The Human Antimicrobial Peptide LL-37, but Not the Mouse Ortholog,mCRAMP, Can Stimulate Signaling by Poly(I:C) through a FPRL1-dependent Pathway

LL-37 is an antimicrobial peptide produced by human cells that can down-regulate the lipopolysaccharide-induced innate immune responses and up-regulate double-stranded (ds) RNA-induced innate responses through Toll-like receptor 3 (TLR3). The murine LL-37 ortholog, mCRAMP, also inhibited lipopolysaccharide-induced responses, but unlike LL-37, it inhibited viral-induced responses in mouse cells. A fluorescence polarization assay showed that LL-37 was able to bind dsRNA better than mCRAMP. In the human lung epithelial cell line BEAS-2B, LL-37, but not mCRAMP, colocalized with TLR3, and the colocalization was increased in the presence of dsRNA. The presence of poly(I:C) increased the accumulation of LL-37 in Rab5 endosomes. Signaling by cells induced with both LL-37 and poly(I:C) was sensitive to inhibitors that affect clathrin-independent trafficking, whereas signaling by poly(I:C) alone was not, suggesting that the LL-37-poly(I:C) complex trafficked to signaling endosomes by a different mechanism than poly(I:C) alone. siRNA knockdown of known LL-37 receptors identified that FPRL1 was responsible for TLR3 signaling induced by LL-37-poly(I:C). These results show that LL-37 and mCRAMP have different activities in TLR3 signaling and that LL-37 can redirect trafficking of poly(I:C) to effect signaling by TLR3 in early endosomes in a mechanism that involves FPRL1.     

Effect of the antimicrobial peptide LL-37 on Toll-like receptors 2-, 3- and 4-triggered expression of IL-6, IL-8 and CXCL10 in human gingival fibroblasts

The antimicrobial peptide LL-37 is known to have a potent LPS-neutralizing activity in monocytes and macrophages. Recently, LL-37 in gingival crevicular fluids is suggested to be the major protective factor preventing infection of periodontogenic pathogens. In this study, we tried to address the effect of LL-37 on proinflammatory responses of human gingival fibroblasts (HGFs) stimulated with Toll-like receptor (TLR)-stimulant microbial compounds. LL-37 potently suppressed LPS-induced gene expression of IL6, IL8 and CXCL10 and intracellular signaling events, degradation of IRAK-1 and IκBα and phosphorylation of p38 MAPK and IRF3, indicating that the LPS-neutralizing activity is also exerted in HGFs. LL-37 also suppressed the expression of IL6, IL8 and CXCL10 induced by the TLR3 ligand poly(I:C). LL-37 modestly attenuated the expression of IL6 and IL8 induced by the TLR2/TLR1 ligand Pam₃CSK₄, but did not affect the expression induced by the TLR2/TLR6 ligand MALP-2. Interestingly, LL-37 rather upregulated the expression of IL6, IL8 and CXCL10 induced by another TLR2/TLR6 ligand FSL-1. Thus, the regulatory effect of LL-37 is differently exerted towards proinflammatory responses of HGFs induced by different microbial stimuli, which may lead to unbalanced proinflammatory responses of the gingival tissue to infection of oral microbes.     

LL37 and cationic peptides enhance TLR3 signaling by viral double-stranded RNAs

Background

Toll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti-microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3.

Methodology/Principal Findings

Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands.

Conclusions/Significance

LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA.     

The Human Antimicrobial Peptide LL-37 Binds Directly to CsrS,a Sensor Histidine Kinase of Group A Streptococcus,to Activate Expression of Virulence Factors

Group A Streptococcus (GAS) responds to subinhibitory concentrations of LL-37 by up-regulation of virulence factors through the CsrRS (CovRS) two-component system. The signaling mechanism, however, is unclear. To determine whether LL-37 signaling reflects specific binding to CsrS or rather a nonspecific response to LL-37-mediated membrane damage, we tested LL-37 fragments for CsrRS signaling and for GAS antimicrobial activity. We identified a 10-residue fragment (RI-10) of LL-37 as the minimal peptide that retains the ability to signal increased expression of GAS virulence factors, yet it has no detectable antimicrobial activity against GAS. Substitution of individual key amino acids in RI-10 reduced or abrogated signaling. These data do not support the hypothesis that CsrS detects LL-37-induced damage to the bacterial cell membrane but rather suggest that LL-37 signaling is mediated by a direct interaction with CsrS. To test whether LL-37 binds to CsrS, we used the purified CsrS extracellular domain to pull down LL-37 in vitro, a result that provides further evidence that LL-37 binds to CsrS. The dissociation of CsrS-mediated signaling from membrane damage by LL-37 fragments together with in vitro evidence for a direct LL-37-CsrS binding interaction constitute compelling evidence that signal transduction by LL-37 through CsrS reflects a direct ligand/receptor interaction.     

