Two novel cyclic lipopeptides, anabaenolysin A and anabaenolysin B, were isolated from two benthic cyanobacterial strains of the genus Anabaena. This novel class of cyanobacterial lipopeptides has a general structure of a small peptide ring consisting of four amino acids from which two are proteinogenic and two unusual; glycine(1), glycine(2), 2-(3-amino-5-oxytetrahydrofuran-2-yl)-2-hydroxyacetic acid(3) and a long unsaturated C(18) β-amino acid(4) with a conjugated triene structure. They are distinguished by the presence of a conjugated dienic structure in the C18 β-amino acid present in anabaenolysin A but not in anabaenolysin B. Conjugated triene structure generates a typical UV spectrum for anabaenolysins for easy recognition. Anabaenolysin A constituted up to 400 ppm of the cyanobacterial dry weight. We found evidence of thirteen variants of anabaenolysins in one cyanobacterial strain. This suggests that the anabaenolysins are an important class of secondary metabolites in benthic Anabaena cyanobacteria. Both anabaenolysin A and B had cytolytic activity on a number of mammalian cell lines.

A series of ultrashort lipopeptides and lipopeptoids were tested for their ability to induce cytokine production in macrophages. Fourteen compounds were found to strongly induce production of chemokines Groα and IL-8, with a structural bias that was absent from previous antibacterial activity investigations. Compounds based on LysGlyLys and NLysGlyNLys sequences did not induce cytokine production, whereas those based on LysLysLys and NLysNLysNLys were active only when linked to a lipid tail at least sixteen carbons long. Three lipopeptides induced high levels of IL-8 production, above that of equivalent concentrations of cathelicidin LL-37, while no compound induced production of the pro-inflammatory cytokine TNF-α at or below 100 µM. Two compounds, peptoids C16OH-NLysNLysNLys and C16OH-NHarNHarNHar, were selective for IL-8 production and did not induce TNF-α or IL-1β. These compounds may prove beneficial for in vivo treatment of infectious disease, with improved bioavailability over LL-37 due to their protease-resistant scaffold.

The development of antibacterial drugs to overcome various pathogenic species, which inhabit the oral cavity, faces several challenges, such as salivary flow and enzymatic activity that restrict dosage retention. Owing to their amphipathic nature, antimicrobial peptides (AMPs) serve as the first line of defense of the innate immune system. The ability to synthesize different types of AMPs enables exploitation of their advantages as alternatives to antibiotics. Sustained release of AMPs incorporated in biodegradable polymers can be advantageous in maintaining high levels of the peptides. In this study, four potent ultra-short lipopeptides, conjugated to an aliphatic acid chain (16C) were incorporated in two different biodegradable polymers: poly (lactic acid co castor oil) (PLACO) and ricinoleic acid-based poly (ester-anhydride) (P(SA-RA)) for sustained release. The lipopeptide and polymer formulations were tested for antibacterial activity during one week, by turbidometric measurements of bacterial outgrowth, anti-biofilm activity by live/dead staining, biocompatibility by hemolysis and XTT colorimetric assays, mode of action by fluorescence-activated cell sorting (FACS) and release profile by a fluorometric assay. The results show that an antibacterial and anti-biofilm effect, as well as membrane disruption, can be achieved by the use of a formulation of lipopeptide incorporated in biodegradable polymer.

A collection of 36 lipopeptides were designed from the cecropin A-melittin hybrid peptide BP100 (H-Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-NH2) previously described with activity against phytopathogenic bacteria. These lipopeptides were synthesized on solid-phase and screened for their antimicrobial activity, toxicity and proteolytic stability. They incorporated a butanoyl, a hexanoyl or a lauroyl group at the N-terminus or at the side chain of a lysine residue placed at each position of the sequence. Their antimicrobial activity and hemolysis depended on the fatty acid length and its position. In particular, lipopeptides containing a butanoyl or a hexanoyl chain exhibited the best biological activity profile. In addition, we observed that the incorporation of the acyl group did not induce the overexpression of defense-related genes in tomato. Best lipopeptides were BP370, BP378, BP381, BP387 and BP389, which were highly active against all the pathogens tested (minimum inhibitory concentration of 0.8 to 12.5 μM), low hemolytic, low phytotoxic and significantly stable to protease degradation. This family of lipopeptides might be promising functional peptides useful for plant protection.

