Host Defense Proteins and Peptides with Lipopolysaccharide-Binding Activity from Marine Invertebrates and Their Therapeutic Potential in Gram-Negative Sepsis.

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.

Expression of membrane beta-barrel protein in E. coli at low temperatures: Structure of Yersinia pseudotuberculosis OmpF porin inclusion bodies.

The recombinant OmpF porin of Yersinia pseudotuberculosis as a model of transmembrane protein of the ß-barrel structural family was used to study low growth temperature effect on the structure of the produced inclusion bodies (IBs). This porin showed a very low expression level in E. coli at a growth temperature below optimal 37 °C. The introduction of a N-terminal hexahistidine tag into the mature porin molecule significantly increased the biosynthesis of the protein at low cultivation temperatures. The recombinant His-tagged porin (rOmpF-His) was expressed in E. coli at 30 and 18 °C as inclusion bodies (IB-30 and IB-18). The properties and structural organization of IBs, as well as the structure of rOmpF-His solubilized from the IBs with urea and SDS, were studied using turbidimetry, electron microscopy, dynamic light scattering, optical spectroscopy, and amyloid-specific dyes. IB-18, in comparison with IB-30, has a higher solubility in denaturants, suggesting a difference between IBs in the conformation of the associated polypeptide chains. The spectroscopic analysis revealed that rOmpF-His IBs have a high content of secondary structure with a tertiary-structure elements, including a native-like conformation, the proportion of which in IB-18 is higher than in IB-30. Solubilization of the porin from IBs is accompanied by a modification of its secondary structure. The studied IBs also contain amyloid-like structures. The results obtained in this study expand our knowledge of the structural organization of IBs formed by proteins of different structural classes and also have a contribution into the new approaches development of producing functionally active recombinant membrane proteins.

Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy.

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.

Porin from Marine Bacterium Marinomonas primoryensis KMM 3633T: Isolation, Physico-Chemical Properties, and Functional Activity.

Marinomonas primoryensis KMM 3633T, extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 °C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin.

Inhibitory Effects of Carrageenans on Endotoxin-Induced Inflammation.

The inhibitory effects of carrageenans (CRGs) on lipopolysaccharide (LPS) induced inflammation in a mouse model of endotoxemia and in complex therapy of patients with enteric infections of Salmonella etiology were studied. The atomic force microscopy (AFM) examination of LPS and its mixture with CRGs showed that the LPS morphology is significantly changed under the action of κ- and κ/ß-CRGs. CRGs were able to increase the synthesis of anti-inflammatory interleukin 10 (IL-10) in vitro, and, at low concentrations, their activity in the mixture with LPS was higher. The protective effect of CRGs against Escherichia coli LPS was studied in vivo by monitoring the biochemical and pathomorphological parameters. The κ- and κ/ß-CRGs and food supplement "Carrageenan-FE" increased the nonspecific resistance of mice to E. coli LPS at the expense of the inhibition of processes of thymus involution, adrenals hypertrophy, thyroid atrophy, hypercorticoidism, glycogenolysis, and lactate acidosis. The estimation of the therapeutic action of food supplement Carrageenan-FE in complex therapy of patients with enteric infections of Salmonella etiology is given. Carrageenan-FE restores the system of hemostasis and corrects some biochemical indicators and parameters in the immune systems of patients. These results allow us to hope for the practical application of CRGs for lowering the endotoxemia level in patients under the development of the infectious process caused by Gram-negative bacteria.

In silico and in vitro analysis of cross-reactivity between Yersinia pseudotuberculosis OmpF porin and thyroid-stimulating hormone receptor.

This work is devoted to the ascertainment of serological cross-reactivity between OmpF porin from Yersinia pseudotuberculosis (YpOmpF) and human thyroid-stimulating hormone receptor (hTSHR). Extracts containing hTSHR were isolated from surgical thyroid tissue of patients with clinical and diagnostic signs of diffuse toxic goiter. Monoclonal antibodies to hTSHR (mAbs) were shown to interact both with antigens in thyroid tissue extracts and with YpOmpF. Models of spatial structures of trimer and monomer complexes of YpOmpF with antibodies to hTSHR were also constructed. According to the results of molecular modeling, YpOmpF, being in monomeric form, can, like hTSHR, interact freely with the mAbs. But when the porin trimer is formed the hydrophobic region that comprises in the porin-antibody interaction zone is closed. This circumstance as well as other spatial rearrangement of amino acid residues that determine the efficiency of binding prevents the interaction between YpOmpF trimer and monoclonal antibody to receptor. These in vitro and in silico results confirmed the existence of the phenomenon of molecular mimicry. Thus, an autoimmune disease of the thyroid gland (Graves' disease) that sometimes arises after suffering pseudotuberculosis may be the consequence of the structural and antigenic similarity between YpOmpF and hTSHR.

