Tomasz Janek

Isolation and characterization of two new lipopeptide biosurfactants produced by Pseudomonas fluorescens BD5 isolated from water from the Arctic Archipelago of Svalbard.

The arctic freshwater bacterium Pseudomonas fluorescens BD5 produces biosurfactants when grown on 2% glucose. Crude biosurfactants were extracted from a cell-free culture supernatant with ethyl acetate and purified by preparative reversed phase high performance liquid chromatography (RP-HPLC). The chemical structure of the purified biosurfactants, pseudofactin I and II, was analyzed by matrix assisted laser desorption/ionization time of flight (MALDI TOF) mass spectrometry and tandem mass spectrometry (MS/MS). Both compounds are novel cyclic lipopeptides with a palmitic acid connected to the terminal amino group of eighth amino acid in peptide moiety. The C-terminal carboxylic group of the last amino acid (Val or Leu) forms a lactone with the hydroxyl of Thr3. Pseudofactin II reduced the surface tension of water from 72 mN/m to 31.5 mN/m at a concentration of 72 mg/l. Its emulsification activity and stability was greater than that of the synthetic surfactants Tween 20 and Triton X-100; pseudofactins thus have a great potential for application in industrial fields such as bioremediation or biomedicine.

Antiadhesive activity of the biosurfactant pseudofactin II secreted by the Arctic bacterium Pseudomonas fluorescens BD5

BackgroundPseudofactin II is a recently identified biosurfactant secreted by Pseudomonas fluorescens BD5, the strain obtained from freshwater from the Arctic Archipelago of Svalbard. Pseudofactin II is a novel compound identified as cyclic lipopeptide with a palmitic acid connected to the terminal amino group of eighth amino acid in peptide moiety. The C-terminal carboxylic group of the last amino acid forms a lactone with the hydroxyl of Thr3.Adhesion is the first stage of biofilm formation and the best moment for the action of antiadhesive and anti-biofilm compounds. Adsorption of biosurfactants to a surface e.g. glass, polystyrene, silicone modifies its hydrophobicity, interfering with the microbial adhesion and desorption processes. In this study the role and applications of pseudofactin II as a antiadhesive compound has been investigated from medicinal and therapeutic perspectives.ResultsPseudofactin II lowered the adhesion to three types of surfaces (glass, polystyrene and silicone) of bacterial strains of five species: Escherichia coli, Enterococcus faecalis, Enterococcus hirae, Staphylococcus epidermidis, Proteus mirabilis and two Candida albicans strains. Pretreatment of a polystyrene surface with 0.5 mg/ml pseudofactin II inhibited bacterial adhesion by 36-90% and that of C. albicans by 92-99%. The same concentration of pseudofactin II dislodged 26-70% of preexisting biofilms grown on previously untreated surfaces. Pseudofactin II also caused a marked inhibition of the initial adhesion of E. faecalis, E. coli, E. hirae and C. albicans strains to silicone urethral catheters. The highest concentration tested (0.5 mg/ml) caused a total growth inhibition of S. epidermidis, partial (18-37%) inhibition of other bacteria and 8-9% inhibition of C. albicans growth.ConclusionPseudofactin II showed antiadhesive activity against several pathogenic microorganisms which are potential biofilm formers on catheters, implants and internal prostheses. Up to 99% prevention could be achieved by 0.5 mg/ml pseudofactin II. In addition, pseudofactin II dispersed preformed biofilms. Pseudofactin II can be used as a disinfectant or surface coating agent against microbial colonization of different surfaces, e.g. implants or urethral catheters.

