Genetic analysis of the biosynthesis of non-ribosomal peptide- and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3

The Bacillus subtilis strain A1/3 shows exceptionally diverse antibiotic capacities compared to other B. subtilis strains. To analyze this phenomenon, mutants for the putative pantotheinyltransferase gene ( pptS), and for several genes involved in non-ribosomal peptide synthesis and polyketide synthesis were constructed and characterized, using bioassays with blood cells, bacterial and fungal cells, and mass spectrometry. Among at least nine distinct bioactive compounds, five antibiotics and one siderophore activity were identified. The anti-fungal and hemolytic activities of strain A1/3 could be eliminated by mutation of the fen and srf genes essential for the synthesis of fengycins and surfactins. Both pptS - and dhb -type mutants were defective in iron uptake, indicating an inability to produce a 2,3-dihydroxybenzoate-type iron siderophore. Transposon mutants in the malonyl CoA transacylase gene resulted in the loss of hemolytic and anti-fungal activities due to the inhibition of bacillomycin L synthesis, and this led to the discovery of bmyLD-LA-LB* genes. In mutants bearing disruption mutations in polyketide ( pksM - and/or pksR -like) genes, the biosynthesis of bacillaene and difficidins, respectively, was inactivated and was accompanied by the loss of discrete antibacterial activities. The formation of biofilms (pellicles) was shown to require the production of surfactins, but no other lipopeptides, indicating that surfactins serve specific developmental functions.Communicated by A. Kondorosi     

Additive and synergistic effects of Bacillus spp. isolates and essential oils on the control of phytopathogenic and saprophytic fungi from medicinal plants and marigold seeds

Contamination of plants and seeds with microorganisms is one of the main problems in the production and distribution of various agricultural products, as well as raw herbal material for the preparation of herbal remedies. In targeting microbial contamination, among other bacteria, Bacillus species showed a significant capacity for biocontrol. The antifungal activity of 14 isolates of Bacillus spp. against 15 fungal isolates from medicinal plants was examined utilizing a dual plate assay. The strongest and broadest antagonistic activity against all fungi tested was exhibited by isolates SS-12.6 and SS-13.1 (from a 43% to 74% reduction in fungal growth), while isolates SS-39.1 and SS-39.3 were effective against the fewest fungus species and also had the weakest antifungal activity. The effect of a crude lipopeptide extract (CLE) of Bacillus sp. SS-12.6 was similar to that achieved by a dual culture with isolate SS-12.6, confirming that the antagonism was the result of the antifungal activities of lipopeptides. In addition, essential oils of thyme (0.55 mg/mL) and savory (0.32 mg/mL) in various combinations with the CLE of SS-12.6 were tested for antifungal activity, and additive and synergistic effects for some of the fungi were obtained. When testing the effect of CLE, oils (0.40 mg/mL for thyme oil and 0.21 mg/mL for savory oil) and combinations in situ on marigold seeds, a reduction of total fungal infection without an adverse effect on germination was accomplished by 6-h treatments with CLE of SS-12.6 (85% reduction of fungal infection and 63% germination), supernatant from liquid culture of SS-12.6 (more than 90% reduction of fungal infection with 69% seed germination) and combinations of CLE and savory oil (77% reduction of fungal infection and 62% seed germination) and CLE with thyme and savory oils (about 75% reduction of fungal infection with 69% seed germination).     

Rhamnolipid inspired lipopeptides effective in preventing adhesion and biofilm formation of Candida albicans

Rhamnolipids are biodegradable low toxic biosurfactants which exert antimicrobial and anti-biofilm properties. They have attracted much attention recently due to potential applications in areas of bioremediation, therapeutics, cosmetics and agriculture, however, the full potential of these versatile molecules is yet to be explored. Based on the facts that many naturally occurring lipopeptides are potent antimicrobials, our study aimed to explore the potential of replacing rhamnose in rhamnolipids with amino acids thus creating lipopeptides that would mimic or enhance properties of the parent molecule. This would allow not only for more economical and greener production but also, due to the availability of structurally different amino acids, facile manipulation of physico-chemical and biological properties.Our synthetic efforts produced a library of 43 lipopeptides revealing biologically more potent molecules. The structural changes significantly increased, in particular, anti-biofilm properties against Candida albicans, although surface activity of the parent molecule was almost completely abolished. Our findings show that the most active compounds are leucine derivatives of 3-hydroxy acids containing benzylic ester functionality. The SAR study demonstrated a further increase in activity with aliphatic chain elongation. The most promising lipopeptides 15, 23 and 36 at 12.5 µg/mL concentration allowed only 14.3%, 5.1% and 11.2% of biofilm formation, respectively after 24 h. These compounds inhibit biofilm formation by preventing adhesion of C. albicans to abiotic and biotic surfaces.     

Further aspects on the hemolytic activity of the antibiotic lipopeptide iturin A

The bacterial lipopeptide iturin A is able to cause hemolysis of human erythrocytes in a dose-dependent manner. Hemolysis takes place at iturin concentrations below its critical micellar concentration. Relative kinetics determinations clearly show that K⁺ leakage occurs prior to hemoglobin release. Furthermore, hemolysis can be prevented by addition to the outer solution of osmotic protectants of appropriate size. Altogether these results indicate that iturin A-induced hemolysis follows a colloid-osmotic mechanism, with the formation of a membrane pore of average diameter 32 Å. Iturin A is capable of inducing leakage of an aqueous fluorescent probe trapped in human erythrocyte ghosts, but not in large unilamellar liposomes made of various lipid compositions. The different permeabilizing effects of iturin A on model and biological membranes are discussed on the light of the presented results.     