The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

Recognition of viral dsRNA by Toll-like receptor 3 (TLR3) leads to induction of interferons (IFNs) and proinflammatory cytokines, and innate antiviral response. Here we identified the RNA-binding protein Mex3B as a positive regulator of TLR3-mediated signaling by expression cloning screens. Cells from Mex3b^−/− mice exhibited reduced production of IFN-β in response to the dsRNA analog poly(I:C) but not infection with RNA viruses. Mex3b^−/− mice injected with poly(I:C) was more resistant to poly(I:C)-induced death. Mex3B was associated with TLR3 in the endosomes. It bound to dsRNA and increased the dsRNA-binding activity of TLR3. Mex3B also promoted the proteolytic processing of TLR3, which is critical for its activation. Mutants of Mex3B lacking its RNA-binding activity inhibited TLR3-mediated IFN-β induction. These findings suggest that Mex3B acts as a coreceptor of TLR3 in innate antiviral response.     

Molecular mechanisms of LL-37-induced receptor activation: An overview

The human cathelicidin peptide LL-37 plays a crucial role in the immune system on many levels, from the first line of defense in epithelial cells to restoring the tissue after infection. On host cells, the majority of the LL-37-induced effects are mediated via the direct or indirect activation of several structurally unrelated cell surface receptors or intracellular targets. How LL-37 is able to affect multiple receptors is currently not well understood. So far, the mechanistic details underlying receptor activation are poorly investigated and evidence for a conventional ligand/receptor interaction is scarce. Over the past few decades, a large number of studies have reported on the activation of a receptor and/or components of the downstream signal transduction pathway induced by LL-37. This review summarizes the current knowledge on molecular mechanisms underlying LL-37-induced receptor activation.     

Tumor-Produced Versican V1 Enhances hCAP18/LL-37 Expression in Macrophages through Activation of TLR2 and Vitamin D3 Signaling to Promote Ovarian Cancer Progression In Vitro

Tumor-associated macrophages have been shown to promote tumor growth. They may have an obligatory function in angiogenesis, invasion, and metastasis through release of inflammatory mediators. Their presence in ovarian cancer has been correlated with poor prognosis in these patients. The human cationic antimicrobial protein-18 (hCAP18)/LL-37 was originally identified as an effector molecule of the innate immune system. It is released by innate immune cells, such as macrophages, to combat microorganisms. Previous studies have characterized the hCAP18/LL-37 as a growth factor that has been shown to promote ovarian tumor progression. However, the role hCAP18/LL-37 has in macrophage-promoted ovarian tumor development and how its expression is controlled in this context remains poorly understood. Here, we demonstrate in co-culture experiments of macrophages and ovarian cancer cells a significant increase in the in vitro proliferation and invasiveness of the tumor cells is observed. These enhanced growth and invasion properties correlated with hCAP18/LL-37 induction. HCAP18/LL-37 expression was diminished by addition of two neutralizing antibodies, TLR2 or TLR6, as well as Cyp27B1 or VDR inhibitors. Furthermore, either the TLR2 or TLR6 antibody reduced vitamin D3 signaling and tumor cell progression in vitro. Addition of Cyp27B1 or VDR inhibitors abrogated TLR2/6 activation-induced expression of hCAP18/LL-37 in macrophages. Knockdown of tumor-produced versican V1 by RNAi in these tumor cells led to a decreased induction of hCAP18/LL-37 in macrophages. Versican V1 knockdown also inhibited TLR2 and vitamin D3 signaling, as well as growth and invasiveness of these tumor cells in the in vitro co-culture. In summary, we have found that versican V1 enhances hCAP18/LL-37 expression in macrophages through activation of TLR2 and subsequent vitamin D-dependent mechanisms which promote ovarian tumor progression in vitro.     

