Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins

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.     

Influence of lipophilic groups in cationic alpha-helical peptides on their abilities to bind with DNA and deliver genes into cells.

For the purpose of achieving gene transfer into cells mediated by peptides with a short chain length, we employed two kinds of amphiphilic alpha-helix peptides, mastoparan (INLK-ALAA-LAKK-IL-NH2) obtained from wasp venom and an alpha-helix model peptide (LARL-LARL-LARL-NH2). Furthermore, to strengthen the hydrophobicity of the peptide required for the formation of the aggregates with the DNA, we modified these peptides using several lipophilic groups, i.e. acyl groups with a single chain, a dialkylcarbamoyl group and a cholesteryloxycarbonyl group. We examined the ability of the peptides and their derivatives to bind and aggregate with plasmid DNA, the structural change in the peptides caused by binding with the DNA and the in vitro gene transfer abilities into COS-7 cells. As a result, mastoparan was found to acquire the DNA binding ability by introduction of the lipophilic group. The conformational change in the peptides depended on the hydrophobicity of the introduced acyl group. The DNA complex of most lipophilic mastoparan derivatives could be incorporated into the cells via the endocytosis pathway. In the case of the helix model peptide, the acyl group with a moderate chain length was required for the formation of the aggregate which is competent for incorporation into the cells. In this study, we succeeded in giving such short peptides sufficient gene transfer ability by modifying them with some lipophilic groups. However, the influence of the modification by the lipophilic groups on the formation of aggregates with DNA and the gene transfer ability depended on the structure of the peptide portion. These results indicate that consideration of total hydrophobicity balance is needed for the design of an efficient gene carrier peptide.     

Structural investigations into the interaction of hemoglobin and part structures with bacterial endotoxins

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.     

Mechanism of interaction of optimized Limulus-derived cyclic peptides with endotoxins: thermodynamic, biophysical and microbiological analysis

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.     

Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides

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.     

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 β ↔ α 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.     

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.     

Biophysical characterization of lipopolysaccharide and lipid A inactivation by lactoferrin

The interaction of bacterial endotoxins (LPS Re and lipid A, the 'endotoxic principle' of LPS) with the endogenous antibiotic lactoferrin (LF) was investigated using various physical techniques and biological assays. By applying Fourier-transform infrared (FTIR) spectroscopy, we find that LF binds to the phosphate group within the lipid A part and induces a rigidification of the acyl chains of LPS. The secondary structure of the protein - as monitored by the amide I band - is, however, not changed. Concomitant with the IR data, scanning calorimetric data indicate a sharpening of the acyl chain phase transition. From titration calorimetric and zeta potential data, saturation of LF binding to LPS was found to lie at a [LF]:[LPS] ratio of 1:3 to 1:5 M from the former and 1:10 M from the latter technique. X-ray scattering data indicate a change of the lipid A aggregate structure from inverted cubic to multilamellar, and with fluorescence (FRET) spectroscopy, LF is shown to intercalate by itself into phospholipid liposomes and may also block the lipopolysaccharide-binding protein (LBP)-induced intercalation of LPS. The LPS-induced cytokine production of human mononuclear cells exhibits a decrease due to LF binding, whereas the coagulation of amebocyte lysate in the Limulus test exhibited concentration-dependent changes. Based on these results, a model for the mechanisms of endotoxin inactivation by LF is proposed.     

The 2004 Biophysical Society-Avanti Award in Lipids address: roles of bilayer structure and elastic properties in peptide localization in membranes

This review details how bilayer structural/elastic properties impact three distinct areas of biological significance. First, the partitioning of melittin into bilayers and melittin-induced bilayer leakage depended strongly on bilayer composition. The incorporation of cholesterol into phosphatidylcholine bilayers decreased melittin-induced leakage from 73 to 3%, and bilayers composed of lipopolysaccharide (LPS), the main lipid on the surface of Gram-negative bacteria, also had low (3%) melittin-induced leakage. Second, transbilayer peptides of different hydrophobic lengths were largely excluded from bilayer microdomains (“rafts”) enriched in sphingomyelin (SM) and cholesterol, even when the length of the transbilayer peptide domain matched the hydrocarbon thickness of the raft bilayer. This is likely due to the large area compressibility modulus of SM:cholesterol bilayers. Third, the major water barrier of skin, the extracellular lamellae of the stratum corneum, was found to contain tightly packed asymmetric lipid bilayers with cholesterol located preferentially on one side of the bilayer and a unique skin ceramide containing an unsaturated acyl chain on the opposite side. We argue that, in each of these three areas, key factors are differences in lipid hydrocarbon chain packing for different lipids, particularly the tight hydrocarbon chain packing caused by cholesterol’s strong interaction with saturated chains.     