Optimization of Sterilization of Salmonella enteritidis in Meat by Surfactin and Iturin Using a Response Surface Method

In this paper, the sterilization of surfactin and iturin to Salmonella enteritidis was observed, and the optimization of the inactivation of surfactin and iturin to S. enteritidis in meat by a response surface methodology was studied. Results showed that surfactin and iturin had high sterilization to S. enteritidis, whose minimal inhibitory concentration was 6.25 and 12.50 μM respectively. The optimization result indicated that S. enteritidis could be inactivated by five orders of magnitude when the temperature was 4.83°C, the action time was 17.20 h, and the concentration (surfactin/iturin molar ratio 1:2) was 0.69 MIC.     

Development of novel membrane active lipidated peptidomimetics active against drug resistant clinical isolates

A new series of small cationic lipidated peptidomimetics have been synthesized and found to be highly active against several susceptible as well as drug resistant clinical isolates of bacteria and fungi. All lipidated peptidomimetics do not cause significant lysis of human erythrocytes (HC₅₀ > 200 μg/mL). Calcein dye leakage experiment revealed membranolytic effect of LPEP08 which was further confirmed by scanning electron microscopy (SEM). The involvement of intracellular targets as an alternate mode of action was precluded by DNA retardation assay. Additionally, LPEP08 exhibit high proteolytic stability and dose not elicit resistance against drug resistant clinical isolate of Staphylococcus aureus, even after 16 rounds of passaging. These results demonstrate the potential of lipidated peptidomimetics as biocompatible anti-infective therapeutics.     

Ultrashort cationic lipopeptides and lipopeptoids: Evaluation and mechanistic insights against epithelial cancer cells

Peptides present an attractive scaffold for the development of new anticancer lead agents due to their accessibility and ease of modification. Synthetic ultrashort cationic lipopeptides, with four amino acids or less conjugated to a fatty acid, were developed to retain the biological activity of longer peptides in a smaller molecular size. Herein, we report the activity of amphiphilic lipotripeptides, lipotripeptoids and lipotetrapeptides against breast (MDA-MB-231, JIMT-1), prostate (DU145) and pancreas (MiaPaCa2) epithelial cancer cell lines. The lipotripeptide C16-KKK-NH₂ and lipotetrapeptide C16-P_CatP_HexP_HexP_Cat-NH₂ were identified to possess anticancer activity. The latter lipotetrapeptide possess a short polyproline scaffold consisting of only two L-4R-aminoproline (P_Cat) and two L-4R-hexyloxyproline (P_Hex). However, all the prepared lipotripeptoids lack anticancer activity. The amphiphilic C16-P_CatP_HexP_HexP_Cat-NH₂ exhibited similar anticancer potency to the surfactant benzethonium chloride while superior activity was observed in comparison to myristylamine. Mechanistic studies revealed that the peptides do not lyse ovine erythrocytes nor epithelial cancer cells, thus ruling out necrosis as the mechanism of cell death. Surprisingly, the two lipopeptides exhibit different mechanisms of action that result in cancer cell death. The lipotripeptide C16-KKK-NH₂ was found to induce caspase-mediated apoptosis while C16-P_CatP_HexP_HexP_Cat-NH₂ kills tumor cells independent of caspases.     

Studies on lipopeptide biosynthesis by Bacillus subtilis: Isolation and characterization of iturin−, surfactin+ mutants

Abstract Two mutant strains, M35 and M89, were obtained by UV irradiation from a wild-type Bacillus subtilis producing iturin and surfactin. Sporulation and surfactin production were similar in both mutants and in the parent strain, while the iturin production of M35 was 300-fold less than that of the wild-type strain; M89 did not produce any iturin. The analysis of the incorporation of sodium [1-¹⁴C]acetate into cellular lipids and lipopeptides showed that M89 still synthesized β-amino fatty acids, the lipid moiety of iturin.     

Improving the biological activity of the antimicrobial peptide anoplin by membrane anchoring through a lipophilic amino acid derivative

The lipophilic amino acid, (S)-2-aminoundecanoic acid, was synthesized and incorporated at a number of specific positions within the peptide sequence of anoplin. These lipophilic anoplin analogs showed to be more active against Escherichia coli and Staphylococcus aureus compared to native anoplin, while the EC₅₀-value of hemolysis was at least one order of magnitude lower than the MIC values. This was in sharp contrast to the N-acylated anoplin derivative, where a gain in activity also led to a complete loss of selectivity. Thus, the incorporation of a lipophilic amino acid residue into anoplin enhanced the antimicrobial activity, while selectivity towards microbial membranes was retained.     

Optimization of Inactivation of Endospores of <Emphasis Type="Italic">Bacillus cereus</Emphasis> in Milk by Surfactin and Fengycin Using a Response Surface Method

In this paper, the sterilization of surfactin and fengycin to Bacillus cereus was observed, and the optimization of the inactivation of surfactin and fengycin to spores of B. cereus by a response surface methodology was studied. Results showed that surfactin and fengycin had high sterilization to B. cereus, whose minimal inhibitory concentration was 31.25 μM and 62.5 μM respectively. The optimization result indicated that spores of B. cereus could be inactivated by two orders of magnitude when the temperature was 20.41°C, the action time was 21.13 h, and the concentration (surfactin/fengycin molar ratio 1:1) was 54.20 μM.