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.The life-threatening clinical consequences of sepsis and septic shock arise from recognition of microbial immunostimulatory molecules by the hosts' professional immune cells and the release of hemodynamically active mediators. The most potent immunostimulatory constituents are part of the microbial cell envelope, such as lipopolysaccharide (LPS) or lipoproteins. They are released continuously due to cell growth and division and massively liberated as a consequence of the attack of the immune system. In the case of Gram-negative bacteria, the most potent factor is LPS, which, therefore, is also called an endotoxin. LPS concentrations in blood serum as low as 1 ng/ml are able to cause sepsis. Septic shock resulting from bacterial infection remains a frequent cause of death, particularly in intensive care units, with more than 200,000 people dying each year in the United States alone. Death by septic shock can happen despite appropriate broad-range antibiotic treatment, which may kill bacteria but is not only incapable of neutralizing immunostimulatory LPS but also may promote its release into circulation (11).The response of mammalian cells to LPS is initiated by its interaction with serum proteins such as lipopolysaccharidebinding protein (LBP) and specific receptors and/or binding proteins of immune cells such as soluble CD14 (sCD14) and membrane-bound CD14 (mCD14), which finally leads to cell activation through the Toll-like receptor 4 (TLR4)-MD-2 pathway (31). The hydrophobic moiety of LPS, lipid A, anchoring LPS to the bacterial outer membrane, constitutes the "endotoxic principle" of LPS (24). Enterobacterial lipid A consists of a diglucosamine backbone phosphorylated at positions 1 and 4Ј, to which six acyl chains are linked at positions 2,3 a...

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.

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 exist 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.

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 β-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.

Background: Pseudomonas aeruginosa is a Gram-negative pathogen that frequently causes life-threatening infections in immunocompromised patients. We previously showed that subinhibitory concentrations of short synthetic peptides permeabilize P. aeruginosa and enhance the lethal action of co-administered antibiotics. Methods: Long-term permeabilization caused by exposure of multidrug-resistant P. aeruginosa strains to peptide P4-9 was investigated by measuring the uptake of several antibiotics and fluorescent probes and by using confocal imaging and atomic force microscopy. Results: We demonstrated that P4-9, a 13-amino acid peptide, induces a growth delay (i.e. post-antibiotic effect) of 1.3 h on a multidrug-resistant P. aeruginosa clinical isolate. Remarkably, when an independently P4-9-treated culture was allowed to grow in the absence of the peptide, cells remained sensitive to subinhibitory concentrations of antibiotics such as ceftazidime, fosfomycin and erythromycin for at least 2 h. We designated this persistent sensitization to antibiotics occurring in the absence of the sensitizing agent as Post-Antibiotic Effect associated Permeabilization (PAEP). Using atomic force microscopy, we showed that exposure to P4-9 induces profound alterations on the bacterial surface and that treated cells need at least 2 h of growth to repair those lesions. During PAEP, P. aeruginosa mutants overexpressing either the efflux pump MexAB-OprM system or the AmpC β-lactamase were rendered sensitive to antibiotics that are known substrates of those mechanisms of resistance. Finally, we showed for the first time that the descendants of bacteria surviving exposure to a membrane disturbing peptide retain a significant level of permeability to hydrophobic compounds, including propidium iodide, even after 20 h of growth in the absence of the peptide. Conclusions: The phenomenon of long-term sensitization to antibiotics shown here may have important therapeutic implications for a combined peptide-antibiotic treatment because the peptide would not need to be present to exert its antibiotic enhancing activity as long as the target organism retains sensitization to the antibiotic.