Biomedical subjects

Jörg Andrä

Publications and source records attributed to Jörg Andrä.

At least 19 recordsLinked to original sources

PubMed2006

Biophysical characterization of synthetic rhamnolipids.

Synthetic rhamnolipids, derived from a natural diacylated glycolipid, RL-2,2(14), produced by Burkholderia (Pseudomonas) plantarii, were analyzed biophysically. Changes in the chemical structures comprised variations in the length, the stereochemistry and numbers of the lipid chains, numbers of rhamnoses, and the occurrence of charged or neutral groups. As relevant biophysical parameters, the gel (beta) to liquid crystalline (alpha) phase behavior of the acyl chains of the rhamnoses, their three-dimensional supramolecular aggregate structure, and the ability of the compounds to intercalate into phospholipid liposomes in the absence and presence of lipopolysaccharide-binding protein were monitored. Their biological activities were examined as the ability to induce cytokines in human mononuclear cells and to induce chemiluminescence in monocytes. Depending on the particular chemical structures, the physicochemical parameters as well as the biological test systems show large variations. This relates to the acyl chain fluidity, aggregate structure, and intercalation ability, as well as the bioactivity. Most importantly, the data extend our conformational concept of endotoxicity, based on the intercalation of naturally originating amphiphilic virulence factors into membranes from immune cells. This 'endotoxin conformation', produced by amphiphilic molecules with a hydrophilic charged backbone and apolar hydrophobic moiety, and adopting inverted cubic aggregate structures, causes high mechanical stress in target immune cells on integral proteins, eventually leading to cell activation. Furthermore, biologically inactive rhamnolipids with lamellar aggregate structures antagonize the endotoxin-induced activity in a way similar to lipid A-derived antagonists.

Animals↗

PubMed2006

Influence of N-acylation of a peptide derived from human lactoferricin on membrane selectivity.

Increasing numbers of bacterial strains being resistant to conventional antibiotics emphasize the urgent need for new antimicrobial agents. One strategy is based on host defence peptides that can be found in every organism including humans. We have studied the antimicrobial peptide LF11, derived from the pepsin cleavage product of human lactoferrin, known for its antimicrobial and lipid A-binding activity, and peptide C12LF11, the N-lauryl-derivative of LF11, which has owing to the attached hydrocarbon chain an additional hydrophobic segment. The influence of this hydrocarbon chain on membrane selectivity was studied using model membranes composed of dipalmitoylphosphatidylglycerol (DPPG), mimicking bacterial plasma membranes, and of dipalmitoylphosphatidylcholine (DPPC), a model system for mammalian membranes. A variety of biophysical techniques was applied. Thereby, we found that LF11 did not affect DPPC bilayers and showed only moderate effects on DPPG membranes in accordance with its non-hemolytic and weak antimicrobial activity. In contrast, the introduction of the N-lauryl group caused significant changes in the phase behaviour and lipid chain packing in both model membrane systems. These findings correlate with the in vitro tests on methicillin resistant S. aureus, E. coli, P. aeruginosa and human red blood cells, showing increased biological activity of C12LF11 towards these test organisms. This provides evidence that both electrostatic and hydrophobic interactions are crucial for biological activity of antimicrobial peptides, whereas a certain balance between the two components has to be kept, in order not to loose the specificity for bacterial membranes.

Acylation↗

PubMed2006

Mechanisms of endotoxin neutralization by synthetic cationic compounds.

A basic challenge in the treatment of septic patients in critical care units is the release of bacterial pathogenicity factors such as lipopolysaccharide (LPS, endotoxin) from the cell envelope of Gram-negative bacteria due to killing by antibiotics. LPS aggregates may interact with serum and membrane proteins such as LBP (lipopolysaccharide-binding protein) and CD14 leading to the observed strong reaction of the immune system. Thus, an effective treatment of patients infected by Gram-negative bacteria must comprise beside bacterial killing the neutralization of endotoxins. Here, data are summarized for synthetic compounds indicating the stepwise development to very effective LPS-neutralizing agents. These data include synthetic peptides, based on the endotoxin-binding domains of natural binding proteins such as lactoferrin, Limulus anti-LPS factor, NK-lysin, and cathelicidins or based on LPS sequestering polyamines. Many of these compounds could be shown to act not only in vitro, but also in vivo (e.g. in animal models of sepsis), and might be useful in future clinical trials and in sepsis therapy.

