Angeles Manresa

Self-aggregation and antimicrobial activity of imidazolium and pyridinium based ionic liquids in aqueous solution.

Two series of long-chain imidazolium and pyridinium based ionic liquids (1-alkyl-3-methylimidazolium and 1-alkylpyridinium bromides) were synthesised and the effect of the alkyl chain length and the nature of the cationic head group on micellization and antimicrobial activity of the ionic liquids (ILs) were investigated. Tensiometry, conductometry, spectrofluorimetry and PGSE-NMR were applied to study the self-aggregation of the amphiphilic ILs in aqueous solution. The ILs investigated displayed surface activity and the characteristic chain length dependence of the micellization process of surfactants. The antimicrobial activity was evaluated against Gram-negative and Gram-positive bacteria and fungi. ILs containing more than eight carbon atoms in the alkyl chain showed antimicrobial activity. Their efficiency as antimicrobial agents increased with the hydrophobicity of the amphiphilic cation being the C(14) homologous the most active compounds.

Aggregation Behavior and Antimicrobial Activity of Ester-Functionalized Imidazolium- and Pyridinium-Based Ionic Liquids in Aqueous Solution

Two series of long chain imidazolium- and pyridinium-based ionic liquids containing an ester functional group in the alkyl side chain, 3-methyl-1-alkyloxycarbonylmethylimidazolium bromides (C(n)EMeImBr) and 1-alkyloxycarbonylmethylpyridinium bromides (C(n)EPyrBr), were synthesized and their thermal stability, aggregation behavior in aqueous medium, and antimicrobial activity investigated. The introduction of an ester group decreased the thermal stability of the functionalized ILs compared to simple alkyl chain containing ILs (1-alkyl-3-methylimidazolium bromides and 1-alkylpyridinium bromides). Tensiometry, conductimetry, and spectrofluorimetry were applied to study the self-aggregation of the amphiphilic ILs in aqueous solution. The ILs investigated displayed surface activity and the characteristic chain length dependence of the micellization process of surfactants. As compared to simple alkyl chain containing ILs bearing the same hydrocarbon chain, ester-functionalized ILs possess higher adsorption efficiency (pC(20)) and significantly lower critical micelle concentration (cmc) and surface tension at the cmc (γ(cmc)), indicating that the incorporation of an ester group promotes adsorption at the air/water interface and micelle formation. The antimicrobial activity was evaluated against Gram-negative and Gram-positive bacteria and fungi. ILs containing more than eight carbon atoms in the alkyl chain showed antimicrobial activity. Their efficiency as antimicrobial agents increased with the hydrophobicity of the amphiphilic cation being the C(12) homologous the most active compounds. The incorporation of an ester group particularly increased the biological activity against fungi.

Cationic surfactants from lysine: Synthesis, micellization and biological evaluation

Biocompatible cationic surfactants from the amino acid lysine (hydrochloride salts of N(epsilon)-lauroyl lysine methyl ester, N(epsilon)-myristoyl lysine methyl ester and N(epsilon)-palmitoyl lysine methyl ester) have been prepared in high yields by lysine acylation in epsilon position with three natural saturated fatty acids. The micellization process of these surfactants has been studied using the PGSE-NMR technique. The compounds were tested as antimicrobial agents against Gram-positive and Gram-negative bacteria. The surfactants show moderate antimicrobial activity against the Gram-positive bacteria but Gram-negative bacteria are resistant to these surfactants in the concentration range tested. The haemolytic activity is considerably lower than those reported for other cationic N(alpha)-acyl amino acid analogues. The acute toxicity against Daphnia magna and biodegradability was studied. The toxicity is clearly lower than that reported for conventional cationic surfactants from quaternary ammonium and the three surfactants from lysine can be classified as ready biodegradable surfactants.

