Yersinia pestis Surface Antigens in Reception of Specific Bacteriophages.

The significance of Yersinia pestis surface antigens in adhesiveness to specific bacteriophages has been studied with the use of two methodological approaches. It was shown that Ail protein immobilized on the surface of polystyrene microspheres (but not in the solution), can bind both the Pokrovskaya phage and pseudotuberculous diagnostic phage. YapF autotransporter interacted with both phages in a water-soluble form, but YapF bound to polystyrene microspheres interacted only with the Pokrovskaya phage. An assumption was made that Ail and YapF proteins can be the primary receptors providing non-specific reversible binding to the phages used in this work.

[DETERMINATION OF PHYLOGENETIC RELATIONSHIP OF YERSINIA PESTIS STRAINS FROM NATURAL PLAGUE FOCI OF THE CAUCASUS BY MULTI-LO- CUS VNTR-ANALYSIS].

AIM: Determination of the degree of phylogenetic relationship of Yersinia pestis strains iso- lated from the territories of natural foci of plague from the Caucasus using VNTR-typing by 25 loci (MLVA25). MATERIALS AND METHODS: 26 strains of Y pestis from Russian natural foci of the Caucasus were used in the study. 25 loci of tandem repeats in Y pestis genome by Le Fleche scheme were used for execution of multi-locus VNTR-analysis. Deciphering of nucleotide sequences was carried out in automatic sequencer ABI 3130 Genetic Analyser. Analysis of confinement of clusters to certain territories, objects and time of isolation of strains was carried out. using Arc GIS 10.1 program. RESULTS: Groups of MLVA25-types of various levels of discrimination were formed: clusters, groups and subgroups. Clusters were formed by strains ofvarious taxonomic membership: main and subspecies of Y pestis. Subgroups reflect membership of strains in certain foci, and MLVA25-types - the degree of genetic relationship. CONCLUSION: Genetic <> of plague causative agents obtained using MLVA25-types circulating in various natural-focal territories allow to solve problems ofboth theoretical and practical character: from interpretation of microevolution processes to the search of the source of infection and ways of its spread during possible epidemic complications.

[COMPARATIVE ANALYSIS OF THE MLVA25- AND MLVA7-TYPING ACCORDING TO THEIR ABILITY TO ASCERTAIN FOCAL AFFILIATION OF YERSINIA PESTIS STRAINS BY THE EXAMPLE OF ISOLATES FROM THE CENTRAL-CAUCASIAN HIGHLAND NATURAL PLAGUE FOCUS].

Comparative analysis of the MLVA25- and MLVA7-typing ability to evaluate focal belonging of Y. pestis strains by the example of bv. medievalis isolates from the Central-Caucasian highland natural plague focus was carried out. The MLVA25-types of-82 isolates from this area were determined and included into the database containing information on 949 Y. pestis strains from other natural foci of Russia and other countries. Categorical-UPGMA dendrograms were created on the bases of the data concerning all 25 VNTR loci or only seven of them, which were recommended by the experts of the Russian Research Anti-Plague Institute "Microbe" for differentiation of the Y. pestis strains according to their affiliation to specific foci. The obtained data indicated greater possibility of diagnostic mistakes in the case of the MLVA7-typing and supported expediency of division of the Central-Caucasian highland natural plague focus into two sub-foci.

[ON THE ORIGIN OF HYPERVIRULENCE OF THE CAUSATIVE AGENT OF PLAGUE].

The attempt to combine Yersinia pseudotuberculosis and Yersinia pestis into one species has been unsupported by microbiologists due to the specific features of the epidemiology and clinical presentations of their induced diseases and to basic differences in their virulence. Pseudotuberculosis is predominantly a relatively mild human intestinal infection transmitted through contaminated food and plague is an acute generalized disease with high mortality, which is most frequently transmitted by the bites of infected fleas. Y. pestis hypervirulence, the ability of single bacteria to ensure the development of predagonal bacteriemia in rodents, which is sufficient to contaminate the fleas, is one of the main events during pathogen adaptation to a new ecological niche. By analyzing the data of molecular typing of the representative kits of naturally occurring Y. pestis isolates, the authois consider the issues of formation of intraspecies groups with universal hypervirulence, as well as biovars that are highly virulent only to their major host. A strategy for searching for selective virulence factors, the potential molecular targets for vaccination and etiotropic treatment of plague, is discussed.