Cations↗

PubMed2006

Endotoxin-like properties of a rhamnolipid exotoxin from Burkholderia (Pseudomonas) plantarii: immune cell stimulation and biophysical characterization.

Here we report on the purification, structural characterization, and biological activity of a glycolipid, 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-alpha(R)-3-hydroxytetradecanoyl-(R)-3-hydroxytetradecanoate (RL-2,2(14)) produced by Burkholderia (Pseudomonas) plantarii. RL-2,2(14) is structurally very similar to a rhamnolipid exotoxin from Pseudomonas aeruginosa and identical to the rhamnolipid of Burkholderia pseudomallei, the causative agent of melioidosis. Interestingly, RL-2,2(14) exhibits strong stimulatory activity on human mononuclear cells to produce tumor necrosis factor alpha, the overproduction of which is known to cause sepsis and the septic shock syndrome. Such a property has not been noted so far for rhamnolipid exotoxins, only for bacterial endotoxins (lipopolysaccharide, LPS). Consequently, we analyzed RL-2,2(14) with respect to its pathophysiological activities as a heat-stable extracellular toxin. Like LPS, the cell-stimulating activity of the rhamnolipid could be inhibited by incubation with polymyxin B. However, immune cell activation by RL-2,2(14) does nor occur via receptors that are involved in LPS (TLR4) or lipopeptide signaling (TLR2). Despite its completely different chemical structure, RL-2,2(14) exhibits a variety of endotoxin-related physicochemical characteristics, such as a cubic-inverted supramolecular structure. These data are in good agreement with our conformational concept of endotoxicity: intercalation of naturally originating virulence factors into the immune cell membrane leads to strong mechanical stress on integral proteins, eventually causing cell activation.

Bacterial Toxins↗

PubMed2005

The NK-lysin derived peptide NK-2 preferentially kills cancer cells with increased surface levels of negatively charged phosphatidylserine.

The NK-lysin derived peptide NK-2 is a potent antibacterial, but non-toxic to a human keratinocyte cell line and of low hemolytic activity. Its target selectivity is based upon a strong binding preference to membranes containing anionic phospholipids, which are normally not found on the surface of human cells. Here, we analyzed the interaction of NK-2 with normal human lymphocytes and seven different human cancer cell lines and demonstrate that some of these cells expose negatively charged surface phosphatidylserine (PS), which presumably facilitates killing of the cells by NK-2. This is underlined by the specific intercalation of the peptide into PS-containing liposomes analyzed by fluorescence-resonance energy transfer spectroscopy.

Antineoplastic Agents↗

PubMed2005

Dissection of the mechanisms of cytolytic and antibacterial activity of lysenin, a defence protein of the annelid Eisenia fetida.

The body fluid of earthworms is known to contain a variety of cytolytic and antibacterial activities to combat potential pathogens that may migrate from the environment into the body cavity. In the annelid Eisenia fetida, a multi-gene family exists that give rise to several isoforms, which display hemolytic and antibacterial activity. The hemolytic activity of lysenin, a major isoform, is known to be strictly dependent on sphingomyelin. As bacteria are devoid of sphingomyelin, the nature of the antibacterial activity of lysenin-like proteins appeared obscure. Here, we report the recombinant expression of lysenin, a defined single entity, which exerted hemolytic, antibacterial and membrane-permeabilizing activity comparable to that of the natural counterpart. Experiments using fluorescence resonance energy transfer spectroscopy with liposomes and planar lipid bilayers demonstrated membrane insertion and single channel fluctuations in the presence of sphingomyelin. By monitoring the lipid specificity of lysenin and its molecular organization on different target cell membranes, it became evident that oligomerization to stable pore-like structures occurs on erythrocytes but does not occur on bacterial membranes. However, bacterial membranes became permeabilized by lysenin, albeit much slower. Accordingly, lysenin appears to display sphingomyelin-dependent and sphingomyelin-independent activities to kill various foreign intruders of the earthworm's coelomic cavity.

Animals↗

PubMed2005

Divalent cations affect chain mobility and aggregate structure of lipopolysaccharide from Salmonella minnesota reflected in a decrease of its biological activity.

