Paper-Strip Blood-Sampling Technique for the Detection of Antibody to the Plague Organism Yersinia pestis

The paper-strip blood-sampling technique was evaluated for efficacy in plague passive hemagglutination tests. It is valuable for widespread serological surveys.     

Draft Genome Sequences of Yersinia pestis Strains from the 1994 Plague Epidemic of Surat and 2002 Shimla Outbreak in India

We report the first draft genome sequences of the strains of plague-causing bacteria, Yersinia pestis, from India. These include two strains from the Surat epidemic (1994), one strain from the Shimla outbreak (2002) and one strain from the plague surveillance activity in the Deccan plateau region (1998). Genome size for all four strains is ~4.49 million bp with 139–147 contigs. Average sequencing depth for all four genomes was 21x.     

A highly specific one-step PCR - assay for the rapid discrimination of enteropathogenic Yersinia enterocolitica from pathogenic Yersinia pseudotuberculosis and Yersinia pestis

Based on differences within the yopT-coding region of Yersinia. enterocolitica, Y pseudotuberculosis and Y pestis, a rapid and sensitive one-step polymerase chain reaction assay with high specificity for pathogenic Y enterocolitica was developed. By this method pathogenic isolates of Y enterocolitica can be easily identified and discriminated from other members of this genus. The entire coding sequence of the yopT effector gene of Y. pseudotuberculosis Y36 was determined.     

The effect of Yersinia pestis EV76 6 MD plasmid on the composition of outer membrane proteins of Yersinia pseudotuberculosis YPIII]

On the basis of Yersinia pseudotuberculosis strain YPIII the isogenic variants containing the different combinations of 47 Md plasmids from Yersinia pestis or Yersinia pseudotuberculosis cells with the 6 Md pYP plasmid from Yersinia pestis EV (intact or having impaired the pla gene determining the synthesis of plasmocoagulase). The degradation of the secreted proteins encoded by the 47 Md plasmids of Yersinia pestis and Yersinia pseudotuberculosis in the cells harbouring the 6Md pYP plasmid has been registered. Yersinia pseudotuberculosis strain YPIII carrying its own 47Md and pYP plasmids also contained no YOP1 protein, in contract to the parent strain. The damage of the pla gene eliminated the destructive effect on the outer membrane proteins. Imposition of the 47Md and 6Md plasmids from Yersinia pestis in Yersinia pseudotuberculosis cells may be used for obtaining and study of the physiological role of low molecular mass proteins resulting from proteolysis of proteins encoded by the 47Md virulence plasmid of Yersinia.     

Draft Genome Sequence of Yersinia pestis Strain 2501, an Isolate from the Great Gerbil Plague Focus in Xinjiang, China

We deciphered the genome of Yersinia pestis strain 2501, isolated from the Junggar Basin, a newly discovered great gerbil plague focus in Xinjiang, China. The total length of assembly was 4,597,322 bp, and 4,265 coding sequences were predicted within the genome. It is the first Y. pestis genome from this plague focus.     

Structure and function of cytidine monophosphate kinase from Yersinia pseudotuberculosis,essential for virulence but not for survival

The need for new antibiotics has become pressing in light of the emergence of antibiotic-resistant strains of human pathogens. Yersinia pestis, the causative agent of plague, is a public health threat and also an agent of concern in biodefence. It is a recently emerged clonal derivative of the enteric pathogen Yersinia pseudotuberculosis. Previously, we developed a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. One such target was cytidine monophosphate (CMP) kinase, which is an essential gene in some organisms. Previously, we had thought CMP kinase was essential for Y. pseudotuberculosis, but by modification of the mutagenesis approach, we report here the production and characterization of a Δcmk mutant. The isogenic mutant had a growth defect relative to the parental strain, and was highly attenuated in mice. We have also elucidated the structure of the CMP kinase to 2.32 Å, and identified three key residues in the active site that are essential for activity of the enzyme. These findings will have implications for the development of novel CMP kinase inhibitors for therapeutic use.     

