Relative biomembrane fusogenicities of the tumor-selective liposomes of RGDK- and CGKRK-lipopeptides.

Cancer is the second leading cause of death globally after heart diseases. Currently used highly cytotoxic anti-cancer drugs not only kill cancer cells but also often kill non-cancerous healthy body cells, causing adverse side effects. Efforts are now being directed towards developing tumor-selective chemotherapy. Tumor/tumor endothelial cell selective peptide ligands are being covalently grafted onto the exo-surfaces of drug carriers such as liposomes, polymers, etc. A number of prior studies used conjugation of tumor/tumor endothelial cell-selective RGDK- or CGKRK-peptide ligands on the outer surfaces of liposomes, metal-based nanoparticles, single walled carbon nanotubes (SWNTs), etc. However, studies aimed at examining the relative cell membrane fusogenicities and the relative degrees of cellular uptake for the RGDK- and CGKRK-ligand-grafted nanometric drug carriers have not yet been undertaken. Herein, using the widely used liposomes of DOPC, DOPE, DOPS and cholesterol (45 : 25 : 20 : 15, w/w ratio) as the model biomembranes and the fluorescence resonance energy transfer (FRET) assay for measuring membrane fusogenicities, we show that the liposomes of the RGDK-lipopeptide are more biomembrane fusogenic than the liposomes of the CGKRK-lipopeptide. Notably, such FRET assay-derived relative biomembrane fusogenicities of the liposomes of RGDK- and CGKRK-lipopeptides were found to be consistent with their relative degrees of cellular uptake in cultured cancer cells. The present findings open the door for undertaking in-depth in vivo studies aimed at evaluating the relative therapeutic potential of different nanocarriers of drugs/genes/siRNA having tumor-targeting RGDK- and CGKRK-peptides on their exo-surfaces.

Two promising natural lipopeptides from Bacillus subtilis effectively induced membrane permeabilization in Candida glabrata.

Candida glabrata is an important opportunistic human pathogen well known to develop resistance to antifungal drugs. Due to their numerous desirable qualities, antimicrobial lipopeptides have gained significant attention as promising candidates for antifungal drugs. In the present study, two bioactive lipopeptides (AF4 and AF5 m/z 1071.5 and 1085.5, respectively), coproduced and purified from Bacillus subtilis RLID12.1, consist of seven amino acid residues with lipid moieties. In our previous studies, the reversed phased-HPLC purified lipopeptides demonstrated broad-spectrum of antifungal activities against over 110 Candida albicans, Candida non-albicans and mycelial fungi. Two lipopeptides triggered membrane permeabilization of C. glabrata cells, as confirmed by propidium iodide-based flow cytometry, with PI uptake up to 99% demonstrating fungicidal effects. Metabolic inactivation in treated cells was confirmed by FUN-1-based confocal microscopy. Together, the results indicate that these lipopeptides have potentials to be developed into a new set of antifungals for combating fungal infections.

Discovery and Derivatization of Tridecaptin Antibiotics with Altered Host Specificity and Enhanced Bioactivity.

The prevalence of multidrug-resistant (MDR) pathogens combined with a decline in antibiotic discovery presents a major challenge for health care. To refill the discovery pipeline, we need to find new ways to uncover new chemical entities. Here, we report the global genome mining-guided discovery of new lipopeptide antibiotics tridecaptin A5 and tridecaptin D, which exhibit unusual bioactivities within their class. The change in the antibacterial spectrum of Oct-TriA5 was explained solely by a Phe to Trp substitution as compared to Oct-TriA1, while Oct-TriD contained 6 substitutions. Metabolomic analysis of producer Paenibacillus sp. JJ-21 validated the predicted amino acid sequence of tridecaptin A5. Screening of tridecaptin analogues substituted at position 9 identified Oct-His9 as a potent congener with exceptional efficacy against Pseudomonas aeruginosa and reduced hemolytic and cytotoxic properties. Our work highlights the promise of tridecaptin analogues to combat MDR pathogens.