Selective Protective Potency of Yersinia pestis ΔnlpD Mutants.

It has recently been shown that the NlpD lipoprotein is essential to Yersinia pestis virulence and that subcutaneous administration of the nlpD mutant could protect mice against bubonic and pneumonic plague better than the EV vaccine strain [PLoS One 2009. V. 4. № 9. e7023]. In this study, similar ΔnlpD mutants were generated on the basis of other Y. pestis parent strains, including strains from the subspecies microtus, which is avirulent to guinea pigs and humans. Comparative testing confirmed that immunization of mice with ΔnlpD mutants induces immunity 105 times more potent than the one induced by the administration of the EV vaccine strain. At the same time, NlpD- bacteria failed to protect guinea pigs in the case of a subcutaneous challenge with Y. pestis, inducing a 106 times less potent protection compared with that conferred by immunization with the EV vaccine strain. The possible causes of the observed phenomena are discussed.

Distinct biological activity of lipopolysaccharides with different lipid A acylation status from mutant strains of Yersinia pestis and some members of genus Psychrobacter.

Correlation between the chemical structure of lipid A from various Gram-negative bacteria and biological activity of their lipopolysaccharide (LPS) as an agonist of the innate immune receptor Toll-like receptor 4 was investigated. Purified LPS species were quantitatively evaluated by their ability to activate the production of tumor necrosis factor (TNF) by murine bone marrow-derived macrophages in vitro. Wild-type LPS from plague-causing bacteria Yersinia pestis was compared to LPS from mutant strains with defects in acyltransferase genes (lpxM, lpxP) responsible for the attachment of secondary fatty acid residues (12:0 and 16:1) to lipid A. Lipid A of Y. pestis double ΔlpxM/ΔlpxP mutant was found to have the chemical structure that was predicted based on the known functions of the respective acyltransferases. The structures of lipid A from two members of the ancient psychrotrophic bacteria of the genus Psychrobacter were established for the first time, and biological activity of LPS from these bacteria containing lipid A fatty acids with shorter acyl chains (C10-C12) than those in lipid A from LPS of Y. pestis or E. coli (C12-C16) was determined. The data revealed a correlation between the ability of LPS to activate TNF production by bone marrow-derived macrophages with the number and the length of acyl chains within lipid A.

[A molecular basis of the plague vaccine development].

Molecular mechanisms of the Yersinia pestis pathogenicity and peculiarities of maturation of specific immunity to plague are reviewed. The history and modern state of the plague vaccine development are described. Special attention is focused on the prospects in the area of the plague vaccine development. The possible approaches to improvement of vaccine preparations are discussed.

[Molecular typing of Yersinia pestis].

Techniques for differentiating single bacterial isolates into intraspecies clusters corresponding to subspecies, biovars, and natural foci are reviewed. The techniques under consideration are reproducible under different laboratory settings. A version of the intraspecies classification of Y. pestis that is in harmony with the International Code of Nomencláture of Bacteria is suggested.

Lipopolysaccharide of Yersinia pestis, the Cause of Plague: Structure, Genetics, Biological Properties.

The present review summarizes data pertaining to the composition and structure of the carbohydrate moiety (core oligosaccharide) and lipid component (lipid A) of the various forms of lipopolysaccharide (LPS), one of the major pathogenicity factors ofYersinia pestis, the cause of plague. The review addresses the functions and the biological significance of genes for the biosynthesis of LPS, as well as the biological properties of LPS in strains from various intraspecies groups ofY. pestis and their mutants, including the contribution of LPS to the resistance of bacteria to factors of the innate immunity of both insect-vectors and mammal-hosts. Special attention is paid to temperature-dependent variations in the LPS structure, their genetic control and roles in the pathogenesis of plague. The evolutionary aspect is considered based on a comparison of the structure and genetics of the LPS ofY. pestis and other enteric bacteria, including otherYersinia species. The prospects of development of live plague vaccines created on the basis ofY. pestis strains with the genetically modified LPS are discussed.

[The phylogeography of the Yersinia pestis vole strains isolated from the natural foci of caucasian region].

