Thomas Gutsmann

The mode of action of the lantibiotic lacticin 3147 - a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II

Lacticin 3147 is a two‐peptide lantibiotic produced by Lactococcus lactis in which both peptides, LtnA1 and LtnA2, interact synergistically to produce antibiotic activities in the nanomolar concentration range; the individual peptides possess marginal (LtnA1) or no activity (LtnA2). We analysed the molecular basis for the synergism and found the cell wall precursor lipid II to play a crucial role as a target molecule. Tryptophan fluorescence measurements identified LtnA1, which is structurally similar to the lantibiotic mersacidin, as the lipid II binding component. However, LtnA1 on its own was not able to substantially inhibit cell wall biosynthesis in vitro; for full inhibition, LtnA2 was necessary. Both peptides together caused rapid K+ leakage from intact cells; in model membranes supplemented with lipid II, the formation of defined pores with a diameter of 0.6 nm was observed. We propose a mode of action model in which LtnA1 first interacts specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. The resulting lipid II:LtnA1 complex is then able to recruit LtnA2 which leads to a high‐affinity, three‐component complex and subsequently inhibition of cell wall biosynthesis combined with pore formation.

Dual Role of Lipopolysaccharide (LPS)-Binding Protein in Neutralization of LPS and Enhancement of LPS-Induced Activation of Mononuclear Cells

ABSTRACT The lipopolysaccharide (LPS)-binding protein (LBP) has a concentration-dependent dual role in the pathogenesis of gram-negative sepsis: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation. In stimulation experiments, we have found that LBP mediates the LPS-induced cytokine release from MNC even under serum-free conditions. In biophysical experiments we demonstrated that LBP binds and intercalates into lipid membranes, amplified by negative charges of the latter, and that intercalated LBP can mediate the CD14-independent intercalation of LPS into membranes in a lipid-specific and temperature-dependent manner. In contrast, prior complexation of LBP and LPS inhibited binding of these complexes to membranes due to different binding of LBP to LPS or phospholipids. This results in a neutralization of LPS and, therefore, to a reduced production of tumor necrosis factor by MNC. We propose that LBP is not only present as a soluble protein in the serum but may also be incorporated as a transmembrane protein in the cytoplasmic membrane of MNC and that the interaction of LPS with membrane-associated LBP may be an important step in LBP-mediated activation of MNC, whereas LBP-LPS complexation in the serum leads to a neutralization of LPS.

Hydramacin-1, Structure and Antibacterial Activity of a Protein from the Basal Metazoan Hydra

Hydramacin-1 is a novel antimicrobial protein recently discovered during investigations of the epithelial defense of the ancient metazoan Hydra. The amino acid sequence of hydramacin-1 shows no sequence homology to any known antimicrobial proteins. Determination of the solution structure revealed that hydramacin-1 possesses a disulfide bridge-stabilized αβ motif. This motif is the common scaffold of the knottin protein fold. The structurally closest relatives are the scorpion oxin-like superfamily. Within this superfamily hydramacin-1 establishes a new family of proteins that all share antimicrobial activity. Hydramacin-1 is potently active against Gram-positive and Gram-negative bacteria including multi-resistant human pathogenic strains. It leads to aggregation of bacteria as an initial step of its bactericidal mechanism. Aggregated cells are connected via electron-dense contacts and adopt a thorn apple-like morphology. Analysis of the hydramacin-1 structure revealed an unusual distribution of amino acid side chains on the surface. A belt of positively charged residues is sandwiched by two hydrophobic areas. Based on this characteristic surface feature and on biophysical analysis of protein-membrane interactions, we propose a model that describes the aggregation effect exhibited by hydramacin-1.

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.

New Antiseptic Peptides To Protect against Endotoxin-Mediated Shock

ABSTRACT 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.

