RESUMO
The interaction of selected endotoxin preparations (lipid A from Erwinia carotovora and LPS Re and Ra from Salmonella enterica sv. Minnesota strains R595 and R60, respectively) with selected bile acids was investigated biophysically. Endotoxin aggregates were analyzed for their gel-to-liquid crystalline phase behavior, the type of their aggregates, the conformation of particular functional groups, and their Zeta potential in the absence and presence of the bile acids by applying Fourier-transform infrared spectroscopy, differential scanning calorimetry, measurements of the electrophoretic mobility, and synchrotron radiation X-ray scattering. In addition, the ability of the endotoxins to induce cytokines in human mononuclear cells was tested in the absence and presence of varying concentrations of bile acids. The data show that the endotoxin:bile acid interaction is not governed by Coulomb forces, rather a hydrophobic interaction takes place. This leads to an enhanced formation of the inherent cubic aggregate structures of the endotoxins, concomitant with a slight disaggregation, as evidenced by freeze-fracture electron microscopy. Parallel to this, the addition of bile acids increased the bioactivity of lipid A and, to a lower degree, also that of the tested rough mutant LPS at lower concentrations of the endotoxin preparation, a finding similar as reported for the interaction of other agents such as hemoglobin. These data imply that there are general mechanisms that govern the expression of biological activities of endotoxins.
Assuntos
Ácidos e Sais Biliares/química , Endotoxinas/química , Biofísica , Varredura Diferencial de Calorimetria , Ácido Quenodesoxicólico/química , Citocinas/biossíntese , Ácido Desidrocólico/química , Ácido Desoxicólico/química , Eletroquímica , Técnica de Fratura por Congelamento , Humanos , Técnicas In Vitro , Lipídeo A/farmacologia , Ácido Litocólico/química , Monócitos/metabolismo , Pectobacterium carotovorum/química , Salmonella enterica/química , Colato de Sódio/química , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Fenômenos Biofísicos , Interações Hidrofóbicas e Hidrofílicas , Lipopolissacarídeos/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Materiais Biomiméticos/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citocinas/metabolismo , Feminino , Caranguejos Ferradura/efeitos dos fármacos , Caranguejos Ferradura/metabolismo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Bicamadas Lipídicas/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/toxicidade , Camundongos , Fosfolipídeos/metabolismo , Ligação ProteicaRESUMO
We report x-ray reflectivity and grazing incidence x-ray diffraction measurements of lipopolysaccharide (LPS) monolayers at the water-air interface. Our investigations reveal that the structure and lateral ordering of the LPS molecules is very different from phospholipid systems and can be modulated by the ionic strength of the aqueous subphase in an ion-dependent manner. Our findings also indicate differential effects of monovalent and divalent ions on the two-dimensional ordering of lipid domains. Na(+) ions interact unspecifically with LPS molecules based on their ability to efficiently screen the negative charges of the LPS molecules, whereas Ca(2+) ions interact specifically by cross-linking adjacent molecules in the monolayer. At low lateral pressures, Na(+) ions present in the subphase lead to a LPS monolayer structure ordered over large areas with high compressibility, nearly hexagonal packing of the hydrocarbon chains, and high density in the LPS headgroup region. At higher film pressures, the LPS monolayer becomes more rigid and results in a less perfect, oblique packing of the LPS hydrocarbon chains as well as a smaller lateral size of highly ordered domains on the monolayer. Furthermore, associated with the increased surface pressure, a conformational change of the sugar headgroups occurs, leading to a thickening of the entire LPS monolayer structure. The effect of Ca(2+) ions in the subphase is to increase the rigidity of the LPS monolayer, leading to an oblique packing of the hydrocarbon chains already at low film pressures, an upright orientation of the sugar moieties, and much smaller sizes of ordered domains in the plane of the monolayer. In the presence of both Na(+)- and Ca(2+) ions in the subphase, the screening effect of Na(+) is predominant at low film pressures, whereas, at higher film pressures, the structure and lateral organization of LPS molecules is governed by the influence of Ca(2+) ions. The unspecific charge-screening effect of the Na(+) ions on the conformation of the sugar moiety becomes less dominant at biologically relevant lateral pressures.