57 Y pestis bv. caucasica strains were assayed using molecular typing. The results of these assays indicated the presence within this biovar of the three separate clonal clusters and necessity of detachment of the Leninakan mountain mesofocus (subfocus) from the structure of Transcaucasian-highland focus into self-supporting one, as well as inclusion of a part of the Pre-Araks low-mountain natural plague focus in the capacity of the subfocus along with Pre-Sevan mountain and Zanzegur-Karabakh mountain subfoci into the structure of Transcaucasian-highland focus. It was shown that the strains circulating in the East-Caucasian highland plague focus were the most ancient branch of bv. caucasica or even of the entire Y pestis phylogenetic tree.

[Evolutionary regularities of somatic polyploidy expansion in salivary glands of gastropod mollusks. V. Subclasses Opisthobranchia and Pulmonata].

Salivary glands of 25 species of euthyneural gastropod mollusks (Opisthobranchia and Pulmonata) have been investigated by means of histochemical methods and DNA cytophotometry in nuclei of cells. The cells of three basic types are distinguished in glandular epithelim: granular cells (with glicoproteid granular inclusions), mucocytes-I (with sulfatic acid mucopolysaccharides) and mucocytes-II (with neutral and acid nonsulfatic polysaccharides and proteins) and so the epithelial ciliated cells and cells of the ducts. It was shown that glandular cells of salivary glands of all discovered mollusks' species are polyploid in different degree. The highest ploidy level estimated by means of DNA content in most of species is 64-128c. The giant polyploidy, attained to 4096c, is discovered in cells of salivary glands of Tritonia diomedea. The functional conditionality connected with features of feeding of different mollusk species and phylogenetic tendencies of expansion of somatic polyploidy in class Gastropoda are discussed. In comparison with allogenic, facultative and small polyploidy manifestation in Prosobranchia the obligatory polyploidization of high degree revealed in cells of salivary glands of Opisthobranchia and Pulmonata is consider to be the original cytological arogenesis. The probable causes of such differences are conneted with euthyneural type of organization of central nervous system and giant polyploidy of neurons in Opisthobranchia and Pulmonata mollusks. The causes, mechanisms and significance of such correlations are unclear for the present.

Functional characterization and biological significance of Yersinia pestis lipopolysaccharide biosynthesis genes.

In silico analysis of available bacterial genomes revealed the phylogenetic proximity levels of enzymes responsible for biosynthesis of lipopolysaccharide (LPS) of Yersinia pestis, the cause of plague, to homologous proteins of closely related Yersinia spp. and some other bacteria (Serratia proteamaculans, Erwinia carotovora, Burkholderia dolosa, Photorhabdus luminescens and others). Isogenic Y. pestis mutants with single or double mutations in 14 genes of LPS biosynthetic pathways were constructed by site-directed mutagenesis on the base of the virulent strain 231 and its attenuated derivative. Using high-resolution electrospray ionization mass spectrometry, the full LPS structures were elucidated in each mutant, and the sequence of monosaccharide transfers in the assembly of the LPS core was inferred. Truncation of the core decreased significantly the resistance of bacteria to normal human serum and polymyxin B, the latter probably as a result of a less efficient incorporation of 4-amino-4-deoxyarabinose into lipid A. Impairing of LPS biosynthesis resulted also in reduction of LPS-dependent enzymatic activities of plasminogen activator and elevation of LD(50) and average survival time in mice and guinea pigs infected with experimental plague. Unraveling correlations between biological properties of bacteria and particular LPS structures may help a better understanding of pathogenesis of plague and implication of appropriate genes as potential molecular targets for treatment of plague.

Pleiotropic effects of the lpxM mutation in Yersinia pestis resulting in modification of the biosynthesis of major immunoreactive antigens.

Deletion mutants in the lpxM gene in two Yersinia pestis strains, the live Russian vaccine strain EV NIIEG and a fully virulent strain, 231, synthesise a less toxic penta-acylated lipopolysaccharide (LPS). Analysis of these mutants revealed they possessed marked reductions in expression and immunoreactivity of numerous major proteins and carbohydrate antigens, including F1, Pla, Ymt, V antigen, LPS, and ECA. Moreover, both mutants demonstrated altered epitope specificities of the antigens as determined in immunodot-ELISAs and immunoblotting analyses using a panel of monoclonal antibodies. The strains also differed in their susceptibility to the diagnostic plague bacteriophage L-413C. These findings indicate that the effects of the lpxM mutation on reduced virulence and enhanced immunity of the Y. pestis EV DeltalpxM is also associated with these pleiotropic changes and not just to changes in the lipid A acylation.

