Jörg Andrä

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.

Biophysical characterization of endotoxin inactivation by NK-2, an antimicrobial peptide derived from mammalian NK-lysin.

ABSTRACT NK-2, a membrane-acting antimicrobial peptide, was derived from the cationic core region of porcine NK-lysin and consists of 27 amino acid residues. It adopts an amphipathic, α-helical secondary structure and has been shown to interact specifically with membranes of negatively charged lipids. We therefore investigated the interaction of NK-2 with lipopolysaccharide (LPS), the main, highly anionic component of the outer leaflet of the outer membrane of gram-negative bacteria, by means of biophysical and biological assays. As model organisms and a source of LPS, we used Salmonella enterica strains with various lengths of the LPS carbohydrate moiety, including smooth LPS, rough LPS, and deep rough LPS (LPS Re) mutant strains. NK-2 binds to LPS Re with a high affinity and induces a change in the endotoxin-lipid A aggregate structure from a cubic or unilamellar structure to a multilamellar one. This structural change, in concert with a significant overcompensation of the negative charges of LPS, is thought to result in the neutralization of the endotoxic LPS activity in a cell culture system. Neutralization of LPS activity by NK-2 as well as its antibacterial activity against the various Salmonella strains strongly depends on the length of the sugar chains of LPS, with LPS Re being the most sensitive. This suggests that a hydrophobic peptide-LPS interaction is necessary for efficient neutralization of the biological activity of LPS and that the long carbohydrate chains, besides their function as a barrier for hydrophobic drugs, also serve as a trap for polycationic substances.

Amoebapores, archaic effector peptides of protozoan origin, are discharged into phagosomes and kill bacteria by permeabilizing their membranes.

Antimicrobial peptides are widespread in animal species and their function as defensive molecules may even have appeared before the evolution of metazoa. The amoeboid protozoon Entamoeba histolytica discharge membrane-permeabilizing polypeptides named amoebapores into the phagosome in which engulfed bacteria are situated as evidenced here by confocal laser microscopy and electron microscopy using specific antibodies. We demonstrate that the purified three isoforms of the amoebic polypeptides exhibit complementary antibacterial activities in vitro. The potency of amoebapores were compared with that of antimicrobial peptides of phylogenetically widespread species by monitoring in parallel their activities against representatives of gram-positive and gram-negative bacteria and liposomes in various assays, and differences in the mechanism of membrane permeabilization became apparent. Northern blot analysis revealed that expression of genes coding for amoebapores and amoebic lysozymes is not dramatically changed upon co-culture of amoebae with bacteria indicating that the antimicrobial arsenal is rather constitutively expressed than induced in these primitive phagocytes.

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.

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

Abstract Here we report on the purification, structural characterization, and biological activity of a glycolipid, 2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-α(R)-3-hydroxytetradecanoyl-(R)-3-hydroxytetradecanoate (RL-2,214) produced by Burkholderia (Pseudomonas) plantarii. RL-2,214 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-2,214 exhibits strong stimulatory activity on human mononuclear cells to produce tumor necrosis factor α, 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-2,214 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-2,214 does nor occur via receptors that are involved in LPS (TLR4) or lipopeptide signaling (TLR2). Despite its completely different chemical structure, RL-2,214 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.

Rationale for the design of shortened derivatives of the NK-lysin-derived antimicrobial peptide NK-2 with improved activity against Gram-negative pathogens.

The peptide NK-2 is an effective antimicrobial agent with low hemolytic and cytotoxic activities and is thus a promising candidate for clinical applications. It comprises the α-helical, cationic core region of porcine NK-lysin a homolog of human granulysin and of amoebapores of pathogenic amoeba. Here we visualized the impact of NK-2 on Escherichia coli by electron microscopy and used NK-2 as a template for sequence variations to improve the peptide stability and activity and to gain insight into the structure/function relationships. We synthesized 18 new peptides and tested their activities on seven Gram-negative and one Gram-positive bacterial strains, human erythrocytes, and HeLa cells. Although all peptides appeared unordered in buffer, those active against bacteria adopted an α-helical conformation in membrane-mimetic environments like trifluoroethanol and negatively charged phosphatidylglycerol (PG) liposomes that mimick the cytoplasmic membrane of bacteria. This conformation was not observed in the presence of liposomes consisting of zwitterionic phosphatidylcholine (PC) typical for the human cell plasma membrane. The interaction was paralleled by intercalation of these peptides into PG liposomes as determined by FRET spectroscopy. A comparative analysis between biological activity and the calculated peptide parameters revealed that the decisive factor for a broad spectrum activity is not the peptide overall hydrophobicity or amphipathicity, but the possession of a minimal positive net charge plus a highly amphipathic anchor point of only seven amino acid residues (two helical turns).

