Search

To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. A similar holin-endolysin system from phage λ containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with maximally hydrolyzed peptidoglycan had a greater protectivity compared to BGs with a preserved peptidoglycan skeleton.

Lipopolysaccharide (LPS), localized in the outer leaflet of the outer membrane, serves as the major surface component of the Gram-negative bacterial cell envelope responsible for the activation of the host’s innate immune system. Variations of the LPS structure utilized by Gram-negative bacteria promote survival by providing resistance to components of the innate immune system and preventing recognition by TLR4. This review summarizes studies of the biosynthesis of Yersinia pseudotuberculosis complex LPSs, and the roles of their structural components in molecular mechanisms of yersiniae pathogenesis and immunogenesis.

Electrophoretic, homogeneous, highly purified preparations of various isoforms of the immunodominant antigen V (LcrV protein) of Yersinia pestis have been obtained via chromatographic purification. A significant similarity in the chromatographic profiles of the LcrV antigen isoforms of different strains with tryptophan (W113) and glutamic acid (E113) residues at position 113, which differed from the isoform profile containing glycine residue LcrV (G113), has been recorded. The latter is characterized by the highest antigenic and protective activity as compared with other protein isoforms. This isoform almost completely protected the immunized group of animals and had the greatest ability to induce the production of antibodies, the titers of which reached 1 : 250 000.

Plague is a zoonotic infection whose pathogenic agent has caused hundreds of million human deaths. A broad range of hosts and vectors, along with the geographical dispersion of natural plague foci characterized by different ecological conditions, contribute to the formation of the polytypic Y. pestis species, the result of selection of the genetic variants specific for certain natural foci. Through the efforts of a world consortium of scientists, a global coordinated phylogram of the SNP types of the plague pathogen has been developed. However, debates on the intraspecies Y. pestis taxonomy still continue on the vast Russian expanses. The work of a taxonomist has many specific, individual features, formed on the basis of individual experience. It is important in this kind of work to follow an old rule which requires that borders should be placed where they have been put by nature, and should not be put where nature has not put them. With that in mind, we suggest here the rational variant of the plague pathogen nomenclature constructed in accordance with the rules set out in the International Code of Bacterial Nomenclature and Evolutionary Taxonomy.

We report the whole-genome sequence of Yersinia pestis subsp. pestis bv. Antiqua strain 231 belonging to the 0.ANT3 phylogroup, the reference strain for testing plague vaccine protection in Russia. Genome sequencing was completed using the Oxford Nanopore MinION and Illumina platforms.

The outer membrane is a key virulence determinant of gram‐negative bacteria. In Yersinia pestis, the deadly agent that causes plague, the protein Ail and lipopolysaccharide (LPS)(6) enhance lethality by promoting resistance to human innate immunity and antibiotics, enabling bacteria to proliferate in the human host. Their functions are highly coordinated. Here we describe how they cooperate to promote pathogenesis. Using a multidisciplinary approach, we identify mutually constructive interactions between Ail and LPS that produce an extended conformation of Ail at the membrane surface, cause thickening and rigidification of the LPS membrane, and collectively promote Y. pestis survival in human serum, antibiotic resistance, and cell envelope integrity. The results highlight the importance of the Ail–LPS assembly as an organized whole, rather than its individual components, and provide a handle for targeting Y. pestis pathogenesis.