Structural diversity and endotoxic activity of the lipopolysaccharide of Yersinia pestis.

The endotoxic activity of the lipopolysaccharides (LPS) with defined chemical structure from Yersinia pestis strains of various subspecies differing in their epidemic potential was studied. The LPS of two strains of Y. pestis ssp. caucasica and ssp. altaica, whose structures have not been studied earlier, were analyzed by high-resolution mass spectrometry. In addition to reported structural changes, an increase in the degree of LPS phosphorylation was observed when strain I-2377 (ssp. altaica) was cultivated at an elevated temperature. A high tumor necrosis factor alpha(TNF-alpha)-inducing activity observed for LPS samples from Y. pestis cultures grown at 25 degrees C correlated with an increased degree of lipid A acylation, particularly, with the presence of the hexaacyl form of lipid A, which was absent from the LPS when bacteria were cultivated at 37 degrees C. No correlation was found between the lethal toxicity of the LPS in vivo and its ability to induce TNF-alpha production in vitro.

A Yersinia pestis lpxM-mutant live vaccine induces enhanced immunity against bubonic plague in mice and guinea pigs.

The lpxM mutant of the live vaccine Yersinia pestis EV NIIEG strain synthesising a less toxic penta-acylated lipopolysaccharide was found to be avirulent in mice and guinea pigs, notably showing no measurable virulence in Balb/c mice which do retain some susceptibility to the parental strain itself. Twenty-one days after a single injection of the lpxM-mutant, 85-100% protection was achieved in outbred mice and guinea pigs, whereas a 43% protection rate was achieved in Balb/c mice given single low doses (10(3) to 2.5 x 10(4) CFU) of this vaccine. A subcutaneous challenge with 2000 median lethal doses (equal to 20,000 CFU) of fully virulent Y. pestis 231 strain, is a 6-10-fold higher dose than that which the EV NIIEG itself can protect against.

[Generation of vaccine strains of gram-negative bacteria with reduced adverse reactions].

The main shortcoming of the modem live and killed vaccines based on gram-negative bacterial strains is their ability to cause adverse reactions. The majority of the adverse reactions are associated with the effect of biological activity of lipopolysaccharide. The report covers the problems concerned with biogenesis of the lipid A, lipopolysaccharide structural component, responsible for its endotoxic activity, as well as with genes determining lipid A synthesis. The special attention is paid to gene-engineering technique for reduction of adverse reactions of vaccine strains that are based on the knock-out mutagenesis of genes waaM and/or waaN responsible for addition of lauroyl and myristoyl residues to the distal glucosamine unit lipid A, generating acyloxyacyl moieties.

Effect of zosterin on protein-synthesizing activity of hepatocytes.

The effect of zosterin (pectin polysaccharide) on the protein-synthesizing function of mouse liver cells was studied after its intragastric administration of 1% gel zosterin. The drug modified the morphology and function of the nucleolar apparatus by changing the number and summary area of the nucleoli, content of Ag proteins, nucleolar/nuclear ratio, and increased protein content in hepatocyte cytoplasm.

[Morpho-functional parameters of nucleoli in polyploid mucous and albumen cells of salivary gland in the snail Succinea lauta].

