Interaction of Yersinia pestis bacteria with bacteriophage mu

Yersinia pestis cells are shown to be sensitive to bacteriophage Mu cts62 infection. Lysis of bacteria has been shown to be more efficient on solid nutrient medium than in LB broth. 10(-5) pfu per ml is the maximal concentration of bacteriophage particles yielded from the broth cultures of bacteria. Moi 0.1 has been used to obtain such yields of bacteriophage. Lysogenization of Yersinia pestis cells has not been achieved when the standard methods of bacteriophage infection have been used. It was accomplished by the conjugal transfer of plasmid RP4::MU cts62 to Yersinia pestis from Escherichia coli. The deficiency of Yersinia pestis in producing bacteriophage Mu cts62 mature particles during the lytic cycle of bacteriophage is discussed.     

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.     

Numerical analysis of the phenotypic properties and total genomic characteristics of strains of Yersinia pestis related to different subspecies

The numerical analysis of the phenotypical properties of Y. pestis strains, classified with 5 subspecies by 60 signs, was carried out. In comparing the properties of strains belonging to the main (nomenclature) subspecies with strains of other subspecies, the similarity index varied within the range 82-95%. A high degree of genetic affinity between 21 Y. pestis strains of five subspecies was demonstrated by the method of molecular DNA-DNA hybridization. The level of DNA homology with respect to the alpha-CTP.[3H] reference mark of Y. pestis P-1300 in strains belonging to different subspecies was found to be 84-97%. The plasmid spectrum of 25 examined strains of these three subspecies proved to be identical and consisted of plasmids similar in their electrophoretic motility to marker plasmids from Y. pestis strains EV from the Research Institute of Epidemiology and Hygiene and Otten.     

The sensitivity of Yersinia to bacteriophage Mu

Various representatives of the genus Yersinia were found to differ in their sensitivity to the lytic action of bacteriophage Mu cts62, which could serve as an auxiliary test for the differentiation of Y. pestis and Y. pseudotuberculosis. Among the strains under study, the causative agents of plague (34 strains) were sensitive to phage Mu cts62, while the causative agents of enteric yersiniosis (42 strains) and pseudotuberculosis (73 strains), except 3 strains with the properties of Y. pestis, were resistant to this phage.     

Comparative and evolutionary genomics of Yersinia pestis

Yersinia pestis is the causative agent of plague, causing three human plague pandemics in history. Comparative and evolutionary genomics of Y. pestis are extensively discussed in this review. Understanding the genomic variability and the adaptive evolution of Y. pestis from the genomic point of view will contribute greatly to plague detection, identification, control and prevention.     

Concepts of macro- and microevolution as related to the problem of origin and global expansion of the plague pathogen Yersinia pestis

The relationship between macro- and microevolutionary processes is considered with reference to the ecological scenario of the origin of the plague pathogen and its subsequent natural and anthropogenic global expansion. The macroevolutionary transformation of the ancestral pseudotuberculosis microbe clone into the initial plague microbe Yersinia pestis tarbagani occurred in Central Asia at the end of the Late Pleistocene by a “vertical” Darwinian way in an inadaptive heterothermal continual intermediate environment—the Mongolian marmot Marmota sibirica—flea Oropsylla silantiewi system—via a sequence of unstable and currently extinct intermediate forms. Its natural geographic expansion on the “oil spot” principle in the postglacial time led to the microevolutionary formation of 20–30 hostal subspecies circulating in populations of the background species of burrowing rodents and pikas in arid areas of Eurasia. The intercontinental spread of the “marmot” and “rat” pathogen subspecies in the past few centuries has been exclusively anthropogenic, with the involvement of synanthropic (ship) rats.     

鼠疫耶尔森氏菌的进化研究:从系统发育学到系统发育基因组学

20世纪90年代末起,基因组学在细菌研究中应用越来越广泛,尤其在进化领域,取得了一系列革命性的发现.本文以鼠疫耶尔森氏菌进化研究为例,介绍了从利用基因组中少数特定片段(等位基因)多态性进行分析的传统系统发育学,到基于大量菌株全基因组序列进行系统发育基因组学的研究发展历程,回顾讨论了基因组学技术的进步为鼠疫菌进化研究领域带来的成果.     