Assuntos
Lipopolissacarídeos/química , Cristalização , Íons , Modelos Moleculares , Pressão , Síncrotrons , Temperatura , Difração de Raios XRESUMO
Lipopolysaccharides (LPSs) from Gram-negative bacteria are strong elicitors of the human immune systems. There is strong evidence that aggregates and not monomers of LPS play a decisive role at least in the initial stages of cell activation of immune cells such as mononuclear cells. In previous reports, it was shown that the biologically most active part of enterobacterial LPS, hexa-acyl bisphosphorylated lipid A, adopts a particular supramolecular conformation, a cubic aggregate structure. However, little is known about the size and morphology of these aggregates, regarding the fact that LPS may have strong variations in the length of the saccharide chains (various rough mutant and smooth-form LPS). Thus, in the present paper, several techniques for the determination of details of the aggregate morphology such as freeze-fracture and cryo-electron microscopy, analytical ultracentrifugation, laser backscattering analysis, and small-angle X-ray scattering were applied for various endotoxin (lipid A and different LPS) preparations. The data show a variety of different morphologies not only for different endotoxins but also when comparing different applied techniques. The data are interpreted with respect to the suitability of the single techniques, in particular on the basis of available literature data.
Assuntos
Biopolímeros/metabolismo , Lipídeo A/metabolismo , Infecções por Salmonella/microbiologia , Salmonella enterica/metabolismo , Salmonella enterica/ultraestrutura , Biopolímeros/química , Biopolímeros/genética , Configuração de Carboidratos , Microscopia Crioeletrônica , Interações Hospedeiro-Patógeno/imunologia , Humanos , Lipídeo A/química , Lipídeo A/genética , Mutação/genética , Infecções por Salmonella/imunologia , Salmonella enterica/genética , Salmonella enterica/patogenicidade , Relação Estrutura-Atividade , Ultracentrifugação , Difração de Raios XRESUMO
Systemic bacterial infections are associated with high mortality. The access of bacteria or constituents thereof to systemic circulation induces the massive release of immunomodulatory mediators, ultimately causing tissue hypoperfusion and multiple-organ failure despite adequate antibiotic treatment. Lipid A, the "endotoxic principle" of bacterial lipopolysaccharide (LPS), is one of the major bacterial immunostimuli. Here we demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the Limulus anti-LPS factor for systemic application. We show efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock in vivo, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. The molecular mechanism of LPS neutralization was elucidated by biophysical techniques. The lipid A part of LPS is converted from its "endotoxic conformation," the cubic aggregate structure, into an inactive multilamellar structure, and the binding affinity of the peptide to LPS exceeds those of known LPS-binding proteins, such as LPS-binding protein (LBP). Our results thus delineate a novel therapeutic strategy for the clinical management of patients with septic shock.
Assuntos
Anti-Infecciosos/farmacologia , Anti-Infecciosos/uso terapêutico , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Choque Séptico/prevenção & controle , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Bactérias/efeitos dos fármacos , Calorimetria , Células Cultivadas , Citocinas/metabolismo , Feminino , Hemólise/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/química , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Peptídeos/síntese química , Peptídeos/química , Choque Séptico/tratamento farmacológico , Choque Séptico/imunologiaRESUMO
An analysis of the interaction of the NK-lysin derived peptide NK-2 and of analogs thereof with bacterial lipopolysaccharide (LPS, endotoxin) was performed to determine the most important biophysical parameters for an effective LPS neutralization. We used microcalorimetry, FTIR spectroscopy, Zeta potential measurements, and small-angle X-ray scattering to analyze the peptide:LPS binding enthalpy, the accessible LPS surface charge, the fluidity of the LPS hydrocarbon chains, their phase transition enthalpy change, the aggregate structure of LPS, and how these parameters are modulated by the peptides. We conclude that (i) a high peptide:LPS binding affinity, which is facilitated by electrostatic and hydrophobic interactions and which leads to a positive Zeta potential, (ii) the formation of peptide-enriched domains, which destabilize the lipid packing, demonstrated by a drastic decrease of phase transition enthalpy change of LPS, and (iii) the multilamellarization of the LPS aggregate structure are crucial for an effective endotoxin neutralization by cationic peptides.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Endotoxinas/antagonistas & inibidores , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Endotoxinas/química , Endotoxinas/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Dados de Sequência Molecular , Transição de Fase , Proteolipídeos/química , Salmonella enterica/imunologia , Espalhamento a Baixo Ângulo , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
The innate immune response provides a critical first-line defense against Mycobacterium tuberculosis, an intracellular pathogen that represents a major health threat world-wide. A synthetic lipopeptide (LP) mimicking the lipid moiety of the cell-wall associated 19-kDa lipoprotein from M. tuberculosis has recently been assigned an important role in the induction of an antibacterial immune response in host macrophages. Here, we present experimental data on the biological activities and the biophysical mechanisms underlying cell activation by synthetic 19-kDa M. tuberculosis-derived lipopeptide (Mtb-LP). Investigation of the geometry of the LP (i.e. the molecular conformation and supramolecular aggregate structure) and the preference for membrane intercalation provide an explanation for the biological activities of the mycobacterial LP. Cell activation by low concentrations of Mtb-LP was enhanced by the lipopolysaccharide-binding protein and CD14. However, surprisingly, we found that activation of human macrophages to induce pro- as well as antiinflammatory mediators (tumor necrosis factor(TNF)-alpha, Interleukin(IL)-6, IL-8, and IL-10) in response to the Mtb-LP is strongly reduced in the presence of serum. This observation could be confirmed for the immune response of murine macrophages which showed a strongly enhanced TNF-alpha release in the absence of serum, suggesting that the molecular mechanisms of immune recognition of the Mtb-LP are tailored to the ambient conditions of the lung.
