Gennadii Alexandrovich Naberezhnykh

Sepsis is a life-threatening complication of an infectious process that results from the excessive and uncontrolled activation of the host’s pro-inflammatory immune response to a pathogen. Lipopolysaccharide (LPS), also known as endotoxin, which is a major component of Gram-negative bacteria’s outer membrane, plays a key role in the development of Gram-negative sepsis and septic shock in humans. To date, no specific and effective drug against sepsis has been developed. This review summarizes data on LPS-binding proteins from marine invertebrates (ILBPs) that inhibit LPS toxic effects and are of interest as potential drugs for sepsis treatment. The structure, physicochemical properties, antimicrobial, and LPS-binding/neutralizing activity of these proteins and their synthetic analogs are considered in detail. Problems that arise during clinical trials of potential anti-endotoxic drugs are discussed.

Sepsis is a life-threatening complication of an infectious process that results from excessive and uncontrolled activation of the host's pro-inflammatory immune response to a pathogen. Lipopolysaccharide (LPS), also known as endotoxin, which is a major component of the Gram-negative bacteria outer membrane, plays a key role in the development of Gram-negative sepsis and septic shock in humans. To date, no specific and effective drug against sepsis has been developed. This review summarizes data on LPS-binding proteins from marine invertebrates (ILBPs), that inhibit LPS toxic effects, and are of interest as potential drugs for the sepsis treatment. The structure, physicochemical properties, antimicrobial and LPS-binding/neutralizing activity of these proteins and their synthetic analogues are considered in details. Problems that arise during clinical trials of potential anti-endotoxic drugs are discussed.

Quantum dots (QDs) are a new generation of fluorochromes with significant advantages over traditional organic dyes. QDs based on CdS are promising materials for optics, optoelectronics, biology, and medicine. QDs in the form of colloidal solutions are of great scientific and practical interest. CdS quantum dots were synthesized by chemical condensation in an aqueous solution using Yersinia pseudotuberculosis porin proteins, positively (chitosan) and negatively (carrageenan, lipopolysaccharide) charged polysaccharides. The maxima of the emission spectra for all samples were 450 nm, which indicates the same QD size; is determined by the size of the "cells" of the grid matrix, which limit the size of the QD. It was shown that the fluorescence intensity of QDs synthesized in LPS solutions was two times higher than that of other samples. The fluorescence of the samples and the maxima of the emission spectra (450 nm) are preserved during intensive dialysis against buffers, which indicates the stability of QDs and the possibility of using the obtained labeled preparations. Keeping samples at pH 3 leads to a significant decrease in fluorescence, especially for acidic polysaccharides. Anionic oxygen of phosphate, hydroxyl groups of sugar, nitrogen atoms can interact with metal ions, which are precursors (precursors) for CdS nanocrystals. It was found that the interaction with porin-specific IgG leads to a significant change in the luminescence intensity of QD-porin samples. This is of interest from the point of view of chemical sensing and opens the prospect of using QD-labeled porin-based nanostructures as biosensors.

Ordered nanostructures of porin from the outer membrane of Yersinia pseudotuberculosis (YpOmpF) were formed in two ways: from proteoliposomes and by direct protein reconstitution in the pre-deposited phospholipid bilayer on mica surface. The morphological analysis of the structures was performed by atomic force microscopy. It was shown that the efficiency of formation, the degree of homogeneity, and the size of porin domains substantially depend on the experimental conditions and the presence of lipopolysaccharide in a porin sample or in the bilayer. It was found that using proteoliposomes resulted in formation of the aggregates of porin nanodomains on the mica surface, with uneven distribution in the bilayer and quite different size ranges (50–250 nm). In the case of direct reconstruction of porin, it was shown that a decrease in pH of the solubilizing buffer promotes the inclusion of a sufficiently large amount of protein as homogeneous domains with an average size of 35–40 nm but does not lead to the formation of extended nanostructured regions in the bilayer. The most efficient incorporation of porin into the lipid bilayer with the formation of clusters of tightly packed protein domains was achieved using a porin sample in combination with peptidoglycan and lipopolysaccharide, which this protein is tightly bound to in the native bacterial membrane.

The commercial species of jellyfishes of Sea of Japan were studied as the sources of proteins that are capable lipopolysaccharides (LPSs) of Gram-negative bacteria. LPS-binding proteins have been found in lysates of jellyfishes mesogloea by means of immunofluorescence assay using LPS labeled with fluorescein (F-LPS). It was shown that lysate from rhopilema demonstrate the major LPS-binding activity in comparison with extracts from aurelia. For isolation and purifying of LPS-binding proteins from lysate of jellyfishes, the cation-exchange chromatography was used. It was shown, that fractions of jellyfishes lysate after cation-exchange chromatography contained several LBP with high-molecular weights. By ligand enzyme solid phase assay it was shown that isolated LBP of jellyfishes was bound to LPS directly by concentration-dependent manner in saturation process.

The interactions of lipopolysaccharide (LPS) with the polycation chitosan and its derivatives — high molecular weight chitosans (300 kDa) with different degree of N-alkylation, its quaternized derivatives, N-monoacylated low molecular weight chitosans (5.5 kDa) — entrapped in anionic liposomes were studied. It was found that the addition of chitosans changes the surface potential and size of negatively charged liposomes, the magnitudes of which depend on the chitosan concentration. Acylated low molecular weight chitosan interacts with liposomes most effectively. The binding of alkylated high molecular weight chitosan with liposomes increases with the degree of its alkylation. The analysis of interaction of LPS with chitoliposomes has shown that LPS-binding activity decreased in the following order: liposomes coated with a hydrophobic chitosan derivatives > coated with chitosan > free liposomes. Liposomes with N-acylated low molecular weight chitosan bind LPS more effectively than liposomes coated with N-alkylated high molecular weight chitosans. The increase in positive charge on the molecules of N-alkylated high molecular weight chitosans at the cost of quaternization does not lead to useful increase in efficiency of binding chitosan with LPS. It was found that increase in LPS concentration leads to a change in surface ζ-potential of liposomes, an increase in average hydrodynamic diameter, and polydispersity of liposomes coated with N-acylated low molecular weight chitosan. The affinity of the interaction of LPS with a liposomal form of N-acylated chitosan increases in comparison with free liposomes. Computer simulation showed that the modification of the lipid bilayer of liposomes with N-acylated low molecular weight chitosan increases the binding of lipopolysaccharide without an O-specific polysaccharide with liposomes due to the formation of additional hydrogen and ionic bonds between the molecules of chitosan and LPS.

A series of four water-soluble chitosan derivatives differing in molecular mass, hydrophobicity, and charge was synthesized and tested for the intensity of their effects on Gram-negative and Gram-positive bacteria. It was shown that the tested compounds allowed the penetration of ethidium bromide into the bacteria, which showed increased permeability of their cell walls under the effect of chitosans. The tolerance to various chitosan derivatives differed in Gram-negative and Gram-positive bacteria. The Gram-negative bacteria were the most responsive to high-molecular chitosan and the Gram-positive ones, to N-,O-carboxypropylchitosan, whereas high-molecular chitosan had little effect. Research on the correlation between the structure and activity of the studied compounds revealed that depolymerization of chitosan reduced, and introduction of hydrophobic substantives in chitosan molecule significantly enhanced its permeability effect on bacterial cell walls. The obtained results provide a basis for the construction of new chitosan derivatives with antimicrobial activities.