the induction of structural alterations in the LPS or cell permeability. These results suggest a novel mechanism of action that may avoid the common route in acquisition of resistance via LPS structural modification.

Tryptophan end-tagging for promoted lipopolysaccharide interactions and anti-inflammatory effects

The objective of the present study is the investigation of possibilities for boosting peptide anti-inflammatory effects by tryptophan end-tagging, including identification of underlying mechanisms for this. In doing so, effects of tryptophan end-tagging of KYE21 (KYEITTIHNLFRKLTHRLFRR), a peptide derived from heparin co-factor II, on membrane and lipopolysaccharide (LPS) interactions were investigated by ellipsometry, NMR, fluorescence spectroscopy, and circular dichroism measurements. Through its N-terminal W stretch, WWWKYE21 displays higher membrane binding, liposome rupture, and bacterial killing than unmodified KYE21. Analogously, W-tagging promotes binding to E. coli LPS and to its endotoxic lipid A moiety. Furthermore, WWWKYE21 causes more stable peptide/LPS complexes than KYE21, as evidenced by detailed NMR studies, adopting a pronounced helical conformation, with a large hydrophobic surface at the N-terminus due to the presence of W-residues, and a flexible C-terminus due to presence of several positively charged arginine residues. Mirroring its increased affinity for LPS and lipid A, WWWKYE21 displays strongly increased anti-inflammatory effect due to a combination of direct lipid A binding, peptide-induced charge reversal of cell membranes for LPS scavenging, and peptide-induced fragmentation of LPS aggregates for improved phagocytosis. Importantly, potent anti-inflammatory effects were observed at low cell toxicity, demonstrated for both monocytes and erythrocytes.

Activation of Contact Pathway of Blood Coagulation on the Lipopolysaccharide Aggregates

Coagulation of blood plasma is a complex network of biochemical reactions that are usually divided into extrinsic and intrinsic pathways. The extrinsic pathway starts from vessel injury, whereas the intrinsic one initiates from a contact with a foreign surface. The latter consists of the proteolytic cleavages of plasma proteins factor XII (FXII) and prekallikrein (PK) promoted by the surface and requiring a cofactor, high molecular weight kininogen (HMWK). One of the frequently occurring events of contact pathway activation is bacterial infection. FXII and PK seem to be activated by components of the outer leaflet of Gram-negative bacteria outer membrane, lipopolysaccharides (LPS). The mechanisms of contact pathway activation by LPS remains unclear.Here we have constructed a mathematical model of contact pathway activation by LPS aggregates based on in vitro experimental data for FXII-PK and FXII-PK-HMWK systems (Morisson and Cochrane, J Exper Med (1974) 140:797-811; Kalter et al., J. Infect. Dis. (1983) 148:682-691). The series of models consisted of 6-11 ordinary differential equations describing interactions of proteins governed by mass action or Michaelis-Menten kinetics. The system was integrated by LSODE method in COPASI (http://copasi.org).The computational models described the phenomenon of apparent inhibition of contact activation by high LPS concentrations and the existence of optimal LPS concentration experimentally observed in human plasma. The computational analysis suggested that, when LPS concentration is higher than the optimal one, surface concentrations of contact pathway factors decrease because of the activating surface excess. In the case when LPS concentrations are smaller than the optimal one, activating surface deficiency is the reason for the rate limitation. Thus, the main mechanism of the contact system activation in presence of LPS is surface-dependent and occurs due to the presence of LPS in plasma in aggregated state.

Lipopolysaccharide Neutralization by Cationic-Amphiphilic Polymers through Pseudoaggregate Formation.

Synthetic polymers incorporating the cationic charge and hydrophobicity to mimic the function of antimicrobial peptides (AMPs) have been developed. These cationic-amphiphilic polymers bind to bacterial membranes that generally contain negatively charged phospholipids and cause membrane disintegration resulting in cell death; however, cationic-amphiphilic antibacterial polymers with endotoxin neutralization properties, to the best of our knowledge, have not been reported. Bacterial endotoxins such as lipopolysaccharide (LPS) cause sepsis that is responsible for a great amount of mortality worldwide. These cationic-amphiphilic polymers can also bind to negatively charged and hydrophobic LPS and cause detoxification. Hence, we envisaged that cationic-amphiphilic polymers can have both antibacterial as well as LPS binding properties. Here we report synthetic amphiphilic polymers with both antibacterial as well as endotoxin neutralizing properties. Levels of proinflammatory cytokines in human monocytes caused by LPS stimulation were inhibited by >80% when coincubated with these polymers. These reductions were found to be dependent on concentration and, more importantly, on the side-chain chemical structure due to variations in the hydrophobicity profiles of these polymers. These cationic-amphiphilic polymers bind and cause LPS neutralization and detoxification. Investigations of polymer interaction with LPS using fluorescence spectroscopy and dynamic light scattering (DLS) showed that these polymers bind but neither dissociate nor promote LPS aggregation. We show that polymer binding to LPS leads to sort of a pseudoaggregate formation resulting in LPS neutralization/detoxification. These findings provide an unusual mechanism of LPS neutralization using novel synthetic cationic-amphiphilic polymers.