Gram-negative bacteria Y. pestis subsp. pestis of 0.ANT-4.ANT, 1.ORI, and 2.MED SNP types are the cause of numerous epidemic outbreaks, epidemics, and three plague pandemics, claiming hundreds of millions of human lives. At the same time, strains of the microtus subspecies that belong to the 0.PE SNP type circulating in populations of different species of voles (Microtus spp.) are able to cause only extremely rare human infections that are not transmitted from person to person. It is suggested that the clinical form of the infection can develop only in individuals with impaired immune status. Strains of Y. pestis bv. caucasica (0.PE2), one of the most ancient phylogenetic groups of subsp. microtus, are isolated on the territory of the following natural foci: the Transcaucasian highland (including the Leninakan (4), Pre-Sevan (5), and Zanzegur- Karabakh (6)) mesofoci and the Dagestan-highland (39). In addition to the enumerated areas, similar strains of Y. pestis are isolated in the territories of the Pre-Araks focus (7) bordering the Transcaucasian highland one. Previously, we showed that passport data on the phenotypic differences of strains of the biovar caucasica, isolated from different foci, corresponded to their MLVA25, CRISPR, and DFR genotypes. In the present work, a comparative analysis of the clustering ability of MLVA25- and CRISPR-typing methods with the “gold standard” of phylogenetic studies—SNP typing—has been conducted on 21 strains of Y. pestis subsp. microtus bv. caucasica (0.PE2). The analysis of the obtained results confirms the existence of three clonal clusters of strains corresponding to the natural plague foci—39, 4, and the group of foci 5–7. Only the SNP-typing method made it possible to separate branch of focus 5 isolates from a group of strains isolated in foci 5–7. In addition, this method made it possible to reveal a greater genetic heterogeneity of strains from focus 39, in contrast to the strains of foci 4–7. When analyzing the genomes of Y. pestis strains isolated in the territory of focus 39, a deletion (~20 kb) was detected in the CRISPR region of the Ypb locus. The absence of this locus can serve as a marker for the determination of a given population of Y. pestis strains.

Species Yersinia pestis includes two subspecies, pestis and microti. Strains of subsp. pestis were the cause of three plague pandemics. Strains of subsp. microti circulate in populations of rodents belonging to the genus Microtus and cause only rare endemic diseases in human beings that are not accompanied by human-to-human transmission. The existence of combined plague foci with strains from different genotypes, biovars, and even both the Y. pestis subspecies circulating in the same area, as well as periodic introductions of Y. pestis subsp. pestis strains into the foci from which only the strains of subsp. microti were isolated previously, require fast and reliable identification of strain subspecies for optimization of plague prevention and control. For differentiation of Y. pestis subspecies among themselves and against Y. pseudotuberculosis, we analyzed the DNA targets (YPDSF_3711 and opgG), selected the primers, and determined optimal concentrations of the components of the reaction mixture and temperature conditions of PCR. The suggested method allows identification according to the amplicon number and size of Y. pestis subsp. pestis (563 bp), two phylogenetic groups of subsp. microti (SNP-types 0.PE2, 0.PE3, 0.PE4, and 0.PE5, 583 and 426 bp; 0.PE7, 779, 515, and 358 bp), the progenitor of the plague microbe Y. pseudotuberculosis (779, 583, and 426 bp) and Y. similis (779 and 426 bp). The study was carried out on a representative set of strains from the State Research Center for Applied Microbiology and Biotechnology (http://obolensk.org/center/state-collection.htm) and nucleotide sequences deposited in the DDBJ/EMBL/GenBank. The study showed 100% specificity and sensitivity of the proposed method.

Aim. Determination of the degree of phylogenetic relationship of Yersinia pestis strains isolated 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 of various 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 «portraits» 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.

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.

Comparative analysis of MLVA25-typing and MLVA7-typing for their ability to identify focal affiliation of the Yersinia pestis strains have been performed on the basis of the bv. Medievalis isolates obtained from the Central Caucasus highland natural plague focus. For purposes of analysis, MLVA25 genotypes were identified for 82 isolates and further introduced into a database containing data on the 949 strains of the plague-causing agent obtained in other natural foci in Russia and countries within the former Soviet Union and beyond. The dendrograms were reconstructed by the UPGMA method with a categorical coefficient using data on all 25 VNTR loci or on only 7 of them, which were recommended by the experts from the Microbe Russian Research Anti-Plague Institute to be used for Y. pestis strains differentiation by their focal affiliation. The results of the analysis indicate the higher possibility of diagnostic errors when using the MLVA7-typing for distinguishing between the Y. pestis strains and suggest that it should be useful to subdivide the Central Caucasus highland plague focus into two subfoci.

A phosphorylated O-polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Photorhabdus temperata subsp. cinerea 3240 and studied by sugar analysis, dephosphorylation, and 1H and 13C NMR spectroscopy. The following structure of the linear trisaccharide repeating unit of the O-polysaccharide was established: →3)-β- d -GalpNAc4PEtN-(1→4)-β- d -GlcpA-(1→3)-β- d -FucpNAc4N-(1→ where GlcA indicates glucuronic acid, FucNAc4N 2-acetamido-4-amino-2,4,6-trideoxygalactose, and PEtN 2-aminoethyl phosphate.