The physicochemical properties and biological activities of rough mutant lipopolysaccharides Re (LPS Re) as preformed divalent cation (Mg2+, Ca2+, Ba2+) salt form or as natural or triethylamine (Ten+)-salt form under the influence of externally added divalent cations were investigated using complementary methods: Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopic (FT-IR) measurements for the beta <--> alpha gel to liquid crystalline phase behaviour of the acyl chains of LPS, synchrotron radiation X-ray diffraction studies for their aggregate structures, electron density calculations of the LPS bilayer systems, and LPS-induced cytokine (interleukin-6) production in human mononuclear cells. The divalent cation salt forms of LPS exhibit considerable changes in physicochemical parameters such as acyl chain mobility and aggregate structures as compared to the natural or monovalent cation salt forms. Concomitantly, the biological activity was much lower in particular for the Ca2+- and Ba2+-salt forms. This decrease in activity results mainly from the conversion of the unilamellar/cubic aggregate structure of LPS into a multilamellar one. The reduced activity also clearly correlates with the higher order--lower mobility--of the lipid A acyl chains. Both effects can be understood by an impediment of the interactions of LPS with binding proteins such as lipopolysaccharide-binding protein (LBP) and CD14 due to the action of the divalent cations.

Calorimetry, Differential Scanning↗

PubMed2005

Synthesis and mesomorphic properties of glycosyl dialkyl- and diacyl-glycerols bearing saturated, unsaturated and methyl branched fatty acid and fatty alcohol chains. Part II. Mesomorphic properties.

The biophysical properties of a series of glycosyl dialkyl- and diacyl-glycerols bearing unsaturated or chiral methyl branched chains in the tail, and di- and trisaccharide carbohydrate headgroups are described. Thermotropism was investigated by polarising microscopy, the lyotropism was investigated by small angle X-ray diffraction and by the contact preparation method, and the gel to liquid crystalline phase transition by FT-IR-spectroscopy. The compounds displayed thermotropic Smectic A (SmA), cubic and columnar phases, whereas in the lyotropic phase diagram lamellar, hexagonal and cubic phases are found. The introduction of unsaturated or methyl branched chains leads to liquid crystallinity at ambient temperature. The difference between the 1,3-oleyl-glycerol maltoside and the corresponding 1,2-oleoyl-glycerol maltoside is small.

Acylation↗

PubMed2005

Structural rearrangement of model membranes by the peptide antibiotic NK-2.

We have developed a novel alpha-helical peptide antibiotic termed NK-2. It efficiently kills bacteria, but not human cells, by membrane destruction. This selectivity could be attributed to the different membrane lipid compositions of the target cells. To understand the mechanisms of selectivity and membrane destruction, we investigated the influence of NK-2 on the supramolecular aggregate structure, the phase transition behavior, the acyl chain fluidity, and the surface charges of phospholipids representative for the bacterial and the human cell cytoplasmic membranes. The cationic NK-2 binds to anionic phosphatidylglycerol liposomes, causing a thinning of the membrane and an increase in the phase transition temperature. However, this interaction is not solely of electrostatic but also of hydrophobic nature, indicated by an overcompensation of the Zeta potential. Whereas NK-2 has no effect on phosphatidylcholine liposomes, it enhances the fluidity of phosphatidylethanolamine acyl chains and lowers the phase transition enthalpy of the gel to liquid cristalline transition. The most dramatic effect, however, was observed for the lamellar/inverted hexagonal transition of phosphatidylethanolamine which was reduced by more than 10 degrees C. Thus, NK-2 promotes a negative membrane curvature which can lead to the collapse of the phosphatidylethanolamine-rich bacterial cytoplasmic membrane.

Anti-Bacterial Agents↗

PubMed2005

Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide.

Antibacterial peptide acylation, which mimics the structure of the natural lipopeptide polymyxin B, increases antimicrobial and endotoxin-neutralizing activities. The interaction of the lactoferricin-derived peptide LF11 and its N-terminally acylated analogue, lauryl-LF11, with different chemotypes of bacterial lipopolysaccharide (LPS Re, Ra and smooth S form) was investigated by biophysical means and was related to the peptides' biological activities. Both peptides exhibit high antibacterial activity against the three strains of Salmonella enterica differing in the LPS chemotype. Lauryl-LF11 has one order of magnitude higher activity against Re-type, but activity against Ra- and S-type bacteria is comparable with that of LF11. The alkyl derivative peptide lauryl-LF11 shows a much stronger inhibition of the LPS-induced cytokine induction in human mononuclear cells than LF11. Although peptide-LPS interaction is essentially of electrostatic nature, the lauryl-modified peptide displays a strong hydrophobic component. Such a feature might then explain the fact that saturation of the peptide binding takes place at a much lower peptide/LPS ratio for LF11 than for lauryl-LF11, and that an overcompensation of the negative LPS backbone charges is observed for lauryl-LF11. The influence of LF11 on the gel-to-liquid-crystalline phase-transition of LPS is negligible for LPS Re, but clearly fluidizing for LPS Ra. In contrast, lauryl-LF11 causes a cholesterol-like effect in the two chemotypes, fluidizing in the gel and rigidifying of the hydrocarbon chains in the liquid-crystalline phase. Both peptides convert the mixed unilamellar/non-lamellar aggregate structure of lipid A, the 'endotoxic principle' of LPS, into a multilamellar one. These data contribute to the understanding of the mechanisms of the peptide-mediated neutralization of endotoxin and effect of lipid modification of peptides.