Bacillus velezensis iturins inhibit the hemolytic activity of Staphylococcus aureus.

Bovine mastitis caused by S. aureus has a major economic impact on the dairy sector. With the crucial need for new therapies, anti-virulence strategies have gained attention as alternatives to antibiotics. Here we aimed to identify novel compounds that inhibit the production/activity of hemolysins, a virulence factor of S. aureus associated with mastitis severity. We screened Bacillus strains obtained from diverse sources for compounds showing anti-hemolytic activity. Our results demonstrate that lipopeptides produced by Bacillus spp. completely prevented the hemolytic activity of S. aureus at certain concentrations. Following purification, both iturins, fengycins, and surfactins were able to reduce hemolysis caused by S. aureus, with iturins showing the highest anti-hemolytic activity (up to 76% reduction). The lipopeptides showed an effect at the post-translational level. Molecular docking simulations demonstrated that these compounds can bind to hemolysin, possibly interfering with enzyme action. Lastly, molecular dynamics analysis indicated general stability of important residues for hemolysin activity as well as the presence of hydrogen bonds between iturins and these residues, with longevous interactions. Our data reveals, for the first time, an anti-hemolytic activity of lipopeptides and highlights the potential application of iturins as an anti-virulence therapy to control bovine mastitis caused by S. aureus.

Multifunctional Lipidated Protein Carrier with a Built-In Adjuvant as a Universal Vaccine Platform Potently Elevates Immunogenicity of Weak Antigens.

Weak antigens represented by MUC1 are poorly immunogenic, which greatly constrains the development of relevant vaccines. Herein, we developed a multifunctional lipidated protein as a carrier, in which the TLR1/2 agonist Pam3CSK4 was conjugated to the N-terminus of MUC1-loaded carrier protein BSA through pyridoxal 5'-phosphate-mediated transamination reaction. The resulting Pam3CSK4-BSA-MUC1 conjugate was subsequently incorporated into liposomes, which biomimics the membrane structure of tumor cells. The results indicated that this lipidated protein carrier significantly enhanced antigen uptake by APCs and obviously augmented the retention of the vaccine at the injection site. Compared with the BSA-MUC1 and BSA-MUC1 + Pam3CSK4 groups, Pam3CSK4-BSA-MUC1 evoked 22- and 11-fold increases in MUC1-specific IgG titers. Importantly, Pam3CSK4-BSA-MUC1 elicited robust cellular immunity and significantly inhibited tumor growth. This is the first time that lipidated protein was constructed to enhance antigen immunogenicity, and this universal carrier platform exhibits promise for utilization in various vaccines, holding the potential for further clinical application.

Noducyclamides A1-A4, B1, and B2 from the Cyanobacterium Nodularia sp. NIES-3585.

A chemical investigation of the hydrophilic fraction of a cultured Nodularia sp. (NIES-3585) afforded six new cyclic lipopeptides, noducyclamides A1-A4 (1-4) containing 10 amino acid residues and dodecapeptides noducyclamides B1 and B2 (5 and 6). The planar structures of these lipopeptides were elucidated based on the combination of HRMS and 1D and 2D NMR spectroscopic data analyses. These peptides are structurally analogous to laxaphycins and contain the nonproteinogenic amino acids 3-hydroxyvaline and 3-hydroxyleucine and a ß-amino decanoic acid residue. The absolute configurations of the noducyclamides (1-6) were determined by acid hydrolysis, followed by advanced Marfey's analysis. Noducyclamide B1 (5) showed cytotoxic activities against MCF7 breast cancer cell lines with an IC50 value of 3.0 µg/mL (2.2 µM).

Production of Lipopeptides from Bacillus velezensis BVQ121 and Their Application in Chitosan Antibacterial Coating.

