Immunochemical characteristics of synthetic peptides incorporating T- and B-cell epitopes nonspecific porins of pathogenic Yersinia

Multiple antigenic peptides (MAPs), a sequence which include common antigenic epitopes of outer membrane porins (OM) bacteria of the genus Yersinia (Y. pseudotuberculosis, Y. enterocolitica, Y. pestis), pathogenic for humans have been synthesized. After immunization of BALB/c mice the antiserum to the peptide have been obtained. With the help of ELISA we showed that these sera interact with porins isolated from OM pathogenic Yersinia, and MAP interact with antibodies in sera from rabbits immunized with individual porins, and with antibodies in sera of patients with intestinal yersiniosis and pseudotuberculosis.     

Changes in the oxygen-dependent metabolic activity of polymorphonuclear leukocytes from the peripheral blood and macrophages from the peritoneal exudate in the experimental infection of mice with the causative agent of plague

The subcutaneous infection of C57BL/6J mice and noninbred white mice with 40 LD100 of Y. pestis virulent strain has been found to produce synchronous changes in the oxygen-dependent metabolism (ODM) of peripheral blood neutrophils in the spontaneous or zymosan-, E. coli- and Y. pestis-stimulated variants of the NBT test. These changes can be divided into three phases: (I) the phase of a sharp drop in ODM activity; (II) the phase of the increase of this activity, occurring simultaneously with the penetration of Y. pestis cells into the blood stream; and (III) the phase of the terminal decrease of ODM activity as the cytotoxic lesion of phagocytic cells occurs. Peritoneal exudate macrophages show a more gradual decrease in ODM activity. The infection of the animals with 40,000 LD100 of Y. pestis has been found to produce an increase in the ODM activity of neutrophils, rapidly followed by its decrease to the zero level. Macrophages show phasic changes in their ODM activity, identical to changes in the ODM values of neutrophils in mice infected with 40 LD100 of Y. pestis.     

Analysis of genomic polymorphism of typical and atypical strains of the plague pathogen using polymerase chain reaction with universal primers

An analysis of genome polymorphism of the Y. pestis strains by using the method of polymerase chain reaction (PCR) for the tandem repeats of bacteriophage M13 DNA revealed a species similarity of both typical and atypical (according to diagnostic signs) plague-microbe strains. Strain Y. pestis A-1726 with the atypical differential-and-diagnostic properties, without the amplicon specified for Y. pestis and sized 1000 b.p., was identified among 27 analyzed Y. pestis strains. The amplicon profiles of the basic Y. pestis subtype were found to be different from such profiles of other Y. pestis subtypes.     

环介导等温扩增技术检测鼠疫耶尔森菌的敏感性和特异性研究

为观察环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术能否适用于我国不同疫源地鼠疫耶尔森菌所有基因组型的检测,本研究建立了一种基于3a靶序列设计特异性引物快速检测鼠疫耶尔森菌的LAMP方法.选择分离自我国11个鼠疫自然疫源地的65株野生代表性鼠疫耶尔森菌株,同...     

Detection of viable Yersinia pestis by fluorescence in situ hybridization using peptide nucleic acid probes

A successful method has been developed for the detection of live Yersinia pestis, the plague bacillus, which incorporates nascent RNA synthesis. A fluorescent in situ hybridization (FISH) assay using peptide nucleic acid (PNA) probes was developed specifically to differentiate Y. pestis strains from closely related bacteria. PNA probes were chosen to target high copy mRNA of the Y. pestis caf1 gene, encoding the Fraction 1 (F1) antigen, and 16S ribosomal RNA. Among Yersinia strains tested, PNA probes Yp-16S-426 and Yp-F1-55 exhibited binding specificities of 100% and 98%, respectively. Y. pestis grown in the presence of competing bacteria, as might be encountered when recovering Y. pestis from environmental surfaces in a post-release bioterrorism event, was recognized by PNA probes and neither hybridization nor fluorescence was inhibited by competing bacterial strains which exhibited faster growth rates. Using fluorescence microscopy, individual Y. pestis bacteria were clearly differentiated from competing bacteria with an average detection sensitivity of 7.9x10(3) cells by fluorescence microscopy. In the current system, this would require an average of 2.56x10(5) viable Y. pestis organisms be recovered from a post-release environmental sample in order to achieve the minimum threshold for detection. The PNA-FISH assays described in this study allow for the sensitive and specific detection of viable Y. pestis bacteria in a timely manner.     