Genotyping Yersinia pestis in Historical Plague: Evidence for Long-Term Persistence of Y. pestis in Europe from the 14th to the 17th Century

Ancient DNA (aDNA) recovered from plague victims of the second plague pandemic (14^th to 17^th century), excavated from two different burial sites in Germany, and spanning a time period of more than 300 years, was characterized using single nucleotide polymorphism (SNP) analysis. Of 30 tested skeletons 8 were positive for Yersinia pestis-specific nucleic acid, as determined by qPCR targeting the pla gene. In one individual (MP-19-II), the pla copy number in DNA extracted from tooth pulp was as high as 700 gene copies/μl, indicating severe generalized infection. All positive individuals were identical in all 16 SNP positions, separating phylogenetic branches within nodes N07_N10 (14 SNPs), N07_N08 (SNP s19) and N06_N07 (s545), and were highly similar to previously investigated plague victims from other European countries. Thus, beside the assumed continuous reintroduction of Y. pestis from central Asia in multiple waves during the second pandemic, long-term persistence of Y. pestis in Europe in a yet unknown reservoir host has also to be considered.     

The Effect of the Cultivation Method and the Growth Phase on the Lipopolysaccharide Composition of Yersinia pseudotuberculosis

Effects of the cultivation method (suspension cultures in a liquid nutrient broth or colonies on a solid agarized medium) and the growth phase on the lipopolysaccharide (LPS) composition of Yersinia pseudotuberculosis(O : Ib serovar, strain KS 3058) grown in cold (5°C) were studied. The amount of the LPS synthesized by cells depended on the bacteria growth phase for both media. The LPS acylation degree was constant, whereas the length of the O-specific polysaccharide chain varied with the culture age and for both media achieved maximum in the stationary growth phase. The bacteria cultivation on the nutrient agar stimulated more intensive synthesis of LPS, which were extracted more easily, had longer polysaccharide O-chains, and were more toxic than LPS of the bacteria cultivated in the liquid medium. It was proposed that the cultivation of Yersinia pseudotuberculosisin cold as colonies on the agar surface increases the bacterial virulence.     

Development of a multiplex PCR for detection of the Yersinia genus with identification of pathogenic species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica)

To identify the Yersinia genus and pathogenic species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) in a single reaction a multiplex PCR technique was developed. It was optimized by five compounds of PCR buffer and temperature of primers annealing. The multiplex PCR provides an improved and rapid method for detection of the Yersinia genus and identification of pathogenic species.     

The effect of temperature on the interaction of Yersinia pseudotuberculosis lipopolysaccharide with chitosan

The mechanism of binding of lipopolysaccharide (LPS) from Yersinia pseudotuberculosis to low-molecular-weight chitosan was investigated using sedimentation analysis, centrifugation in glycerol and percoll density gradients, and isopicnic centrifugation in cesium chloride. The LPS interaction with chitosan was shown to be a multistage process that depended on time and reaction temperature. A stable LPS-chitosan complex could be formed only after preliminary incubation of the initial components at an elevated temperature (37 degrees C). This temperature caused the LPS dissociation and promoted its binding to chitosan. The LPS binding to chitosan results in further dissociation of the endotoxin and formation of the complex with a molecular weight that is tens of times less than the initial molecular weight of LPS. The obtained complex remained stable in solutions of high ionic strength.     

Phenotypic features of a strain of Yersinia pseudotuberculosis with the pVM82 plasmid, detected at pseudotuberculosis outbreak foci]

The phenotypic properties conferred to Yersinia pseudotuberculosis cells by the genetical determinants of a 25Md fragment of the plasmid pVM82 coding for the modified cellular immune response in the infected organism. The fragment was shown to determine the conjugative properties of the plasmid, the resistance of bacterial cells to a number of hydrophobic agents and cellular ability to absorb the Congo red dye. The latter confirms the presence of additional structural components in the cell wall of the strain harbouring the plasmid pVM82. The increased resistance of the plasmid-containing strain to bactericidal effect of the blood plasma was demonstrated as compared with the resistance of the strains harbouring the p57 plasmid lacking the 25Md fragment or no plasmid at all.     