Assuntos
Proteínas de Bactérias/imunologia , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/imunologia , Soro/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Biomimética , Células da Medula Óssea/imunologia , Meios de Cultura Livres de Soro , Citocinas/análise , Citocinas/biossíntese , Transferência Ressonante de Energia de Fluorescência , Humanos , Imunidade Celular/imunologia , Imunidade Inata/imunologia , Indicadores e Reagentes , Inflamação/imunologia , Lipídeos/química , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Lipopolysaccharides (LPS, endotoxins) are main constituents of the outer membranes of Gram-negative bacteria, with the 'endotoxic principle' lipid A anchoring LPS into the membrane. When LPS is removed from the bacteria by the action of the immune system or simply by cell dividing, it may interact strongly with immunocompetent cells such as mononuclear cells. This interaction may lead, depending on the LPS concentration, to beneficial (at low) or pathophysiological (at high concentrations) reactions, the latter frequently causing the septic shock syndrome. There is a variety of endogenous LPS-binding proteins. To this class belong lactoferrin (LF) and hemoglobin (Hb), which have been shown to suppress and enhance the LPS-induced cytokine secretion in mononuclear cells, respectively. To elucidate the interaction mechanisms of endotoxins with these proteins, we have investigated in an infrared reflection-absorption spectroscopy (IRRAS) study the interaction of LPS or lipid A monolayers at the air/water interface with LF and Hb proteins, injected into the aqueous subphase. The data are clearly indicative of completely different interaction mechanisms of the endotoxins with the proteins, with the LF acting only at the LPS backbone, whereas Hb incorporates into the lipid monolayer. These data allow an understanding of the different reactivities in the biomedicinal systems.
Assuntos
Lipídeo A/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/metabolismo , Absorção , Animais , Hemoglobinas/metabolismo , Humanos , Lactoferrina/metabolismo , Salmonella , Espectrofotometria InfravermelhoRESUMO
Lipopolysaccharides (LPS, endotoxins) belong to the strongest elicitors of the mammalian immune system due to the induction of a series of cytokines such as tumor-necrosis-factor-alpha (TNFalpha) in immunocompetent cells like mononuclear cells. Since the effects of LPS on human health may be pathologically at too high concentrations (e.g., septic shock syndrome), it is of uttermost importance to have a reliable assay for measuring the concentrations of endotoxins in vitro and in vivo (human body fluids). The activation of the clotting cascade from the horseshoe crab (Limulus polyphemus), the Limulus amoebocyte lysate test (LAL), has been the standard and most sensitive assay to detect bacterial endotoxins. However, there are restrictions with this test. It was found in some clinical trials that the results from the LAL test did not correlate with the presence of bacteremia due to Gram-negative organisms or with the mortality but correlated with the presence of fungal bloodstream infections. This resulted from the fact that the LAL assay does not only respond to bacterial endotoxins but is activated also by (1-->3)-beta-D-glucan. Furthermore, in extensive studies the structural requirements for activation of the LAL test were analyzed, and it was found that the LAL activity correlated with pyrogenicity but not with activation of the complement cascade. Furthermore, there was no correlation of the LAL activity with cytokine expression (for example tumor-necrosis-factor-alpha and interleulkins-1 and 6) in mononuclear cells when the 4/2 acyl chain pattern of enterobacterial lipid A was changed, or when the cytokine production induced by LPS from various different species in the whole blood assay was compared with the response from the LAL test. To clarify the questions raised by the different experimental findings, data from literature are summarized to get a more closer insight where the Limulus test confidentially monitors the endotoxicity of LPS and other compounds and where this is not the case, and which are the decisive epitopes for recognition of the LPS molecules. These data are very crucial for example in clinical tests, whether the LAL assay can reliably describe the effectivity of an antibacterial therapy.