The extracellular substance of Bacillus has antibacterial effects inhibiting multiple foodborne pathogens and plays important roles in food production. This study found one Bacillus velezensis BVQ121 strain producing antibacterial lipopeptides (BVAL). After optimization of the fermentation conditions, the BVAL yield was the highest at 1.316 ± 0.03 g/L in reality with the initial pH 6.0, temperature 31 °C, and shaker speed 238 rpm when the optimal nitrogen and carbon sources were used in Landy medium for fermentation. The antibacterial components were identified as iturin, surfactin, and fengycin by HPLC and MALDI-TOF-MS. The MIC was at 2 mg/mL and MBC was at 5 mg/mL. The 6% weight ratio of nanocellulose dosage in chitosan solution could improve the tensile length and strength of the film, and the antibacterial performance was enhanced by the addition of BVAL. The addition of BVAL had no effect on the color and ductility of the film and improved its antibacterial effect. The shelf life of pigeon eggs can be extended by more than 10 days to resist bacterial infections after coating with the chitosan-nanocellulose-BVAL film solution.

Total synthesis of antifungal lipopeptide iturin A analogues and evaluation of their bioactivity against F. graminearum.

The pursuit of novel antifungal agents is imperative to tackle the threat of antifungal resistance, which poses major risks to both human health and to food security. Iturin A is a cyclic lipopeptide, produced by Bacillus sp., with pronounced antifungal properties against several pathogens. Its challenging synthesis, mainly due to the laborious synthesis of the ß-amino fatty acid present in its structure, has hindered the study of its mode of action and the development of more potent analogues. In this work, a facile synthesis of bioactive iturin A analogues containing an alkylated cysteine residue is presented. Two analogues with opposite configurations of the alkylated cysteine residue were synthesized, to evaluate the role of the stereochemistry of the newly introduced amino acid on the bioactivity. Antifungal assays, conducted against F. graminearum, showed that the novel analogues are bioactive and can be used as a synthetic model for the design of new analogues and in structure-activity relationship studies. The assays also highlight the importance of the ß-amino acid in the natural structure and the role of the stereochemistry of the amino fatty acid, as the analogue with the D configuration showed stronger antifungal properties than the one with the L configuration.

A Fluorous Peptide Amphiphile with Potent Antimicrobial Activity for the Treatment of MRSA-induced Sepsis and Chronic Wound Infection.

The rising prevalence of global antibiotic resistance evokes the urgent need for novel antimicrobial candidates. Cationic lipopeptides have attracted much attention due to their strong antimicrobial activity, broad-spectrum and low resistance tendency. Herein, a library of fluoro-lipopeptide amphiphiles was synthesized by tagging a series of cationic oligopeptides with a fluoroalkyl tail via a disulfide spacer. Among the lipopeptide candidates, R6F bearing six arginine moieties and a fluorous tag shows the highest antibacterial activity, and it exhibits an interesting fluorine effect as compared to the non-fluorinated lipopeptides. The high antibacterial activity of R6F is attributed to its excellent bacterial membrane permeability, which further disrupts the respiratory chain redox stress and cell wall biosynthesis of the bacteria. By co-assembling with lipid nanoparticles, R6F showed high therapeutic efficacy and minimal adverse effects in the treatment of MRSA-induced sepsis and chronic wound infection. This work provides a novel strategy to design highly potent antibacterial peptide amphiphiles for the treatment of drug-resistant bacterial infections.

N-Desmethylmajusculamide B, a lipopeptide isolated from the Okinawan cyanobacterium Okeania hirsuta.

A new lipopeptide, N-desmethylmajusculamide B (1), was isolated from the Okinawan cyanobacterium Okeania hirsuta along with 2 known compounds majusculamide A (2) and majusculamide B (3). The planar structure of (1) was elucidated by a detailed analysis of mass spectrometry and nuclear magnetic resonance spectra. The absolute configurations of the amino acid residues were determined using Marfey's analysis. The configuration of C-16 in the α-methyl-ß-keto-decanoyl moiety was determined unambiguously to be S by conducting a semisynthesis of N-desmethylmajusculamide B from 3. The cytotoxicity against mouse L1210 leukemia cells was evaluated for majusculamides (1-3).