Characterization of the O-antigen gene clusters of Yersinia pseudotuberculosis and the cryptic O-antigen gene cluster of Yersinia pestis shows that the plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotype O:1b

One of the most virulent and feared bacterial pathogens is Yersinia pestis, the aetiologic agent of bubonic plague. Characterization of the O-antigen gene clusters of 21 serotypes of Yersinia pseudotuberculosis and the cryptic O-antigen gene cluster of Y. pestis showed that the plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotype O:1b. The nucleotide sequences of both gene clusters (about 20.5 kb each) were determined and compared to identify the differences that caused the silencing of the Y. pestis gene cluster. At the nucleotide sequence level, the loci were 98.9% identical and, of the 17 biosynthetic genes identified from the O:1b gene cluster, five were inactivated in the Y. pestis cluster, four by insertions or deletions of one nucleotide and one by a deletion of 62 nucleotides. Apparently, the expression of the O-antigen is not beneficial for the virulence or to the lifestyle of Y. pestis and, therefore, as one step in the evolution of Y. pestis, the O-antigen gene cluster was inactivated.     

Loss of a biofilm-inhibiting glycosyl hydrolase during the emergence of Yersinia pestis

Yersinia pestis, the bacterial agent of plague, forms a biofilm in the foregut of its flea vector to produce a transmissible infection. The closely related Yersinia pseudotuberculosis, from which Y. pestis recently evolved, can colonize the flea midgut but does not form a biofilm in the foregut. Y. pestis biofilm in the flea and in vitro is dependent on an extracellular matrix synthesized by products of the hms genes; identical genes are present in Y. pseudotuberculosis. The Yersinia Hms proteins contain functional domains present in Escherichia coli and Staphylococcus proteins known to synthesize a poly-beta-1,6-N-acetyl-D-glucosamine biofilm matrix. In this study, we show that the extracellular matrices (ECM) of Y. pestis and staphylococcal biofilms are antigenically related, indicating a similar biochemical structure. We also characterized a glycosyl hydrolase (NghA) of Y. pseudotuberculosis that cleaved beta-linked N-acetylglucosamine residues and reduced biofilm formation by staphylococci and Y. pestis in vitro. The Y. pestis nghA ortholog is a pseudogene, and overexpression of functional nghA reduced ECM surface accumulation and inhibited the ability of Y. pestis to produce biofilm in the flea foregut. Mutational loss of this glycosidase activity in Y. pestis may have contributed to the recent evolution of flea-borne transmission.     

Modern concepts on the relationship between the agents causing plague and pseudotuberculosis

The authors present published data and their own findings on the relationship between Yersinia pestis and Y. pseudotuberculosis and on the origination of Y. pestis from Y. pseudotuberculosis. Study of microbiological and biochemical characteristics, external membrane protein spectra, and stability of chromosomal region of pigmentation brought the authors to a hypothesis that Y. pestis minor subspecies (ssp. caucasica, altaica, hissarica, ulegeica) which are characterized by selective virulence occupy an intermediate position between Y. pseudotuberculosis and basic species of Y. pestis.     

Temperature-dependent variations and intraspecies diversity of the structure of the lipopolysaccharide of Yersinia pestis

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 weak interaction of LcrV and TLR2 does not contribute to the virulence of Yersinia pestis

Yersinia pestis and the enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica share the virulence-antigen LcrV. Previously, using reverse genetics we have proven that LcrV contributes to the virulence of Y. enterocolitica serotype O:8 by inducing IL-10 via Toll-like receptor 2 (TLR2). However, both the ability of Y. pestis LcrV to activate TLR2 and a possible role of TLR2-dependent IL-10 induction by LcrV in Y. pestis are not yet known. To eliminate interference from additional protein sequences, we produced LcrVs without affinity tags from Y. pestis and from Y. enterocolitica O:8 (LcrVO:8). LcrVO:8 was much more potent in TLR2-activity than Y. pestis LcrV. To analyse the role of TLR2 in plague, we infected both wild-type and TLR2-/- mice subcutaneously with Y. pestis GB. While TLR2-/- mice exhibited lower blood levels of IL-10 (day 2 post-infection) and of the pro-inflammatory cytokines TNF-alpha, IFN-gamma and MCP-1 (day 4) than wild-type mice, there was no significant difference in survival. The low TLR2-activity of Y. pestis LcrV and associated cytokine expression might explain why - in contrast to Y. enterocolitica O:8 infection - TLR2-deficient mice are not more resistant than wild-type mice in a bubonic plague model.     