Assuntos
Citocinas/metabolismo , Teste do Limulus/métodos , Acilação , Citocinas/análise , Endotoxinas/análise , Endotoxinas/toxicidade , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/análise , Lipopolissacarídeos/toxicidadeRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs) represent non-specific inhibitors of the cycloxygenase pathway of inflammation, and therefore an understanding of the interaction process of the drugs with membrane phospholipids is of high relevance. We have studied the interaction of the NSAIDs with phospholipid membranes made from dimyristoylphosphatidylcholine (DMPC) by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). FTIR data obtained via attenuated total reflectance (ATR) show that the interaction between DMPC and NSAIDs is limited to a strong interaction of the drugs with the phosphate region of the lipid head group. The FTIR transmission data furthermore are indicative of a strong effect of the drugs on the hydrocarbon chains inducing a reduction of the chain-chain interactions, i.e., a fluidization effect. Parallel to this, from the DSC data beside the decrease of T(m) a reduction of the peak height of the melting endotherm connected with its broadening is observed, but leaving the overall phase transition enthalpy constant. Additionally, phase separation is observed, inducing the formation of a NSAID-rich and a NSAID-poor phase. This is especially pronounced for Diclofenac. Despite the strong influence of the drugs on the acyl chain moiety, FRET data do not reveal any evidence for drug incorporation into the lipid matrix, and ITC measurements performed do not exhibit any heat production due to drug binding. This implies that the interaction process is governed by only entropic reactions at the lipid/water interface.
Assuntos
Anti-Inflamatórios não Esteroides/química , Diclofenaco/química , Dimiristoilfosfatidilcolina/química , Ibuprofeno/química , Lecitinas/química , Naproxeno/química , Calorimetria , Varredura Diferencial de Calorimetria , Transferência Ressonante de Energia de Fluorescência , Lipossomos/química , Espectrofotometria Infravermelho , Espectroscopia de Infravermelho com Transformada de Fourier , TermodinâmicaRESUMO
Pseudomonas aeruginosa produces the quorum signal 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal), which is important for stimulating outer membrane vesicle (MV) formation. Here we describe the importance of the 3-hydroxyl and 2-alkyl chain for MV production and the length of the 2-alkyl chain for association with MVs.
Assuntos
Pseudomonas aeruginosa/metabolismo , Quinolonas/química , Quinolonas/metabolismo , Vesículas Secretórias/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Modelos Moleculares , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.
Assuntos
Hemoglobinas/farmacologia , Fatores de Virulência/farmacologia , Animais , Carboidratos/química , Citocinas/biossíntese , Técnica de Fratura por Congelamento , Humanos , Técnicas In Vitro , Lipídeos/química , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Monócitos/metabolismo , Salmonella/química , Ovinos , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-Atividade , Temperatura , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Fatores de Virulência/química , Difração de Raios XRESUMO
The physico-chemical properties of three fully hydrated monoacyl maltoside glycolipids were investigated with Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS). The different synthesized maltoside glycoconjugates vary in the length and saturation of the fatty acid moiety, whereas the constant head group region contains a beta-linked maltose with a OC(2)-NH spacer. The compounds with saturated acyl chains showed a complex pattern of temperature-dependent behaviour, regarding the adopted three-dimensional aggregate structure of the molecules and the main phase transition from the gel to liquid crystalline phase of the acyl chains. A substitution of the saturated acyl chain with an unsaturated acyl chain led to a complete change of the structural preferences, from a high ordered stacking of the bilayers to an unilamellar arrangement of completely disordered and fluid membranes. The presence of the NH group in the spacer, compared to the compounds lacking the NH group allows the formation of a hydrogen bonding network, which influences the observed phase properties. The results of these studies of the hydrated monoacylated maltose glycolipids are discussed in relation to the thermotropic phase properties of the pure compounds in the absence of water.