The rhizosphere isolate Pseudomonas putida BW11M1 produces a mixture of cyclic lipopeptide congeners, designated xantholysins. Properties of the major compound xantholysin A, shared with several other Pseudomonas lipopeptides, include antifungal activity and toxicity to Gram-positive bacteria, a supportive role in biofilm formation, and facilitation of surface colonization through swarming. Atypical is the lipopeptide's capacity to inhibit some Gram-negative bacteria, including several xanthomonads. The lipotetradecadepsipeptides are assembled by XtlA, XtlB and XtlC, three co-linearly operating non-ribosomal peptide synthetases (NRPSs) displaying similarity in modular architecture with the entolysin-producing enzymes of the entomopathogenic Pseudomonas entomophila L48. A shifted serine-incorporating unit in the eight-module enzyme XtlB elongating the central peptide moiety not only generates an amino acid sequence differing at several equivalent positions from entolysin, but also directs xantholysin's macrocyclization into an octacyclic structure, distinct from the pentacyclic closure in entolysin. Relaxed fatty acid specificity during lipoinitiation by XtlA (acylation with 3-hydroxydodec-5-enoate instead of 3-hydroxydecanoate) and for incorporation of the ultimate amino acid by XtlC (valine instead of isoleucine) account for the production of the minor structural variants xantholysin C and B, respectively. Remarkably, the genetic backbones of the xantholysin and entolysin NRPS systems also bear pronounced phylogenetic similarity to those of the P. putida strains PCL1445 and RW10S2, albeit generating the seemingly structurally unrelated cyclic lipopeptides putisolvin (undecapeptide containing a cyclotetrapeptide) and WLIP (nonapeptide containing a cycloheptapeptide), respectively. This similarity includes the linked genes encoding the cognate LuxR-family regulator and tripartite export system components in addition to individual modules of the NRPS enzymes, and probably reflects a common evolutionary origin. Phylogenetic scrutiny of the modules used for selective amino acid activation by these synthetases indicates that bacteria such as pseudomonads recruit and reshuffle individual biosynthetic units and blocks thereof to engineer reorganized or novel NRPS assembly lines for diversified synthesis of lipopeptides.

Natural killer (NK) cells are lymphocyte effectors that are activated to control certain microbial infections and tumors. Many NK-activating and regulating receptors are involved in regulating NK cell function. In addition, activation of naïve NK cells is fundamentally triggered by cytokines or myeloid dendritic cells (mDC) in various modes. In this study, we synthesized 16 S-[2,3-bis(palmitoyl)propyl]cysteine (Pam2Cys) lipopeptides with sequences designed from lipoproteins of Staphylococcus aureus, and assessed their functional properties using mouse (C57BL/6) bone marrow-derived DC (BMDC) and NK cells. NK cell activation was evaluated by three criteria: IFN-gamma production, up-regulation of NK activation markers and cytokines, and NK target (B16D8 cell) cytotoxicity. The diacylated lipopeptides acted as TLR2 ligands, inducing up-regulation of CD25/CD69/CD86, IL-6, and IL-12p40, which represent maturation of BMDC. Strikingly, the Pam2Cys lipopeptides induced mouse NK cell activation based on these criteria. Cell-cell contact by Pam2Cys peptide-stimulated BMDC and NK cells rather than soluble mediators released by stimulated BMDC induced activation of NK cells. For most lipopeptides, the BMDC TLR2/MyD88 pathway was responsible for driving NK activation, while some slightly induced direct activation of NK cells via the TLR2/MyD88 pathway in NK cells. The potential for NK activation was critically regulated by the peptide primary sequence. Hydrophobic or proline-containing sequences proximal to the N-terminal lipid moiety interfered with the ability of lipopeptides to induce BMDC-mediated NK activation. This mode of NK activation is distinctly different from that induced by polyI:C, which is closely associated with type I IFN-inducing pathways of BMDC. These results imply that the MyD88 pathway of BMDC governs an alternative NK-activating pathway in which the peptide sequence of TLR2-agonistic lipopeptides critically affects the potential for NK activation.

