[Molecular aspects of anthrax pathogenesis].

A model of anthrax infection with the role determined for main pathogenicity factors of Bacillus anthracis exotoxin and capsule is presented. After spore phagocytosis by macrophages, synthesis of the main exotoxin component begins - a protective antigen that in oligomeric form disrupts phagosome membrane. This accelerates the transition of the pathogen from phagosome into the macrophage cytoplasm. Poly-D-glutamine capsule synthesized by the pathogen triggers the exit (exocytosis) of vegetative cells from macrophages and protects them from re-phagocytosis in lymphatic node lumen. The vegetative cells, that actively and freely replicate in lymphatic node, secret an exotoxin that disrupts endothelial septum between lymph and blood due to cytotoxic activity. As a result the vegetative cells get into blood and bacteremia develops. Pathogenetic pattern during anthrax (multiple hemorrhages in various organs etc.) is associated with local microcirculation disorders of various organs caused by the effect of bacterial exoproteases via activation of Willebrand factor. This results in a rapid local increase of microbial mass and consequent powerful cytotoxic effect of exotoxin on the tissue cells of the affected organ. Death of the infected organism takes place at the final stage of infec- tion due to toxic shock caused by the exotoxin. A reduction of body temperature takes place after death and the process of spore formation begins in the dead animal: capsule depolymerization, chain shortening, peptidoglycan cortex formation. Spores in this form are the prolonged source of infectious agent conservation and spread of infection in nature.

Protective Immune Response against Bacillus anthracis Induced by Intranasal Introduction of a Recombinant Adenovirus Expressing the Protective Antigen Fused to the Fc-fragment of IgG2a.

Anthrax is a particularly dangerous infectious disease that affects humans and livestock. It is characterized by intoxication, serosanguineous skin lesions, development of lymph nodes and internal organs, and may manifest itsself in either a cutaneous or septic form. The pathogenic agent is Bacillus anthracis, a grampositive, endospore-forming, rod-shaped aerobic bacterium. Efficacious vaccines that can rapidly induce a long-term immune response are required to prevent anthrax infection in humans. In this study, we designed three recombinant human adenovirus serotype-5-based vectors containing various modifications of the fourth domain of the B. anthracis protective antigen (PA). Three PA modifications were constructed: a secretable form (Ad-sPA), a non-secretable form (Ad-cPA), and a form with the protective antigen fused to the Fc fragment of immunoglobulin G2a (Ad-PA-Fc). All these forms exhibited protective properties against Bacillus anthracis. The highest level of protection was induced by the Ad-PA-Fc recombinant adenovirus. Our findings indicate that the introduction of the Fc antibody fragment into the protective antigen significantly improves the protective properties of the Ad-PA-Fc adenovirus against B. anthracis.

[Molecular model of anthrax toxin translocation into target-cells].

Anthrax toxin is formed from three components: protective antigen (PA), lethal (LF) and edema (EF) factors. PA83 is cleaved by cell surface protease furin to produce a 63-kDa fragment (PA63). PA63 and LF/EF molecules are assembled to anthrax toxin complexes: oligomer PA63 x 7 + LF/EF x 3. Assembly is occurred during of binding with cellular receptor or near surface of target-cell. This toxin complex forms pore and induces receptor-mediated endocytosis. Formed endosome consists extracellular liquid with LF/EF and membrane-associated ferments (H+ and K+/Na+-ATPases) and proteins (receptors and others). H+ concentration is increased into endosome as result of K/Na-ATPase-dependent- activity of H+-ATPase. Difference of potentials (between endosome and intracellular liquid) is increased and LF/EF molecules are moved to pore and bound with PA63-oligomer to PA63 x 7 + LF/EF x 7 and full block pore (ion-selective channel). Endosome is increased in volume and induces increasing of PA63-oligomer pore to.size of effector complex: LF/EF x 7 + PAl7 x 7 = 750 kDa. Effector complex is translocated from endosome to cytosol by means high difference of potentials (H+) and dissociates from PA47 x 7 complex after cleavage of FFD315-sait by intracellular chymotrypsin-like proteases in all 7 molecules PA63. PA47 x 7 complex (strongly fixed in membrane with debris of hydrophobic loops) return into endosome and pore is destroyed. Endosome pH is decreased rapidly and PA47 x 7 complex is destroyed by endosomal/lysosomal proteases. Receptor-mediated endocytosis is ended by endosome recycling in cell-membrane.

[Molecular mechanism of AB5 toxin A-subunit translocation into the target cells].