Assuntos
Glicolipídeos/química , Maltose/química , Acetilação , Varredura Diferencial de Calorimetria , Carboidratos/química , Físico-Química/métodos , Ligação de Hidrogênio , Lipídeos/química , Modelos Químicos , Espalhamento de Radiação , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Água/química , Difração de Raios X , Raios XRESUMO
Bacteria have evolved elaborate communication strategies to co-ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4'-phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side-chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome-like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signalling functions.
Assuntos
Membrana Celular/fisiologia , Lipídeos de Membrana/metabolismo , Pseudomonas aeruginosa/fisiologia , Quinolonas/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Lipopolissacarídeos/metabolismo , Lipossomos/metabolismo , Estrutura Molecular , Fosfolipídeos/metabolismoRESUMO
The neutralization of endotoxin structures such as the active 'endotoxic principle' lipid A by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of Gram-negative bacteria which frequently may lead to the septic shock syndrome. An effective antimicrobial peptide, originally found in the skin of an African frog, is magainin 2. Here, the interaction of magainin 2-amide and a peptide derived thereof, M2V, with chemically defined and homogeneous hexaacyl and heptaacyl lipids A isolated from LPS of Erwinia carotovora, was investigated. By using Fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid A and the conformation of their phosphate groups due to peptide binding was investigated. The former parameter was also determined by using differential scanning calorimetry. The electrophoretic mobility of lipid A aggregates under the influence of the peptides was studied to determine the Zeta potential, and small-angle X-ray scattering was applied for the elucidation of the types of aggregate structures in the absence and presence of the peptides. The lipid A-induced cytokine production in human mononuclear cells shows that the ability of the two peptides to inhibit a tumor necrosis factor-alpha production correlates with characteristic changes of the biophysical parameters. These are much stronger expressed for the peptide M2V than for magainin 2-amide, which apparently is connected with the higher number of positive as well as more hydrophobic amino acids, leading to a stronger amphiphilicity necessary to neutralize the amphiphilic lipid A aggregates.
Assuntos
Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Lipídeo A/química , Pectobacterium carotovorum/química , Proteínas de Xenopus/química , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Humanos , Leucócitos Mononucleares/imunologia , Magaininas , Espectroscopia de Infravermelho com Transformada de Fourier , Fator de Necrose Tumoral alfa/imunologia , Proteínas de Xenopus/genéticaRESUMO
An understanding of details of the interaction mechanisms of bacterial endotoxins (lipopolysaccharide, LPS) with the oxygen transport protein hemoglobin is still lacking, despite its high biological relevance. Here, a biophysical investigation into the endotoxin:hemoglobin interaction is presented which comprises the use of various rough mutant LPS as well as free lipid A; in addition to the complete hemoglobin molecule from fetal sheep extract, also the partial structure alpha-chain and the heme-free sample are studied. The investigations comprise the determination of the gel-to-liquid crystalline phase behaviour of the acyl chains of LPS, the ultrastructure (type of aggregate structure and morphology) of the endotoxins, and the incorporation of the hemoglobins into artificial immune cell membranes and into LPS. Our data suggest a model for the interaction between Hb and LPS in which hemoglobins do not react strongly with the hydrophilic or with the hydrophobic moiety of LPS, but with the complete endotoxin aggregate. Hb is able to incorporate into LPS with the longitudinal direction parallel to the lipid A double-layer. Although this does not lead to a strong disturbance of the LPS acyl chain packing, the change of the curvature leads to a slightly conical molecular shape with a change of the three-dimensional arrangement from unilamellar into cubic LPS aggregates. Our previous results show that cubic LPS structures exhibit strong endotoxic activity. The property of Hb on the physical state of LPS described here may explain the observation of an increase in LPS-mediating endotoxicity due to the action of Hb.