The increasing emergence of multidrug-resistant microorganisms represents one of the greatest challenges in the clinical management of infectious diseases, and requires the development of novel antimicrobial agents. To this aim, we de novo designed a library of Arg-rich ultra-short cationic antimicrobial lipopeptides (USCLs), based on the Arg-X-Trp-Arg-NH2 peptide moiety conjugated with a fatty acid, and investigated their antibacterial potential. USCLs exhibited an excellent antimicrobial activity against clinically pathogenic microorganisms, in particular Gram-positive bacteria, including multidrug resistant strains, with MIC values ranging between 1.56 and 6.25 μg/mL. The capability of the two most active molecules, Lau-RIWR-NH2 and Lau-RRIWRR-NH2, to interact with the bacterial membranes has been predicted by molecular dynamics and verified on liposomes by surface plasmon resonance. Both compounds inhibited the growth of S. aureus even at sub MIC concentrations and induced cell membranes permeabilization by producing visible cell surface alterations leading to a significant decrease in bacterial viability. Interestingly, no cytotoxic effects were evidenced for these lipopeptides up to 50-100 μg/mL in hemolysis assay, in human epidermal model and HaCaT cells, thus highlighting a good cell selectivity. These results, together with the simple composition of USCLs, make them promising lead compounds as new antimicrobials.

In this research, a strain with broad-spectrum antimicrobial activities was isolated from the gastrointestinal tract of hairtail (Trichiurus haumela) and identified as Bacillus siamensis JFL15 through morphological, 16S rRNA, and average nucleotide identity analyses. The genome of B. siamensis JFL15 was sequenced, and three gene clusters involved in the biosynthesis of surfactin (srf), bacillibactin (dhb), and fengycin (fen) were predicted through antiSMASH analysis. The combined genomics-metabolics profiling of the strain revealed 20 active compounds, which belong to four main types of cyclic lipopeptides produced by Bacillus species: bacillibactin, iturin, fengycin, and surfactin. Among these lipopeptides, two high-purity antifungal components, namely, components b and c, were successfully identified as iturin A and bacillomycin F. The minimum inhibitory concentrations (MICs) of iturin A for Magnapothe grisea, Rhizoctorzia solani, and Colletotrichum gloeosporioides were 125.00, 62.50, and 125.00 μg/ml, respectively, whereas the MICs of bacillomycin F for these three organisms were 62.50, 31.25, and 62.50 μg/ml, respectively. The mechanism of bacillomycin F and iturin A against M. grisea was also investigated. Scanning electron microscopy (SEM) indicated that the surface of the hypha treated with iturin A or bacillomycin F became sunk, lumpy, and wrinkled. The diversity of the identified and predicted compounds from B. siamensis JFL15 suggested that this strain might be a promising biocontrol agent for an effective and environmentally friendly control of pathogenic microorganisms. To the best of our knowledge, this study is the first to describe cyclic lipopeptides purified and identified from B. siamensis.

Toll-like receptor (TLR)-2/TLR1 heterodimers recognize bacterial lipopeptides and initiate the production of inflammatory mediators. Adaptors and co-receptors that mediate this process, as well as the mechanisms by which these adaptors and co-receptors function, are still being discovered.

A pepducin is a lipopeptide containing a peptide sequence that is identical to one of the intracellular domains of the G-protein coupled receptor (GPCR) assumed to be the target. Neutrophils express two closely related formyl peptide receptors belonging to the family of GPCRs; FPR1 and FPR2 in human and their respective orthologue Fpr1 and Fpr2 in mouse. By applying the pepducin concept, we have earlier identified FPR2 activating pepducins generated from the third intracellular loop of FPR2. The third intracellular loop of FPR2 differs in two amino acids from that of FPR1, seven from Fpr2 and three from Fpr1. Despite this, we found that pepducins generated from FPR1, FPR2, Fpr1 and Fpr2 all targeted FPR2 in human neutrophils and Fpr2 in mouse, but with different modulating outcomes. Whereas the FPR1/Fpr1 derived pepducins inhibited the FPR2 function in human neutrophils, they activated Fpr2 in mouse. The FPR2 derived pepducin activated FPR2/Fpr2, whereas the pepducin generated from Fpr2 inhibited both FPR2 and Fpr2. In summary, our data demonstrate that pepducins generated from the third intracellular loop of human FPR1/2 and mouse Fpr1/2, all targeted FPR2 in human and Fpr2 in mouse. With respect to the modulating outcomes, pepducin inhibitors identified for FPR2 are in fact activators for Fpr2 in mouse neutrophils. Our data thus questions the validity of pepducin concept regarding their receptor selectivity but supports the notion that FPR2/Fpr2 may recognize a lipopeptide molecular pattern, and highlight the differences in ligand recognition profile between FPR2 and its mouse orthologue Fpr2.