AB5 toxins are pore-forming protein complexes, which destroy eukaryotic target cells inactivating essential enzyme complexes through protein ADP-ribosylation or glycosylation by enzymatically active A1 subunits. The B-subunit pentamer interacts with the target cell receptor, induces membrane pore formation, and initiates receptor-mediated endocytosis. In the present article, we propose a model of A1-subunit translocation in the form of a globular structure, as opposed to the generally accepted hypothesis of A-subunit unfolding in the acidic milieu of the endosome followed by its transport in the form of unfolded polypeptide and refolding in the cytoplasm. This model is based on physical-chemical processes and explains why an endosome, but not an exosome, is formed. A-subunit translocation into the cytosol is driven by the proton potential difference generated by K/Na- and H(+)-ATPases. After reduction of the disulphide bond between A1 and A2 fragments by intracellular enzymes, B-subunit returns back into the endosome, where they are destroyed by endosomal proteases, and the pore is closed. Endosome integrates into the cellular membrane, and membrane-bound enzymatic complexes (ATPases and others) return back to their initial position. The proposed model of receptor-mediated endocytosis is a universal molecular mechanism of translocation of effector toxin molecule subunits or any other proteins into the target cell, as well as of cell membrane reparation after any cell membrane injury by pore-forming complexes.

[Changing the protective properties of the receptor domain of protective antigen anthrax exotoxin, depending on the orientation of the presentation on nanoparticles].

Polysterene particles bearing on its surface recombinant protein receptor domain of protective antigen anthrax exotoxin, exposed in different orientations have been constructed. Particles with exposed COOH region of receptor domain induced the highest protective immunity in mice anthrax model (95%). We revealed that immunization with these particles causes a specific induction of Th1-response, characterized by increased levels ofcytokine TNF-alpha and IL-2.

[Anthrax: early steps of the intracellular stage of infection development].

It was shown that spore germination of different Bacillus anthracis strains in macrophage-like cells J774A.1 depended on the genotype of the strains. The virulent B. anthracis strains contain plasmids pXO1 and pX02 responsible for the synthesis of a toxin and a capsule, respectively. The loss of one of the plasmids results in the reduction of strain virulence. It was shown that effective survival of germinating spores in macrophages occurred in the presence of plasmid pXO1 only. The spores of the B. anthracis strains ?Ames and STI-Rif deprived of plasmid pXO1 were least adapted to passing through the intracellular stage. The B. anthracis strains 81/1 and 71/12 (carrying plasmids pXO1 and pXO2 and synthesizing the toxin and capsule) less effectively survived in the cytoplasm of macrophages than the strain STI-1 which has only the plasmid pXO1. It was found that the rate of synthesis of the capsule consisting of polymer gamma-D-glutamic acid depended on the ability of bacterial cells to escape from macrophages. In the B. anthracis strains carrying plasmid pXO2, capsule synthesis by vegetative cells was activated within macrophages that promoted a rapid escape of the vegetative cells from the macrophages. On the contrary, most of capsule-free cells of the vaccine strain STI-1 remained inside macrophages during the whole period of observation. Thus, integrated regulation of two processes, namely synthesis of the toxin components participating in the transition of the germinating cell from phagosome into cytoplasm, and synthesis of the capsule whose presence promotes rapid escape of bacterial cells from macrophages by presently unknown mechanism play the key role in anthrax development at early stages.

[Molecular mechanisms underlying bacillus anthracis infection at early stages and search for novel vaccines]. / Molekuliarnye mekhanizmy nachal'nykh étapov sibireiazvennoí infektsii i poisk novykh vaktsin.

The developmental mechanisms of anthrax immunity were studied. Immunization was found to generally generate specific antibodies and lysozyme. Collectively, all the factors are responsible for suppressing the development of spores in the body. This proves the fact that the immunity is directed not only towards the exotoxin of B. anthracis, but it affects mainly the formation of vegetative cells. On entering the immuned body, vegetative cells may cause B. anthracis infection because antitoxic antibodies have no effect on encapsulated cells. The findings indicate that any anti-anthrax vaccine strain must show a complete immunological response in the body, as well as constitute immunity to all pathogenetic factors of B anthracis.

[Elucidation of functionally active domains in molecules of protective antigen bacillus anthracis's toxin]. / Vyiavlenie funktsional'no-aktivnykh domentov v molekulakh protektivnogo antigena i letal'nogo faktora sibireiazvennogo ékzotoksina.