Peculiarities of thermal denaturation of OmpF porin from Yersinia ruckeri.

Irreversible denaturation of membrane proteins in detergent solutions is similar to unfolding of water-soluble multidomain proteins and represents a complex, multistage process. Pore-forming proteins of Gram-negative bacteria are heat-modifiable proteins, i.e., proteins altering their molecular forms (trimers or monomers), and accordingly, their electrophoretic mobilities depending upon denaturation conditions. There are still some contradictory data on the peculiarities of the conformational changes in the porin structure with temperature. Some authors demonstrated the loss of the porin trimeric structure only after unfolding of monomer subunits. Other researchers initially observed the dissociation of porin oligomers into the folded monomers. Using SDS-PAGE, spectroscopic methods and differential scanning calorimetry, a detailed study of thermally induced changes in the spatial structure of OmpF porin from the fish pathogen Yersinia ruckeri (Yr-OmpF) was carried out. The data obtained allowed us to conclude unambiguously that changes in the spatial structure of the monomers of Yr-OmpF precede the dissociation of the porin trimer.

Modified and Mutant Porins in the Study on Molecular Basis of Non- Specific Diffusion.

Site-directed mutagenesis allows elucidation of the basic principles of the porin-driven membrane permeability and opens the possibility for the modulation of functional states of porin channels. The review is aimed to show the advantages of using mutant and chemically modified porins for obtaining detailed information about molecular mechanisms that underlie the non-specific transmembrane diffusion. We summarized data regarding the effects of the point substitutions and the external loop deletions on electrophysiological properties of general porins. The influence of charges inside the pore eyelet and the roles of external loops in ion conductance, ion selectivity, and voltage gating were described.

Influence of red algal sulfated polysaccharides on blood coagulation and platelets activation in vitro.

The influence of sulfated polysaccharides (λ-, κ-, and κ/ß-carrageenan and porphyran) - on platelet activation was studied. Carrageenans were much weaker inhibitors of a coagulation process than heparin, while porphyran had not that effect. Results of the aPTT and PT assays suppose that carrageenans affected mostly intrinsic pathway of coagulation, while their effect on the extrinsic pathway is extremely low (λ and κ/ß) or absent (κ, LMW derivative of κ-carrageenan). λ-Carrageenan was the most potent anticoagulant agent in TT, aPTT, PT, and anti-factor Xa activity. This sample was also the strongest inhibitor of collagen-induced platelet aggregation in PRP. Generally, the correlation of anticoagulant and antithrombotic action in PRP is preserved for carrageenans but not for heparin. Carrageenans and porphyran affected platelet adhesion to collagen by influencing glycoprotein VI. Low molecular weight κ-carrageenan had a similar effect on platelet adhesion mediated with both major collagen receptors: integrin α2 ß1 and glycoprotein VI as native polysaccharide had. Carrageenans resulted in activation of platelets under platelet adhesion mediated by integrin αIIb ß3 with less degree than heparin. The least sulfated κ/ß-carrageenan that possessed an inhibiting effect on thrombin- and collagen-induced aggregation of washed platelets and on the PT test but it had no significant effect on TT was the weakest promoter of integrin αIIb ß3 mediated platelet activation. In summary, our study showed that the polysaccharide action was complex, since it depended on its molecular mass, sulfation degree, and monosaccharide contents (3,6-anhydrogalactose).

Marine compounds with therapeutic potential in gram-negative sepsis.

This paper concerns the potential use of compounds, including lipid A, chitosan, and carrageenan, from marine sources as agents for treating endotoxemic complications from Gram-negative infections, such as sepsis and endotoxic shock. Lipid A, which can be isolated from various species of marine bacteria, is a potential antagonist of bacterial endotoxins (lipopolysaccharide (LPSs)). Chitosan is a widespread marine polysaccharide that is derived from chitin, the major component of crustacean shells. The potential of chitosan as an LPS-binding and endotoxin-neutralizing agent is also examined in this paper, including a discussion on the generation of hydrophobic chitosan derivatives to increase the binding affinity of chitosan to LPS. In addition, the ability of carrageenan, which is the polysaccharide of red alga, to decrease the toxicity of LPS is discussed. We also review data obtained using animal models that demonstrate the potency of carrageenan and chitosan as antiendotoxin agents.

Effect of phenol-induced changes in lipid composition on conformation of OmpF-like porin of Yersinia pseudotuberculosis.