Cholesterol as an inbuilt immunoadjuvant for a lipopeptide vaccine against group A Streptococcus infection.

Peptide-based vaccines can trigger highly specific immune responses, although peptides alone are usually unable to confer strong humoral or cellular immunity. Consequently, peptide antigens are administered with immunostimulatory adjuvants, but only a few are safe and effective for human use. To overcome this obstacle, herein a peptide antigen was lipidated to effectively anchor it to liposomes and emulsion. A peptide antigen B cell epitope from Group A Streptococcus M protein was conjugated to a universal T helper epitope, the pan DR-biding epitope (PADRE), alongside a lipidic moiety cholesterol. Compared to a free peptide antigen, the lipidated version (LP1) adopted a helical conformation and self-assembled into small nanoparticles. Surprisingly, LP1 alone induced the same or higher antibody titers than liposomes or emulsion-based formulations. In addition, antibodies produced by mice immunized with LP1 were more opsonic than those induced by administering the antigen with incomplete Freund's adjuvant. No side effects were observed in the immunized mice and no excessive inflammatory immune responses were detected. Overall, this study demonstrated how simple conjugation of cholesterol to a peptide antigen can produce a safe and efficacious vaccine against Group A Streptococcus - the leading cause of superficial infections and the bacteria responsible for deadly post-infection autoimmune disorders.

An Engineered Microbial Consortium Provides Precursors for Fengycin Production by Bacillus subtilis.

Fengycin has great potential for applications in biological control because of its biosafety and degradability. In this study, the addition of exogenous precursors increased fengycin production by Bacillus subtilis. Corynebacterium glutamicum was engineered to produce high levels of precursors (Thr, Pro, Val, and Ile) to promote the biosynthesis of fengycin. Furthermore, recombinant C. glutamicum and Yarrowia lipolytica providing amino acid and fatty acid precursors were co-cultured to improve fengycin production by B. subtilis in a three-strain artificial consortium, in which fengycin production was 2100 mg·L-1. In addition, fengycin production by the consortium in a 5 L bioreactor reached 3290 mg·L-1. Fengycin had a significant antifungal effect on Rhizoctonia solani, which illustrates its potential as a food preservative. Taken together, this work provides a new strategy for improving fengycin production by a microbial consortium and metabolic engineering.

Lipopeptides from Bacillus: unveiling biotechnological prospects-sources, properties, and diverse applications.

Bacillus sp. has proven to be a goldmine of diverse bioactive lipopeptides, finding wide-range of industrial applications. This review highlights the importance of three major families of lipopeptides (iturin, fengycin, and surfactin) produced by Bacillus sp. and their diverse activities against plant pathogens. This review also emphasizes the role of non-ribosomal peptide synthetases (NRPS) as significant enzymes responsible for synthesizing these lipopeptides, contributing to their peptide diversity. Literature showed that these lipopeptides exhibit potent antifungal activity against various plant pathogens and highlight their specific mechanisms, such as siderophore activity, pore-forming properties, biofilm inhibition, and dislodging activity. The novelty of this review comes from its comprehensive coverage of Bacillus sp. lipopeptides, their production, classification, mechanisms of action, and potential applications in plant protection. It also emphasizes the importance of ongoing research for developing new and enhanced antimicrobial agents. Furthermore, this review article highlights the need for future research to improve the production efficiency of these lipopeptides for commercial applications. It recognizes the potential for these lipopeptides to expand the field of biological pest management for both existing and emerging plant diseases.

A stapled lipopeptide platform for preventing and treating highly pathogenic viruses of pandemic potential.

