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.

Characterization of a Transposon Tn5-Generated Mutant of Yersinia pestis Defective in Lipooligosaccharide Biosynthesis.

To identify Yersinia pestis genes involved in the microbe's resistance to cationic antimicrobial peptides, the strategy of random transposon mutagenesis with a Tn5 minitransposon was used, and the library was screened for detecting polymyxin B (PMB) susceptible mutants. The mutation responsible for PMB-sensitive phenotype and the lipopolysaccharide (LPS) structure were characterized for the Y. pestis strain KM218-A3. In this strain the mini-Tn5 was located in an open reading frame with the product homologous to the E. coli protein GmhB (82% identity) functioning as d-glycero-d-manno-heptose-1,7-diphosphate phosphatase. ESI FT ICR mass spectrometry of anions was used to study the structure of the unmodified LPS of Y. pestis KM218-A3, and molecules were revealed with the full-size LPS core or with two types of an incomplete core: consisting of Kdo-Kdo or Ko-Kdo disaccharides and Hep-(Kdo)-Kdo or Hep-(Ko)-Kdo trisaccharides. The performed complementation confirmed that the defect in the biological properties of the mutant strain was caused by inactivation of the gmhB gene. These findings indicated that the gmhB gene product of Y. pestis is essential for production of wild-type LPS resistant to antimicrobial peptides and serum.

[Trials of the domestically produced nutrient medium «Agar Muller-Hinton II - Obolensk¼.]

The results of the comparative tests of the «Agar Muller-Hinton II - Obolensk¼ nutrient medium developed in SRCAMB, Obolensk, and the control nutrient medium imported «Mueller Hinton II Agar¼ are presented in the study. The susceptibility of bacterial clinical strains to antimicrobial agents (AMP) was determined by the disc diffusion method and the method of gradient diffusion (E-test). The carbapenemase activity of the strains carrying the carbapenemase genes was determined by CIM-test. Total 173 characterized bacterial strains of species Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Serratia marcescens, Enterobacter aerogenes, Escherichia coli; Photorhabdus spp., Staphylococcus aureus, Enterococcus spp. were used in the study, including producers of OXA- and NDM-types carbapenemases for gram negative bacteria. A high degree of coincidence of the results obtained on both nutrient media was shown. The consistency index of the strain sensitivity categories to AMPs (S, I, and R) was 98.2% for the disc diffusion method, and 94.4-100% - for E-test and CIM-test methods. Thus, within the framework of the Import Substitution Program, the domestic nutrient medium «MHA II-Obolensk¼ has been successfully developed. The nutrient medium meets the requirements of GOST R ISO 20776-2-2010 «Clinical laboratory testing and in vitro diagnostic test systems - Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices¼.

Structure and gene cluster of the O-polysaccharide of Yersinia rohdei H274-36/78.

A branched O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Yersinia rohdei H274-36/78 and found to contain d-rhamnose, d-mannose, and 3,6-dideoxy-4-C-[(S)-1-hydroxyethyl]-d-xylo-hexose called yersiniose A (Yer). Partial acid hydrolysis of the O-polysaccharide eliminated Yer residues to give a modified linear polysaccharide. Studies by sugar analysis and 1H and 13C NMR spectroscopy, including computational NMR analysis, enabled structure elucidation of a hexasaccharide repeating unit of the O-polysaccharide having two Yer residues attached as monosaccharide side chains. The O-antigen gene cluster of Y. rohdei H274-36/78 located between JUMPStart and galF genes contained putative genes for synthesis of precursors of two O-antigen constituents, GDP-d-Man and GDP-d-Rha, whereas genes responsible for synthesis of CDP-Yer were within the chromosome outside the O-antigen gene cluster. Glycosyltransferase genes and ABC 2 transporter genes were present in the O-antigen gene cluster, and hence the structure established is consistent with the polysaccharide synthesis gene content of the genome.

Structure and gene cluster of a tyvelose-containing O-polysaccharide of an entomopathogenic bacterium Yersinia entomophaga MH96T related to Yersinia pseudotuberculosis.

An O-polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Yersinia entomophaga MH96T by mild acid hydrolysis and studied by 2D NMR spectroscopy. The following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: where Tyv indicates 3,6-dideoxy-d-arabino-hexose (tyvelose). The structure established is consistent with the gene content of the O-antigen gene cluster. The O-polysaccharide structure and gene cluster of Y. entomophaga are related to those of some Y. pseudotuberculosis serotypes.

MOLECULAR TYPING OF SHIGELLA STRAINS.

Shigelloses still provide one of the toughest health challenges in many countries, especially in developing ones. Current molecular-genetic techniques are increasingly replacing conventional phenotypic approaches to investigating Shigella-associated infections. The review describes available methods of typing Shigella bacteria, their potential applications and selection criteria depending on the research task. It also deals with their advantages and disadvantages. Such methods as PFGE (pulsed field gel electrophoresis), the "gold standard" in typing various pathogens, and MLVA (multi-locus variable number of tandem repeat analysis) are the most widely used Shigella typing procedures. These methods are often combined or used along with some other genotyping tools. In the nearest future, with the advent of web platforms allowing express processing data, whole genome sequence will possibly become more widely used in lab diagnostics to evaluate outbreaks and to perform epidemiological monitoring.

[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.

[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.

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.