In the last decade intensive research has been conducted to determine the role of innate immunity host defense peptides (also termed antimicrobial peptides) in the killing of prokaryotic and eukaryotic cells. Many antimicrobial peptides damage the cellular membrane as part of their killing mechanism. However, it is not clear what makes cancer cells more susceptible to some of these peptides, and what the molecular mechanisms underlying these activities are. Two general mechanisms were suggested: (i) plasma membrane disruption via micellization or pore formation, and (ii) induction of apoptosis via mitochondrial membrane disruption. To be clinically used, these peptides need to combine high and specific anticancer activity with stability in serum. Although so far very limited, new studies have paved the way for promising anticancer host defense peptides with a new mode of action and with a broad spectrum of anticancer activity.
Binding of lipopolysaccharide (LPS) to macrophages results in proinflammatory cytokine secretion. In extreme cases it leads to endotoxic shock. A few innate immunity antimicrobial peptides (AMPs) neutralize LPS activity. However, the underlying mechanism and properties of the peptides are not yet clear. Toward meeting this goal we investigated four AMPs and their fluorescently labeled analogs. These AMPs varied in composition, length, structure, and selectivity toward cells. The list included human LL-37 (37-mer), magainin (24-mer), a 15-mer amphipathic ␣-helix, and its D,L-amino acid structurally altered analog. The peptides were investigated for their ability to inhibit LPS-mediated cytokine release from RAW264.7 and bone marrow-derived primary macrophages, to bind LPS in solution, and when LPS is already bound to macrophages (fluorescence spectroscopy and confocal microscopy), to compete with LPS for its binding site on the CD14 receptor (flow cytometry) and affect LPS oligomerization. We conclude that a strong binding of a peptide to LPS aggregates accompanied by aggregate dissociation prevents LPS from binding to the carrier protein lipopolysaccharide-binding protein, or alternatively to its receptor, and hence inhibits cytokine secretion. Lipopolysaccharide (LPS),2 also termed endotoxin, is an integral structural component of the outer membrane of Gram-negative bacteria (1). LPS is released from the bacteria during cell division, cell death, or in particular, as a result of antibiotic treatment against bacterial infection (2, 3). Upon its release, LPS is recognized by mononuclear phagocytes (monocytes and macrophages), which are part of the innate immunity of the host, and activates them. This results in an increase in their phagocytic activity and significantly enhances the secretion of proinflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣), interleukin-6 (IL-6), and others (4 -6). Although pro-inflammatory cytokine secretion is essential for the development of the local inflammatory response, an unbalanced and overproduction of such cytokines may lead to septic shock characterized by endothelial damage, loss of vascular tone, coagulopathy, and multiple system organ failure, often resulting in death (7,8). The activation mechanism of macrophages by LPS starts when LPS (through its toxic entity, lipid A) binds with LPSbinding protein (LBP), accelerating the binding of LPS to CD14, the primary receptor of LPS, which is expressed mainly on macrophages (9 -11). The LPS-CD14 complex initiates intracellular signaling by interacting with the transmembrane protein Toll-like receptor-4 (TLR-4), which activates the NF-B transcription factor, resulting in the production and secretion of pro-inflammatory cytokines (12-16).In an attempt to understand the mechanism of macrophage stimulation by LPS, two major approaches have been reported. The first one utilized LPS receptor antagonists including anti-CD14 antibodies, anti-LBP antibodies, and lipid A analogs, all of which bind to essential components particip...
Lytic peptides comprise a large group of membrane-active peptides used in the defensive and offensive systems of all organisms. Differentiating between their modes of interaction with membranes is crucial for understanding how these peptides select their target cells. Here we utilized SPR to study the interaction between lytic peptides and lipid bilayers (L1 sensor chip). Using studies also on hybrid monolayers (HPA sensor chip) revealed that SPR is a powerful tool for obtaining a real-time monitoring of the steps involved in the mode of action of membrane-active peptides, some of which previously could not be detected directly by other techniques and reported here for the first time. We investigated the mode of action of peptides that represent two major families: (i) the bee venom, melittin, as a model of a non-cell-selective peptide that forms transmembrane pores and (ii) magainin and a diastereomer of melittin (four amino acids were replaced by their D enantiomers), as models of bacteria-selective non-pore-forming peptides. Fitting the SPR data to different interaction models allows differentiating between two major steps: membrane binding and membrane insertion. Melittin binds to PC/cholesterol approximately 450-fold better than its diastereomer and magainin, mainly because it is inserted into the inner leaflet (2/3 of the binding energy), whereas the other two are not. In contrast, there is only a slight difference in the binding of all the peptides to negatively charged PE/PG mono- and bilayer membranes (in the first and second steps), indicating that the inner leaflet contributes only slightly to their binding to PE/PG bilayers. Furthermore, the 100-fold stronger binding of the cell-selective peptides to PE/PG as compared with PC/cholesterol resulted only from electrostatic attraction to the negatively charged headgroups of the outer leaflet. These results clearly differentiate between the two general mechanisms: pore formation by melittin only in zwitterionic membranes and a detergent-like effect (carpet mechanism) for all the peptides in negatively charged membranes, in agreement with their biological function.
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 ␣-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.
