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.

The main limitation for practical implementation of quantum dots-based sensors and biosensors is the possible contamination of sensing media with quantum dots (QDs) moved out from the sensor structure, being critical for living systems measurements. Numerous efforts have addressed the challenge of pre-synthesized QDs incorporation into porous matrix provide, on the one hand, proper fixation of quantum dots in its volume and preserving a free analyte transfer from the sensing media to them - on the other hand. Here, we propose an alternative insight into this problem. Instead of using preliminary synthesized particles for doping a matrix, we have in situ synthesized cadmium sulfide QDs in porous biopolymeric matrices, both in an aqueous solution and on a mica substrate. The proposed technique allows obtaining QDs in a matrix acting simultaneously as a ligand passivating surface defects and preventing QDs aggregation. The conjugates were used as a photoluminescence sensor for the metal ions and glutathione detection in an aqueous media. Different kinds of sensor responses have been found depending on the analyte nature. Zinc ions' presence initiates the intraband QDs emission increases due to the reduction of non-radiative processes. The presence of copper ions, in contrast, leads to a gradual photoluminescence decrease due to the formation of the non-luminescent copper-based alloy in the QDs structure. Finally, the presence of glutathione initiates a ligand exchange process followed by some QDs surface treatment enhancing defect-related photoluminescence. As a result, three different kinds of sensor responses for three analytes allow claiming development of a new selective QD-based sensor suitable for biomedical applications.

Белковые молекулы обладают уникальным свойством передачи материала и/или информации через непроницаемый барьер, которым является липидный бислой. Эта своеобразная роль клеточных «привратников» особенно интересна для целей нанобиотехнологии, поскольку мембранные белки могут с успехом использоваться для связи между двумя различными средами, разделенными липидным бислоем. В настоящем обзоре описаны структурные особенности и свойства трансмембранных белков как наиболее перспективных компонентов современных биосенсорных устройств. Приведены также методы формирования липидных бислоев, прикрепленных к твердой поверхности, и способы получения жесткой белковой «арматуры», которые позволяют реконструировать и сохранять используемые белки в функционально активной конформации по отношению к широкому спектру определяемых аналитов. Высокая селективность и специфичность белковых молекул в распознавании рецепторов или лигандов делает их незаменимыми для биомедицинского анализа и молекулярной диагностики.

Protein molecules have a unique property of transferring material or information across the impenetrable barrier of a lipid bilayer. This peculiar role of cell “gatekeepers” is especially interesting for the purposes of nanobiotechnology because membrane proteins can be successfully used for communication between two different media separated by a lipid bilayer. This review describes the structural features and properties of transmembrane proteins as the most promising components of modern biosensor devices. Another focus is the methods to produce lipid bilayers attached to a solid surface and to obtain a rigid protein “armature.” These methods make it possible to reconstruct and maintain proteins in the conformation that is functionally active toward a wide range of analytes to be determined. Being highly selective and specific in recognizing receptors or ligands, protein molecules provide an indispensable tool for biomedical analysis and molecular diagnosis.

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.

Mature pore-forming OmpF protein from the outer membrane of Yersinia pseudotuberculosis was expressed in Escherichia coli in the form of inclusion bodies (IBs) under different cultivation conditions. The properties and structural organization of the IBs as well as the structure of the recombinant porin (rOmpF) solubilized from the IBs were investigated using electron microscopy, dynamic light scattering, optical spectroscopy, and specific hydrophobic dyes. The size, shape, and stability of the IBs under denaturing solutions were determined. It was found that the IBs were readily soluble in SDS and more resistant to urea. Dissolution of the IBs in both denaturing agents led to formation of a heterogeneous in size population of oligomeric particles. The IBs contained an intermediate form of the rOmpF with native-like secondary structure and elements of tertiary structure, which was able to penetrate a lipid bilayer and adopt a functionally active conformation. There were no significant differences in the properties and structure between the examined IBs formed at different concentrations of the inducer (IPTG). However, the content of amyloids in the IBs increased with increasing concentration of the inducer. These results contribute to the development of new approaches for the production of active proteins from IBs, as well as biologically and functionally active IBs.

This study investigates the spreading of the Gram-negative bacterium Yersinia ruckeri , pathogenic for fish, among representatives of the marine flora and fauna of the Sea of Okhotsk. The enzyme-linked immunosorbent assay (ELISA) revealed the presence of Y. ruckeri outer membrane antigens in 9 of the 29 samples obtained by washing the internal organs of fish, shellfish, crustaceans, and surfaces of algal thalli. Using lipopolysaccharide (LPS) containing a fluorescent label we detected LPS-binding proteins, which are a component of the innate immune system of invertebrates, in half of the samples studied. However, the simultaneous presence of Y. ruckeri and LPS-binding proteins was found only in brachiopods, scallops, and shrimps. It is assumed that this may be indicative of a particular response of the immune system of these animals to infection and/or manifestation of a toxic effect of Y. ruckeri .

To gain a mechanistic insight in the functioning of the OmpF-like porin from Yersinia pseudotuberculosis (YOmpF), we compared the effect of pH variation on the ion channel activity of the protein in planar lipid bilayers and its binding to lipid membranes. The behavior of YOmpF channels upon acidification was similar to that previously described for Escherichia coli OmpF. In particular, a decrease in pH of the bathing solution resulted in a substantial reduction of YOmpF single channel conductance, accompanied by the emergence of subconductance states. Similar subconductance substates were elicited by the addition of lysophosphatidylcholine. This observation, made with porin channels for the first time, pointed to the relevance of lipid-protein interactions, in particular, the lipid curvature stress, to the appearance of subconductance states at acidic pH. Binding of YOmpF to membranes displayed rather modest dependence on pH, whereas the channel-forming potency of the protein tremendously decreased upon acidification.