Limited proteolysis has established that the protective antigen (PA) molecule consists of four functional-active domains. So, the shielding domain borrows an area in the linear structure of the PA molecule with NH2 of the end up to Lys 166 and plays a conducting role in the proteolytic activation of PA. The associative domain, engaging in the area Arg 167-Met266, plays a key role in the interaction with LF or EF at self-assembly toxic complexes LT or ET. The stabilizing domain borrows in the linear structure of the PA molecule are with Gly351 up to Met434. On the one hand, this area promotes formation with LF conformationally steady toxic complex's, and, on the other, takes a direct participation in the formation of a hydrophobic channel by which the molecule LF or EF enters the target cell. The receptor domain, representing a COOH-terminal area, starting from Leu663 amino acid, begins to interact with specific receptors on the macrophages and thus delivers the toxic complex to the target cell. It has been found that in the molecule of lethal factor there are 3 functionally active domains located in the linear structure of the molecule as follows: the associative domain borrows an area from Lys39 up to Met242, stabilizing and effector domains occupy areas from Leu517 to Lys614 and from that point to Lys COOH-terminal amino acid.

[Tularemia vaccine strain is a potential vector]. / Tuliaremiínyí vaktsinnyí shtamm-potentsial'nyí bakterial'nyí vektor.

The immunological and genetic properties of Francisella tularensis vaccine strain are discussion with regard to its use to produce recombinant vaccines. This bacterium-based vector is supposed to be an excellent object for investigating the role of protective antigens in the development of immunity against intracellular bacteria.

[Detection of the functionally active domains in the molecule of the lethal factor of the anthrax exotoxin]. / Vyiavlenie funktsional'no aktivnykh domenov v molekule letal'nogo faktora sibireiazvennogo ékzotoksina.

Three functional domains were revealed in the molecule of the lethal factor of B. anthracis. They are located in the linear structure of the molecula as follows: the associative domain occupies the area from Lys39 to Met242, the stabilizing domain from Leu517 to Lys614, and the effector domain still further to the COOH-terminal Lys mino acid.

[Detection of the functionally active domains in the molecule of protective antigen of the anthrax exotoxin]. / Vyiavlenie funktsional'no aktivnykh domenov v molekule protektivnogo antigena sibireiazvennogo ékzotoksina.

Using the limited proteolysis method, we established that the protective antigen (PA) molecule consists of four functionally active domains. The shielding domain occupies an area in the linear structure of the molecule PA with NH4-terminal up to Lys166 and plays an important role in the proteolytic activation of PA. The associative domain situated in the Arg167-Met266 region is responsible for interactions with either lethal or edematous factors in self-assembly of the toxic complexes of the lethal or edematous toxin. The stabilizing domain occupies the Gly351 to Met434 area. On the one hand, this area promotes the formation of conformationally stable toxic complexes with the lethal factor, on the other, directly participates in the formation of the hydrophobic canal, through which the molecule of the lethal or edematous factor and, evidently, a fragment of PA molecule as well (from Arg167 to Gly314), including the associative gene, gets inside the target cell. The receptor domain representing a COOH-terminal region, starting from Leu663 amino acid, interacts with the specific receptors on macrophages and thus delivers the toxic complex to the target cell.

[Specific proteolysis by fibrinolysin-coagulase from Yersinia pestis of Yersinia pseudotuberculosis outer membrane proteins coded by the Ca(2+)-dependence plasmid]. / Spetsificheskii proteoliz fibrinolizinokoagulazoi Yersinia pestis belkov vneshnei membrany, kodiruemykh plazmidoi Ca(2+)-zavisimosti v Yersinia pseudotuberculosis.

The pesticinogenicity 9.5 kb plasmid from Yersinia pestis strain EV76 has been marked by the kanamycin phosphotransferase gene inserted into PstI site and designated pP3. The obtained plasmid pP3 determines the synthesis of 45 kd pesticin, alpha and beta-forms of fibrinolysin coagulase (37 and 35 kd) and the 29, 19 and 13 kd proteins in Escherichia coli mini cells. When transferred into Yersinia pseudotuberculosis strain 6933 the plasmid causes the proteolysis of outer membrane proteins. The 150 kd protein is reduced to 138 kd, the 48.5 kd protein is reduced to 45 kd. The proteins secreted into the cultural medium (51 and 38 kd) are also cleaved. The proteolysis of the 150 kd protein was found to occur at the stage of secretion via the inner membrane. The purified fibrinolysin coagulase from Escherichia coli strain JM83 harbouring the plasmid pP3 induces the proteolysis in vitro of the isolated membrane proteins from Yersinia pseudotuberculosis strain 6953 similar to the proteolysis registered in vivo.

[Use of filter-adsorbed enterotoxins for isolating human immune interferon]. / Ispol'zovanie énterotoksinov, adsorbirovannykh na fil'trakh, dlia polucheniia immunnogo interferona cheloveka.

The use of A and B enterotoxins of St. aureus adsorbed on Millipore nitrocellulose filters for induction of donor blood leukocytes resulted in production of immune interferon free of the inducer. The interferon produced by this method is acid-labile and inactivated by antiserum to human immune interferon but not to human leukocyte interferon. The advantage of this type of induction consists in the possibility of multiple use of the filters with adsorbed enterotoxins.