The continued emergence of highly pathogenic viruses, which either thwart immune- and small molecule-based therapies or lack interventions entirely, mandates alternative approaches, particularly for prompt and facile pre- and post-exposure prophylaxis. Many highly pathogenic viruses, including coronaviruses, employ the six-helix bundle heptad repeat membrane fusion mechanism to achieve infection. Although heptad-repeat-2 decoys can inhibit viral entry by blocking six-helix bundle assembly, the biophysical and pharmacologic liabilities of peptides have hindered their clinical development. Here, we develop a chemically stapled lipopeptide inhibitor of SARS-CoV-2 as proof-of-concept for the platform. We show that our lead compound blocks infection by a spectrum of SARS-CoV-2 variants, exhibits mucosal persistence upon nasal administration, demonstrates enhanced stability compared to prior analogs, and mitigates infection in hamsters. We further demonstrate that our stapled lipopeptide platform yields nanomolar inhibitors of respiratory syncytial, Ebola, and Nipah viruses by targeting heptad-repeat-1 domains, which exhibit strikingly low mutation rates, enabling on-demand therapeutic intervention to combat viral outbreaks.

Self-Assembly and Antimicrobial Activity of Lipopeptides Containing Lysine-Rich Tripeptides.

The conformation and self-assembly of two pairs of model lipidated tripeptides in aqueous solution are probed using a combination of spectroscopic methods along with cryogenic-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS). The palmitoylated lipopeptides comprise C16-YKK or C16-WKK (with two l-lysine residues) or their respective derivatives containing d-lysine (k), i.e., C16-Ykk and C16-Wkk. All four molecules self-assemble into spherical micelles which show structure factor effects in SAXS profiles due to intermicellar packing in aqueous solution. Consistent with micellar structures, the tripeptides in the coronas have a largely unordered conformation, as probed using spectroscopic methods. The molecules are found to have good cytocompatibility with fibroblasts at sufficiently low concentrations, although some loss of cell viability is noted at the highest concentrations examined (above the critical aggregation concentration of the lipopeptides, determined from fluorescence dye probe measurements). Preliminary tests also showed antimicrobial activity against both Gram-negative and Gram-positive bacteria.

Modifying the Catalytic Activity of Lipopeptide Assemblies with Nucleobases.

Biohybrid catalysts that operate in aqueous media are intriguing for systems chemistry. In this paper, we investigate whether control over the self-assembly of biohybrid catalysts can tune their properties. As a model, we use the catalytic activity of functional hybrid molecules consisting of a catalytic H-dPro-Pro-Glu tripeptide, derivatized with fatty acid and nucleobase moieties. This combination of simple biological components merged the catalytic properties of the peptide with the self-assembly of the lipid, and the structural ordering of the nucleobases. The biomolecule hybrids self-assemble in aqueous media into fibrillar assemblies and catalyze the reaction between butanal and nitrostyrene. The interactions between the nucleobases enhanced the order of the supramolecular structures and affected their catalytic activity and stereoselectivity. The results point to the significant control and ordering that nucleobases can provide in the self-assembly of biologically inspired supramolecular catalysts.

Studies of antibacterial activity (in vitro and in vivo) and mode of action for des-acyl tridecaptins (DATs).

Tridecaptins comprise a class of linear cationic lipopeptides with an N-terminal fatty acyl moiety. These 13-mer antimicrobial peptides consist of a combination of d- and l-amino acids, conferring increased proteolytic stability. Intriguingly, they are biosynthesized by non-ribosomal peptide synthetases in the same bacterial species that also produce the cyclic polymyxins displaying similar fatty acid tails. Previously, the des-acyl analog of TriA1 (termed H-TriA1) was found to possess very weak antibacterial activity, albeit it potentiated the effect of several antibiotics. In the present study, two series of des-acyl tridecaptins were explored with the aim of improving the direct antibacterial effect. At the same time, overall physico-chemical properties were modulated by amino acid substitution(s) to diminish the risk of undesired levels of hemolysis and to avoid an impairment of mammalian cell viability, since these properties are typically associated with highly hydrophobic cationic peptides. Microbiology and biophysics tools were used to determine bacterial uptake, while circular dichroism and isothermal calorimetry were used to probe the mode of action. Several analogs had improved antibacterial activity (as compared to that of H-TriA1) against Enterobacteriaceae. Optimization enabled identification of the lead compound 29 that showed a good ADMET profile as well as in vivo efficacy in a variety of mouse models of infection.