Identification of a thermo-regulated glutamine-binding protein from Yersinia pestis

Here we present modeling and NMR spectroscopic evidence that the function of a Yersinia pestis pMT1 plasmid protein, designated as orf38, is most likely a glutamine binding protein. The modeling was homology-based at a very low level of sequence identity ( approximately 16%) and involved structural comparison of multiple templates, as well as template-substrate interaction analyses. Transferred nuclear Overhauser and saturation transfer difference experiments were used to characterize the binding of sugars and amino acids to orf38. The identification and characterization of an unknown protein function using the strategy presented here has applicability to a variety of research areas, including functional genomics and proteomics efforts.     

Pleiotropic effects of a Yersinia pestis fur mutation.

A Yersinia pestis fur mutation was constructed by insertionally disrupting the fur open reading frame. Analysis of a Fur-regulated beta-galactosidase reporter gene revealed a loss of iron regulation as a result of the fur mutation. trans complementation with the cloned Y. pestis fur gene restored iron regulation. The expression of most iron-regulated proteins was also deregulated by this mutation; however, a number of iron-repressible and two iron-inducible polypeptides retained normal regulation. Mutations in fur or hmsH, a gene encoding an 86-kDa surface protein required for hemin storage, increased the sensitivity of Y. pestis cells to the bacteriocin pesticin. Interestingly, the Y. pestis fur mutant lost temperature control of hemin storage; however, expression of the HmsH polypeptide was not deregulated. When grown with excess iron, a Y. pestis fur mutant possessing the 102-kb pigmentation locus exhibited severe growth inhibition and a dramatic increase in the number of spontaneous nonpigmented chromosomal deletion mutants present at late log phase. These results suggest that the Fur protein of Y. pestis is an important global regulator and that a separate Fur-independent iron regulatory system may exist.     

Factors providing the blocking activity of Yersinia pestis

Discusses published data on the specific mechanism of Y. pestis transfer by "blocked" fleas. Special attention is paid to individual phenotypical signs and genetic determinants of Y. pestis whose expression correlates with the blocking activity of bacteria. Prospects for further research are outlined.     

Magnetic biosensor for the detection of Yersinia pestis

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.     

Proteomic characterization of host response to Yersinia pestis and near neighbors

Host-pathogen interactions result in protein expression changes within both the host and the pathogen. Here, results from proteomic characterization of host response following exposure to Yersinia pestis, the causative agent of plague, and to two near neighbors, Yersinia pseudotuberculosis and Yersinia enterocolitica, are reported. Human monocyte-like cells were chosen as a model for macrophage immune response to pathogen exposure. Two-dimensional electrophoresis followed by mass spectrometry was used to identify host proteins with differential expression following exposure to these three closely related Yersinia species. This comparative proteomic characterization of host response clearly shows that host protein expression patterns are distinct for the different pathogen exposures, and contributes to further understanding of Y. pestis virulence and host defense mechanisms. This work also lays the foundation for future studies aimed at defining biomarkers for presymptomatic detection of plague.     

Biological and physico-chemical properties of Yersinia pseudotuberculosis bacterial culture having the fra-operon Yersinia pestis

The biological and physico-chemical properties of cultures of two isogenous recombinant variants of Yersinia pseudotuberculosis were studied. The cell genomes of the cultures are distinguished from one another only by the presence or by the absence of the fra-operon, which is a determined attribute of the plague microbe capsule-forming process. The expression of the attribute is amplified by rising the microbial biomass cultivation temperature and stimulates the decrease in the viability of the bacteria and adaptation potential in vitro. In the warm-blooded owner organism the microbes of the capsule-forming recombinant variant are characterized by the greater residual pathogenicity and immunogenic ability to the experimental plague of the laboratory animals as compared to the reference-variant cells. These specific features could be explained by more expressed colonizing ability of the capsule-forming microbes provided by owner cells' stability to the phagocyte process.     