Interaction between tachyplesin I,an antimicrobial peptide derived from horseshoe crab,and lipopolysaccharide

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program.CD and NMR measurements revealed that binding to LPS slightly extends the two β-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS.     

Interaction of antimicrobial peptides with lipopolysaccharides

We study the interaction of antimicrobial peptides with lipopolysaccharide (LPS) bilayers to understand how antimicrobial peptides interact with the LPS monolayer on the outer membrane of Gram-negative bacteria. LPS in water spontaneously forms a multilamellar structure composed of symmetric bilayers. We performed X-ray lamellar diffraction and wide-angle in-plane scattering to study the physical characteristics of LPS multilayers. The multilayer alignment of LPS is comparable to phospholipids. Thus, it is suitable for the application of oriented circular dichroism (OCD) to study the state of peptides in LPS bilayers. At high hydration levels, the chain melting temperature in multilamella detected by X-ray diffraction is the same as that of LPS aqueous dispersions, as measured by calorimetry. LPS has a strong CD, but with a careful subtraction of the lipid background, the OCD of peptides in LPS is measurable. The method was tested successfully with melittin. It was then applied to two representative antimicrobial peptides, magainin and protegrin. At peptide concentrations comparable to the physiological conditions, both peptides penetrate transmembrane in LPS bilayers. The results imply that antimicrobial peptides readily penetrate the LPS monolayer of the outer membrane.     

Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharide.

Endotoxin-neutralizing protein (ENP) of the horseshoe crab is one of the most potent neutralizers of endotoxins [bacterial lipopolysaccharide (LPS)]. Here, we report on the interaction of LPS with recombinant ENP using a variety of physical and biological techniques. In biological assays (Limulus amebocyte lysate and tumour necrosis factor-alpha induction in human mononuclear cells), ENP causes a strong reduction of the immunostimulatory ability of LPS in a dose-dependent manner. Concomitantly, the accessible negative surface charges of LPS and lipid A (zeta potential) are neutralized and even converted into positive values. The gel to liquid crystalline phase transitions of LPS and lipid A shift to higher temperatures indicative of a rigidification of the acyl chains, however, the only slight enhancement of the transition enthalpy indicates that the hydrophobic moiety is not strongly disturbed. The aggregate structure of lipid A is converted from a cubic into a multilamellar phase upon ENP binding, whereas the secondary structure of ENP does not change due to the interaction with LPS. ENP contains a hydrophobic binding site to which the dye 1-anilino-8-sulfonic acid binds at a K(d) of 19 micro m, which is displaced by LPS. Because lipopolysaccharide-binding protein (LBP) is not able to bind to LPS when ENP and LPS are preincubated, tight binding of ENP to LPS can be deduced with a K(d) in the low nonomolar range. Importantly, ENP is able to incorporate by itself into target phospholipid liposomes, and is also able to mediate the intercalation of LPS into the liposomes thus acting as a transport protein in a manner similar to LBP. Thus, LPS-ENP complexes might enter target membranes of immunocompetent cells, but are not able to activate due to the ability of ENP to change LPS aggregates from an active into an inactive form.     

A molecular mechanism for lipopolysaccharide protection of Gram-negative bacteria from antimicrobial peptides

Cationic antimicrobial peptides serve as the first chemical barrier between all organisms and microbes. One of their main targets is the cytoplasmic membrane of the microorganisms. However, it is not yet clear why some peptides are active against one particular bacterial strain but not against others. Recent studies have suggested that the lipopolysaccharide (LPS) outer membrane is the first protective layer that actually controls peptide binding and insertion into Gram-negative bacteria. In order to shed light on these interactions, we synthesized and investigated a 12-mer amphipathic alpha-helical antimicrobial peptide (K(5)L(7)) and its diastereomer (4D-K(5)L(7)) (containing four d-amino acids). Interestingly, although both peptides strongly bind LPS bilayers and depolarize bacterial cytoplasmic membranes, only the diastereomer kills Gram-negative bacteria. Attenuated total reflectance Fourier transform infrared, CD, and surface plasmon resonance spectroscopies revealed that only the diastereomer penetrates the LPS layer. In contrast, K(5)L(7) binds cooperatively to the polysaccharide chain and the outer phosphate groups. As a result, the self-associated K(5)L(7) is unable to traverse through the tightly packed LPS molecules, revealed by epifluorescence studies with LPS giant unilamellar vesicles. The difference in the peptides' modes of binding is further demonstrated by the ability of the diastereomer to induce LPS miscellization, as shown by transmission electron microscopy. In addition to increasing our understanding of the molecular basis of the protection of bacteria by LPS, this study presents a potential strategy to overcome resistance by LPS, and it should help in the design of antimicrobial peptides for future therapeutic purposes.     