Antimicrobial Cathelicidin Peptide LL-37 Inhibits the LPS/ATP-Induced Pyroptosis of Macrophages by Dual Mechanism

Pyroptosis is a caspase-1 dependent cell death, associated with proinflammatory cytokine production, and is considered to play a crucial role in sepsis. Pyroptosis is induced by the two distinct stimuli, microbial PAMPs (pathogen associated molecular patterns) and endogenous DAMPs (damage associated molecular patterns). Importantly, cathelicidin-related AMPs (antimicrobial peptides) have a role in innate immune defense. Notably, human cathelicidin LL-37 exhibits the protective effect on the septic animal models. Thus, in this study, to elucidate the mechanism for the protective action of LL-37 on sepsis, we utilized LPS (lipopolysaccharide) and ATP (adenosine triphosphate) as a PAMP and a DAMP, respectively, and examined the effect of LL-37 on the LPS/ATP-induced pyroptosis of macrophage-like J774 cells. The data indicated that the stimulation of J774 cells with LPS and ATP induces the features of pyroptosis, including the expression of IL-1β mRNA and protein, activation of caspase-1, inflammasome formation and cell death. Moreover, LL-37 inhibits the LPS/ATP-induced IL-1β expression, caspase-1 activation, inflammasome formation, as well as cell death. Notably, LL-37 suppressed the LPS binding to target cells and ATP-induced/P2X₇-mediated caspase-1 activation. Together these observations suggest that LL-37 potently inhibits the LPS/ATP-induced pyroptosis by both neutralizing the action of LPS and inhibiting the response of P2X₇ to ATP. Thus, the present finding may provide a novel insight into the modulation of sepsis utilizing LL-37 with a dual action on the LPS binding and P2X₇ activation.     

The human host defense peptide LL-37 interacts with Neisseria meningitidis capsular polysaccharides and inhibits inflammatory mediators release

Capsular polysaccharides (CPS) are a major virulence factor in meningococcal infections and form the basis for serogroup designation and protective vaccines. Our work has identified meningococcal CPS as a pro-inflammatory ligand that functions through TLR2 and TLR4-MD2-dependent activation. We hypothesized that human cationic host defense peptides interact with CPS and influence its biologic activity. Accordingly, the interaction of meningococcal CPS with the human-derived cationic peptide LL-37, which is expressed by phagocytic and epithelial cells that interface with meningococci during infection, was investigated. LL-37 neutralized the pro-inflammatory activity of endotoxin-free CPS as assessed by TLR2 and TLR4-MD-2-dependent release of TNFα, IL-6 and IL-8 from human and murine macrophages. The cationic and hydrophobic properties of LL-37 were crucial for this inhibition, which was due to binding of LL-37 to CPS. LL-37 also inhibited the ability of meningococcal CPS to induce nitric oxide release, as well as TNFα and CXCL10 (IP-10) release from TLR4-sufficient and TLR4-deficient murine macrophages. Truncated LL-37 analogs, especially those that retained the antibacterial domain, inhibited vaccine grade CPS and meningococcal CPS prepared from the major serogroups (A, B C, Y and W135). Thus, LL-37 interaction with CPS was independent of specific glucan structure. We conclude that the capacity of meningococcal CPS to activate macrophages via TLR2 and TLR4-MD-2 can be inhibited by the human cationic host defense peptide LL-37 and propose that this impacts CPS-based vaccine responses.     

Modulation of double-stranded RNA recognition by the N-terminal histidine-rich region of the human toll-like receptor 3

Toll-like receptors (TLRs) are an essential component of the innate immune response to microbial pathogens. TLR3 is localized in intracellular compartments, such as endosomes, and initiates signals in response to virus-derived double-stranded RNA (dsRNA). The TLR3 ectodomain (ECD), which is implicated in dsRNA recognition, is a horseshoe-shaped solenoid composed of 23 leucine-rich repeats (LRRs). Recent mutagenesis studies on the TLR3 ECD revealed that TLR3 activation depends on a single binding site on the nonglycosylated surface in the C-terminal region, comprising H539 and several asparagines within LRR17 to -20. TLR3 localization within endosomes is required for ligand recognition, suggesting that acidic pH is the driving force for TLR3 ligand binding. To elucidate the pH-dependent binding mechanism of TLR3 at the structural level, we focused on three highly conserved histidine residues clustered at the N-terminal region of the TLR3 ECD: His39 in the N-cap region, His60 in LRR1, and His108 in LRR3. Mutagenesis of these residues showed that His39, His60, and His108 were essential for ligand-dependent TLR3 activation in a cell-based assay. Furthermore, dsRNA binding to recombinant TLR3 ECD depended strongly on pH and dsRNA length and was reduced by mutation of His39, His60, and His108, demonstrating that TLR3 signaling is initiated from the endosome through a pH-dependent binding mechanism, and that a second dsRNA binding site exists in the N-terminal region of the TLR3 ECD characteristic solenoid. We propose a novel model for the formation of TLR3 ECD dimers complexed with dsRNA, which incorporates this second binding site.     