16 S-[2,3-bis(palmitoyl)propyl]cysteine (Pam2) lipopeptides act as toll-like receptor (TLR)2/6 ligands and activate natural killer (NK) cells and dendritic cells (DCs) to produce inflammatory cytokines and cytotoxic NK activity in vitro. However, in this study, we found that systemic injection of Pam2 lipopeptides was not effective for the suppression of NK-sensitive B16 melanomas in vivo. When we investigated the immune suppressive mechanisms, systemic injection of Pam2 lipopeptides induced IL-10 in a TLR2-dependent manner. The Pam2 lipopeptides increased the frequencies of Foxp3(+)CD4(+) regulatory T (T reg) cells in a TLR2- and IL-10- dependent manner. The T reg cells from Pam2-lipopeptide injected mice maintained suppressor activity. Pam2 lipopeptides, plus the depletion of T reg with an anti-CD25 monoclonal antibody, improved tumor growth compared with Pam2 lipopeptides alone. In conclusion, our data suggested that systemic treatment of Pam2 lipopeptides promoted IL-10 production and T reg function, which suppressed the effective induction of anti-tumor immunity in vivo. It is necessary to develop an adjuvant that does not promote IL-10 and T reg function in vivo for the future establishment of an anti-cancer vaccine.

This study examined the contribution of GltB on biofilm formation and biocontrol efficiency of B. subtilis Bs916.

A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.

Glyco-lipopeptides, a form of lipid-tailed glyco-peptide, are currently under intense investigation as B- and T-cell based vaccine immunotherapy for many cancers. However, the cellular and molecular mechanisms of glyco-lipopeptides (GLPs) immunogenicity and the position of the lipid moiety on immunogenicity and protective efficacy of GLPs remain to be determined.

The echinocandins are lipopeptides that can be employed as antifungal drugs that inhibit the synthesis of 1,3-β-glucans within the fungal cell wall. Anidulafungin and caspofungin are echinocandins used in the treatment of Candida infections and have activity against other fungi including Aspergillus fumigatus. The echinocandins are generally considered fungistatic against Aspergillus species.

In the case of melanoma, advances in therapies are slow, which raises the need to evaluate new therapeutic strategies and natural products with potential cancer cell inhibiting effect. Pseudofactin II (PFII), a novel cyclic lipopeptide biosurfactant has been isolated from the Arctic strain of Pseudomonas fluorescens BD5. The aim of this study was to investigate the effect of PFII on A375 melanoma cells compared with the effect of PFII on Normal Human Dermis Fibroblast (NHDF) cells and elucidate the underlying mechanism of PFII cytotoxic activity. Melanoma A375 cells and NHDF cells were exposed to PFII or staurosporine and apoptotic death was assessed by monitoring caspase 3-like activity and DNA fragmentation. From time-dependent monitoring of lactate dehydrogenase (LDH) release, Ca(2+) influx, and a correlation between Critical Micelle Concentration (CMC) we concluded that cell death is the consequence of plasma membrane permeabilisation by micelles. This finding suggests that pro-apoptotic mechanism of PFII is different from previously described cyclic lipopeptides. The mechanism of PFII specificity towards malignant cells remains to be discovered. The results of this study show that PFII could be a new promising anti-melanoma agent.

Antimicrobial peptides (AMPs) are conserved evolutionary components of the innate immune system that are being tested as alternatives to antibiotics. Slow release of AMPs using biodegradable polymers can be advantageous in maintaining high peptide levels for topical treatment, especially in the oral environment in which dosage retention is challenged by drug dilution with saliva flow and by drug inactivation by salivary enzymatic activity. Enterococcus faecalis is a multidrug resistant nosocomial pathogen and a persistent pathogen in root canal infections. In this study, four ultra-short lipopeptides (C16-KGGK, C16-KLLK, C16-KAAK and C16-KKK) and an amphipathic α-helical antimicrobial peptide (Amp-1D) were tested against E. faecalis. The antibacterial effect was determined against planktonic bacteria and bacteria grown in biofilm. Of the five tested AMPs, C16-KGGK was the most effective. Next C16-KGGK was formulated with one of two polymers poly (lactic acid co castor oil) (DLLA) or ricinoleic acid-based poly (ester-anhydride) P(SA-RA). Peptide-synthetic polymer conjugates, also referred to as biohybrid mediums were tested for antibacterial activity against E. faecalis grown in suspension and in biofilms. The new formulations exhibited strong and improved anti-E. faecalis activity.

