Marcin Łukaszewicz

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.

Capric Acid Secreted by S. boulardii Inhibits C. albicans Filamentous Growth, Adhesion and Biofilm Formation

Candidiasis are life-threatening systemic fungal diseases, especially of gastro intestinal track, skin and mucous membranes lining various body cavities like the nostrils, the mouth, the lips, the eyelids, the ears or the genital area. Due to increasing resistance of candidiasis to existing drugs, it is very important to look for new strategies helping the treatment of such fungal diseases. One promising strategy is the use of the probiotic microorganisms, which when administered in adequate amounts confer a health benefit. Such a probiotic microorganism is yeast Saccharomyces boulardii, a close relative of baker yeast. Saccharomyces boulardii cells and their extract affect the virulence factors of the important human fungal pathogen C. albicans, its hyphae formation, adhesion and biofilm development. Extract prepared from S. boulardii culture filtrate was fractionated and GC-MS analysis showed that the active fraction contained, apart from 2-phenylethanol, caproic, caprylic and capric acid whose presence was confirmed by ESI-MS analysis. Biological activity was tested on C. albicans using extract and pure identified compounds. Our study demonstrated that this probiotic yeast secretes into the medium active compounds reducing candidal virulence factors. The chief compound inhibiting filamentous C. albicans growth comparably to S. boulardii extract was capric acid, which is thus responsible for inhibition of hyphae formation. It also reduced candidal adhesion and biofilm formation, though three times less than the extract, which thus contains other factors suppressing C. albicans adherence. The expression profile of selected genes associated with C. albicans virulence by real-time PCR showed a reduced expression of HWP1, INO1 and CSH1 genes in C. albicans cells treated with capric acid and S. boulardii extract. Hence capric acid secreted by S. boulardii is responsible for inhibition of C. albicans filamentation and partially also adhesion and biofilm formation.

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.

14-3-3 Protein regulation of the antioxidant capacity of transgenic potato tubers

The 14-3-3 proteins are known to regulate a large number of processes. In this study we use the chemiluminescence method to show the impact of 14-3-3 proteins on antioxidant activity in potato plants. Tuber epidermis analysis in transgenic potato plants with overexpression of 14-3-3 proteins showed a 45% higher antioxidant activity than control plants. The opposite effect was found in transgenic plants with repressed 14-3-3 proteins. The changes in antioxidant activity were correlated with the amount of phenolic acids and anthocyanins present. The most abundant of the phenolics was chlorogenic acid (about 40% of total), while petunidine was the most common of the anthocyanins (about 50% of total). The phenolic and anthocyanin level was highly correlated (0.92). A regulatory role of the 14-3-3 proteins in antioxidant compound biosynthesis is postulated.

Methanogenic archaea database containing physiological and biochemical characteristics

The methanogenic archaea are a group of micro-organisms that have developed a unique metabolic pathway for obtaining energy. There are 150 characterized species in this group; however, novel species continue to be discovered. Since methanogens are considered a crucial part of the carbon cycle in the anaerobic ecosystem, characterization of these micro-organisms is important for understanding anaerobic ecology. A methanogens database (MDB; http://metanogen.biotech.uni.wroc.pl/), including physiological and biochemical characteristics of methanogens, was constructed based on the descriptions of isolated type strains. Analysis of the data revealed that methanogens are able to grow from 0 to 122 °C. Methanogens growing at the same temperature may have very different growth rates. There is no clear correlation between the optimal growth temperature and the DNA G+C content. The following substrate preferences are observed in the database: 74.5% of archaea species utilize H2+CO2, 33% utilize methyl compounds and 8.5% utilize acetate. Utilization of methyl compounds (mainly micro-organisms belonging to the genera Methanosarcina and Methanolobus ) is seldom accompanied by an ability to utilize H2+CO2. Very often, data for described species are incomplete, especially substrate preferences. Additional research leading to completion of missing information and development of standards, especially for substrate utilization, would be very helpful.

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.

The antagonistic effect of Saccharomyces boulardii on Candida albicans filamentation, adhesion and biofilm formation

The dimorphic fungus Candida albicans is a member of the normal flora residing in the intestinal tract of humans. In spite of this, under certain conditions it can induce both superficial and serious systemic diseases, as well as be the cause of gastrointestinal infections. Saccharomyces boulardii is a yeast strain that has been shown to have applications in the prevention and treatment of intestinal infections caused by bacterial pathogens. The purpose of this study was to determine whether S. boulardii affects the virulence factors of C. albicans. We demonstrate the inhibitory effect of live S. boulardii cells on the filamentation (hyphae and pseudohyphae formation) of C. albicans SC5314 strain proportional to the amount of S. boulardii added. An extract from S. boulardii culture has a similar effect. Live S. boulardii and the extract from S. boulardii culture filtrate diminish C. albicans adhesion to and subsequent biofilm formation on polystyrene surfaces under both aerobic and microaerophilic conditions. This effect is very strong and requires lower doses of S. boulardii cells or concentrations of the extract than serum-induced filamentation tests. Saccharomyces boulardii has a strong negative effect on very important virulence factors of C. albicans, i.e. the ability to form filaments and to adhere and form biofilms on plastic surfaces.

The effect of Saccharomyces boulardii on Candida albicans-infected human intestinal cell lines Caco-2 and Intestin 407

Saccharomyces boulardii is a probiotic strain that confers many benefits to human enterocolopathies and is used against a number of enteric pathogens. Candida albicans is an opportunistic pathogen that causes intestinal infections in immunocompromised patients, and after translocation into the bloodstream, is responsible for serious systemic candidiasis. In this study, we investigated the influence of S. boulardii cells and its culture extract on C. albicans adhesion to Caco-2 and Intestin 407 cell lines. We also tested the proinflammatory IL-1beta, IL-6 and IL-8 cytokine expression by C. albicans-infected Caco-2 cells, using real-time RT-PCR. We found that both S. boulardii and its extract significantly inhibited C. albicans adhesion to epithelial cell lines. The IL-8 gene expression by C. albicans-infected Caco-2 cells was suppressed by the addition of S. boulardii extract. Our results indicate that S. boulardii affects C. albicans adhesion and reduces cytokine-mediated inflammatory host response.

Effect of Antioxidants on Saccharomyces cerevisiae Mutants Deficient in Superoxide Dismutases

S. cerevisiae strain Δsod1 lacking Cu,Zn-superoxide dismutase and Δsod1Δsod2 mutant lacking both Cu,Zn-SOD and Mn-superoxide dismutase displayed strongly reduced aerobic growth on glucose, glycerol and lactate; Δsod2 deletion had no effect on aerobic growth on glucose and largely precluded growth on glycerol and lactate. The oxygen-induced growth defects and their alleviation by antioxidants depended on growth conditions, in particular on oxygen supply to cells. Under strong aeration, vitamins A and E had a low effect, 100 µmol/L quercetin alleviated the growth defects of all three mutants while β-carotene had no growth-restoring effect. The superoxide producer paraquat inhibited the aerobic growth of all three mutants in a concentration-dependent manner. Low concentrations of antioxidants had no effect on paraquat toxicity while higher concentrations supported the toxic effect of the agent.