Deacylation of lipopolysaccharide in whole Escherichia coli during destruction by cellular and extracellular components of a rabbit peritoneal inflammatory exudate

Deacylation of purified lipopolysaccharides (LPS) markedly reduces its toxicity toward mammals. However, the biological significance of LPS deacylation during infection of the mammalian host is uncertain, particularly because the ability of acyloxyacyl hydrolase, the leukocyte enzyme that deacylates purified LPS, to attack LPS residing in the bacterial cell envelope has not been established. We recently showed that the cellular and extracellular components of a rabbit sterile inflammatory exudate are capable of extensive and selective removal of secondary acyl chains from purified LPS. We now report that LPS as a constituent of the bacterial envelope is also subject to deacylation in the same inflammatory setting. Using Escherichia coli LCD25, a strain that exclusively incorporates radiolabeled acetate into fatty acids, we quantitated LPS deacylation as the loss of radiolabeled secondary (laurate and myristate) and primary fatty acids (3-hydroxymyristate) from the LPS backbone. Isolated mononuclear cells and neutrophils removed 50% and 20-30%, respectively, of the secondary acyl chains of the LPS of ingested whole bacteria. When bacteria were killed extracellularly during incubation with ascitic fluid, no LPS deacylation occurred. In this setting, the addition of neutrophils had no effect, but addition of mononuclear cells resulted in removal of >40% of the secondary acyl chains by 20 h. Deacylation of LPS was always restricted to the secondary acyl chains. Thus, in an inflammatory exudate, primarily in mononuclear phagocytes, the LPS in whole bacteria undergoes substantial and selective acyloxyacyl hydrolase-like deacylation, both after phagocytosis of intact bacteria and after uptake of LPS shed from extracellularly killed bacteria. This study demonstrates for the first time that the destruction of Gram-negative bacteria by a mammalian host is not restricted to degradation of phospholipids, protein, and RNA, but also includes extensive deacylation of the envelope LPS.     

A new pathway of exogenous fatty acid incorporation proceeds by a classical phosphoryl transfer reaction

The Firmicute bacteria readily incorporate exogenous fatty acids into their phospholipids. In some (but not all) family members incorporation of the fatty acids present in human serum precludes the use of fatty acid synthesis inhibitors to treat infections. However, the pathway(s) of exogenous fatty acid incorporation in these bacteria remained unknown, although it was thought to differ from known pathways. Parsons and co‐workers show that in Staphylococcus aureus exogenous fatty acids are activated by phosphoryl transfer from ATP to form acyl‐phosphates, a mixed anhydride suggested as a potential intermediate 70 years ago. This finding has important ramifications for the efficacy of treatment of S. aureus infections using inhibitors of fatty acid synthesis.     

Shape change of human erythrocytes induced by phosphatidylcholine and lysophosphatidylcholine species with various acyl chain lengths

Intact human erythrocytes were treated, under non-haemolytic conditions at 37 degrees C, with synthetic phosphatidylcholine which has homologous, saturated acyl chains of 8-18 even-numbered carbon atoms (C8-C18-PC) or with lysophosphatidylcholine which has a saturated acyl chain of 8-18 carbon atoms (C8-C18-lysoPC). The C8-C14-PC and C12-C18-lysoPC species were rapidly incorporated into the erythrocytes and induced a shape change of the crenation (echinocyte formation) type. The site of the incorporation was found to be most probably on the outer leaflet of the membrane lipid bilayer. The extent of the shape change was dependent on the amount of each lipid incorporated. When the same amount of a PC or lysoPC species was incorporated into the membrane, about the same extent of crenation was induced, independent of acyl chain length. However, C16-PC, C18-PC, C8-lysoPC and C10-lysoPC, which were not incorporated into the erythrocytes, did not induce any shape change. It is therefore suggested that the hydrophobic moiety of these amphiphilic lipids may greatly contribute to their transfer from the outer medium into the erythrocyte membrane, but do not influence so much the perturbation of the membrane lipid bilayer which may be responsible for induction of the shape change.     

Phase diagram of deep rough mutant lipopolysaccharide from Salmonella minnesota R595.