Bacillus spp. produce a broad spectrum of lipopeptide biosurfactants, among which surfactin, iturin and fengycin are widely studied families. The goals of this study were to characterize the biosurfactant activity of Bacillus spp. and to investigate their motility and biofilm formation capabilities. In addition, we extracted lipopeptides from these bacteria to assess their antifungal activities and analyzed these products by mass spectrometry (MS). B. amyloliquefaciens FZB42, Bacillus sp. NH 217 and B. subtilis NH-100 exhibited excellent biosurfactant and surface spreading activities, whereas B. atrophaeus 176s and Paenibacillus polymyxa C1225 showed moderate activity, and B. subtilis 168 showed no activity. Strains FZB42, NH-100, NH-217, 176s and CC125 exhibited excellent biofilm formation capabilities. Lipopeptide extracts displayed good antifungal activity against various phytopathogens and their associated diseases, such as Fusarium moniliforme (rice bakanae disease), Fusarium oxysporum (root rot), Fusarium solani (root rot) and Trichoderma atroviride (ear rot and root rot). Lipopeptide extracts of these strains also showed hemolytic activity, demonstrating their strong potential to produce surfactants. LCMS-ESI analyses identified the presence of surfactin, iturin and fengycin in the extracts of Bacillus strains. Thus, the strains assayed in this study show potential as biocontrol agents against various Fusarium and Trichoderma species.

Research addressing the in vivo effects of T cell activation by lipids, glycolipids, and lipopeptides is hampered by the absence of a suitable animal model. Mice and rats do not express CD1a, CD1b, and CD1c molecules that present pathogen-derived lipid antigens in humans. In cattle, two CD1A and three CD1B genes are transcribed. The proteins encoded by these genes differ in their antigen binding domains and in their cytoplasmic tails, suggesting that they may traffic differently in the cell and thus have access to different antigens. In the current study, we describe the genomic organization of the bovine CD1 locus and transcription of bovine CD1 genes in freshly isolated dendritic cells and B cells from different tissues. After determining the specificity of previously only partly characterized anti-CD1 antibodies by testing recombinant single chain bovine CD1 proteins and CD1-transfected cells, we were able to determine cell surface protein expression on freshly isolated cells. Our study suggests that CD1b1 and CD1b3 are more broadly expressed than CD1b5, and CD1a2 is more broadly expressed than CD1a1. Pseudoafferent lymph dendritic cells express CD1B genes, but no transcription is detected in lymph nodes. Even though B cells transcribe CD1B genes, there is no evidence of protein expression at the cell surface. Thus, patterns of CD1 protein expression are largely conserved among species.

Pseudomonas putida is a member of the fluorescent pseudomonads known to produce the yellow-green fluorescent pyoverdine siderophore. P. putida W15Oct28, isolated from a stream in Brussels, was found to produce compound(s) with antimicrobial activity against the opportunistic pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and the plant pathogen Pseudomonas syringae, an unusual characteristic for P. putida. The active compound production only occurred in media with low iron content and without organic nitrogen sources. Transposon mutants which lost their antimicrobial activity had the majority of insertions in genes involved in the biosynthesis of pyoverdine, although purified pyoverdine was not responsible for the antagonism. Separation of compounds present in culture supernatants revealed the presence of two fractions containing highly hydrophobic molecules active against P. aeruginosa. Analysis of the draft genome confirmed the presence of putisolvin biosynthesis genes and the corresponding lipopeptides were found to contribute to the antimicrobial activity. One cluster of ten genes was detected, comprising a NAD-dependent epimerase, an acetylornithine aminotransferase, an acyl CoA dehydrogenase, a short chain dehydrogenase, a fatty acid desaturase and three genes for a RND efflux pump. P. putida W15Oct28 genome also contains 56 genes encoding TonB-dependent receptors, conferring a high capacity to utilize pyoverdines from other pseudomonads. One unique feature of W15Oct28 is also the presence of different secretion systems including a full set of genes for type IV secretion, and several genes for type VI secretion and their VgrG effectors.