The search for antibiotics with a new mode of action led to numerous studies on antibacterial peptides. Most of the studies were carried out with L-amino acid peptides possessing amphipathic ␣-helix or -sheet structures, which are known to be important for biological activities. Here we compared the effect of significantly altering the sequence of an amphipathic ␣-helical peptide (15 amino acids long) and its diastereomer (composed of both L-and D-amino acids) regarding their structure, function, and interaction with model membranes and intact bacteria. Interestingly, the effect of sequence alteration on biological function was similar for the L-amino acid peptides and the diastereomers, despite some differences in their structure in the membrane as revealed by attenuated total reflectance Fourier-transform infrared spectroscopy. However, whereas the all L-amino acid peptides were highly hemolytic, had low solubility, lost their activity in serum, and were fully cleaved by trypsin and proteinase K, the diastereomers were nonhemolytic and maintained full activity in serum. Furthermore, sequence alteration allowed making the diastereomers either fully, partially, or totally protected from degradation by the enzymes. Transmembrane potential depolarization experiments in model membranes and intact bacteria indicate that although the killing mechanism of the diastereomers is via membrane perturbation, it is also dependent on their ability to diffuse into the inner bacterial membrane. These data demonstrate the advantage of the diastereomers over their all L-amino acid counterparts as candidates for developing a repertoire of new target antibiotics with a potential for systemic use.The widespread use of antibiotics led to the development of numerous antibiotic-resistant strains, resulting in an urgent need for new antibiotics (1-4). The search for antibiotics with a new mode of action has increased the interest in antibacterial peptides as potential therapeutic agents. Isolation from biological sources (bacteria, insects, amphibians, mammals, etc.) has served as a common means for discovering novel antibacterial peptides such as gramicidins, magainins, defensins, cecropins, dermaseptins, and indolicidins (5-9). The amphipathic ␣-helix or -sheet structures and a net positive charge are common characteristics found to be crucial for most native antibacterial peptides that act by perturbing the barrier function of membranes (3, 10 -12). These features were used as building blocks to modify native sequences as well as to create many de novo designed antimicrobial peptides (13)(14)(15)(16)(17)(18)(19)(20). Another important group of peptide antibiotics composed of both L-and D-amino acids includes gramicidins, actinomycins, bacitracin, lantibiotics, and bombinins H 3-5 (21-24). The coexistence of L-and D-amino acids causes the formation of unique structures and properties completely different from all L-amino acid peptides. Unfortunately, these peptide antibiotics are not cell-selective and are highly toxic to normal euk...
Despite significant advances in cancer therapy, there is an urgent need for drugs with a new mode of action that will preferentially kill cancer cells. Several cationic antimicrobial peptides, which bind strongly to negatively charged membranes, were shown to kill cancer cells slightly better than normal cells. This was explained by a slight increase (3-9%) in the level of the negatively charged membrane phosphatidylserine (PS) in many cancer cells compared to their normal counterparts. Unfortunately, however, these peptides are inactivated by serum components. Here we synthesized and investigated the anticancer activity and the role of peptide charge, peptide structure, and phospholipid headgroup charge on the activity of a new group of diastereomeric lytic peptides (containing D- and L-forms of leucine and lysine; 15-17 amino acids long). The peptides are highly toxic to cancer cells, to a degree similar to or larger than that of mitomycin C. However, compared with mitomycin C and many native antimicrobial peptides, they are more selective for cancer cells. The peptides were investigated for (i) their binding to mono- and bilayer membranes by using the surface plasmon resonance (SPR) technique, (ii) their ability to permeate membranes by using fluorescence spectroscopy, (iii) their structure and their effect on the lipid order by using ATR-FTIR spectroscopy, and (iv) their ability to bind to cancer versus normal cells by using confocal microscopy. The data suggest that the peptides disintegrate the cell membrane in a detergent-like manner. However, in contrast to native antimicrobial peptides, the diastereomers bind and permeate similarly zwitterionic and PS-containing model membranes. Therefore, cell selectivity is probably determined mainly by improved electrostatic attraction of the peptides to acidic components on the surface of cancer cells (e.g., O-glycosylation of mucines). The simple composition of the diastereomeric peptides and their stability regarding enzymatic degradation by serum components make them excellent candidates for new chemotherapeutic drugs.
Antimicrobial peptides are produced by all organisms in response to microbial invasion and are considered as promising candidates for future antibiotics. There is a wealth of evidence that many of them interact and increase the permeability of bacterial membranes as part of their killing mechanism. However, it is not clear whether this is the lethal step. To address this issue, we studied the interaction of the antimicrobial peptide temporin L with Escherichia coli by using fluorescence, confocal and electron microscopy. The peptide previously isolated from skin secretions of the frog Rana temporaria has the sequence FVQWFSKFLGRIL-NH2. With regard to fluorescence microscopy, we applied, for the first time, a triple-staining method based on the fluorochromes 5-cyano-2,3-ditolyl tetrazolium chloride, 4',6-diamidino-2-phenylindole and FITC. This technique enabled us to identify, in the same sample, both living and total cells, as well as bacteria with altered membrane permeability. These results reveal that temporin L increases the permeability of the bacterial inner membrane in a dose-dependent manner without destroying the cell's integrity. At low peptide concentrations, the inner membrane becomes permeable to small molecules but does not allow the killing of bacteria. However, at high peptide concentrations, larger molecules, but not DNA, leak out, which results in cell death. Very interestingly, in contrast with many antimicrobial peptides, temporin L does not lyse E. coli cells but rather forms ghost-like bacteria, as observed by scanning and transmission electron microscopy. Besides shedding light on the mode of action of temporin L and possibly that of other antimicrobial peptides, the present study demonstrates the advantage of using the triple-fluorescence approach combined with microscopical techniques to explore the mechanism of membrane-active peptides in general.
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