Early interaction of Yersinia pestis with APCs in the lung

Despite the importance of pneumonic plague, little is known of the early pulmonary immune responses that occur following inhalation of Yersinia pestis. Therefore, we conducted studies to identify the early target cells for uptake of Y. pestis in the lungs following intratracheal or i.v. inoculation. Following intratracheal inoculation, Y. pestis was rapidly internalized primarily by a distinctive population of CD11c+DEC-205+CD11b- cells in the airways, whereas i.v. inoculation resulted in uptake primarily by CD11b+CD11c- macrophages and granulocytes in lung tissues. The airway cells internalized and were infected by Y. pestis, but did not support active replication of the organism. Intratracheal inoculation of Y. pestis resulted in rapid activation of airway CD11c+ cells, followed within 24 h by the selective disappearance of these cells from the airways and lungs and the accumulation of apoptotic CD11c+ cells in draining lymph nodes. When CD11c+ cells in the airways were depleted using liposomal clodronate before infection, this resulted in a significantly increased replication of Y. pestis in the lungs and dissemination to the spleen and draining lymph nodes. These findings suggest that CD11c+ cells in the airways play an important role in suppressing the initial replication and dissemination of inhaled Y. pestis, although these results will also require confirmation using fully virulent strains of Y. pestis. Depletion of these airway cells by Y. pestis may therefore be one strategy the organism uses to overcome pulmonary defenses following inhalation of the organism.     

Technical note: a rapid diagnostic test detects plague in ancient human remains: an example of the interaction between archeological and biological approaches (southeastern France, 16th-18th centuries)

A rapid diagnostic test (RDT) that detects Yersinia pestis F1 antigen was applied to 28 putative plague victims exhumed from seven burial sites in southeastern France dating to the 16th-18th centuries. Yersinia pestis F1 antigen was detected in 19 of the 28 (67.9%) samples. The 27 samples used as negative controls yielded negative results. Soil samples taken from archeological sites related to both positive and negative samples tested negative for F1 antigen. The detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a preliminary retrospective diagnosis of Y. pestis infection in human remains. The high specificity and sensitivity of the assay were confirmed. For two sites positive to F1 antigen (Lambesc and Marseille), Y. pestis-specific DNA (pla gene) had been identified previously by PCR-sequence based analyses. Specifically, the positive results for two samples, from the Lambesc cemetery and the Marseille pit burial, matched those previously reported using PCR. Independent analyses in Italy and France of different samples taken from the same burial sites (Draguignan and Martigues) led to the identification of both Y. pestis F1 antigen and Y. pestis pla and gplD genes. These data are clear evidence of the presence of Y. pestis in the ancient human remains examined in this study.     

Analysis of the pesticin receptor from Yersinia pestis: role in iron-deficient growth and possible regulation by its siderophore.

We have sequenced a region from the pgm locus of Yersinia pestis KIM6+ that confers sensitivity to the bacteriocin pesticin to certain strains of Escherichia coli and Y. pestis. The Y. pestis sequence is 98% identical to the pesticin receptor from Yersinia enterocolitica and is homologous to other TonB-dependent outer membrane proteins. Y. pestis strains with an in-frame deletion in the pesticin receptor gene (psn) were pesticin resistant and no longer expressed a group of iron-regulated outer membrane proteins, IrpB to IrpD. In addition, this strain as well as a Y. pestis strain with a mutation constructed in the gene (irp2) encoding the 190-kDa iron-regulated protein HMWP2 could not grow at 37 degrees C in a defined, iron-deficient medium. However, the irp2 mutant but not the psn mutant could be cross-fed by supernatants from various Yersinia cultures grown under iron-deficient conditions. An analysis of the proteins synthesized by the irp2 mutant suggests that HMWP2 may be indirectly required for maximal expression of the pesticin receptor. HMWP2 likely participates in synthesis of a siderophore which may induce expression of the receptor for pesticin and the siderophore.     

The population variability of Yersinia pestis in soil samples from the natural focus of plague

Three Y. pestis strains were found to exist in the experimental soil ecosystem at a temperature of 4 degrees - 8 degrees C for a longer period (10 months, the term of observation) than at room temperature (3.5 months). Y. pestis population structure was characterized by relative stability in strains of the subspecies altaica and heterogeneity in the strain of the main subspecies, manifested by the loss of the pgm locus by vegetative cells and the preservation of pgm+ variants in the latent (uncultivable) form (LF). In the populations of all strains uniformity in calcium dependence, the tendency towards a decrease in the synthesis of factor 1, nutritional requirements in amino acids was observed. An important factor of the preservation of Y. pestis in the soil was LF formation. At room temperature this process quickly resulted in the death of the population. At 4 degrees - 8 degrees C A. pestis altaica avirulent strain could be inoculated onto solid nutrient media for a two-fold longer period (for 4 month) than the strain with selective virulence and for 5.5 months longer than Y. pestis pestis highly virulent strain.     