Oxydation of oleic acid to (E)-10-hydroperoxy-8-octadecenoic and (E)-10-hydroxy-8-octadecenoic acids by Pseudomonas sp. 42A2

Biotransformation of oleic acid with Pseudomonas sp. 42A2 has been found to produce(E)-10-hydroxy-8-octadecenoic acid (2a), (E)-10-hydroperoxy-8-octadecenoic acid (3a), and (E)-7,10-dihydroxy-8-octadecenoic acid (4a). Structures of the metabolites were fully characterized by infrared and 1H and 13C NMR spectra of the acids, by fast atom bombardment (FAB) and electron impact (EI) and chemical ionization (CI) mass spectrometry of the corresponding methyl esters. This is the first time that the two former compounds of trans stereochemistry have been described to have originated from a Pseudomonas sp. cell culture. Time course of products accumulation showed that biotransformation started with bacterial growth, the amount of products 2a (5.58 g/l) and 4a (2.63 g/l) being optimum after 24 h of incubation while hydroperoxide 3a (1.15 g/l) reached its maximum after 16 h of the biotransformation process. Experiments conducted to ascertain whether the conversion enzyme(s) was cell-bound or extracellular, showed that the enzyme(s) is cell bound, located in the periplasmic space and has lipoxygenase activity.

Rapid flow cytometry - Nile red assessment of PHA cellular content and heterogeneity in cultures of Pseudomonas aeruginosa 47T2 (NCIB 40044) grown in waste frying oil

The accumulation of cytoplasmic polyhydroxyalkanoates (PHAs) and the heterogeneity of bacterial populations were analysed by flow cytometry and SYTO-13 and Nile red staining in rhamnolipid-producing Pseudomonas aeruginosa cultures grown in waste frying oil as carbon source. A combination of SYTO-13 and Nile red fluorescence with cytometric forward and side scatter values may allow increases in the final production of polyhydroxyalkanoates (PHA) by two basic mechanisms: (i) rapid assessment of polyhydroxyalkanoate content and (ii) definition of flow cytometric cell sorting protocols to select high polyhydroxyalkanoate (PHA)-producing strains. We report a rapid (less than 30 min) flow cytometric assessment of PHAs in Pseudomonas aeruginosa 47T2 following Nile red staining: (i) to estimate cellular PHAs content; (ii) to study heterogeneity of the batch cultures producing PHAs and (iii) to establish the basis for sorting sub-populations with a high capacity to accumulate PHAs.

Permeabilization of biological and artificial membranes by a bacterial dirhamnolipid produced by Pseudomonas aeruginosa.

Pseudomonas aeruginosa, when cultured under the appropriate conditions, secretes rhamnolipids to the external medium. These glycolipids constitute one of the most interesting classes of biosurfactants so far. A dirhamnolipid fraction was isolated and purified from the crude biosurfactant, and its action on model and biological membranes was studied. Dirhamnolipid induced leakage of internal contents, as measured by the release of carboxyfluorescein, in phosphatidylcholine unilamellar vesicles, at concentrations below its CMC. Membrane solubilization was not observed within this concentration range. The presence of inverted cone-shaped lipids in the membrane, namely lysophosphatidylcholine, accelerated leakage, whereas cone-shaped lipids, like phosphatidylethanolamine, decreased leakage rate. Increasing concentrations of cholesterol protected the membrane against dirhamnolipid-induced leakage, which was totally abolished by the presence of 50 mol% of the sterol. Dirhamnolipid caused hemolysis of human erythrocytes through a lytic mechanism, as shown by the similar rates of K(+) and hemoglobin leakage, and by the absence of effect of osmotic protectants. Scanning electron microscopy showed that the addition of the biosurfactant changed the usual disc shape of erythrocytes into that of spheroechinocytes. The results are discussed within the frame of the biological actions of dirhamnolipid, and the possible future applications of this biosurfactant.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

Self-assembly and antimicrobial activity of long-chain amide-functionalized ionic liquids in aqueous solution