The interaction of endotoxin (lipopolysaccharide — LPS) with low molecular weight chitosan (5.5 kDa), its N-acylated derivative and chitoliposomes was studied using a gravimetric piezoelectric quartz crystal microbalance biosensor. The optimal conditions for the formation of a biolayer based on immobilized LPS on the resonator surface and its regeneration were elaborated. The association and dissociation rate constants for LPS binding to chitosans were determined and the affinity constants (Kaf) were calculated based on the data on changes in the oscillation frequency of the quartz crystal resonator. The Kaf values correlated with the ones obtained using other methods. The affinity of N-acylated chitosan binding to LPS was higher than that of the parent chitosan binding to LPS. Based on the results obtained, we suggest that water-soluble N-acylated derivatives of chitosan with low degree of substitution of amino groups could be useful compounds for endotoxin binding and neutralization.

Invertebrates from the Sea of Okhotsk were studied as a source of proteins that are capable of binding to lipopolysaccharides (LPSs) of gram-negative bacteria. A DOT analysis using dansyl-labeled LPS that we developed revealed LPS-binding proteins in blood-cell lysates of 21 out of 33 investigated species of invertebrates. Most of the investigated species with positive LPS-binding activity were decapod crustaceans (class Malacostraca, phylum Arthropoda). Hemocyte lysates from the red king crab Paralithodes camtschaticus and the sculptured shrimp Sclerocrangon boreas contained several LPS-binding proteins with different molecular weights. LPS-binding proteins were found for the first time in echinoderms (classes Holothuroidea, Asteroidea, and Echinoidea), sipunculans, and brachiopods.

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.

Amphiphilic low-molecular-weight chitosan N-acylated with 3-hydroxytetradecanoic acid was synthesized. It was shown that addition of the acylated chitosan changes the surface potential and size of negatively charged liposomal particles, the magnitudes of which depend on the chitosan concentration. Charge neutralization and aggregation of the liposomes occurs at low chitosan concentration. Increasing its concentration produces positive charge on the surface of the liposomes and decreases their size. This increases the stability of the liposomal particles in solution. Liposomes coated with chitosan may be of interest as carriers for genes, vaccines, and drugs.

A low-molecular-weight cationic protein that can bind human and rabbit immunoglobulins G has been isolated from Yersinia pseudotuberculosis cells. This immunoglobulin binding protein (IBP) interacts with IgG Fc-fragment, the association constant of the resulting complex being 3.1 μM−1. MALDI-TOF mass spectrometry analysis of IBP revealed its molecular mass of 16.1 kDa, and capillary isoelectrofocusing analysis showed pI value of 9.2. N-Terminal sequence determination by Edman degradation revealed the sequence of the 15 terminal amino acid residues (ADKIAIVNVSSIFQ). Tryptic hydrolysate of IBP was subjected to MALDI-TOF mass spectrometry for proteolytic peptide profiling. Based on the peptide fingerprint, molecular mass, pI, and N-terminal sequence and using bioinformatic resources, IBP was identified as Y. pseudotuberculosis periplasmic chaperone Skp. Using the method of comparative modeling a spatial model of Skp has been built. This model was then used for modeling of Skp complexes with human IgG1 Fc-fragment by means of molecular docking.

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.

The interactions of lipopolysaccharide (LPS) with the natural polycation chitosan and its derivatives—high molecular weight chitosans (80 kD) with different degree of acetylation, low molecular weight chitosan (15 kD), acylated oligochitosan (5.5 kD) and chitooligosaccharides (biose, triose, and tetraose)—were studied using ligand-enzyme solid-phase assay. The LPS-binding activity of chitosans (80 kD) decreased with increase in acetylation degree. Affinity of LPS interaction with chitosans increased after introduction of a fatty acid residue at the reducing end of chitosan. Activity of N-monoacylated chitooligosaccharides decreased in the order: oligochitosan → tetra-→ tri-→ disaccharides. The three-dimensional structures of complexes of R-LPS and chitosans with different degree of acetylation, chitooligosaccharides, and their N-monoacylated derivatives were generated by molecular modeling. The number of bonds stabilizing the complexes and the energy of LPS binding with chitosans decreased with increase in acetate group content in chitosans and resulted in changing of binding sites. It was shown that binding sites of chitooligosaccharides on R-LPS overlapped and chitooligosaccharide binding energies increased with increase in number of monosaccharide residues in chitosan molecules. The input of the hydrophobic fragment in complex formation energy is most prominent for complexes in water phase and is due to the hydrophobic interaction of chitooligosaccharide acyl fragment with fatty acid residues of LPS.

The interaction of endotoxins—lipopolysaccharides (LPS) different in degree of the O-specific chain polymerization—with 20-and 130-kD chitosan was studied using the competitive binding of LPS with the complex of chitosananionic dye (tropaeolin 000-2) and the direct binding of 125I-labeled LPS with chitosan immobilized on Sepharose 4B. The interaction of 20-kD chitosan with LPS was non-cooperative, and immobilization of the polycation on Sepharose resulted in its binding to 125I-labeled LPS with a positive cooperativity. The interaction of LPS possessing a long O-specific chain with 130-kD chitosan was characterized by negative cooperativity. Binding constants of LPS with the polycation and the number of binding sites per amino group of chitosan were determined. The interaction affinity and stoichiometry of the LPS-chitosan complexes significantly depend on the LPS structure and concentration in the reaction mixture. The increase in the length of carbohydrate chains of LPS results in increase in the binding constants and decrease in the bound endotoxin amount.