Identification and characterization of biosurfactants produced by the Arctic bacterium Pseudomonas putida BD2

One hundred and thirty bacterial strains, isolated from Arctic soil on the Svalbard Archipelago, were screened for biosurfactant production. Among them, an isolate identified as Pseudomonas putida BD2 was selected as a potential biosurfactant-producer based on the surface/interfacial activity of the culture supernatant. The ability of the strain to produce simultaneously phosphatidylethanolamines and rhamnolipid, using glucose as a sole carbon source, was demonstrated. The rhamnolipid Rha-Rha-C10-C10 and two homologs of phosphatidylethanolamine were extracted from cell-free supernatant of P. putida BD2 culture with ethyl acetate and identified by UPLC-MS analysis. For Rha-Rha-C10-C10 the surface tension decreased from 72 to 31mN/m and the critical micelle concentration was 0.130mg/mL. The Rha-Rha-C10-C10 was able to form stable aggregates (80-121nm). Pretreatment of a polystyrene surface with 0.5mg/mL rhamnolipid inhibited bacterial adhesion by 43-79% and that of the pathogenic fungal species C. albicans by 89-90%. The same concentration of phosphatidylethanolamines inhibited bacterial adhesion by 23-72% and that of C. albicans by 96-98%. To our knowledge, this is the first report where one type rhamnolipid and two homologs of phospholipid biosurfactants were produced by P. putida isolated from Arctic soil.

Lipopeptide biosurfactant pseudofactin II induced apoptosis of melanoma A 375 cells by specific interaction with the plasma membrane.

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, Ca2+ 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.

Screening concepts, characterization and structural analysis of microbial-derived bioactive lipopeptides: a review

Lipopeptide biosurfactants are surface active biomolecules that are produced by a variety of microorganisms. Microbial lipopeptides have gained the interest of microbiologists, chemists and biochemists for their high biodiversity as well as efficient action, low toxicity and good biodegradability in comparison to synthetic counterparts. In this report, we review methods for the production, isolation and screening, purification and structural characterization of microbial lipopeptides. Several techniques are currently available for each step, and we describe the most commonly utilized and recently developed techniques in this review. Investigations on lipopeptide biosurfactants in natural products require efficient isolation techniques for the characterization and evaluation of chemical and biological properties. A combination of chromatographic and spectroscopic techniques offer opportunities for a better characterization of lipopeptide structures, which in turn can lead to the application of lipopeptides in food, pharmaceutical, cosmetics, agricultural and bioremediation industries.

Spectroscopic and nonlinear optical properties of new chalcone fluorescent probes for bioimaging applications: a theoretical and experimental study

AbstractIn this study, the newly synthesized non-centrosymmetric, 4-dimethylamino-3′-isothiocyanatochalcone (PKA) compound was presented. This compound belongs to the chalcone group, and its main purpose is to be used in biomedical imaging as a fluorescence dye. For this reason, the linear and nonlinear properties in solvents of different polarity were thoroughly studied. In accordance with the requirements for a fluorochrome, the PKA compound is characterized by strong absorption, large Stokes’ shifts, relatively high fluorescence quantum yields and high nonlinear optical response. Moreover, the isothiocyanate reactive probe was conjugated with Concanavalin A. Conventional fluorescence microscopy imaging of Candida albicans cells incubated with the PKA-Concanavalin A, is presented. The results of this study show that the novel conjugate PKA-Concanavalin A could be a promising new probe for cellular labelling in biological and biomedical research. Graphical abstractSpectroscopic behavior of the PKA dye

Structure and mode of action of cyclic lipopeptide pseudofactin II with divalent metal ions

