Cathelicidin and PMB neutralize endotoxins by multifactorial mechanisms including LPS interaction and targeting of host cell membranes.

Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell-directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.

Influence of disulfide bonds in human beta defensin-3 on its strain specific activity against Gram-negative bacteria.

Antimicrobial peptides (AMPs) play an important role in the host defense against various microbes. One of the most efficient human AMPs is the human beta defensin-3 (hBD-3) which is produced by, e.g. keratinocytes and lung epithelial cells. However, the structure-function relationship for AMPs and in particular for defensins with their typical three disulfide bonds is still poorly understood. In this study the importance of the three disulfide bonds for the activity of the AMPs is investigated with biological assays and with biophysical experiments utilizing different membrane reconstitution systems. The activities of natural hBD-3, hBD-3-c (cyclic variant with one disulfide bond), and hBD-3-l (linear variant without disulfide bonds) and fragments thereof were tested against specific Gram-negative bacteria. Furthermore, hemolytic and cytotoxic activities were analyzed as well as the potency to neutralize immune cell stimulation of lipopolysaccharide (LPS). Experiments using reconstituted lipid matrices composed of phospholipids or LPS purified from the respective Gram-negative bacteria, showed that the membrane activity of all three hBD-3 peptides is decisive for their capability to kill bacteria and to neutralize LPS. In most of the test systems the linear hBD-3-l showed the highest activity. It was also the only peptide significantly active against polymyxin B-resistant Proteus mirabilis R45. However, the stability of hBD-3 against protease activity decreases with decreasing number of disulfide bonds. This study demonstrates that the refining of AMP structures can generate more active compounds against certain strains.

Antibacterial action of synthetic antilipopolysaccharide peptides (SALP) involves neutralization of both membrane-bound and free toxins.

Increasing failure of conventional antibiotics to combat bacterial infections requires the urgent development of new antibacterial drugs; a promising class of new drugs based on antimicrobial peptides. Here, we studied the molecular interaction of polycationic synthetic antilipopolysaccharide peptides (SALPs) with various gram-negative and gram-positive bacteria, including resistant strains. The analysis of antimicrobial activity by conventional techniques and atomic force microscopy showed a strict dependence on amino acid (aa) sequences, with the type of amino acid, its position within the primary structure, and the sequence length being critical parameters. By monitoring lipopolysaccharide (LPS)- or bacteria-induced cytokine production in human mononuclear cells and whole blood, we found a direct link between the binding of the lead compound Pep19-2.5 to Salmonella enterica and the anti-inflammatory activity of the peptide. Thermodynamic analysis of Pep19-2.5 binding to the bacterial cell envelope showed an exothermic reaction with saturation characteristics, whereas small-angle X-ray scattering data indicated a direct attachment of Pep19-2.5 to the bacterial cell envelope. This binding preferentially takes place to the LPS outer monolayer, as evidenced by the change in the LPS acyl chain and phosphate vibrational bands seen by Fourier-transform infrared spectroscopy. We report here that the anti-inflammatory activity of Pep19-2.5 is not only connected with neutralization of cell-free bacterial toxins but also with a direct binding of the peptide to the outer leaflet of the bacterial outer membrane.

Enhancing actions of peptides derived from the γ-chain of fetal human hemoglobin on the immunostimulant activities of monophosphoryl lipid A.

Hemoglobin and its structures have been described since the 1990s to enhance a variety of biological activities of endotoxins (LPS) in a dose-dependent manner. To investigate the interaction processes in more detail, the system was extended by studying the interactions of newly designed peptides from the γ-chain of human hemoglobin with the adjuvant monophosphoryl lipid A (MPLA), a partial structure of lipid A lacking its 1-phosphate. It was found that some selected Hbg peptides, in particular two synthetic substructures designated Hbg32 and Hbg35, considerably increased the bioactivity of MPLA, which alone was only a weak activator of immune cells. These findings hold true for human mononuclar cells, monocytes and T lymphocytes. To understand the mechanisms of action in more detail, biophysical techniques were applied. These showed a peptide-induced change of the MPLA aggregate structure from multilamellar into a non-lamellar, probably inverted, cubic structure. Concomitantly, the peptides incorporated into the tightly packed MPLA aggregates into smaller units down to monomers. The fragmentation of the aggregates was an endothermic process, differing from a complex formation but rather typical for a catalytic reaction.

Self-Organisation, Thermotropic and Lyotropic Properties of Glycolipids Related to their Biological Implications.