O-Polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Photorhabdus temperata subsp. temperata XlNach(T). Sugar analysis after full acid hydrolysis of the polysaccharide revealed D-glucose, D-mannose, D-galactose, D-GalNAc, and a branched monosaccharide, 3,6-dideoxy-4-C-[(S)-1',2'-dihydroxyethyl]-D-xylo-hexose (Sug), which was isolated as a 1,2'-anhydro furanose derivative. The following structure of the polysaccharide was established by 1D and 2D 1H and 13C NMR spectroscopy

Comparative analysis of rhamnose-positive Y. pestis strains, which are traditionally recognized as the biovar altaica, was performed with the use of molecular typing. The strains were isolated from the natural plague foci of the Changay mountainous system and the Sailugem natural plague focus in Mongolia. The Russian part of the latter is known as the Altai Mountains focus (focus 36). All isolates were compared with the other rhamnose-positive vole strains from the natural plague foci located in the grassland of Xilingol (focus L, China), the plateau of Qinghai-Tibet (focus M, China), the Gissarian Ridge (focus 34, Tadjikistan and Uzbekistan), and the Talassian Ridge (focus 40, Kyrgyzstan). The strains studied formed a single cluster with two branches, of which the first branch included the talassica (0.PE4?), qinghaiensis (0.PE4ab), and xilingolensis (0.PE4cd) phylogenetic groups, while the second included the hissarica (0.PE9) and altaica (0.PE1) phylogenetic groups. The results of molecular typing suggest that several strains from the Qinghai group of the Mongolian foci belong to the qinghaiensis and xilingolensis biovars but not to the altaica biovar.

We here report the draft genome sequences of 8 Yersinia pestis subsp. microtus bv. caucasica strains isolated from the East Caucasian (previous name, Dagestan) mountain focus (no. 39), representing the most ancient branch of the 0.PE2 phylogroup circulating in populations of common voles ( Microtus arvalis ).

Plague, one of the most devastating infectious diseases in human history, is caused by the bacterial species Yersinia pestis. A live attenuated Y. pestis strain (EV76) has been widely used as a plague vaccine in various countries around the world. Here we compared the whole genome sequence of an EV76 strain used in China (EV76-CN) with the genomes of Y. pestis wild isolates to identify genetic variations specific to the EV76 lineage. We identified 6 SNPs and 6 Indels (insertions and deletions) differentiating EV76-CN from its counterparts. Then, we screened these polymorphic sites in 28 other strains of EV76 lineage that were stored in different countries. Based on the profiles of SNPs and Indels, we reconstructed the parsimonious dissemination history of EV76 lineage. This analysis revealed that there have been at least three independent imports of EV76 strains into China. Additionally, we observed that the pyrE gene is a mutation hotspot in EV76 lineages. The fine comparison results based on whole genome sequence in this study provide better understanding of the effects of laboratory passages on the accumulation of genetic polymorphisms in plague vaccine strains. These variations identified here will also be helpful in discriminating different EV76 derivatives.

Studied have been Yersinia pestis strains isolated in Central-Caucasian high-mountain natural plague focus within the period of 1971–2012 using MLVA typing on 25 VNTR-loci. Analysis of dendrogram cluster association with certain areas, objects, and periods of the strain isolation has been carried out by means of Arc GIS 10.1 software. Established has been the connection between 25 MLVA Y. pestis types and spatial and temporal characteristics of strains isolation in the Central-Caucasian high-mountain natural plague focus, and proline requirement too. Clusters, groups and subgroups of the strains are confined to particular territories as it follows: groups are attributed to landscape-epizootiological regions, subgroups – to mountain souslik settlements. Occasionally groups of the strains disseminate onto the other landscape-epizootiological regions of the focus. Genetic variations of plague agent strains within the limits of mountain souslik settlements evolve over time which can be viewed as indirect evidence of microevolution mechanism effect.

The molecular mechanisms of pathogenicity and properties of the formation of specific plague immunity are reviewed. The history and modern state of vaccine-prevention of plague are described. Special attention is focused on the prospects in the plague vaccine development, and possible approaches to the improvement of vaccine preparations are considered.