Surface active amide-functionalized ionic liquids (ILs) consisting of a long alkyl chain (C6C14) connected to a polar head group (methylimidazolium or pyridinium cation) via an amide functional group were synthesized and their thermal stability, micellar properties and antimicrobial activity in aqueous solution investigated. The incorporation of an amide group increased the thermal stability of the functionalized ionic liquids compared to simple alkyl chain substituted ionic liquids. The surface activity and aggregation behaviour in aqueous solution of amide-functionalized ionic liquids were examined by tensiometry, conductivity and spectrofluorimetry. Amide-functionalized ILs displayed surface activity and their critical micelle concentration (cmc) in aqueous media decreased with the elongation of the alkyl side chain as occurs for typical surfactants. Compared to non-functionalized ILs bearing the same alkyl chain, ionic liquids with an amide moiety possess higher surface activity (pC20) and lower cmc values. The introduction of an amide group in the hydrophobic chain close to the polar head enhances adsorption at the air/water interface and micellization which could be attributed to the H-bonding in the headgroup region. The antimicrobial activity was evaluated against a panel of representative Gram-negative and Gram-positive bacteria and fungi. Amide-functionalized ILs with more than eight carbon atoms in the side chain showed broad antimicrobial activity. Antibacterial activities were found to increase with the alkyl chain length being the C12 homologous the most effective antimicrobial agents. The introduction of an amide group enhanced significantly the antifungal activity as compared to non-functionalized ILs.

Membrane Vesicles: A Common Feature in the Extracellular Matter of Cold-Adapted Antarctic Bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter, which has potential biotechnology applications. We examined the ultrastructure of extracellular matter from five Antarctic bacteria (Shewanella livingstonensis NF22T, Shewanella vesiculosa M7T, Pseudoalteromonas sp. M4.2, Psychrobacter fozii NF23T, and Marinobacter guineae M3BT) by transmission electron microscopy after high-pressure freezing and freeze substitution. All analyzed extracellular matter appeared as a netlike mesh composed of a capsular polymer around cells and large numbers of membrane vesicles (MVs), which have not yet been described for members of the genera Psychrobacter and Marinobacter. MVs showed the typical characteristics described for these structures, and seemed to be surrounded by the same capsular polymer as that found around the cells. The analysis of MV proteins from Antarctic strains by SDS-PAGE showed different banding profiles in MVs compared to the outer membrane, suggesting some kind of protein sorting during membrane vesicle formation. For the psychrotolerant bacterium, S. livingstonensis NF22T, the growth temperature seemed to influence the amount and morphology of MVs. In an initial attempt to elucidate the functions of MVs for this psychrotolerant bacterium, we conducted a proteomic analysis on membrane vesicles from S. livingstonensis NF22T obtained at 4 and 18°C. At both temperatures, MVs were highly enriched in outer membrane proteins and periplasmic proteins related to nutrient processing and transport in Gram-negative bacteria suggesting that MVs could be related with nutrient sensing and bacterial survival. Differences were observed in the expression of some proteins depending on incubation temperature but further studies will be necessary to define their roles and implications in the survival of bacteria in the extreme Antarctic environment.

Bacterial lipoxygenases, a new subfamily of enzymes? A phylogenetic approach

Lipoxygenases (EC. 1.13.11.12) are a non-heme iron enzymes consisting of one polypeptide chain folded into two domains, the N-terminal domain and the catalytic moiety β-barrel domain. They catalyze the dioxygenation of 1Z,4Z-pentadiene moieties of polyunsaturated fatty acids obtaining hydroperoxy fatty acids. For years, the presence of lipoxygenases was considered a eukaryotic feature, present in mammals, plants, small marine invertebrates, and fungi, but now, some lipoxygenase sequences have been detected on prokaryotic organisms, changing the idea that lipoxygenases are exclusively a eukaryotic affair. Lipoxygenases are involved in different types of reactions on eukaryote organisms where the biological role and the structural characteristics of these enzymes are well studied. However, these aspects of the bacterial lipoxygenases have not yet been elucidated and are unknown. This revision discusses biochemical aspects, biological applications, and some characteristics of these enzymes and tries to determine the existence of a subfamily of bacterial lipoxygenases in the context of the phylogeny of prokaryotic lipoxygenases, supporting the results of phylogenetic analyzes with the comparison and discussion of structural information of the first prokaryotic lipoxygenase crystallized and other eukaryotic lipoxygenases structures.