Glycolipids are amphiphilic molecules which bear an oligo- or polysaccharide as hydrophilic head group and hydrocarbon chains in varying numbers and lengths as hydrophobic part. They play an important role in life science as well as in material science. Their biological and physiological functions are quite diverse, ranging from mediators of cell-cell recognition processes, constituents of membrane domains or as membrane-forming units. Glycolipids form an exceptional class of liquid-crystal mesophases due to the fact that their self-organisation obeys more complex rules as compared to classical monophilic liquid-crystals. Like other amphiphiles, the supra-molecular structures formed by glycolipids are driven by their chemical structure; however, the details of this process are still hardly understood. Based on the synthesis of specific glycolipids with a clearly defined chemical structure, e.g., type and length of the sugar head group, acyl chain linkage, substitution pattern, hydrocarbon chain lengths and saturation, combined with a profound physico-chemical characterisation of the formed mesophases, the principles of the organisation in different aggregate structures of the glycolipids can be obtained. The importance of the observed and formed phases and their properties are discussed with respect to their biological and physiological relevance. The presented data describe briefly the strategies used for the synthesis of the used glycolipids. The main focus, however, lies on the thermotropic as well as lyotropic characterisation of the self-organised structures and formed phases based on physico-chemical and biophysical methods linked to their potential biological implications and relevance.

Intestinal mucus affinity and biological activity of an orally administered antibacterial and anti-inflammatory peptide.

OBJECTIVE: Antimicrobial peptides (AMP) provide protection from infection by pathogenic microorganisms and restrict bacterial growth at epithelial surfaces to maintain mucosal homeostasis. In addition, they exert a significant anti-inflammatory activity. Here we analysed the anatomical distribution and biological activity of an orally administered AMP in the context of bacterial infection and host-microbial homeostasis. DESIGN: The anatomical distribution as well as antibacterial and anti-inflammatory activity of the endogenous AMP cryptdin 2 and the synthetic peptide Pep19-2.5 at the enteric mucosal surface were analysed by immunostaining, functional viability and stimulation assays, an oral Salmonella enterica subsp. enterica sv. Typhimurium (S. Typhimurium) model and comparative microbiota analysis. RESULTS: Endogenous cryptdin 2 was found attached to bacteria of the enteric microbiota within the intestinal mucus layer. Similarly, the synthetic peptide Pep19-2.5 attached rapidly to bacterial cells, exhibited a marked affinity for the intestinal mucus layer in vivo, altered the structural organisation of endotoxin in a mucus matrix and demonstrated potent anti-inflammatory and antibacterial activity. Oral Pep19-2.5 administration induced significant changes in the composition of the enteric microbiota as determined by high-throughput 16S rDNA sequencing. This may have contributed to the only transient improvement of the clinical symptoms after oral infection with S. Typhimurium. CONCLUSIONS: Our findings demonstrate the anti-inflammatory activity and mucus affinity of the synthetic AMP Pep19-2.5 and characterise the influence on microbiota composition and enteropathogen infection after oral administration.

Galleria mellonella native and analogue peptides Gm1 and ΔGm1. I) biophysical characterization of the interaction mechanisms with bacterial model membranes.

Natural occurring antimicrobial peptides (AMPs) are important components of the innate immune system of animals and plants. They are considered to be promising alternatives to conventional antibiotics. Here we present a comparative study of two synthetic peptides: Gm1, corresponding to the natural overall uncharged peptide from Galleria mellonella (Gm) and ΔGm1, a modified overall positively charged Gm1 variant. We have studied the interaction of the peptides with lipid membranes composed of different kinds of lipopolysaccharides (LPS) and dimyristoylphosphatidylglycerol (DMPG), in some cases also dimyristoylphosphatidylethanolamine (DMPE) as representative lipid components of Gram-negative bacterial membranes, by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). Gm1 generates a destabilizing effect on the gel to liquid crystalline phase transition of the acyl chains of the lipids, as deduced from a decrease in the phase transition temperature and enthalpy, suggesting a fluidization, whereas ΔGm1 led to the opposite behavior. Further, FTIR analysis of the functional groups of the lipids participating in the interaction with the peptides indicated a shift in the band position and intensity of the asymmetric PO2(-) stretching vibration originating from the lipid phosphate groups, a consequence of the sterical changes in the head group region. Interestingly, FRET spectroscopy showed a similar intercalation of both peptides into the DMPG and LPS, but much less into the DMPE membrane systems. These results are discussed in the light of a possible use of the peptides as antimicrobial and anti-endotoxin drugs.