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 Nomenclature of Bacteria is suggested.

The Yersinia pestis outer membrane porin F (OmpF) is a transmembrane protein located in the outer membrane of this Gram-negative bacterium which is the causative agent of plague, where it plays a significant role in controlling the selective permeability of the membrane. The amino acid sequences of OmpF proteins from 48 Y. pestis strains representing all currently available phylogenetic groups of this Gram-negative bacterium were recently deduced. Comparison of these amino acid sequences revealed that the OmpF can be present in four isoforms, the pestis-pestis type, and the pestis-microtus types I, II, and III. OmpF of the most recent pestis-pestis type has an alanine residue at the position 148, where all the pestis-microtus types have threonine there (T148A polymorphism). The variability of different pestis-microtus types is caused by an additional polymorphism at the 193rd position, where the OmpFs of the pestis-microtus type II and type III have isoleucine-glycine (IG+193) or isoleucine-glycine-isoleu...

Plague still poses a significant threat to human health and as a reemerging infection is unfamiliar to the majority of the modern medical doctors. In this chapter, the plague is described according to Dr. Nikiforov’s experiences in the diagnosis and treatment of patients, and also a review of the relevant literature on this subject is provided. The main modern methods and criteria for laboratory diagnosis of plague are briefly described. The clinical presentations include the bubonic and pneumonic form, septicemia, rarely pharyngitis, and meningitis. Early diagnosis and the prompt initiation of treatment reduce the mortality rate associated with bubonic plague and septicemic plague to 5–50 %; although a delay of more than 24 h in the administration of antibiotics and antishock treatment can be fatal for plague patients. Most human cases can successfully be treated with antibiotics.

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 of Yersinia 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 of Y. 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 of Y. pestis and other enteric bacteria, including other Yersinia species. The prospects of development of live plague vaccines created on the basis of Y. pestis strains with the genetically modified LPS are discussed.

Comparative analysis of 57 strains of Y. pestis subsp. microtus bv. caucasica was carried out using molecular typing. The results obtained indicate the presence of three independent phylogenetic groups and indicate the advisability of isolation of the Leninakan mountain mesofocus from the Transcaucasian highland focus into an independent focus, as well as inclusion of part of the Pre-Araks low-mountain focus as the mesofocus along with the Pre-Sevan mountain and Zangezur-Karabakh mountain mesofoci into the Transcaucasian highland plague focus. It is shown that the strains circulating in the East Caucasus highland focus of plague are the most ancient branch of the caucasica biovar, and possibly of the entire phylogenetic tree of Y. pestis.

The O-polysaccharide from the lipopolysaccharide of a symbiotic bacterium Photorhabdus luminescens subsp. laumondii TT01 from an insect-pathogenic nematode was studied by sugar analysis and 1 H and 13 C NMR spectroscopy and found to contain d - glycero - d - manno -heptose ( dd Hep) and 3,6-dideoxy-3-formamido- d -glucose ( d -Qui3NFo). The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established: Download full-size image

The etiologic agent of plague, Yersinia pestis , includes two subspecies, of which Y. pestis subsp. microtus contains the strains that cause only occasional diseases in humans that are not accompanied by human-to-human transmission. Here, we report the draft genome sequences of 19 Y. pestis strains (across 6 biovars of Y. pestis subsp. microtus ).

φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis , the etiologic agent of plague, and used by the CDC in the identification of Y. pestis . φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc ( l - glycero - d - manno- heptose/ d -glucopyranose)-Kdo/Ko (3-deoxy- d - manno -oct-2-ulopyranosonic acid/ d - glycero - d - talo- oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis .

