Antigen biosynthesis during the limitation of Yersinia pestis growth

The effect of the ingredients of a semisynthetic culture medium on the synthesis of Y. pestis antigens (F1, LPS, "mouse" toxin) under the conditions of batch cultivation at 28 degrees C was studied. The study revealed that the amount of antigens produced by bacterial cells depended on the character of the limitation of growth.     

The role of IS-elements of Yersinia pestis (Lehmann, Neumann) in the emergence of calcium-independent mutations

Calcium independent mutants of two Yersinia pestis strains were studied. Insertions of IS100 element at three different sites of plasmid pCad within calcium dependence region were detected in Y. pestis EV, as well as two extensive deletions covering the whole region. It was shown that IS100 carries no HindIII sites. Novel IS element of Y. pestis designated IS101 was discovered in strain 358, in addition to IS100. It is distinguished by a slightly smaller size, HindIII site presence and high specificity of integration.     

Early Host Cell Targets of Yersinia pestis during Primary Pneumonic Plague

Inhalation of Yersinia pestis causes primary pneumonic plague, a highly lethal syndrome with mortality rates approaching 100%. Pneumonic plague progression is biphasic, with an initial pre-inflammatory phase facilitating bacterial growth in the absence of host inflammation, followed by a pro-inflammatory phase marked by extensive neutrophil influx, an inflammatory cytokine storm, and severe tissue destruction. Using a FRET-based probe to quantitate injection of effector proteins by the Y. pestis type III secretion system, we show that these bacteria target alveolar macrophages early during infection of mice, followed by a switch in host cell preference to neutrophils. We also demonstrate that neutrophil influx is unable to limit bacterial growth in the lung and is ultimately responsible for the severe inflammation during the lethal pro-inflammatory phase.     

Isolation of pathogens other than Yersinia pestis during plague investigations.

From 1975 to 1978, 37 isolates of Pasteurella multocida, 1 of Salmonella enteriditis, and 5 of Francisella tularensis were recovered from 42 mammalian specimens and 1 flea pool submitted for examination for evidence of infection with Yersinia pestis. Most of the specimens were collected during investigations of either a human plague infection or a reported epizootic among rodent populations. All specimens were of species regularly or occasionally involved in plague or tularemia cycles in nature and most were collected in areas of known plague or tularemia activity.     

Genome sequence of Yersinia pestis KIM

We present the complete genome sequence of Yersinia pestis KIM, the etiologic agent of bubonic and pneumonic plague. The strain KIM, biovar Mediaevalis, is associated with the second pandemic, including the Black Death. The 4.6-Mb genome encodes 4,198 open reading frames (ORFs). The origin, terminus, and most genes encoding DNA replication proteins are similar to those of Escherichia coli K-12. The KIM genome sequence was compared with that of Y. pestis CO92, biovar Orientalis, revealing homologous sequences but a remarkable amount of genome rearrangement for strains so closely related. The differences appear to result from multiple inversions of genome segments at insertion sequences, in a manner consistent with present knowledge of replication and recombination. There are few differences attributable to horizontal transfer. The KIM and E. coli K-12 genome proteins were also compared, exposing surprising amounts of locally colinear "backbone," or synteny, that is not discernible at the nucleotide level. Nearly 54% of KIM ORFs are significantly similar to K-12 proteins, with conserved housekeeping functions. However, a number of E. coli pathways and transport systems and at least one global regulator were not found, reflecting differences in lifestyle between them. In KIM-specific islands, new genes encode candidate pathogenicity proteins, including iron transport systems, putative adhesins, toxins, and fimbriae.     

Immunobiological properties of Yersinia pestis antigens

The present review contains information concerning immunobiological properties of plague microbe antigens. All of the identified antigens are evaluated in relation to pathogenicity of Yersinia pestis namely a resistance to phagocytosis, toxicity, adhesiveness etc. as well as persistence ability and adaptation to variable environment. In addition, the role of antigens in immunogenicity of living plague microbe for experimental animals is considered. The data concerning mechanisms of antigenic contribution to the development of adaptive immunity are presented.     

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.     

Proteomic characterization of Yersinia pestis virulence

The Yersinia pestis proteome was studied as a function of temperature and calcium by two-dimensional differential gel electrophoresis. Over 4,100 individual protein spots were detected, of which hundreds were differentially expressed. A total of 43 differentially expressed protein spots, representing 24 unique proteins, were identified by mass spectrometry. Differences in expression were observed for several virulence-associated factors, including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as several putative virulence factors and membrane-bound and metabolic proteins. Differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants.     