Alkylation↗

PubMed2005

Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity.

Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria belongs to the most potent activators of the mammalian immune system. Its lipid moiety, lipid A, the 'endotoxic principle' of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition. The two negative charges of lipid A, represented by the two phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups. We hypothesized that the phosphate groups could be substituted by other negatively charged groups without changing the endotoxic properties of lipid A. To test this hypothesis, we synthesized carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-CM-506) and of tetraacyl lipid A (Bis-CM-406) and correlated their physicochemical with their endotoxic properties. We found that, similarly to compounds 506 and 406, also for their carboxymethyl derivatives a particular molecular ('endotoxic') conformation and with that, a particular aggregate structure is a prerequisite for high cytokine-inducing capacity and antagonistic activity, respectively. In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3-deoxy-D-manno-oct-2-ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed. These data allow a better understanding of endotoxic activity and its structural prerequisites.

Antibodies, Monoclonal↗

PubMed2005

Investigation into the interaction of the phosphoporin PhoE with outer membrane lipids: physicochemical characterization and biological activity.

Outer membrane pore proteins such as phosphoporin (PhoE) are important constituents of Gram-negative bacteria such as Escherichia coli. We have studied the interaction of PhoE with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein:lipid interaction corresponding to the outer membrane system as well as the activity of LPS:PhoE complexes in the infected host after release from the bacterial surface. The interaction of the lipids PE, PG, and LPS with PhoE was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface groups (ester), and polar groups (phosphates) applying Fourier-transform infrared spectroscopy (FTIR), and by analysing the phase transition behaviour of the lipids using FTIR and differential scanning calorimetry (DSC). The activity of PhoE and LPS:PhoE complexes was investigated in biological test systems (human mononuclear cells and Limulus amebocyte lysate assay) and with phospholipid model membranes using fluorescence resonance energy transfer spectroscopy (FRET). The results show a strong influence of PhoE on the mobility of the lipids leading to a considerable fluidization of the acyl chains of LPS, but much less to those from phospholipids: PhoE released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which is not found in the LPS-specific Limulus amebocyte lysate test, indicates an LPS-independent mechanism of cell activation.

Bacterial Outer Membrane Proteins↗

PubMed2004

Temperature dependence of the binding of endotoxins to the polycationic peptides polymyxin B and its nonapeptide.

The interaction between endotoxins-free lipid A and various lipopolysaccharide (LPS) chemotypes with different sugar chain lengths-and the polycationic peptides polymyxin B and polymyxin nonapeptide has been investigated by isothermal titration calorimetry between 20 and 50 degrees C. The results show a strong dependence of the titration curves on the phase state of the endotoxins. In the gel phase (<30 degrees C for LPS and <45 degrees C for lipid A), an endothermic reaction is observed, for which the driving force is an entropically driven endotoxin-polymyxin interaction, due to disruption of the ordered water structure and cation assembly in the lipid A backbone and adjacent molecules. In the liquid crystalline phase (>35 degrees C for LPS and >47 degrees C for lipid A) an exothermic reaction takes place, which is mainly due to the strong electrostatic interaction of the polymyxins with the negative charges of the endotoxins, i.e., the entropic change DeltaS is much lower than in the gel phase. For endotoxins with short sugar chains (lipid A, LPS Re, LPS Rc) the stoichiometry of the polymyxin binding corresponds to pure charge neutralization; for the compounds with longer sugar chains (LPS Ra, LPS S-form) this is no longer valid. This can be related to the lower susceptibility of the corresponding bacterial strains to antibiotics.

Anti-Bacterial Agents↗

PubMed2004

Complement inactivation by recombinant human C3 derivatives.

