Dmitry G. Deryabin

Facile preparation of amine and amino acid adducts of [60]fullerene using chlorofullerene C60Cl6 as a precursor.

We report a general synthetic approach to the preparation of highly functionalized amine and amino acid derivatives of [60]fullerene starting from readily available chlorofullerene C(60)Cl(6). The synthesized water-soluble amino acid derivative of C(60) demonstrated pronounced antiviral activity, while the cationic amine-based compound showed strong antibacterial action in vitro.

The activity of [60]fullerene derivatives bearing amine and carboxylic solubilizing groups against Escherichia coli : a comparative study

We report a comparative investigation of the antibacterial activity of two water-soluble fullerene derivatives bearing protonated amine (AF) and deprotonated carboxylic (CF) groups appended to the fullerene cage via organic linkers. The negatively charged fullerene derivative CF showed no tendency to bind to the bacterial cells and, consequently, no significant antibacterial activity. In contrast, the compound AF loaded with cationic groups showed strong and partially irreversible binding to the negatively charged Escherichia coli K12 TG1 cells and to human erythrocytes, also possessing negative zeta potential. Adsorption of AF on the bacterial surface was visualized by atomic force microscopy revealing the formation of specific clusters (AF aggregates) surrounding the bacterial cell. Incubation of E. coli K12 TG1 with AF led to a dose-dependent bactericidal effect with LD50 = 79.1 µM. The presence of human erythrocytes in the test medium decreased the AF antibacterial activity. Thus we reveal that the water-soluble cationic fullerene derivative AF possesses promising antibacterial activity, which might be utilized in the development of novel types of chemical disinfectants.

A zeta potential value determines the aggregate’s size of penta-substituted [60]fullerene derivatives in aqueous suspension whereas positive charge is required for toxicity against bacterial cells

BackgroundThe cause–effect relationships between physicochemical properties of amphiphilic [60]fullerene derivatives and their toxicity against bacterial cells have not yet been clarified. In this study, we report how the differences in the chemical structure of organic addends in 10 originally synthesized penta-substituted [60]fullerene derivatives modulate their zeta potential and aggregate’s size in salt-free and salt-added aqueous suspensions as well as how these physicochemical characteristics affect the bioenergetics of freshwater Escherichia coli and marine Photobacterium phosphoreum bacteria. Dynamic light scattering, laser Doppler micro-electrophoresis, agarose gel electrophoresis, atomic force microscopy, and bioluminescence inhibition assay were used to characterize the fullerene aggregation behavior in aqueous solution and their interaction with the bacterial cell surface, following zeta potential changes and toxic effects.ResultsDynamic light scattering results indicated the formation of self-assembled [60]fullerene aggregates in aqueous suspensions. The measurement of the zeta potential of the particles revealed that they have different surface charges. The relationship between these physicochemical characteristics was presented as an exponential regression that correctly described the dependence of the aggregate’s size of penta-substituted [60]fullerene derivatives in salt-free aqueous suspension from zeta potential value. The prevalence of DLVO-related effects was shown in salt-added aqueous suspension that decreased zeta potential values and affected the aggregation of [60]fullerene derivatives expressed differently for individual compounds. A bioluminescence inhibition assay demonstrated that the toxic effect of [60]fullerene derivatives against E. coli cells was strictly determined by their positive zeta potential charge value being weakened against P. phosphoreum cells in an aquatic system of high salinity. Atomic force microscopy data suggested that the activity of positively charged [60]fullerene derivatives against bacterial cells required their direct interaction. The following zeta potential inversion on the bacterial cells surface was observed as an early stage of toxicity mechanism that violates the membrane-associated energetic functions.ConclusionsThe novel data about interrelations between physicochemical parameters and toxic properties of amphiphilic [60]fullerene derivatives make possible predicting their behavior in aquatic environment and their activity against bacterial cells.

Investigation of Copper Nanoparticles Antibacterial Mechanisms Tested by Luminescent Escherichia coli Strains

The electrostatic interaction between positively charged copper nanoparticles aggregates (ζ = +15.9 ± 8.63 mV) and negatively charged surface of E. coli K12 TG1 cells (ζ = -50.0 ± 9.35 mV) has been established. The time-dependent decline of bacterial cells zeta potential and the coupled inhibition of constitutive bioluminescence level are the results of this interaction. The development of oxidative stress, probably defined by the electron transfer from the cytoplasmic membrane respiratory chains through membrane-integrated copper nanoparticle to the molecular oxygen, is shown as luminescence induction in superoxide- and peroxide-inducible E. coli K-12 MG1655 pSoxS::lux and pKatG::lux reporter strains. The final result of this process, which is responsible for the development of the bactericidal effect of copper nanoparticles, is DNA damage by active oxygen species detected by SOS-inducible E. coli pRecA::lux luminescent strain.