LL-37 attenuates inflammatory impairment via mTOR signaling-dependent mitochondrial protection

The human cationic antimicrobial protein LL-37 is a multifunctional host defense peptide with a wide range of immunomodulatory activities. Previous work has shown that LL-37 exerts both pro- and anti-inflammatory effects. The role of mitochondria in the skin inflammatory effects of LL-37 has not been well studied. Therefore, our aim was to investigate the immunomodulatory effect of LL-37 in HaCaT cells and to delineate the underlying mechanisms related to mitochondrial function. Immunohistochemistry results from tissue microarrays showed strong cytoplasmic LL-37 staining in inflammatory cells in chronic dermatic inflammation. Using exogenous LL-37 stimulation and LL-37 knockdown and overexpression, LL-37 was demonstrated to dramatically reduce the mRNA levels and protein secretion of inflammatory cytokines including IL-6, IL-8, IL-1α and tumor necrosis factor-α (TNF-α), which are induced by lipopolysaccharides (LPS). The anti-inflammatory effects of LL-37 are dependent upon its ability to increase mitochondrial biogenesis and to maintain mitochondrial homeostasis. Furthermore, we observed that LL-37 enhances the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2) and mammalian target of rapamycin (mTOR). The mTOR inhibitor rapamycin can neutralize the protective effects of LL-37 on mitochondria. In conclusion, these results suggest that high LL-37 expression levels correlate with chronic skin inflammation; mitochondrial dysfunction occurs in HaCaT cells during inflammation; and LL-37 attenuates inflammatory impairment by stimulating mitochondrial biogenesis and protecting mitochondrial function, which are dependent upon mTOR signaling. These findings provide new insights into targeting mitochondria with LL-37 to prevent skin inflammatory reactions.     

Overexpression of CXCR4 synergizes with LL-37 in the metastasis of breast cancer cells

Chemokine receptor CXCR4 was involved in the progression of breast cancer to a metastatic phenotype, leading to the major cause of death in patients. A more in-depth understanding of signaling mechanism underlying CXCR4 is critical to develop effective therapies toward metastasis. Recently, the role of antimicrobial peptide LL-37 in contributing to the metastasis of breast cancer cells was observed. Clinical analysis of data herein demonstrated for the first time that overexpression of LL-37 and CXCR4 co-existed in human primary breast tumors with lymph node metastases. Further study disclosed that forced expression of CXCR4 led to the enhancement of pro-migratory signaling and migration rate induced by LL-37 in breast cancer cells. Moreover, LL-37 affected tumor microenvironment including induction of migration of mesenchymal stem cells and CXCR4-dependent capillary-like tubule formation. Functional analysis showed that LL-37 induced the internalization of CXCR4 through approaching Glu268, the residue of CXCR4, independent of the binding pocket (Asp171, Asp262, and Glu288) for CXCR4 inhibitor AMD3100, signifying that LL-37 is a distinct agonist of CXCR4. These results suggest the reciprocal roles of LL-37 and CXCR4 in promoting breast cancer cell migration and provide new insight into the design of CXCR4 inhibitor for intervention of metastatic breast cancer.     

Sequence determinants in the cathelicidin LL-37 that promote inflammation via presentation of RNA to scavenger receptors

Cathelicidins such as the human 37-amino acid peptide (LL-37) are peptides that not only potently kill microbes but also trigger inflammation by enabling immune recognition of endogenous nucleic acids. Here, a detailed structure–function analysis of LL-37 was performed to understand the details of this process. Alanine scanning of 34-amino acid peptide (LL-34) showed that some variants displayed increased antimicrobial activity against Staphylococcus aureus and group A Streptococcus. In contrast, different substitutions clustered on the hydrophobic face of the LL-34 alpha helix inhibited the ability of those variants to promote type 1 interferon expression in response to U1 RNA or to present U1 to the scavenger receptor (SR) B1 on the keratinocyte cell surface. Small-angle X-ray scattering experiments of the LL-34 variants LL-34, F5A, I24A, and L31A demonstrated that these peptides form cognate supramolecular structures with U1 characterized by inter-dsRNA spacings of approximately 3.5 nm, a range that has been previously shown to activate toll-like receptor 3 by the parent peptide LL-37. Therefore, while alanine substitutions on the hydrophobic face of LL-34 led to loss of binding to SRs and the complete loss of autoinflammatory responses in epithelial and endothelial cells, they did not inhibit the ability to organize with U1 RNA in solution to associate with toll-like receptor 3. These observations advance our understanding of how cathelicidin mediates the process of innate immune self-recognition to enable inert nucleic acids to trigger inflammation. We introduce the term “innate immune vetting” to describe the capacity of peptides such as LL-37 to enable certain nucleic acids to become an inflammatory stimulus through SR binding prior to cell internalization.     