The O-polysaccharides were isolated from the lipopolysaccharides of emerging human pathogens Photorhabdus asymbiotica subsp. asymbiotica US-86 and US-87 and subsp. australis AU36, AU46, and AU92. Studies by sugar analysis and (1)H and (13)C NMR spectroscopy before and after O-deacetylation showed that the O-polysaccharide structures are essentially identical within, and only slightly different between, the subspecies. The following structures of the repeating units of the O-polysaccharides were established: →3)-β-d-Quip4NGlyFo-(1→4)-α-d-GalpNAcAN3Ac-(1→4)-α-d-GalpNAcA3R-(1→3)-α-d-QuipNAc-(1→ where GalNAcA stands for 2-acetamido-2-deoxygalacturonic acid, GalNAcAN for amide of GalNAcA, QuiNAc for 2-acetamido-2,6-dideoxyglucose, and Qui4NGlyFo for 4,6-dideoxy-4-(N-formylglycyl)aminoglucose; R=Ac in subsp. asymbiotica or H in subsp. australis. The structures established resemble those of a number of taxonomically remote bacteria including Francisella tularensis (Vinogradov, E. V.; Shashkov, A. S.; Knirel, Y. A.; Kochetkov, N. K.; Tochtamysheva, N. V.; Averin, S. P.; Goncharova, O. V.; Khlebnikov, V. S. Carbohydr. Res.1991, 214, 289-297), which differs in (i) the presence of a formyl group on Qui4N rather than the N-formylglycyl group, (ii) the mode of the linkage between the repeating units (β1→2 vs α1→3), (iii) amidation of both GalNAcA residues rather than one residue, and iv) the lack of O-acetylation.

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.

Everyone has heard about the plague, a deadly infectious disease. Pandemics of this frightful disease in the Middle Ages and in later times caused the deaths of millions of people. According to molecular-genetic studies, its causative agent is very young compared to other pathogenic bacteria: it originated from a pseudotuberculosis microbe no more than 20000 years ago. Human economic activity probably played a definite role in the formation of natural pestholes of plague and in its epidemic propagation. Studies of the specific properties of the malicious plague microbe and works targeted at creating a highly efficient, harmless, and safe vaccine of a new generation are discussed.

The Pla surface protease of Yersinia pestis activates human plasminogen and is a central virulence factor in bubonic and pneumonic plague. Pla is a transmembrane -barrel protein and member of the omptin family of outer membrane proteases which require bound lipopolysaccharide (LPS) to be proteolytically active. Plasminogen activation and autoprocessing of Pla were dramatically higher in Y. pestis cells grown at 37°C than in cells grown at 20°C; the difference in enzymatic activity by far exceeded the increase in the cellular content of the Pla protein. Y. pestis modifies its LPS structure in response to growth temperature. We purified His6-Pla under denaturing conditions and compared various LPS types for their capacity to enhance plasmin formation by His6-Pla solubilized in detergent. Reactivation of His6-Pla was higher with Y. pestis LPSs isolated from bacteria grown at 37°C than with LPSs from cells grown at 25°C. Lack of O antigens and the presence of the outer core region as well as a lowered level of acylation in LPS were found to enhance the Pla-LPS interaction. Genetic substitution of arginine 138, which is part of a three-dimensional protein motif for binding to lipid A phosphates, decreased both the enzymatic activity of His6-Pla and the amount of Pla in Y. pestis cells, suggesting the importance of the Pla-lipid A phosphate interaction. The temperature-induced changes in LPS are known to help Y. pestis to avoid innate immune responses, and our results strongly suggest that they also potentiate Pla-mediated proteolysis. Plague is a zoonosis that primarily affects rodents but also has caused three waves of plague pandemics in the human population, leading to tens of millions of deaths (51). The etiological agent of plague, Yersinia pestis, is transmitted to humans primarily by the bite of infected fleas. The common form of the disease is bubonic plague, where the bacteria spread from the intradermal fleabite site into the lymphatic system, multiply in lymph nodes, and cause formation of bubos (51, 71). Bubonic plague may progress to systemic infection and reach the lungs, causing pneumonic plague. A high level of bacteremia is essential for the blood feeding by fleas and the efficient transmission of Y. pestis from host to host (51).