Physicochemical characterization of the endotoxins from Coxiella burnetii strain Priscilla in relation to their bioactivities.

BackgroundCoxiella burnetii is the etiological agent of Q fever found worldwide. The microorganism has like other Gram-negative bacteria a lipopolysaccharide (LPS, endotoxin) in its outer membrane, which is important for the pathogenicity of the bacteria. In order to understand the biological activity of LPS, a detailed physico-chemical analysis of LPS is of utmost importace.ResultsThe lipid A moiety of LPS is tetraacylated and has longer (C-16) acyl chains than most other lipid A from enterobacterial strains. The two ester-linked 3-OH fatty acids found in the latter are lacking. The acyl chains of the C. burnetii endotoxins exhibit a broad melting range between 5 and 25°C for LPS and 10 and 40°C for lipid A. The lipid A moiety has a cubic inverted aggregate structure, and the inclination angle of the D-glucosamine disaccharide backbone plane of the lipid A part with respect to the membrane normal is around 40°. Furthermore, the endotoxins readily intercalate into phospholipid liposomes mediated by the lipopolysaccharide-binding protein (LBP). The endotoxin-induced tumor necrosis factor α (TNFα) production in human mononuclear cells is one order of magnitude lower than that found for endotoxins from enterobacterial strains, whereas the same activity as in the latter compounds is found in the clotting reaction of the Limulus amebocyte lysate assay.ConclusionsDespite a considerably different chemical primary structure of the C. burnetii lipid A in comparison with enterobacterial lipid A, the data can be well understood by applying the previously presented conformational concept of endotoxicity, a conical shape of the lipid A moiety of LPS and a sufficiently high inclination of the sugar backbone plane with respect to the membrane plane. Importantly, the role of the acyl chain fluidity in modulating endotoxicity now becomes more evident.

Candidacidal activity of shortened synthetic analogs of amoebapores and NK-lysin

Abstract Natural antimicrobial peptides and synthetic analogs thereof have emerged as compounds with potentially significant therapeutical application against human pathogens. Amoebapores are 77-residue peptides with cytolytic and antibacterial activity considered to act by forming ion channels in cytoplasmic membranes of the victim cells. A functionally and structurally similar peptide named NK-lysin exists in mammalian lymphocytes. Several synthetic analogs of amoebapores and NK-lysin, which are substantially reduced in size compared to the parent molecules, were tested for their ability to inhibit the growth of and to kill Candida albicans. Some of the peptides displayed potent activity against a clinical isolate as well as against defined culture strains. Among the most active peptides found are some shortened substitution analogs of amoebapore C and a cationic core region of NK-lysin. As these peptides are also highly active against Gram-positive and Gram-negative bacteria but are of low cytotoxicity towards a human keratinocyte cell line they may provide promising templates for the design of broad-spectrum peptide antibiotics.

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.

Multiple peptide resistance factor (MPRF)-mediated resistance of staphylococcus aureus against antimicrobial peptides coincides with a modulated peptide interaction with artificial membranes comprising lysyl-phosphatidylglycerol

Modification of the membrane lipid phosphatidylglycerol (PG) of Staphylococcus aureus by enzymatic transfer of a l-lysine residue leading to lysyl-PG converts the net charge of PG from −1 to +1 and is thought to confer resistance to cationic antimicrobial peptides (AMPs). Lysyl-PG synthesis and translocation to the outer leaflet of the bacterial membrane are achieved by the membrane protein MprF. Consequently, mutants lacking a functional mprF gene are in particular vulnerable to the action of AMPs. Hence, we aim at elucidating whether and to which extent lysyl-PG modulates membrane binding, insertion, and permeabilization by various AMPs. Lysyl-PG was incorporated into artificial lipid bilayers, mimicking the cytoplasmic membrane of S. aureus. Moreover, we determined the activity of the peptides against a clinical isolate of S. aureus strain SA113 and two mutants lacking a functional mprF gene and visualized peptide-induced ultrastructural changes of bacteria by transmission electron microscopy. The studied peptides were: (i) NK-2, an α-helical fragment of mammalian NK-lysin, (ii) arenicin-1, a lugworm β-sheet peptide, and (iii) bee venom melittin. Biophysical data obtained by FRET spectroscopy, Fourier transform infrared spectroscopy, and electrical measurements with planar lipid bilayers were correlated with the biological activities of the peptides. They strongly support the hypothesis that peptide-membrane interactions are a prerequisite for eradication of S. aureus. However, degree and mode of modulation of membrane properties such as fluidity, capacitance, and conductivity were unique for each of the peptides. Altogether, our data support and underline the significance of lysyl-PG for S. aureus resistance to AMPs.