OmpA Binding Mediates the Effect of Antimicrobial Peptide LL-37 on Acinetobacter baumannii

Multidrug-resistant Acinetobacter baumannii has recently emerged as an important pathogen in nosocomial infection; thus, effective antimicrobial regimens are urgently needed. Human antimicrobial peptides (AMPs) exhibit multiple functions and antimicrobial activities against bacteria and fungi and are proposed to be potential adjuvant therapeutic agents. This study examined the effect of the human cathelicidin-derived AMP LL-37 on A. baumannii and revealed the underlying mode of action. We found that LL-37 killed A. baumannii efficiently and reduced cell motility and adhesion. The bacteria-killing effect of LL-37 on A. baumannii was more efficient compared to other AMPs, including human ß–defensin 3 (hBD3) and histatin 5 (Hst5). Both flow cytometric analysis and immunofluorescence staining showed that LL-37 bound to A. baumannii cells. Moreover, far-western analysis demonstrated that LL-37 could bind to the A. baumannii OmpA (AbOmpA) protein. An ELISA assay indicated that biotin-labelled LL-37 (BA-LL37) bound to the AbOmpA_74-84 peptide in a dose-dependent manner. Using BA-LL37 as a probe, the ~38 kDa OmpA signal was detected in the wild type but the ompA deletion strain did not show the protein, thereby validating the interaction. Finally, we found that the ompA deletion mutant was more sensitive to LL-37 and decreased cell adhesion by 32% compared to the wild type. However, ompA deletion mutant showed a greatly reduced adhesion defect after LL-37 treatment compared to the wild strain. Taken together, this study provides evidence that LL-37 affects A. baumannii through OmpA binding.     

Binding of LL-37 to model biomembranes: Insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X = 0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Vesicular LL-37 Contributes to Inflammation of the Lesional Skin of Palmoplantar Pustulosis

“Pustulosis palmaris et plantaris”, or palmoplantar pustulosis (PPP), is a chronic pustular dermatitis characterized by intraepidermal palmoplantar pustules. Although early stage vesicles (preceding the pustular phase) formed in the acrosyringium contain the antimicrobial peptides cathelicidin (hCAP-18/LL-37) and dermcidin, the details of hCAP-18/LL-37 expression in such vesicles remain unclear. The principal aim of the present study was to clarify the manner of hCAP-18/LL-37 expression in PPP vesicles and to determine whether this material contributed to subsequent inflammation of lesional skin. PPP vesicle fluid (PPP-VF) induced the expression of mRNAs encoding IL-17C, IL-8, IL-1α, and IL-1β in living skin equivalents, but the level of only IL-8 mRNA decreased significantly upon stimulation of PPP vesicle with depletion of endogenous hCAP-18/LL-37 by affinity chromatography (dep-PPP-VF). Semi-quantitative dot-blot analysis revealed higher concentrations of hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09 µM). This concentration of hCAP-18/LL-37 in PPP-VF could upregulate expression of IL-17C, IL-8, IL-1α, and IL-1β at both the mRNA and protein levels. Recombinant hCAP-18 was incubated with dep-PPP-VF. Proteinase 3, which converts hCAP-18 to the active form (LL-37), was present in PPP-VF. Histopathological and immunohistochemical examination revealed that early stage vesicles contained many mononuclear cells but no polymorphonuclear cells, and the mononuclear cells were CD68-positive. The epidermis surrounding the vesicle expresses monocyte chemotactic chemokine, CCL2. In conclusion, PPP-VF contains the proteinase required for LL-37 processing and also may directly upregulate IL-8 in lesional keratinocytes, in turn contributing to the subsequent inflammation of PPP lesional skin.     

Binding of LL-37 to model biomembranes: insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X=0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.