The present work aimed to compare the effects of different lysophosphatidylethanolamine (LPE) content in lipids derived from Yersinia pseudotuberculosis cells exposed and not exposed to phenol on the conformation of OmpF-like porin of these bacteria. Differential scanning calorimetry and intrinsic protein fluorescence showed that the 2.5-fold increase of LPE content and the corresponding increase in the phase transition temperature of bacterial lipids were accompanied by enhanced protein thermostability. Integral conformational rearrangement of protein was supported by drastic changes in the microenvironment of the tryptophan residues, likely resulting in a convergence of monomers in trimeric porin and exposure of outer tryptophan residues to the water environment. These conformational changes may impede the porin channel permeability under stress conditions in bacteria.

Effects of structural peculiarities of carrageenans on their immunomodulatory and anticoagulant activities.

Biological activity of five carrageenan types - kappa, kappa/beta, kappa/iota, lambda and new type - iks - isolated from the most abundant species belonging to Gigartinaceae and Tichocarpaceae collected from the Pacific coast was investigated. The ability of carrageenans to influence on the cytokine production by human cells is greatly dependent on concentration and structure of polysaccharides. At high concentrations all types of carrageenans increased the level of pro-inflammatory IL-6 and TNF-α, while at low concentration (1-10ng/mL) their activity was insignificant. All types of carrageenans induced the secretion of anti-inflammatory IL-10 in dose-dependent manner. Hybrid kappa/beta-carrageenan showed fairly high activity independent on concentration. At low concentrations (10ng/mL) its activity was more than that of LPS. The structural analysis of polysaccharides suggests that additional sulphate ester residue of lambda-carrageenan increases the concentration of calcium in macrophage cytoplasm and may have an important role in the activation process of the formation of active oxygen forms. Kappa/iota carrageenan possessed for the potential anticoagulant activity, which was extremely strong in low concentration. These results suggest that the immunomodulation and anticoagulant activity of carrageenans depends on the monosaccharide composition of polysaccharides, number, position and distribution of sulphate groups along galactan chain.

Forming and immunological properties of some lipopolysaccharide-chitosan complexes.

The complex formation of lipopolysaccharide (LPS) with chitosan (Ch) was demonstrated using sedimentation velocity analysis in the analytical ultracentrifuge, centrifugation in glycerol gradient and isopicnic centrifugation in cesium chloride. An addition of Ch to the Escherichia coli and Yersinia pseudotuberculosis LPS solutions was found to result in formation of the stable LPS-Ch complexes. The interaction is a complicated process and depends on time and reaction temperature, as well as on the molecular weight of chitosan. A stable LPS-Ch complex could be formed only after preliminary incubation of the initial components at an elevated temperature (37 degrees C). It should be noted that process of LPS complexation with Ch is accompanied by additional dissociating of LPS. The complex formation was shown to be a result not only of ionic binding, but also of other types of interactions. The interaction of Ch with LPS was shown to modulate significantly the biological activity of LPS. The LPS-Ch complex (1:5 w/w) was shown to possess much lower toxicity in a comparison with the parent LPS at injection to mice in the similar concentration. The LPS-Ch complex was shown to maintain an ability to induce of IL-8 and TNF, but induction of IL-8 and TNF biosynthesis by the LPS-Ch complex was lower than that by the parent LPS. The complex LPS-Ch, similarly to the parent LPS, was found stimulated the formation of the IL-8 in the dose-dependent manner in the human embryonal kidney cells (HEK 293 cells) transfected with TLR4 in combination with MD2.

Detailed structure of lipid A isolated from lipopolysaccharide from the marine proteobacterium Marinomonas vaga ATCC 27119.

The chemical structure of a novel lipid A, the major component of the lipopolysaccharide from the marine gamma-proteobacterium Marinomonas vaga ATCC 27119(T), was determined by compositional analysis, NMR spectroscopy, and MS. It was found to be beta-1,6-glucosaminobiose 1-phosphate acylated with (R)-3-[dodecanoyl(dodecenoyl)oxy]decanoic acid [C10 : 0 (3O-C12 : 0 [3O-C12 : 1])] or (R)-3-(decanoyloxy)decanoic acid [C10 : 0 (3O-C10 : 0)], (R)-3-hydroxydecanoic acid [C10 : 0 (3OH)], and (R)-3-[(R)-3-hydroxydecanoyloxy]decanoic acid (C10 : 0 [3O-[C10 : 0 (3OH)]]) at the 2, 3, and 2' positions, respectively. It showed low lethal toxicity, which is probably related to specific structural attributes. The absence of a fatty acid at the 3' position and a phosphoryl group at the 4' position and also the presence of an amide-linked (R)-3-hydroxyalkanoic acid that is further O-acylated with another (R)-3-hydroxyalkanoic acid, distinguish M. vaga lipid A from other such molecules.