NH2-terminal localization of that part of the staphylococcal enterotoxins polypeptide chain responsible for binding with membrane receptor and mitogenic effect.

It is established that the part of the SEA and SEC2 polypeptide chain responsible for the binding of these toxin proteins with the membrane receptor on the surface of rabbit thymocytes and mitogenic effect is localised in the NH2-terminal region of the molecule. The SEC2 splits in two fragments T1 (17 kdalton) and T2 (12.5 kdalton) under limited proteolysis by trypsin in the presence of 2-ME. The amino acid terminal residues of SEA, SEC2 and their proteolytic fragments are also studied.

[Isolation and characteristics of staphylococcal alpha-toxin]. / Vydelenie i kharakteristika stafilokokkovogo alpha-toksina.

A method for isolation of staphylococcal alpha-toxin preparations has been elaborated. Characteristics of the toxin isolated by the method are as follows: mol. mass = 35 Kd; HU = 0.1 microgram; DnD= 0.1 microgram; LD50 = 2 micrograms. It is for the first time that alpha-toxin was fragmented by papain and digested by alpha, gamma-chemotrypsin. The papain fragments (18.5 and 15 Kd) retained lethal activity but lost hemolytic and dermonecrotic activities. Alpha, gamma-chemotryptic digested fragments (18 and 15 Kd) retained hemolytic and lethal effects, but lost their dermonecrotic activity.

[Isolation and characteristics of membrane fraction of rabbit T lymphocytes specifically binding the staphylococcal enterotoxin type A]. / Vydelenie i kharakteristika membrannoi fraktsii T-limfotsitov krolika, spetsificheski sviazyvaiushchei stafilokokkobyi énterotoksin tipa A.

The lymphocyte membranes from rabbit thymus were shown to bind specifically the staphylococcal enterotoxin type A (SEA). The glycolipid components were demonstrated to be absent from the SEA receptor complex on the surface of T-lymphocytes. The mild conditions were elaborated for the receptor membrane fraction solubilization by triton X-100. The affinity chromatography method was used to isolate the SEA binding membrane fraction, the major component of which is a protein with a 42,000 mol mass. The isolated preparation inhibits the specific binding of [125I]-SEA on the cellular (by T-lymphocytes) and subcellular (by membranes) levels.

Membrane protein from rabbit T-lymphocytes, specifically binding staphylococcal enterotoxin A (SEA).

The presence of specific binding of SEA with membranes of lymphocytes from rabbit thymus is established. Components of a glycolipid nature are absent in the composition of the receptor complex for SEA on T-lymphocytes. Suitable conditions for the solubilization of the receptor membrane fraction by Triton X-100 are described. The SEA-binding membrane fraction is isolated by means of an affinity-chromatography method. The main component of the fraction is a protein with molecular mass 42 kd. The isolated protein inhibits the specific binding of [125I] SEA on cell (T-lymphocytes) and subcell (membrane) levels.

Topology of the functions in molecule of staphylococcal enterotoxin Type A.

Four fragments (F1-F4) of SEA, obtained via papain proteolysis were separated and isolated as individual components by means of the SDS-electrophoresis in polyacrilamide gel. Molecular masses of the pairs F1 + F4 and F2 + F3 are equal to the mass of the intact toxin--a fact that supposes a cleavage of polypeptide chain in two regions of "disulphide loop" in a SEA molecule. Neither fragment possesses any enterpathogenic properties. It was established, that interferonogenic and mitogenic activity of SEA is connected only with the part of molecule corresponding to F1(17,500) and F3(15,000). Two kinds of antigenic determinants in the SEA molecule were found: one was attributed to F1 and F3 fragments, the other was localised in F2 and F4. Proteolysis by trypsin led to cleavage of a small peptide from the N-terminal end of toxin molecule. Trypsinized SEA displayed all kinds of biological activity characterizing the native toxin.

Properties of staphylococcal enterotoxin A under limited proteolysis.

1. Staphylococcal enterotoxin A (SEA) was exposed in a state of limited proteolysis to five kinds of proteolytic enzymes: papain, pepsin, pronase, trypsin and alpha-chymotrypsin. SEA was found to be sensitive to the action of three of them: papain, pepsin and pronase. 2. Four fragments were produced after papain proteolysis of SEA in the presence of beta-mercaptoethanol. 3. Papain processing of SEA does not influence its capacity to interact with antibodies to intact toxin, but the capacity of SEA to hydrolyse NAD to nicotinamide (NA) and adenosinediphosphatribose (ADP-ribose) completely disappears. 4. SEA under the action of pepsin and pronase has been hydrolysed to small peptides, which appear to be moving with the front of leading dye in disc-electrophoresis.