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We report the complete genome assembly of Yersinia pestis subsp. pestis bv. Medievalis SCPM-O-B-6530, a strain belonging to the most ancient phylogenetic group (group 2.MED0) of Y. pestis subsp. pestis bv. Medievalis. This proline-dependent strain, carrying an additional plasmid (pCKF), was isolated from the Central-Caucasian high-mountain plague focus in Kabardino-Balkar Republic, Russia.

Yersinia kristensenii is one of the Yersinia enterocolitica -like bacterial species, which are considered nonpathogenic to humans. In this work, we reported the draft genome sequences of six Yersinia kristensenii strains. These draft genomes will help to better characterize Yersinia kristensenii at the genomic level.

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.

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.

Mild acid degradation of the lipopolysaccharide of Yersinia kristensenii C-134 afforded a glycerol teichoic acid-like O-polysaccharide, which was studied by sugar analysis, O-deacetylation and dephosphorylation along with 1D and 2D NMR spectroscopy. The following structure of the O-polysaccharide was established: This structure is related to those of other Y. kristensenii O-polysaccharides studied earlier. The O-antigen gene cluster of Y. kristensenii С-134 was analyzed and found to be consistent with the O-polysaccharide structure established.

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.

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.

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.

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.

Lipopolysaccharide was isolated from bacteria Yersinia intermedia H9-36/83 (O:17) and degraded with mild acid to give an O-specific polysaccharide, which was isolated by GPC on Sephadex G-50 and studied by sugar analysis and 1D and 2D NMR spectroscopy. The polysaccharide was found to contain 3-deoxy-3-[( R )-3-hydroxybutanoylamino]- d -fucose ( d -Fuc3N R 3Hb) and the following structure of the heptasaccharide repeating unit was established: Download high-res image (83KB) Download full-size image The structure established is consistent with the gene content of the O-antigen gene cluster. The O-polysaccharide structure and gene cluster of Y. intermedia are related to those of Hafnia alvei 1211 and Escherichia coli O:103.

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: Download : Download high-res image (52KB) Download : Download full-size image 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.

Aiming at creation of the chemical basis for serotyping of strains of Yersinia spp., we studied the O-specific polysaccharide (OPS) of Yersinia frederiksenii H56-36/81 belonging to serotype O:60. A branched polysaccharide was obtained by mild alkaline degradation of the lipopolysaccharide (LPS) isolated from bacterial cells by the phenol—water extraction and found to contain d-ribose, l-rhamnose, and 4-deoxy d-arabino-hexose (d-ara4dHex). The last monosaccharide occurs rarely in nature and earlier has been found only in OPS of Citrobacter spp. Mild acid hydrolysis of the LPS resulted in cleavage of the d-ara4dHex side-chains to give a linear polysaccharide. Structure of the hexasaccharide repeating unit of the OPS was established by studies of both isolated polysaccharides by 1D and 2D 1H and 13C NMR spectroscopy.

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.

Here, we report the draft genome sequences of six Yersinia pestis subsp. microtus bv. ulegeica strains isolated from the territory of Mongolia and representing the 0.PE5 phylogroup circulating in populations of voles and picas.

We report here the draft genome sequences of nine Yersinia pestis subsp. microtus bv. Altaica strains isolated from the Altai Mountain plague focus (no. 36), which represent the 0.PE4 phylogroup circulating in populations of Mongolian pika ( Ochotona pallasi ).

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.

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 nlpD 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 nlpD 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.

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 ).

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...

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 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 .

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.

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.

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.

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.

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].

The O-polysaccharide was isolated by hydrolysis of the lipopolysaccharide of Yersinia pseudotuberculosis O:2b, and studied by 1D and 2D NMR spectroscopy. The following structure of the polysaccharide was established: alpha-Abep. 1--3. --2) alpha-D-Manp-(1--3)-alpha-L-Fucp-(1--3)-beta-D-GalpNAc-(1--. where Abe stands for 3,6-dideoxy-D-xylo-hexose (abequose).

The structure of the long-chain O-antigen of Yersinia pseudotuberculosis O:4b containing two uncommon deoxy sugars, tyvelose (3,6-dideoxy-d-arabino-hexose, Tyv) and 6-deoxy-d-manno-heptose (D-6dmanHep), was established by 1D and 2D NMR spectroscopy as alpha-Tyvp-(1--3)-beta-D-6dmanHepp 1--4 --3)-alpha-D-Galp-(1--3)-beta-D-GlcpNac-(1--.

Structures of the O-antigens of Yersinia pseudotuberculosis O2c and O3 were reinvestigated by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments. The following revised structure of the O2c tetrasaccharide repeating unit was established, which differs from the structure proposed earlier in the glycosylation pattern of the mannose residue at the branching point: Download : Download full-size image where Abe stands for 3,6-dideoxy- d -xylo-hexose. The structure of the Y. pseudotuberculosis O3 antigen reported earlier was confirmed.

Yersinia pestis Caf1 is a multifunctional protein responsible for antiphagocytic activity and is a key protective antigen. It is generally conserved between globally distributed Y. pestis strains, but Y. pestis subsp. microtus biovar caucasica strains circulating within populations of common voles in Georgia and Armenia were reported to carry a single substitution of alanine to serine. We investigated polymorphism of the Caf1 sequences among other Y. pestis subsp. microtus strains, which have a limited virulence in guinea pigs and in humans. Sequencing of caf1 genes from 119 Y. pestis strains belonging to different biovars within subsp. microtus showed that the Caf1 proteins exist in three isoforms, the global type Caf1NT1 (Ala48 Phe117), type Caf1NT2 (Ser48 Phe117) found in Transcaucasian-highland and Pre-Araks natural plague foci #4–7, and a novel Caf1NT3 type (Ala48 Val117) endemic in Dagestan-highland natural plague focus #39. Both minor types are the progenies of the global isoform. In this report, Caf1 polymorphism was analyzed by comparing predicted intrinsic disorder propensities and potential protein-protein interactivities of the three Caf1 isoforms. The analysis revealed that these properties of Caf1 protein are minimally affected by its polymorphism. All protein isoforms could be equally detected by an immunochromatography test for plague at the lowest protein concentration tested (1.0 ng/mL), which is the detection limit. When compared to the classic Caf1NT1 isoform, the endemic Caf1NT2 or Caf1NT3 had lower immunoreactivity in ELISA and lower indices of self- and cross-protection. Despite a visible reduction in cross-protection between all Caf1 isoforms, our data suggest that polymorphism in the caf1 gene may not allow the carriers of Caf1NT2 or Caf1NT3 variants escaping from the Caf1NT1-mediated immunity to plague in the case of a low-dose flea-borne infection.