Assuntos
Hemoglobinas/metabolismo , Lipopolissacarídeos/metabolismo , Modelos Químicos , Salmonella enterica , Animais , Bovinos , Feminino , Hemoglobinas/química , Hemoglobinas/ultraestrutura , Lipopolissacarídeos/química , Lipopolissacarídeos/ultraestrutura , Microscopia Eletrônica , Gravidez , Ligação Proteica , Espalhamento a Baixo Ângulo , Ovinos , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
The structural polymorphism of two selected disaccharide glycolipids with a maltose (DMMA) and a melibiose (DMME) carbohydrate headgroup linked to dimyristyl alkyl chains were investigated by FTIR-spectroscopy, differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS) and film-balance measurements. The compounds displayed thermotropic multilamellar phases. In the gel phase, DMMA formed also a crystalline phase of orthorhombic symmetry, and DMME an interdigitated phase. The gel to liquid crystalline phase transition temperature T(c) of DMMA depended on the storage and hydration conditions, a precooled sample having a T(c) around 45 degrees C, and a freshly prepared sample around 33 degrees C. In contrast, the phase transition temperature for the gel to liquid crystalline phase of DMME was always found at 24 degrees C. Surface pressure isotherms of the lipids on water and buffer showed that DMMA covers only a small surface area (approximately 35A(2)) whereas DMME requires 50 A(2) of space on the surface. Films of DMMA can be compressed up to a maximum compressibility Pi(max) of 54 mN m(-1) whereas the tilted DMME forms less stable films with Pi(max) of 34 mN m(-1). These different structural characteristics reflect the different conformations of the disaccharide head groups. The presence of the alpha1-->4 linked maltose head group in DMMA and an alpha1-->6 linked melibiose head group in DMME induces geometrical structures ranging from a slightly wedge-shaped towards a more tilted structure, and as a consequence of Israelachvilis packing model, to the formation of different phases. In addition, the structural constraints of DMME allow the formation of a phase with interdigitated hydrocarbon chains.
Assuntos
Dissacarídeos/química , Glicolipídeos/química , Varredura Diferencial de Calorimetria , Espalhamento a Baixo Ângulo , Espectroscopia de Infravermelho com Transformada de Fourier , Lipossomas Unilamelares , Difração de Raios XRESUMO
The structural preferences of 1,2-dioleoyl-sn-glycerol glycolipids with glucose, galactose, maltose, and cellobiose as sugar head group were investigated under near physiological conditions with Fourier-transform infrared spectroscopy (FT-IR) and synchrotron radiation small-angle X-ray scattering (SAXS). Whereas all glycolipids have a very high fluidity at temperatures above 0 degrees C, the mono- and disaccharide compounds differ considerably in their aggregate structures. The monosaccharide compounds adopt only inverted hexagonal (H(II)) structures in the temperature range 5-70 degrees C, while the disaccharide compounds adopt only multilamellar structures. Since these and similar glycolipids are frequently found in nature, these data should be of relevance for the function of their host cell membranes.
Assuntos
Glicolipídeos/química , Dissacarídeos/química , Estrutura Molecular , Monossacarídeos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Difração de Raios XRESUMO
To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step.
Assuntos
Antígenos de Diferenciação de Linfócitos T/química , Toxinas Bacterianas/química , Endotoxinas/química , Lipopolissacarídeos/química , Peptídeos/química , Sequência de Aminoácidos , Citocinas/biossíntese , Humanos , Lipossomos/química , Dados de Sequência MolecularRESUMO
On the basis of formerly investigated peptides corresponding to the endotoxin-binding domain from LALF [Limulus anti-LPS (lipopolysaccharide) factor], a protein from Limulus polyphemus, we have designed and synthesized peptides of different lengths with the aim of obtaining potential therapeutic agents against septic shock syndrome. For an understanding of the mechanisms of action, we performed a detailed physicochemical and biophysical analysis of the interaction of rough mutant LPS with these peptides by applying FTIR (Fourier-transform infrared) spectroscopy, SAXS (small-angle X-ray scattering), calorimetric techniques [DSC (differential scanning calorimetry) and ITC (isothermal titration calorimetry)] and FFTEM (freeze-fracture transmission electron microscopy). Also, the action of the peptides on bacteria of different origin in microbial assays was investigated. Using FTIR and DSC, our results indicated a strong fluidization of the lipid A acyl chains due to peptide binding, with a decrease in the endothermic melting enthalpy change of the acyl chains down to a complete disappearance in the 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. Via ITC, it was deduced that the binding is a clearly exothermic process which becomes saturated at a 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. The results obtained with SAXS indicated a drastic change of the aggregate structures of LPS into a multilamellar stack, which was visualized in electron micrographs as hundreds of lamellar layers. This can be directly correlated with the inhibition of the LPS-induced production of tumour necrosis factor alpha in human mononuclear cells, but not with the action of the peptides on bacteria.