Biophysical analysis of the interaction of the serum protein human ß2GPI with bacterial lipopolysaccharide.

There are several human serum proteins for which no clear role is yet known. Among these is the abundant serum protein beta2-glycoprotein-I (ß2GPI), which is known to bind to negatively charged phospholipids as well as to bacterial lipopolysaccharides (LPS), and was therefore proposed to play a role in the immune response. To understand the details of these interactions, a biophysical analysis of the binding of ß2GPI to LPS and phosphatidylserine (PS) was performed. The data indicate only a moderate tendency of the protein (1) to influence the LPS-induced cytokine production in vitro, (2) to react exothermally with LPS in a non-saturable way, and (3) to change its local microenvironment upon LPS association. Additionally, we found that the protein binds more strongly to phosphatidylserine (PS) than to LPS. Furthermore, ß2GPI converts the LPS bilayer aggregates into a stronger multilamellar form, and reduces the fluidity of the hydrocarbon moiety of LPS due to a rigidification of the acyl chains. From these data it can be concluded that ß2GPI plays a role as an immune-modulating agent, but there is much less evidence for a role in immune defense against bacterial toxins such as LPS.

Biophysical mechanisms of the neutralization of endotoxins by lipopolyamines.

Endotoxins (lipopolysaccharides, LPS) are one of the strongest immunostimulators in nature, responsible for beneficial effects at low, and pathophysiological effects at high concentrations, the latter frequently leading to sepsis and septic shock associated with high mortality in critical care settings. There are no drugs specifically targeting the pathophysiology of sepsis, and new therapeutic agents are therefore urgently needed. The lipopolyamines are a novel class of small molecules designed to sequester and neutralize LPS. To understand the mechanisms underlying the binding and neutralization of LPS toxicity, we have performed detailed biophysical analyses of the interactions of LPS with candidate lipopolyamines which differ in their potencies of LPS neutralization. We examined gel-to-liquid crystalline phase behavior of LPS and of its supramolecular aggregate structures in the absence and presence of lipopolyamines, the ability of such compounds to incorporate into different membrane systems, and the thermodynamics of the LPS:lipopolyamine binding. We have found that the mechanisms which govern the inactivation process of LPS obey similar rules as found for other active endotoxin neutralizers such as certain antimicrobial peptides.

Cellular distribution of lipid A and LPS R595 after in vitro application to isolated human monocytes by freeze-fracture replica immunogold-labelling.

We have performed freeze-fracture replica immunogold labelling of endotoxin preparations (lipid A and deep rough mutant LPS Re from Salmonella enterica sv. Minnesota), i.e. adding the endotoxins to human monocytes, labelling with monoclonal Abs recognizing either lipid A or LPS Re (A6 and A20 respectively), and fixing with immunogold secondary Ab. We have found that the endotoxins intercalated into the cell membranes with subsequent internalization by the cells. Surprisingly, membrane uptake took place only in the inner, plasmic leaflet of the plasma membrane, but there was no uptake of the outer leaflet for both compounds. Remarkable labelling could be also found for the two membranes of the nuclear envelope-in the case of lipid A only at the plasmic leaflet, but in the case of LPS Re on both leaflets. Isothermal calorimetric titration of the AB A20 with LPS and phospholipids showed that the Ab may bind not only to LPS but also to negatively charged phosphatidylserine. These results are discussed in the frame of the published concepts of cell activation induced by the endotoxins, i.e. how they are able to cause a conformational change of signalling proteins, such as the TLR4/MD2 complex.

Preclinical investigations reveal the broad-spectrum neutralizing activity of peptide Pep19-2.5 on bacterial pathogenicity factors.

Bacterial infections are known to cause severe health-threatening conditions, including sepsis. All attempts to get this disease under control failed in the past, and especially in times of increasing antibiotic resistance, this leads to one of the most urgent medical challenges of our times. We designed a peptide to bind with high affinity to endotoxins, one of the most potent pathogenicity factors involved in triggering sepsis. The peptide Pep19-2.5 reveals high endotoxin neutralization efficiency in vitro, and here, we demonstrate its antiseptic/anti-inflammatory effects in vivo in the mouse models of endotoxemia, bacteremia, and cecal ligation and puncture, as well as in an ex vivo model of human tissue. Furthermore, we show that Pep19-2.5 can bind and neutralize not only endotoxins but also other bacterial pathogenicity factors, such as those from the Gram-positive bacterium Staphylococcus aureus. This broad neutralization efficiency and the additive action of the peptide with common antibiotics makes it an exceptionally appropriate drug candidate against bacterial sepsis and also offers multiple other medication opportunities.