The structural polymorphism of deep rough mutant lipopolysaccharide--in many biological systems the most active endotoxin--from Salmonella minnesota strain R595 was investigated as function of temperature, water content, and Mg2+ concentration. Differential scanning calorimetry was used to determine the amount of bound water and the enthalpy change at the beta<==>alpha gel to liquid crystalline acyl chain melting. The onset, midtemperature Tc, and completion of the beta<==>alpha phase transition were studied with Fourier-transform infrared spectroscopy. Synchrotron radiation X-ray diffraction was used to characterize the supramolecular three-dimensional structures in each phase state. The results indicate an extremely complex dependence of the structural behavior of LPS on ambient conditions. The beta<==>alpha acyl chain melting temperature Tc lying at 30 degrees C at high water content (95%) increases with decreasing water content reaching a value of 50 degrees C at 30% water content. Concomitantly, a broadening of the transition range takes place. At still lower water content, no distinct phase transition can be observed. This behavior is even more clearly expressed in the presence of Mg2+. In the lower water concentration range (< 50%) at temperatures below 70 degrees C, only lamellar structures can be observed independent of the Mg2+ concentration. This correlates with the absence of free water. Above 50% water concentration, the supramolecular structure below 70 degrees C strongly depends on the [LPS]:[Mg2+] ratio. For large [LPS]:[Mg2+] ratios, the predominant structure is nonlamellar, for smaller [LPS]:[Mg2+] ratios there is a superposition of lamellar and nonlamellar structures. At an equimolar ratio of LPS and Mg2+ a multibilayered organization is observed. The nonlamellar structures can be assigned in various cases to structures with cubic symmetry with periodicities between 12 and 16 nm. Under all investigated conditions, a transition into the hexagonal II structure takes place between 70 and 80 degrees C. These observations are discussed in relation to the biological importance of LPS as constituent of the outer membrane of gram-negative bacteria and as potent inducer of biological effects in mammals.     

Thermodynamic analysis of the lipopolysaccharide-dependent resistance of gram-negative bacteria against polymyxin B

Cationic antimicrobial cationic peptides (CAMP) have been found in recent years to play a decisive role in hosts' defense against microbial infection. They have also been investigated as a new therapeutic tool, necessary in particular due to the increasing resistance of microbiological populations to antibiotics. The structural basis of the activity of CAMPs has only partly been elucidated and may comprise quite different mechanism at the site of the bacterial cell membranes or in their cytoplasm. Polymyxin B (PMB) is a CAMP which is effective in particular against Gram-negative bacteria and has been well studied with the aim to understand its interaction with the outer membrane or isolated membrane components such as lipopolysaccharide (LPS) and to define the mechanism by which the peptides kill bacteria or neutralize LPS. Since PMB resistance of bacteria is a long-known phenomenon and is attributed to structural changes in the LPS moiety of the respective bacteria, we have performed a thermodynamic and biophysical analysis to get insights into the mechanisms of various LPS/PMB interactions in comparison to LPS from sensitive strains. In isothermal titration calorimetric (ITC) experiments considerable differences of PMB binding to sensitive and resistant LPS were found. For sensitive LPS the endothermic enthalpy change in the gel phase of the hydrocarbon chains converts into an exothermic reaction in the liquid crystalline phase. In contrast, for resistant LPS the binding enthalpy change remains endothermic in both phases. As infrared data show, these differences can be explained by steric changes in the headgroup region of the respective LPS.     

Effects of aromatic residues at the ends of transmembrane alpha-helices on helix interactions with lipid bilayers

We have studied the effects of aromatic residues at the ends of peptides of the type Ac-KKGL(n)()WL(m)()KKA-amide on their interactions with lipid bilayers as a function of lipid fatty acyl chain length, physical phase, and charge. Peptide Ac-KKGFL(6)WL(8)FKKA-amide (F(2)L(14)) incorporated into bilayers of phosphatidylcholines containing monounsaturated fatty acyl chains of lengths C14-C24 at a peptide:lipid molar ratio of 1:100 in contrast to Ac-KKGL(7)WL(9)KKA-amide (L(16)) which did not incorporate at all into dierucoylphosphatidylcholine [di(C24:1)PC]; Ac-KKGYL(6)WL(8)YKKA-amide (Y(2)L(14)) incorporated partly into di(C24:1)PC. Lipid-binding constants relative to that for dioleoylphosphatidylcholine (C18:1)PC were obtained using a fluorescence quenching method. For Y(2)L(14) and F(2)L(14), relative lipid-binding constants increased with increasing fatty acyl chain length from C14 to C24; strongest binding did not occur at the point where the hydrophobic length of the peptide equalled the hydrophobic thickness of the bilayer. For Ac-KKGYL(9)WL(11)YKKA-amide (Y(2)L(20)), increasing chain length from C18 to C24 had little effect on relative binding constants. Anionic phospholipids bound more strongly than zwitterionic phospholipids to Y(2)L(14) and Y(2)L(20) but effects of charge were relatively small. In two phase (gel and liquid crystalline) mixtures, all the peptides partitioned more strongly into liquid crystalline than gel phase; effects were independent of the structure of the peptide or of the lipid (dipalmitoylphosphatidylcholine or bovine brain sphingomyelin). Addition of cholesterol had little effect on incorporation of the peptides into lipid bilayers. It is concluded that the presence of aromatic residues at the ends of transmembrane alpha-helices effectively buffers them against changes in bilayer thickness caused either by an increase in the chain length of the phospholipid or by the presence of cholesterol.     

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.