The LcrV protein is a multifunctional virulence factor and protective antigen of the plague bacterium and is generally conserved between the epidemic strains of Yersinia pestis. We investigated the diversity in the LcrV sequences among non-epidemic Y. pestis strains which have a limited virulence in selected animal models and for humans. Sequencing of lcrV genes from 19 Y. pestis strains belonging to different phylogenetic groups (“subspecies”) showed that the LcrV proteins possess four major variable hotspots at positions 18, 72, 273, and 324–326. These major variations, together with other minor substitutions in amino acid sequences, allowed us to classify the LcrV alleles into five sequence types (A-E). We observed that the strains of different Y. pestis “subspecies” can have the same type of LcrV, including that conserved in epidemic strains, and different types of LcrV can exist within the same natural plague focus. Therefore, the phenomenon of “selective virulence” characteristic of the strains of the microtus biovar is unlikely to be the result of polymorphism of the V antigen. The LcrV polymorphisms were structurally analyzed by comparing the modeled structures of LcrV from all available strains. All changes except one occurred either in flexible regions or on the surface of the protein, but local chemical properties (i.e. those of a hydrophobic, hydrophilic, amphipathic, or charged nature) were conserved across all of the strains. Polymorphisms in flexible and surface regions are likely subject to less selective pressure, and have a limited impact on the structure. In contrast, the substitution of tryptophan at position 113 with either glutamic acid or glycine likely has a serious influence on the regional structure of the protein, and these mutations might have an effect on the function of LcrV. The polymorphisms at positions 18, 72 and 273 were accountable for differences in the oligomerization of LcrV.

Background Yersinia pestis, the causative agent of plague, showed a temperature-dependent change in lipid A composition, with a reduced degree of acylation when bacteria were grown at 37 degrees C (tetraacylated) versus ambient temperature (hexaacylated). Methods Human monocytes and monocyte-derived dendritic cells (DCs) were exposed to Y. pestis grown at 26 degrees C or 37 degrees C, to their corresponding lipopolysaccharides (LPS-26 degrees C or LPS-37 degrees C), and to ligands of different Toll-like receptors (TLRs), such as LPS from Escherichia coli (TLR4), lipoprotein (TLR2), polyinosinic-polycytidylic acid (poly-IC) (TLR9), and their combinations. Production of cytokines was measured, along with expression of surface markers of DC maturation. Results Y. pestis grown at 37 degrees C or LPS-37 degrees C induced much lower production of cytokines (such as tumor necrosis factor alpha and interleukins 1beta, 10, and 12) by DCs than did Y. pestis grown at 26 degrees C or LPS-26 degrees C. Expression of the surface markers HLA-DR, CD86, and CD40 by DCs was also reduced in response to treatment with LPS-37 degrees C compared with LPS-26 degrees C. Pretreatment of DCs with LPS-37 degrees C inhibited subsequent stimulation with LPS-26 degrees C, control LPS from E. coli, lipoprotein, or poly-IC. Conclusions LPS-37 degrees C can inhibit stimulation of DCs not only via TLR4 signaling but also via TLR2 and TLR3. [corrected]

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.

The O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Yersinia pseudotuberculosis O:4a and studied by NMR spectroscopy, including 2D ROESY and (1)H, (13)C HMBC experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established, which differs from the structure reported earlier [Gorshkova, R. P. et al., Bioorg. Khim. 1983, 9, 1401-1407] in the linkage modes between the monosaccharides: [carbohydrate sequence see in text] where Tyv stands for 3,6-dideoxy-D-arabino-hexose (tyvelose). The structure of the Y. pseudotuberculosis O:4a antigen resembles that of Y. pseudotuberculosis O:2c, which differs in the presence of abequose (3,6-dideoxy-D-xylo-hexose) in place of tyvelose only.

Two isogenic sets of Yersinia pestis strains were generated, composed of wild-type strains 231 and I-1996, their non-polar pH 6− mutants with deletions in the psaA gene that codes for its structural subunit or the whole operon, as well as strains with restored ability for temperature- and pH-dependent synthesis of adhesion pili or constitutive production of pH 6 antigen. The mutants were generated by site-directed mutagenesis of the psa operon and subsequent complementation in trans. It was shown that the loss of synthesis or constitutive production of pH 6 antigen did not influence Y. pestis virulence or the average survival time of subcutaneously inoculated BALB/c naïve mice or animals immunized with this antigen.