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.     

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.     

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.     

Ligand bound structures of a glycosyl hydrolase family 30 glucuronoxylan xylanohydrolase

Xylanases of glycosyl hydrolase family 30 (GH30) have been shown to cleave β-1,4 linkages of 4-O-methylglucuronoxylan (MeGX_n) as directed by the position along the xylan chain of an α-1,2-linked 4-O-methylglucuronate (MeGA) moiety. Complete hydrolysis of MeGX_n by these enzymes results in singly substituted aldouronates having a 4-O-methylglucuronate moiety linked to a xylose penultimate from the reducing terminal xylose and some number of xylose residues toward the nonreducing terminus. This novel mode of action distinguishes GH30 xylanases from the more common xylanase families that cleave MeGX_n in accessible regions. To help understand this unique biochemical function, we have determined the structure of XynC in its native and ligand-bound forms. XynC structure models derived from diffraction data of XynC crystal soaks with the simple sugar glucuronate (GA) and the tetrameric sugar 4-O-methyl-aldotetrauronate resulted in models containing GA and 4-O-methyl-aldotriuronate, respectively. Each is observed in two locations within XynC surface openings. Ligand coordination occurs within the XynC catalytic substrate binding cleft and on the structurally fused side β-domain, demonstrating a substrate targeting role for this putative carbohydrate binding module. Structural data reveal that GA acts as a primary functional appendage for recognition and hydrolysis of the MeGX_n polymer by the protein. This work compares the structure of XynC with a previously reported homologous enzyme, XynA, from Erwinia chrysanthemi and analyzes the ligand binding sites. Our results identify the molecular interactions that define the unique function of XynC and homologous GH30 enzymes.     

New perspectives on the endo-beta-glucanases of glycosyl hydrolase Family 17

Isozymes of glycosyl hydrolase Family 17 hydrolyze 1,3-beta-glucan polysaccharides found in the cell wall matrix of plants and fungi, enabling these plant enzymes to serve diverse roles in plant defense and plant development. Fourteen genes from Family 17 have been characterized in the genome of rice. A sequence dendrogram analysis divided these genes into four subfamilies. The recombinant GNS1 enzyme from subfamily B had 1,3;1,4-beta-glucanase activity, suggesting a role for this isozyme in plant development.     

鼠疫菌毒力调控研究

鼠疫菌通过一系列转录调控子(如CRP、PhoP、RovA和Fur)控制着一些关键毒力因子(如Pla、强毒力岛、III型分泌系统等)的基因表达。鼠疫菌可感应宿主体内信号刺激,紧密调控毒力因子的表达。在这个紧密调控过程中,调控子、毒力相关基因构成了一个动态网络。鼠疫菌在从假结核菌祖先演化的进程中,基因表达调控网络的重塑在鼠疫菌毒力进化过程中发挥着不可取代的作用。     

Magnetic immunoenzyme analysis of Yersinia pestis antigens

The detection of Y. pestis cells in magnetic enzyme immunoassay is carried out with the use of magnetic polyacrylamide microgranules. In the assay system for the determination of the antigen commercial Y. pestis antigens, peroxidase-labeled antibodies, the substrate mixture consisting of sodium salt of 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid and H2O2 in citrate-phosphate buffer solution, pH 4.5, are used. The sensitivity of the method is 5 X 10(4) microbial bodies per ml.     

Survival of Yersinia pestis on environmental surfaces

The survival of two strains of Yersinia pestis (avirulent A1122 and virulent Harbin) on the surfaces of four materials was investigated. Viability was evaluated with epifluorescence microscopy by using the metabolic stain cyanoditolyl tetrazolium chloride and plate counts. Small numbers of cells suspended in phosphate buffer survived 2 to 4 h after visible drying on stainless steel, polyethylene, or glass and beyond 48 h on paper. Cells suspended in brain heart infusion broth (BHI) persisted more than 72 h on stainless steel, polyethylene, and glass. Small numbers of cells suspended in BHI were still viable at 120 h on paper. These data suggest that Y. pestis maintains viability for extended periods (last measured at 5 days) under controlled conditions.