X-Linked Agammaglobulinemia Patients Are Not Infected with Epstein-Barr Virus: Implications for the Biology of the Virus

Epstein-Barr virus (EBV) infects both B lymphocytes and squamous epithelial cells in vitro, but the cell type(s) required to establish primary and persistent infection in vivo has not been definitively elucidated. The aim of this study was to investigate a group of individuals who lack mature B lymphocytes due to the rare heritable disorder X-linked agammaglobulinemia in order to determine the role of the B cell in the infection process. The results show that none of these individuals harbored EBV in their blood or throat washings. Furthermore, no EBV-specific memory cytotoxic T lymphocytes were found, suggesting that they had not undergone infection in the past. In contrast, 50% of individuals were found to carry human herpesvirus 6, showing that they are infectible by another lymphotropic herpesvirus. These results add weight to the theory that B lymphocytes, and not oropharyngeal epithelial cells, may be required for primary infection with EBV.     

The core structure of the lipopolysaccharide from the causative agent of plague, Yersinia pestis

The rough-type lipopolysaccharide (LPS) of the plague pathogen, Yersinia pestis, was studied after mild-acid and strong-alkaline degradations by chemical analyses, NMR spectroscopy and electrospray-ionization mass spectrometry, and the following structure of the core region was determined:where L-alpha-D-Hep stands for L-glycero-alpha-D-manno-heptose, Sug1 for either 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (alpha-Kdo) or D-glycero-alpha-D-talo-oct-2-ulosonic acid (alpha-Ko), and Sug2 for either beta-D-galactose or D-glycero-alpha-D-manno-heptose. A minority of the LPS molecules lacks GlcNAc.     

The significance of pesticin 1 for the virulence and immunogenicity of Yersinia pestis]

The work deals with the study of the virulent and immunogenic properties of Y. pestis strains which lost their capacity for producing pesticine 1 as the result of the insertion of a Tn-like element into the 6-MD plasmid responsible for this property. The "switching-off" of gene pst induced a decrease in the virulence of Y. pestis injected subcutaneously into white mice and guinea pigs and had no influence on its level of immunogenicity for white mice. A suggestion was made that pesticine 1 played no essential role in the expression of the virulence and immunogenicity of Y. pestis penetrating into the body by subcutaneous route.     

Proteolytic cleavage of the FlhB homologue YscU of Yersinia pseudotuberculosis is essential for bacterial survival but not for type III secretion

Pathogenic Yersinia species employ a type III secretion system (TTSS) to target antihost factors, Yop proteins, into eukaryotic cells. The secretion machinery is constituted of ca. 20 Ysc proteins, nine of which show significant homology to components of the flagellar TTSS. A key event in flagellar assembly is the switch from secreting-assembling hook substrates to filament substrates, a switch regulated by FlhB and FliK. The focus of this study is the FlhB homologue YscU, a bacterial inner membrane protein with a large cytoplasmic C-terminal domain. Our results demonstrate that low levels of YscU were required for functional Yop secretion, whereas higher levels of YscU lowered both Yop secretion and expression. Like FlhB, YscU was cleaved into a 30-kDa N-terminal and a 10-kDa C-terminal part. Expression of the latter in a wild-type strain resulted in elevated Yop secretion. The site of cleavage was at a proline residue, within the strictly conserved amino acid sequence NPTH. A YscU protein with an in-frame deletion of NPTH was cleaved at a different position and was nonfunctional with respect to Yop secretion. Variants of YscU with single substitutions in the conserved NPTH sequence--i.e., N263A, P264A, or T265A--were not cleaved but retained function in Yop secretion. Elevated expression of these YscU variants did, however, result in severe growth inhibition. From this we conclude that YscU cleavage is not a prerequisite for Yop secretion but is rather required to maintain a nontoxic fold.