Variation of some characteristics of nucleoli of polyploid mucous and albumen cells was examined in salivary glands of the snail Succinea lauta. The number, total area and Ag-protein content of nucleoli, and DNA content in each nucleus were estimated on squashed preparations incubated with AgNO3, decolorized and then Feulgen stained. The ultrastructure of nucleoli was studied by electron microscopy. Differentiated mucous cells had 4c-8c-16c-32c nuclei; albumen cells had 8c-16c-32c-64c-128c nuclei. The ultrastructure of nucleoli of the two cell types was essentially the same. Normally, a large fibrous to granular zone was observed in the nucleoli, without a clear distinction between fibrous and granular components. At the same time, aggregations of granular matter could be discerned at the periphery of nucleoli. No fibrous centers were observed. Occassionally, nucleolonema-like structures occurred. Normally each nucleolus contacted several chromosomes. On squashed preparations, the least size of nucleoli was 2-3 microm, and the largest size amounted to 14 microm in mucous cells, and to 50-80 microm in albumen cells. The number of nucleoli rose from 1-2 in tetraploid nuclei to 2-3 in 32c-nuclei, and to 5-7 in 128c-nuclei. The disparity between the ploidy levels of nuclei and the numbers of nucleoli may be due, presumably, to aggregation of chromosome NORs. The Ag-protein content in the nucleoli, and the total nucleolar area displayed a strong mutual correlation. Both parameters differed significantly by 1.5-2.2 times in mucous and albumen cells of the same ploidy level. Thus, in albumen and mucous cells the total Ag-protein content in octaploid nuclei was 3.3 and 2.2 relative units (r. u.), respectively. In 16c- and 32c-nuclei of albumen cells, it was 7.6 and 15.1 r. u.; and in the same nuclei of mucous cells--3.8 and 6.8 r. u., respectively. On the whole, in albumen cells, in the course of 4 endocycles (4c-128c), the total Ag-protein content increased by 17 times. Therefore, the mean multiplication factor for this parameter was found to be 2.05 per endocycle. In mucous cells, in the course of 3 endocycles (4c-32c), the total Ag-protein content increased by 5.2 times against 8 times expected, with the mean multiplication factor equal to 1.75 per endocycle. Thus, in the course of polyploidization of albumen and mucous cell nuclei, the gene dosage effect was fully pronounced in the former, and only partly in the latter. This differtence is due obviously to peculiarities of differentiation of the two cell types, in particular, to differences in the number of activated ribosomal genes.

[Somatic polyploidy in neurons of the gastropod mollusca. III. Mitosis and endomitosis in the postnatal development of neurons in the Succinea snail central nervous system]. / Somaticheskaia poliploidiia v neironakh briukhonogikh molliuskov. III. Mitoz i éndomitoz v postnatal'nom razvitii neironov TsNS ulitki iantarki.

General morphology of chromatin, the number of chromosomes and chromocenters in normal condition and at the increase of bivalent cation (Ca2+, Mg2+) concentration were studied with the purpose to reveal mechanisms of polyploidization of neuron nuclei in the snail Succinea lauta (Gastropoda, Pulmonata). The morphology of nuclei was studied on squashed preparations. Normal diploid mitoses are described in the cerebral ganglia. A possibility is supposed that part of neurons or neuroblasts in the central nervous system (CNS) of succineid snail may divide mitotically. It has been shown that the basic mechanism of neuron postnatal growth is endomitotic polyploidization of nuclei. The transition from ordinary mitosis to polyploid cycles occurs via restitutional (polyploidizing) mitosis (4c2n-->4c4n). The next endocycles are carried out by means of classic endomitosis up to reaching the highest ploidy levels--4096n--16,384n. The study of general morphology of chromatin and chromocenters at normal condition and at artificial compactization enabled us to exclude any probability of polyteny in the CNS of lauta.

[Yersinia pestis factors, assuring circulation and maintenance of the plague pathogen in natural foci ecosystems. Report 1]. / Faktory Yersinia pestis, obespechivaiushchie tsirkuliatsiiu i sokhranenie vozbutelia chumy v ékosistemakh prirodnykh ochagov. Soobshchenie 1.

Everlasting reproduction of Yersinia pestis, plague bacillus in natural pestholes needs virulent causative agent to invade into the host entity, be potent to overcome protection powers of the rodent organism and to pullulate to entail bacteriemia for subsequent conveyance the plague bacillus to the new host by fleas. All of legs of life cyclic patterns of Yersinia pestis are maintained by a number of plague bacillus factors acting jointly or separately, participating at the different stages of infectious process or conveyance. However these factors provide the perpetuation of the plague bacillus in the ecosystems of natural pestholes only acting in conjunction independently on their distinct contributions. Not only biomolecules, organellas and bacteria systems ensured the pursuance of virulent properties, but other factors, essential for survival of Yersinia pestis and the relationship between separate virulence factors and expression of the different genes of housekeeping and virulence of plague bacillus are considered in this review. The report I covers the problems concerned with adaptational plasticity of Yersinia pestis, it represents the classification of plague causative factors, securing its perpetuation in the environmental space, and discussion the factors promoting plague bacillus survival in the host entity. Not only wellknown publications, but papers in out-of-the-way or hard-to-reach, especially for English-reading experts, editions, also were used to compile this communication. The English version of this review may be requested from Alerton Press.