Unraveling the genome of Bacillus velezensis MEP218, a strain producing fengycin homologs with broad antibacterial activity: comprehensive comparative genome analysis.

Bacillus sp. MEP218, a soil bacterium with high potential as a source of bioactive molecules, produces mostly C16-C17 fengycin and other cyclic lipopeptides (CLP) when growing under previously optimized culture conditions. This work addressed the elucidation of the genome sequence of MEP218 and its taxonomic classification. The genome comprises 3,944,892 bp, with a total of 3474 coding sequences and a G + C content of 46.59%. Our phylogenetic analysis to determine the taxonomic position demonstrated that the assignment of the MEP218 strain to Bacillus velezensis species provides insights into its evolutionary context and potential functional attributes. The in silico genome analysis revealed eleven gene clusters involved in the synthesis of secondary metabolites, including non-ribosomal CLP (fengycins and surfactin), polyketides, terpenes, and bacteriocins. Furthermore, genes encoding phytase, involved in the release of phytic phosphate for plant and animal nutrition, or other enzymes such as cellulase, xylanase, and alpha 1-4 glucanase were detected. In vitro antagonistic assays against Salmonella typhimurium, Acinetobacter baumanii, Escherichia coli, among others, demonstrated a broad spectrum of C16-C17 fengycin produced by MEP218. MEP218 genome sequence analysis expanded our understanding of the diversity and genetic relationships within the Bacillus genus and updated the Bacillus databases with its unique trait to produce antibacterial fengycins and its potential as a resource of biotechnologically useful enzymes.

Synthesis of silver nanoparticles using Bacillus velezensis M3-7 lipopeptides: Enhanced antifungal activity and potential use as a biocontrol agent against Fusarium crown rot disease of wheat seedlings.

Bacillus velezensis M3-7 is a hyperactive mutant, 12-fold improved in its antifungal activity, obtained during a previous study from the wild strain BLB371 after a combination of random mutagenesis and medium component optimization. This study explores the use of this mutant in synthesizing silver nanoparticles (Ag-NPs) for the control of Fusarium crown rot disease (FCR) in wheat seedlings. LC-MS/MS analysis proved that both strains co-produced different families of lipopeptides and that mutagenesis caused the hyper-production of iturin A C14 and C15, the liberation of iturin A C10 and C12, and the inhibition of fengycin release. Our aim was a further improvement in the antifungal activity of the wild strain and the mutant M3-7 in order to control Fusarium crown rot disease (FCR) in wheat seedlings. Therefore, a nanotechnology approach was adopted, and different lipopeptide concentrations produced by the wild strain and the mutant M3-7 were used as capping agents to synthesize silver nanoparticles (Ag-NPs) with enhanced antifungal activity. Ag-NPs formed using 3 mg·mL-1 of the mutant lipopeptides were found to exhibit a good distribution, improved antifungal activity, a promising potential to be used as a biofortified agent for seed germination, and an effective compound to control FCR in wheat seedlings.

Development of truncated Battacin antimicrobials featuring novel N-terminal fatty acids with an excellent safety profile.

Octapeptin B5 peptides containing a novel fatty acids have been found to have enhanced antibacterial activity against Staphylococcus aureus and also have an excellent safety profile. Cyclic lipopeptides such as the polymyxins and battacin are potent antibacterial agents. It has been shown that truncated, non-linear, versions of these agents (e.g. octapeptin B5) can retain the activity of the more complex cyclic compounds. In this work the synthesis of Octapeptin B5 peptides containing a range of novel fatty acids is reported. Many of these lipopeptides have been found to have enhanced antibacterial activity against Staphylococcus aureus compared to Octapeptin B5 whilst also having an excellent safety profile in haemolytic and cytotoxicity assays.