Bacterial cell wall compounds as promising targets of antimicrobial agents II. Immunological and clinical aspects.

The bacterial cell wall represents the primary target for antimicrobial agents. Microbial destruction is accompanied by the release of potent immunostimulatory membrane constituents. Both Gram-positive and Gram-negative bacteria release a variety of lipoproteins and peptidoglycan fragments. Gram-positive bacteria additionally provide lipoteichoic acids, whereas Gram-negative bacteria also release lipopolysaccharide (LPS, endotoxin), essential component of the outer leaflet of the bacterial cell wall and one of the most potent immunostimulatory molecules known. Immune activation therefore can be considered as an adverse effect of antimicrobial destruction and killing during anti-infective treatment. In contrast to antibiotics, the use of cationic amphiphilic antimicrobial peptides allows both effective bacterial killing and inhibition of the immunostimulatory effect of the released bacterial membrane constituents. The administration of antimicrobial peptides alone or in combination with antibiotic agents thus represents a novel strategy in the antiinfective treatment with potentially important beneficial aspects. Here, data are presented which describe immunological and clinical aspects of the use of antimicrobial peptides (AMPs) as therapeutic agents to treat bacterial infection and neutralize the immunostimulatory activity of released cell wall constituents.

Bacterial cell wall compounds as promising targets of antimicrobial agents I. Antimicrobial peptides and lipopolyamines.

The first barrier that an antimicrobial agent must overcome when interacting with its target is the microbial cell wall. In the case of Gram-negative bacteria, additional to the cytoplasmic membrane and the peptidoglycan layer, an outer membrane (OM) is the outermost barrier. The OM has an asymmetric distribution of the lipids with phospholipids and lipopolysaccharide (LPS) located in the inner and outer leaflets, respectively. In contrast, Gram-positive bacteria lack OM and possess a much thicker peptidoglycan layer compared to their Gram-negative counterparts. An additional class of amphiphiles exists in Gram-positives, the lipoteichoic acids (LTA), which may represent important structural components. These long molecules cross-bridge the entire cell envelope with their lipid component inserting into the outer leaflet of the cytoplasmic membrane and the teichoic acid portion penetrating into the peptidoglycan layer. Furthermore, both classes of bacteria have other important amphiphiles, such as lipoproteins, whose importance has become evident only recently. It is not known yet whether any of these amphiphilic components are able to stimulate the immune system under physiological conditions as constituents of intact bacteria. However, all of them have a very high pro-inflammatory activity when released from the cell. Such a release may take place through the interaction with the immune system, or with antibiotics (particularly with those targeting cell wall components), or simply by the bacterial division. Therefore, a given antimicrobial agent must ideally have a double character, namely, it must overcome the bacterial cell wall barrier, without inducing the liberation of the pro-inflammatory amphiphiles. Here, new data are presented which describe the development and use of membrane-active antimicrobial agents, in particular antimicrobial peptides (AMPs) and lipopolyamines. In this way, essential progress was achieved, in particular with respect to the inhibition of deleterious consequences of bacterial infections such as severe sepsis and septic shock.

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.

Physicochemical and biological characterization of anti-endotoxin peptides and their influence on lipid properties.

We have synthesized a series of short peptides (17 to 20 amino acids), originally derived from Limulus anti-lipopolysaccharide factor LALF, which were primarily designed to act as antimicrobial agents as well as neutralizers of bacterial endotoxin (lipopolysaccharide, LPS), Here, two selected peptides, a 17- and a 19-mer, were characterized physicochemically and in biological test systems. The secondary structure of the peptides indicates essentially a ß-sheet including antiparallel strands, the latter being reduced when the peptides bind to LPS. A very strong exothermic binding due to attractive Coulomb interactions governs the LPS-peptide reaction, which additionally leads to a fluidization of the acyl chains of LPS. A comparison of the interaction of the peptide with negatively charged phosphatidylserine shows in contrast a rigidification of the acyl chains of the lipid. Finally, the biological assays reveal a diverging behaviour of the two peptides, with higher antibacterial activity of the 17-mer, but a much higher activity of the 19-mer in its ability to inhibit the LPS-induced cytokine production in human mononuclear cells.

New antiseptic peptides to protect against endotoxin-mediated shock.

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.