The live plague vaccine strain Y. pestis EV line NIIEG widely used for human and animal immunisation proves a high level of specific immunity against both bubonic and pneumonic plague. We constructed an EV NIIEG mutant in the acyltransferase gene lpxM that resulted in the production of a less toxic penta-acylated lipid A of lipopolysaccharide (LPS) of Y. pestis , tested the synthesis and immunoreactivity of the major known antigens involved in virulence and eliciting immunity against plague, and also evaluated the protective properties of the lpxM derivative. The expression of antigens was determined by dot-ELISA, immunoblotting, the indirect hemagglutination test, electron microscopy (capsule), and fibrinolytic and plasma-coagulase activities (plasminogen activator Pla). Stumulation of TNF-alpha production with different types of LPS was revealed by using the J774 macrophage-like cell line. The antibody immune response, followed by immunisation, was estimated by ELISA. Protective efficacy was evaluated in mice and guinea pigs by subcutaneous challenge with the wild-type Y. pestis 231. In comparison with the parental strain, the lpxM mutant was found to produce a reduced amount of capsular antigen F1, multifunctional virulence factor LcrV and murine toxin Ymt as well as had an altered activity of Pla. Moreover, this mutant possessed a modified cell surface as jusjed by the immunoreactivity with monoclonal antibodies to different carbohydrate epitopes and by the resistant phenotype to the plague diagnostic bacteriophage L-413C. The penta-acylated LPS of Y. pestis revealed a reduced capacity of stimulating TNF-alpha in vitro . Immunisation with the lpxM mutant provided an enhanced protection over the original Y. pestis EV NIIEG in both mice and guinea pigs. Thus, the pleiotropic effect of the lpxM mutation led to the construction of the live plague candidate vaccine strain with improved characteristics toward safety, reactogenicity and protective properties.

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.

Yersinia pestisundergoes an obligate flea–rodent–flea enzootic life cycle. The rapidly fatal properties ofY. pestisare responsible for the organism's sustained survival in natural plague foci. Lipopolysaccharide (LPS) plays several roles inY. pestispathogenesis, prominent among them being resistance to host immune effectors and induction of a septic-shock state during the terminal phases of infection. LPS is acylated with 4–6 fatty acids, the number varying with growth temperature and affecting the molecule's toxic properties.Y. pestismutants were constructed with a deletion insertion in thelpxMgene in both virulent and attenuated strains, preventing the organisms from synthesizing the most toxic hexa-acylated lipid A molecule when grown at 25 °C. The virulence and/or protective potency of pathogenic and attenuatedY. pestisΔlpxMmutants were then examined in a mouse model. The ΔlpxMmutation in a virulent strain led to no change in the LD50value compared to that of the parental strain, while the ΔlpxMmutation in attenuated strains led to a modest 2.5–16-fold reduction in virulence. LPS preparations containing fully hexa-acylated lipid A were ten times more toxic in actinomycin D-treated mice then preparations lacking this lipid A isoform, although this was not significant (P>0.05). The ΔlpxMmutation in vaccine strain EV caused a significant increase in its protective potency. These studies suggest there is little impact from lipid A modifications on the virulence ofY. pestisstrains but there are potential improvements in the protective properties in attenuated vaccine strains.

Antibiotic therapy of plague is hampered by the recent isolation of Yersinia pestis strain resistant to all of antibiotics recommended for cure. This has constrained a quest for new antimicrobials taking aim at alternative targets. Recently Y. pestis cysteine protease YopT has been explored as a potential drug target. Targets conserved in the pathogen populations should be more efficacious; therefore, we evaluated intraspecies variability in yopT genes and their products. 114 Y. pestis isolates were screened. Only two YopT full-size isoforms were found among them. The endemic allele (N149) was present in biovar caucasica from Dagestan-highland natural plague focus # 39. The biovar caucasica strains from Transcaucasian highland (# 4-6) and Pre-Araks (# 7) plague foci also contained the N149 allele. These strains from foci # 4 7 possessed a truncated version of YopT that was a consequence of a frame-shift due to the deletion of a single nucleotide at position 71 bp. Computational analyses showed that although the SNP at the position 149 has a very minimal effect of the intrinsic disorder propensity of YopT proteins, whereas the N-terminal truncations of the YopT detected in bv. caucasica strains Pestoides F_YopT1 and F_YopT2, and Pestoides G generated isoforms with the significantly modified intrinsic disorder propensities and with reduced capability to interact with lost ability to utilize their N-terminal tail for the disorder-based interactions with biological partners. Considering that representatives of biovar caucasica were reported to be the reason of sporadic cases of human plague, this study supports the necessity of additional testing of globally disseminated YopT (S149) isoform as a potential target for treatment of plague caused by the strains producing different YopT isoforms.