The interaction of natural lipopeptide pseudofactin II with a series of doubly charged metal cations was examined by matrix-assisted laser-desorption ionization-time of flight (MALDI-TOF) mass spectrometry and molecular modelling. The molecular modelling for metal-pseudofactin II provides information on the metal-peptide binding sites. Overall, Mg(2+), Ca(2+) and Zn(2+) favor the association with oxygen atoms spanning the peptide backbone, whereas Cu(2+) is coordinated by three nitrogens. Circular dichroism (CD) results confirmed that Zn(2+) and Cu(2+) can disrupt the secondary structure of pseudofactin II at high concentrations, while Ca(2+) and Mg(2+) did not essentially affect the structure of the lipopeptide. Interestingly, our results showed that the addition of Zn(2+) and Cu(2+) helped smaller micelles to form larger micellar aggregates. Since pseudofactin II binds metals, we tested whether this phenomena was somehow related to its antimicrobial activity against Staphylococcus epidermidis and Proteus mirabilis. We found that the antimicrobial effect of pseudofactin II was increased by supplementation of culture media with all tested divalent metal ions. Finally, by using Gram-positive and Gram-negative bacteria we showed that the higher antimicrobial activity of metal complexes of pseudofactin II is attributed to the disruption of the cytoplasmic membrane.

Physicochemical study of biomolecular interactions between lysosomotropic surfactants and bovine serum albumin

The interactions between two cationic lysosomotropic surfactants (2-dodecanoyloxyethyl)trimethylammonium bromide (DMM-11) and (2-dodecanoyloxypropyl)trimethylammonium bromide (DMPM-11) with bovine serum albumin (BSA) in Hepes buffer (pH=7.4) were systematically studied by surface tension, fluorescence and circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC). Furthermore, the size of the micellar aggregates and the polydispersity indexes of both cationic surfactants were studied by dynamic light scattering technique (DLS). The hydrodynamic radii, micellar volumes and aggregation numbers were calculated using a method based on density functional theory (DFT). The results showed that, in both cases, the surface tension was modified upon addition of BSA, and the critical micelle concentration (CMC) values of DMM-11 and DMPM-11 were higher in the presence of BSA. The fluorescence intensity of BSA decreased significantly as the concentration of both cationic surfactants increased and this effect was attributed to the formation of surfactant-BSA complexes. Synchronous fluorescence spectrometry showed the binding-induced conformational changes in BSA. Finally, CD and DLS results revealed the occurrence of changes in the secondary structure of the protein in the presence of both surfactants. In conclusion, understanding the interactions between lysosomotropic surfactants and BSA is required to explore their potential applications in medicine.

Trehalose Lipid Biosurfactant Reduces Adhesion of Microbial Pathogens to Polystyrene and Silicone Surfaces: An Experimental and Computational Approach

Rhodococcus fascians BD8, isolated from Arctic soil, was found to produce biosurfactant when grown on n-hexadecane as the sole carbon source. The glycolipid product was identified as the trehalose lipid with a molecular mass of 848 g mol−1. The purified biosurfactant reduced the surface tension of water from 72 to 34 mN m−1. The critical micelle concentration of trehalose lipid was 0.140 mg mL−1. To examine its potential for biomedical applications, the antimicrobial and antiadhesive activity of the biosurfactant was evaluated against several pathogenic microorganisms. Trehalose lipid showed antimicrobial activity against resistant pathogens. The largest antimicrobial activities of trehalose lipid were observed against Vibrio harveyi and Proteus vulgaris. The highest concentration tested (0.5 mg mL−1) caused a partial (11–34%) inhibition of other Gram-positive and Gram-negative bacteria and 30% inhibition of Candida albicans growth. The trehalose lipid also showed significant antiadhesive properties against all of the tested microorganisms to polystyrene surface and silicone urethral catheters. The biosurfactant showed 95 and 70% antiadhesive activity against C. albicans and Escherichia coli, respectively. Finally, the role and application of trehalose lipid as an antiadhesive compound was investigated by the modification of the polystyrene and silicone surfaces. The intermolecular interaction energy calculations were performed for investigated complexes at the density functional level of theory. The results indicate that the presence of aromatic moieties can be substantial in the stabilization of trehalose lipid-surface complexes. The antimicrobial and antiadhesive activities of trehalose lipid make them promising alternatives to synthetic surfactants in a wide range of medical applications. Based on our findings, we propose that, because of its ability to inhibit microbial colonization of polystyrene and silicone surfaces, trehalose lipid can be used as a surface coating agent.