From the implications of the complement system in a large number of diseases, an urgent need for therapeutics effecting reduced complement activity in vivo has emerged. In this study we report the design of a novel class of enzymes of human origin that obliterate functional complement by a noninhibitory, catalytic mechanism. Combining the framework of human C3 and the enzymatic mechanism of cobra venom factor, a nontoxic snake venom protein, we established molecules capable of forming stable C3 convertase complexes. Although the half-life of naturally occurring C3 convertase complexes ranges between 1 and 2 min, these complexes exhibit a half-life of up to several hours. Because the overall identity to human C3 could be extended to >90%, the novel C3 derivatives can be assumed to exhibit low immunogenicity and, therefore, represent promising candidates for therapeutic reduction of complement activity in vivo.

Animals↗

PubMed2004

Cyclic antimicrobial peptides based on Limulus anti-lipopolysaccharide factor for neutralization of lipopolysaccharide.

Bacterial endotoxin (lipopolysaccharide, LPS) is responsible for the septic shock syndrome. As potential therapeutic agents cyclic cationic antimicrobial peptides of different length, based on the Limulus anti-lipopolysaccharide factor (LALF), were synthesized, and their interaction with LPS was characterized physico-chemically and related to results in biological assays. All peptides inhibited the LPS-induced cytokine production in human mononuclear cells and the Limulus amebocyte lysate in a concentration-dependent way, with the peptide comprising the complete LPS-binding loop of the LALF (cLALF22) being the most effective. The peptides were neither cytotoxic nor hemolytic, except a slight effect of cLALF22. The peptides were able to displace Ca(2+) cations from a LPS monolayer, with cLALF22 being again most effective in accordance with results from isothermal titration calorimetry, in which saturation of binding was observed at an equimolar [cLALF22]:[LPS] ratio, and at a ratio 2-2.5 for the other peptides. For cLALF22, zeta (xi) potential experiments exhibited a complete compensation of the negative charges of LPS, whereas for the other peptides a residual negative potential of -20 to -40mV was found. X-ray diffraction experiments showed that the mixed unilamellar/cubic inverted aggregate structure of the lipid A part of LPS was converted into a multilamellar one. The gel to liquid crystalline phase transition of the acyl chains of LPS was changed upon cLALF22 binding, leading to a clear fluidization, which was not observed or only to a lesser degree for the other peptides. The affinity of the peptides for LPS led to a reduced binding of lipopolysaccharide-binding protein (LBP) to target membranes and hence to an inhibition of cytokine induction in human mononuclear cells.

Antimicrobial Cationic Peptides↗

PubMed2004

Complement C3 is required for the progression of cutaneous lesions and neutrophil attraction in Leishmania major infection.

To elucidate the role of complement-mediated uptake in Leishmania major infection in vivo, transgenic BALB/c mice that express the cobra venom factor (CVF) under control of the alpha1-antitrypsin promoter were infected. CVF expression in these mice leads to a continuous activation and subsequent consumption of complement C3 in the serum. In contrast to susceptible non-transgenic BALB/c mice, CVF-transgenic mice are highly resistant to L. major infection and show a significantly reduced parasite dissemination. Transient depletion of C3 in wild-type BALB/c mice delays progression of lesions for some days. Both CVF-transgenic and non-transgenic mice exhibit similar T cell responses upon infection. However, in CVF-transgenic mice, no infiltration of neutrophils, which were the prominent infiltrating cells at the site of infection in normal susceptible mice, could be detected. We conclude that C3 cleavage is required for the attraction of neutrophils that participate in parasite dissemination.

Animals↗

PubMed2004

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes.

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein-lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.

Animals↗

PubMed2004

Membrane lipid composition protects Entamoeba histolytica from self-destruction by its pore-forming toxins.

The protozoan parasite and human pathogen Entamoeba histolytica is protected against killing by its own lytic effector proteins. Amoebae withstand doses of amoebapores, their pore-forming polypeptides, that readily kill human Jurkat T cells. Moreover, the polypeptides do not bind to the amoebic surface membrane as evidenced by using fluorescently labelled amoebapores and confocal laser microscopy. Experiments employing liposomes as a minimalistic membrane system and the major isoform amoebapore A revealed that the lipid composition of amoebic membranes prevents binding of the cytolytic molecule and that both the phospholipid ingredients and the high content of cholesterol contributes to the protection of the toxin-producing cell.

Adenosine Triphosphate↗