Interleukin-1-inducing activity of the polysaccharide-containing antigens of the cell wall in Yersinia pestis

The induction of the synthesis of interleukin-1 (IL-1) in human monocytes under the influence of the endotoxic preparations (LPS) and Y. pestis basis somatic antigen has been experimentally studied. The results obtained in this study make it possible to come to the conclusion that the capacity of the endotoxin of Y. pestis cell wall, consisting of LPS of type R, for inducing the synthesis of IL-1 in human monocytes is not different from the corresponding capacity of Salmonella and Shigella LPS, type S. Y. pestis O-specific polysaccharide in a discrete state has considerably greater IL-1-inducing activity in comparison with other preparations used in this experimental study. Such typical changes, characteristic of the initial stage of Y. pestis infection, as a sharp rise in temperature, transitory neutropenia, significant primary suppression of the oxygen-dependent metabolism of polymorphonuclear neutrophils are probably due to the induction of the synthesis of IL-1 by the polysaccharide-containing antigen of Y. pestis cell wall (LPS, basic somatic antigen) in cells of the mononuclear phagocytizing system.     

Development of a diagnostic test for Yersinia pestis by the polymerase chain reaction

A 501 bp caf1 gene fragment and a 443 bp of pla gene fragment carried by 100 kb (pFra) and 10 kb (pPst) species-specific extrachromosomal replicons, respectively, were used as targets to study the conditions under which DNA amplification by polymerase chain reaction (PCR) may be applied to detect and identify Yersinia pestis DNA in cell lysates of pure cultures and biological samples. The sensitivity limit of PCR with the crude cell lysates of Y. pestis EV was estimated as 10–50 cfu in reaction mixture. When target Y. pestis EV cells were mixed with fresh blood of white mice, which contained 0.4% potassium citrate, the PCR detection level varied from 400 to 100 cfu ml^-1 of blood depending on the method used for preparing the sample. In our tests PCR was effective for the detection of yersinia in the blood of white laboratory mice experimentally infected with virulent Y. pestis KM638 strain. This method can be considered convenient for routine detection and identification of Y. pestis.     

A rapid diagnostic test for plague detects Yersinia pestis F1 antigen in ancient human remains

A rapid diagnostic dipstick test (RDT) that detects Yersinia pestis F1 antigen has been recently applied on 18 putative plague victims exhumed from four archaeological burial sites in southeastern France dating back to the 16(th), 17(th) and 18(th) centuries. The Y. pestis antigen F1 was detected in 12 ancient samples out of 18 (67%). Negative controls confirmed their negativity (100%). Our results emphasize that the detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a first retrospective diagnosis of Y. pestis infection in ancient remains, and confirm the high specificity and sensitivity of the assay. Double-blind analyses performed by using two different techniques (RDT and 'suicide PCR') led us to the identification of the Y. pestis F1 antigen and the Y. pestis pla and gplD genes. These data provide clear evidence of the presence of Y. pestis in the examined specimens.     

Consequences of aspartase deficiency in Yersinia pestis.

Growing cells of Yersinia pseudotuberculosis, but not those of closely related Yersinia pestis, rapidly destroyed exogenous L-aspartic and L-glutamic acids, thus prompting a comparative study of dicarboxylic amino acid catabolism. Rates of amino acid metabolism by resting cells of both species were determined at pH 5.5, 7.0, and 8.5. Regardless of pH, Y. pseudotuberculosis destroyed L-glutamic acid, L-glutamine, L-aspartic acid, and L-asparagine at rates greater than those observed for Y. pestis. Although rates of proline degardation were similar, its metabolism by Y. pestis at pH 8.5 resulted in excretion of glutamic and aspartic acids. Similarly, Y. pestis excreted aspartic acid when incubated with L-glutamic acid (pH 8.5) or L-asparagine (pH 5.5, 7.0, and 8.5). Aspartase activity was not detected in extracts of 10 strains of Y. pestis but was present in all 11 isolates of Y. pseudotuberculosis. The latter contained significantly more glutaminase, asparaginase, and L-glutamate-oxalacetate transminase activity than did extracts of Y. pestis; specific activities of L-glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase were similar. The observed differences in dicarboxylic amino acid metabolism are traceable to asparatase deficiency in Y. pestis and may account for the slow doubling time of this organism relative to Y. pseudotuberculosis.     

No evidence of persistent Yersina pestis infection at prairie dog colonies in north-central Montana

Sylvatic plague is a flea-borne zoonotic disease caused by the bacterium Yersinia pestis, which can cause extensive mortality among prairie dogs (Cynomys) in western North America. It is unclear whether the plague organism persists locally among resistant host species or elsewhere following epizootics. From June to August 2002 and 2003 we collected blood and flea samples from small mammals at prairie dog colonies with a history of plague, at prairie dog colonies with no history of plague, and from off-colony sites where plague history was unknown. Blood was screened for antibody to Y. pestis by means of enzyme-linked immunosorbent assay or passive hemagglutination assay and fleas were screened for Y. pestis DNA by polymerase chain reaction. All material was negative for Y. pestis including 156 blood samples and 553 fleas from colonies with a known history of plague. This and other studies provide evidence that Y. pestis may not persist at prairie dog colonies following an epizootic.