Structure and mode of action of the antimicrobial peptide arenicin

The solution structure and the mode of action of arenicin isoform 1, an antimicrobial peptide with a unique 18-residue loop structure, from the lugworm Arenicola marina were elucidated here. Arenicin folds into a two-stranded antiparallel beta-sheet. It exhibits high antibacterial activity at 37 and 4 degrees C against Gram-negative bacteria, including polymyxin B-resistant Proteus mirabilis. Bacterial killing occurs within minutes and is accompanied by membrane permeabilization, membrane detachment and release of cytoplasm. Interaction of arenicin with reconstituted membranes that mimic the lipopolysaccharide-containing outer membrane or the phospholipid-containing plasma membrane of Gram-negative bacteria exhibited no pronounced lipid specificity. Arenicin-induced current fluctuations in planar lipid bilayers correspond to the formation of short-lived heterogeneously structured lesions. Our results strongly suggest that membrane interaction plays a pivotal role in the antibacterial activity of arenicin.

A New Class of Synthetic Peptide Inhibitors Blocks Attachment and Entry of Human Pathogenic Viruses

Many enveloped viruses, including herpes viruses, hepatitis B virus (HBV), and hepatitis C virus (HCV), and human immunodeficiency virus (HIV), are among the most important human pathogens and are often responsible for coinfections involving ≥2 types of viruses. However, therapies that are effective against multiple virus classes are rare. Here we present a new class of synthetic anti-lipopolysaccharide peptides (SALPs) that bind to heparan sulfate moieties on the cell surface and inhibit infection with a variety of enveloped viruses. We demonstrate that SALPs inhibit entry of human immunodeficiency virus type 1 (HIV-1), herpes simplex virus (HSV) 1 and 2, HBV, and HCV to their respective host cells. Despite their high antiviral efficiency, SALPs were well tolerated, and neither toxicity nor measurable inhibitor-induced adverse effects were observed. Since these broad-spectrum antiviral peptides target a host cell rather than a viral component, they may also be useful for suppression of viruses that are resistant to antiviral drugs.

Structure-activity analysis of the dermcidin-derived peptide DCD 1L, an anionic antimicrobial peptide present in human sweat

Background: The anionic DCD-1L is an antimicrobial peptide active in human sweat. Results: DCD-1L forms cation stabilized oligomeric ion channels. Conclusion: DCD-1L kills bacteria by forming oligomeric ion channels. Significance: The anionic antimicrobial peptide DCD-1L is optimally adapted to the conditions in human sweat. Dermcidin encodes the anionic amphiphilic peptide DCD-1L, which displays a broad spectrum of antimicrobial activity under conditions resembling those in human sweat. Here, we have investigated its mode of antimicrobial activity. We found that DCD-1L interacts preferentially with negatively charged bacterial phospholipids with a helix axis that is aligned flat on a lipid bilayer surface. Upon interaction with lipid bilayers DCD-1L forms oligomeric complexes that are stabilized by Zn2+. DCD-1L is able to form ion channels in the bacterial membrane, and we propose that Zn2+-induced self-assembly of DCD-1L upon interaction with bacterial lipid bilayers is a prerequisite for ion channel formation. These data allow us for the first time to propose a molecular model for the antimicrobial mechanism of a naturally processed human anionic peptide that is active under the harsh conditions present in human sweat.

Interaction of CAP18-Derived Peptides with Membranes Made from Endotoxins or Phospholipids

Antimicrobial peptides with alpha-helical structures and positive net charges are in the focus of interest with regard to the development of new antibiotic agents, in particular against Gram-negative bacteria. Interaction between seven polycationic alpha-helical CAP18-derived peptides and different types of artificial membranes composed of phosphatidylcholine or lipopolysaccharide of the Gram-negative bacterium Escherichia coli were investigated using different biophysical techniques. Results obtained from fluorescence energy transfer spectroscopy with liposomes, monolayer measurements on a Langmuir trough, and electrophysiological measurements on planar reconstituted asymmetric bilayer membranes including the lipid matrix of the outer membrane of E. coli were correlated, and these data were, furthermore, correlated with structural parameters of the peptides (net charge, alpha-helical content, hydrophobic moment, and hydrophobicity). All peptides induced current fluctuations in planar membranes due to the formation of transient lesions above a peptide- and lipid-specific minimal clamp voltage. Antibacterial activity was exhibited only by those peptides that induced lesion formation in the reconstituted outer membrane at clamp voltages below the transmembrane potential of the natural membrane. Thus, we propose that the physicochemical properties of both the peptides as well as of the target membranes are important for antibacterial activity.

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.