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.

Data on the structure and temperature-dependent variations of the lipopolysaccharide (LPS) of Yersinia pestis are summarized and compared with data of other enteric bacteria, including other Yersinia spp. A correlation between the LPS structure and properties of the LPS and bacterial cultures as well as the LPS biosynthesis control are briefly discussed.

Data on the structure and temperature-dependent variations of the lipopolysaccharide (LPS) of Yersinia pestis are summarized and compared with data of other enteric bacteria, including other Yersinia spp. A correlation between the LPS structure and properties of the LPS and bacterial cultures as well as the LPS biosynthesis control are briefly discussed.

Yersinia pestis spread throughout the Americas in the early 20th century, and it occurs predominantly as a single clone within this part of the world. However, within Eurasia and parts of Africa there is significant diversity among Y. pestis strains, which can be classified into different biovars (bv.) and/or subspecies (ssp.), with bv. orientalis/ssp. pestis most closely related to the American clone. To determine one aspect of the relatedness of these different Y. pestis isolates, the structure of the lipopolysaccharide (LPS) of four wild-type and one LPS-mutant Eurasian/African strains of Y. pestis was determined, evaluating effects of growth at mammalian (37 degrees C) or flea (25 degrees C) temperatures on the structure and composition of the core oligosaccharide and lipid A. In the wild-type clones of ssp. pestis, a single major core glycoform was synthesized at 37 degrees C whereas multiple core oligosaccharide glycoforms were produced at 25 degrees C. Structural differences occurred primarily in the terminal monosaccharides. Only tetraacyl lipid A was made at 37 degrees C, whereas at 25 degrees C additional pentaacyl and hexaacyl lipid A structures were produced. 4-Amino-4-deoxyarabinose levels in lipid A increased with lower growth temperatures or when bacteria were cultured in the presence of polymyxin B. In Y. pestis ssp. caucasica, the LPS core lacked D-glycero-D-manno-heptose and the content of 4-amino-4-deoxyarabinose showed no dependence on growth temperature, whereas the degree of acylation of the lipid A and the structure of the oligosaccharide core were temperature dependent. A spontaneous deep-rough LPS mutant strain possessed only a disaccharide core and a slightly variant lipid A. The diversity and differences in the structure of the Y. pestis LPS suggest important contributions of these variations to the pathogenesis of this organism, potentially related to innate and acquired immune recognition of Y. pestis and epidemiologic means to detect, classify, control and respond to Y. pestis infections.

SUMMARYIncreased interest in the pathogenic potential ofYersinia pestishas emerged because of the potential threats from bioterrorism. Pathogenic potential is based on genetic factors present in a population of microbes, yet most studies evaluating the role of specific genes in virulence have used a limited number of strains. ForY. pestisthis issue is complicated by the fact that most strains available for study in the Americas are clonally derived and thus genetically restricted, emanating from a strain ofY. pestisintroduced into the United States in 1902 via marine shipping and subsequent spread of this strain throughout North and South America. In countries from the former Soviet Union (FSU), Mongolia, and China there are large areas of enzootic foci ofY. pestisinfection containing genetically diverse strains that have been intensely studied by scientists in these countries. However, the results of these investigations are not generally known outside of these countries. Here we describe the variety of methods used in the FSU to classifyY. pestisstrains based on genetic and phenotypic variation and show that there is a high level of diversity in these strains not reflected by ones obtained from sylvatic areas and patients in the Americas.

The O-polysaccharide of Yersinia pseudotuberculosis O:1b was reinvestigated using 1H and 13C NMR spectroscopy, and the anomeric configuration of the mannose residue at the branching point was revised. The following is the revised structure of the O-polysaccharide: Download : Download full-size image where Par stands for 3,6-dideoxy- d -ribo-hexose (paratose). The revised structure of the main chain is shared by the O-polysaccharide of Y. pseudotuberculosis O:11, which differs in the presence of a lateral α- l -6-deoxyaltrofuranose residue in place of the β-paratofuranose residue [Cunneen, M. M.; de Castro, C.; Kenyon, J.; Parrilli, M.; Reeves, P. R.; Molinaro, A.; Holst, O.; Skurnik, M. Carbohydr. Res.2009, 344, 1533–1540].