鼠疫耶尔森氏菌LcrV基因的克隆及序列分析

为了研究鼠疫耶尔森氏菌（Y.pestis）保护性抗原V蛋白,从基因库中查得Y.pestis LcrV基因DNA序列,针对序列设计合成了一对PCR扩增引物,以本所保存的Y.pestis菌种为模板进行基因扩增,结果获得长约980bp的DNA片段。将扩增产物回收纯化,克隆至pGEM-T载体,构建重组载体pGEN-T/ypV,经过PCR,酶切鉴定,并对pGEM-T/ypV中的V基因片段进行测序,分析测序结果与己知序列相同,表明获得了LcrV基因。     

Susceptibility of the Siberian polecat to subcutaneous and oral Yersinia pestis exposure.

To determine if the Siberian polecat (Mustela eversmannii) represents a suitable model for the study of plague pathogenesis and prevention in the black-footed ferret (Mustela nigripes), polecats were exposed to 10(3), 10(7), or 10(10) Yersinia pestis organisms by subcutaneous injection; an additional group was exposed to Y. pestis via ingestion of a plague-killed mouse. Plague killed 88% of polecats exposed to Y. pestis (71% mortality in the 10(3) group, 100% mortality in the 10(7) and 10(10) groups, and 83% mortality in the mouse-fed group). Within the challenged group, mean day of death post-challenge ranged from 3.6 to 7.6 days; all polecats died on or before day 12 post-challenge. Animals receiving the lowest parenteral dose survived significantly longer than those receiving higher parenteral doses. Within challenged animals, mean survival time was lower in those presenting with significant weight loss by day 3, lethargy, and low fecal output; time to onset of lethargy and other signs was also related to risk of dying and/or plague dose. Six polecats developed serum antibody titers to the Y. pestis F1 protein. Three seropositive polecats survived the initial challenge and a subsequent exposure to a plague-killed mouse, while two seropositive animals later died. This study confirms that the Siberian polecat is susceptible to plague and suggests that this species will offer an appropriate surrogate for black-footed ferrets in future plague studies and related vaccine trials.     

Ail provides multiple mechanisms of serum resistance to Yersinia pestis

Ail, a multifunctional outer membrane protein of Yersinia pestis, confers cell binding, Yop delivery and serum resistance activities. Resistance to complement proteins in serum is critical for the survival of Y. pestis during the septicemic stage of plague infections. Bacteria employ a variety of tactics to evade the complement system, including recruitment of complement regulatory factors, such as factor H, C4b‐binding protein (C4BP) and vitronectin (Vn). Y. pestis Ail interacts with the regulatory factors Vn and C4BP, and Ail homologs from Y. enterocolitica and Y. pseudotuberculosis recruit factor H. Using co‐sedimentation assays, we demonstrate that two surface‐exposed amino acids, F80 and F130, are required for the interaction of Y. pestis Ail with Vn, factor H and C4BP. However, although Ail‐F80A/F130A fails to interact with these complement regulatory proteins, it still confers 10,000‐fold more serum resistance than a Δail strain and prevents C9 polymerization, potentially by directly interfering with MAC assembly. Using site‐directed mutagenesis, we further defined this additional mechanism of complement evasion conferred by Ail. Finally, we find that at Y. pestis concentrations reflective of early‐stage septicemic plague, Ail weakly recruits Vn and fails to recruit factor H, suggesting that this alternative mechanism of serum resistance may be essential during plague infection.     

Cold temperature-induced modifications to the composition and structure of the lipopolysaccharide of Yersinia pestis

Following a report of variations in the lipopolysaccharide (LPS) structure of Yersinia pestis at mammalian (37 degrees C) and flea (25 degrees C) temperatures, a number of changes to the LPS structure were observed when the bacterium was cultivated at a temperature of winter-hibernating rodents (6 degrees C). In addition to one of the known Y. pestis LPS types, LPS of a new type was isolated from Y. pestis KM218 grown at 6 degrees C. The core of the latter differs in: (i) replacement of terminal galactose with terminal d-glycero-d-manno-heptose; (ii) phosphorylation of terminal oct-2-ulosonic acid with phosphoethanolamine; (iii) a lower content of GlcNAc, and; (iv) the absence of glycine; lipid A differs in the lack of any 4-amino-4-deoxyarabinose and presumably partial (di)oxygenation of a fatty acid(s). The data obtained suggest that cold temperature switches on an alternative mechanism of control of the synthesis of Y. pestis LPS.     

Retracing the Evolutionary Path that Led to Flea-Borne Transmission of Yersinia pestis

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