Antibacterial and quorum sensing regulatory activities of some traditional Eastern-European medicinal plants

Abstract The objective of this study was to screen extracts of twenty Eastern European medicinal plants, using wild-type and reporter Chromobacterium violaceum bioassays, for novel components that target bacterial cells and their quorum sensing (QS) communication systems. Three types of activity and their combinations were revealed: (i) direct antimicrobial growth-inhibitory activity, (ii) non-specific and specific pro-QS activities, (iii) anti-QS activity. Among seven plant extracts showing direct growth-inhibitory activity, the strongest effect was shown by Arctostaphylos uva- -ursi (bearberry) leaves. Many plants stimulated violacein production by wild-type C. violaceum ATCC 31532 in a non-specific manner, and only the herb Bidens tripartita (three-lobe beggarticks) contained compounds that mimic acyl-homoserine lactone and operated as a QS agonist. Anti-QS activity was found in eleven plants including Quercus robur (oak) cortex, Betula verrucosa (birch) buds and Eucalyptus viminalis (Manna Gum) leaves. Subsequent statistical analysis showed differences between antimicrobial and anti-QS activities, whereas both activities were defined by phylogenetic position of medical resource plant. Finally, extract from Quercus robur cortex revealed at least two fractions, showing different anti-QS mechanisms. These data confirm that multicomponent anti-infectious mechanisms are used by plants, which may be useful for drug development

Humidity-Dependent Bacterial Cells Functional Morphometry Investigations Using Atomic Force Microscope

The effect of a relative humidity (RH) in a range of 93–65% on morphological and elastic properties of Bacillus cereus and Escherichia coli cells was evaluated using atomic force microscopy. It is shown that gradual dehumidification of bacteria environment has no significant effect on cell dimensional features and considerably decreases them only at 65% RH. The increasing of the bacteria cell wall roughness and elasticity occurs at the same time. Observed changes indicate that morphological properties of B. cereus are rather stable in wide range of relative humidity, whereas E. coli are more sensitive to drying, significantly increasing roughness and stiffness parameters at RH ≤ 84% RH. It is discussed the dependence of the response features on differences in cell wall structure of gram-positive and gram-negative bacterial cells.

Application of the inhibition of bacterial bioluminescence test for assessment of toxicity of carbon-based nanomaterials

A method for assessment of integral biological toxicity of carbon-based nanomaterials (CBN) using a bacterial luminescent biosensor was developed, which accounted for the properties of the objects under study. The proposed approach includes a special procedure for the preparation of the analyzed CBN samples aimed at obtaining highly dispersed suspensions, an extended period of dynamic monitoring of the luminescence of the contacting sensor microorganism, and the special algorithm for quantification of the results of the bioluminescence analysis, in order to exclude the effect of the optical properties of CBN on the results of investigation. This method was used for assessment of the biological toxicity of a broad spectrum of CBN, such as single- and multi-walled nanotubes, nanofibres, and C60- and C70-fullerenes, in order to compare them in this respect to amorphous (nonstructured) carbon.

An investigation into the interaction between carbon-based nanomaterials and Escherichia coli cells using atomic force microscopy

Using atomic force microscopy (AFM), the nature of the contact between a wide range of carbonbased nanomaterials (CBNs) and Escherichia coli cells is described and the consequences of such an interaction are estimated. It is shown that the contact of multiwall and a number of single-wall carbon nanotubes with the surface of a model microorganism carries a probabilistic nature and is not accompanied by changes in the morphology and viability of bacterial cells. The damage of the surface structures and the consequent destruction of Escherichia coli are observed upon contact with single-wall carbon nanotubes with low degrees of purification, which is presumably determined by the presence of technological impurities in CBNs. A significant enhancement of the affinity of C60 fullerenes functionalized by amine groups to the surface of model microorganisms, which also leads to the development of a bactericidal effect, is shown.

Atomic Force Microscopy Reveals a Morphological Differentiation of Chromobacterium violaceum Cells Associated with Biofilm Development and Directed by N-Hexanoyl-L-Homoserine Lactone

Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceums adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceums cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure.

Antibacterial and Anti-Quorum Sensing Molecular Composition Derived from Quercus cortex (Oak bark) Extract

Quercus cortex (Oak bark) has been used in European folk medicine since medieval times for treatment of diarrhea, stomatitis, pharyngitis and skin inflammations. Its antimicrobial activity is a well-known therapeutic property of oak bark, and its novel anti-quorum sensing (QS) ability has also been described recently. In this study, we examined the bioactive compounds of Quercus cortex extract and compared their direct antibacterial and regulatory anti-QS effects against Chromobacterium violaceum CV026 in a biotest. Evaluation of the original Quercus cortex extract showed weak antibacterial and prominent anti-QS activities that were retained and completely restored when the samples were dried and re-hydrated. The one-step liquid chromatography result indicated that the anti-QS activity might be determined by hydrophobic compounds; however, the subsequent reverse phase high performance liquid chromatography led to dissipation and loss of the activity. The gas chromatography–mass spectrometry gave excellent resolution between a majority of the compounds. Based on this result, 10 of the 35 identified small molecules were selected for further screening. The subsequent investigation indicated several compounds determined both the antibacterial and anti-QS activities of the Quercus cortex extract. Direct antibacterial activity was shown for 1,2,3-benzenetriol and 4-propyl-1,3-benzenediol, while sub-inhibitory concentrations of these compounds led to anti-QS effects. Five compounds: 4-(3-hydroxy-1-propenyl)-2-methoxy-phenol; 3,4,5-trimethoxyphenol; 4-hydroxy-3-methoxybenzaldehyde; 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one and 2H-1-benzopyran-2-one were characterized as QS inhibitors independent of any effect on bacterial growth. Biologically relevant concentrations of each single component showed weak activity only while reconstruction of the small molecule composition derived from the Quercus cortex extract provided comparable complementary activity against C. violaceum CV026 in the biotest as the crude extract.