Adhesines of the Plague Agent

Plague agent has a complex of adhesines providing for anchoring of the pathogen to target cells in a host organism and in many ways defining the onset, character, and development of the disease. The presence of adhesines ensures translocation of effector proteins into target cells of mammalians. The review covers the literature data, both on the most studied Yersinia pestis adhesines (Ail proteins and pH6 antigen), and on recently identified auto transporting proteins of various classes, involved in adhesion processes (YadBC, Yaps, IlpP). Their significance for plague pathogenesis, genetic determinacy, structure and localization in a cell are also described in the paper. It is noted that plague agent adhesines work at different phases of infection process, have multiple functions and take part not only in anchoring to host cells, but provide for resistance to influence of immune mechanisms of a host too

Plasminogen Activator – Multifunctional Protein of Plague Pathogen

Yersinia pestis plasminogen activator, a surface protease (Pla) is encoded by species-specific plasmid pPla. This enzyme belongs to outer membrane protein family and has a ß-barrel structure. Pla protease interacts complexly with homeostasis system of a hostorganism and is an important factor of plague agent virulence. Represented are the literature data on the part of plasminogen activator in the development of bubonic and pneumonic plague. Described is the role of R-form lipopolysaccharide in protease Pla activity manifestation. Plasminogen activator directly contributes to fibrinolysis by activating plasminogen and inactivating plasminogen-1 activator inhibitor (PAI-1) and ά2-anti-plasmin, as well as promoted by thrombin fibrinolysis inhibitor (TAFI). Pro-coagulant Pla protein activity is caused by the destruction of tissue factor pathway inhibitor (TFPI). In addition, given is the information on non-proteolytic functions of plasminogen activator

Historical and Modern Classifications of the Plague Agent

The review presents the data on domestic and foreign phenotypic classifications of Yersinia pestis strains developed in the XX century; genetic classifications of the XXI century; as well as on the genealogy of ancient strains of the plague microbe, reconstructed using paleogenomic technologies. Since the discovery of the plague agent in 1894, many classifications were created that corresponded to the level of development of microbiology at that time. The intraspecific classification schemes of the XX century were based on three principles: phenotypic differences between strains, features of the species composition of carriers, and geographical affiliation. With the development of molecular microbiology early on in the XXI century, a genetic nomenclature of the branches of the pathogen evolution was developed and a number of classifications based on the analysis of the population structure of Y. pestis were created. Through the prism of the genetic diversity of Y. pestis strains from natural plague foci in Russia, near and far abroad countries, an improved classification with a division into seven subspecies has been developed: pestis, tibetica, caucasica, qinghaica, angolica, central asiatica, ulegeica, which allocates the subspecies according to the phylogenetic principle and epidemic significance. With the advancements in paleomicrobiology, prehistoric lineages of evolution have been included in the genealogy of Y. pestis, which expand the data on the intraspecific diversity of the plague microbe

CONSTRUCTION OF A DNA-MICROARRAY FOR DIFFERENTIATION BETWEEN THE MAIN AND NON-MAIN SUBSPECIES AND BIOVARS OF THE MAIN SUBSPECIES OF YERSINIA PESTIS

Objective of the study is to design the DNA-microarray for differentiation of Y. pestis strains of the main and non-main subspecies and biovars of the main subspecies. Materials and methods. Efficiency analysis for the devised means was conducted using 62 Y. pestis strains of various subspecies and biovars, isolated in the natural foci of Russia and neighboring countries. Results and conclusions. Selected have been the DNA-targets, probes and primers – calculated. Enhanced is the method of sub-specific and biovar differentiation of Y. pestis strains by means of DNA-microarray. DNA-chip with “Med24”, “glpD(-93)”, and “45” targets allows for prompt differentiation of the strains of the main and non-main subspecies and biovars of the main subspecies based on the presence and absence of fluorescent signal by the specific for the main subspecies and its biovars DNA-targets

Bacterial Biofilm and Peculiarities of Its Formation in Plague Agent and in Other Pathogenic Yersinia

This paper represents a review of the current literature data concerning general principles of bacterial biofilm formation, stages of biofilm production and its structural and functional organization, as well as the data concerning involvement of different enzyme systems with the process of biofilm functioning. Carried out is the analysis of the data on the peculiarities of biofilm formation by pathogenic Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. Displayed are the data on the role of hmsHFRS-operon genes, located in pigmentation chromosomal region of Y. pestis, in the process of biofilm structure formation, and the data on its regulation by hmsP and hmsT genes. Summarized are the results of the recent years' investigations devoted to the genetic determinancy of the plague agent biofilm formation processes, and in particular to the involvement of genes encoding synthesis of lipopolysaccharide (waaA, yrbH and gmhA) and polyamines (speA and speC), as well as rcsA and phoP-phoQ regulatory genes with biofilm formation procedures. Represented are the results of our own investigations concerning the studies of peculiarities of biofilm formation by plague agent strains of different subspecies on the abiotic surfaces and on the nematode model - Caenorhabditis elegans. Discussed is the role of the biofilm in the complex life span of the plague agent. Noted is biofilm significance not only for facilitation in plague transmission by fleas, but also for Y. pestis preservation in the environment, out of the organism of a warm-blooded host

Identification of Genovariants of Yersinia pestis Strains Belonging to Main Subspecies Using PCR with Hybridization-Fluorescent Registration of Results

For the first time ever developed has been the method of identification of genovariants in Yersinia pestis strains belonging to the main subspecies using PCR with hybridization-fluorescent registration of results. A complex of DNA-targets, the most of which are newly detected ones, have been utilized to differentiate between genovariants of antique (0.ANT, 1.ANT, 2.ANT, 4.ANT), medievalis (2.MED, 2.MED0), and orientalis (1.ORI) biovars. Application of these targets in PCR with hybridization-fluorescent registration of results provides for a rapid, effective and reliable classifying of the strains of the genovariants, circulating in various geographical regions. Efficacy of the method has been validated via analysis of 110 Y. pestis strains, isolated in the territory of the Russian Federation, neighboring countries and beyond, including 37 strains of antique, 53 strains of medievalis, and 20 strains of orientalis biovars

Comparative Analysis of the Nutrient Requirements among <I>Yersinia pestis</I> Strains of the Main and Non-Main Subspecies as well as Genetic Causes of Their Auxotrophy

Studied is the nutrient demand for growth factors among 185 Yersinia pestis strains of the main and non-main subspecies ( altaica, caucasica, hissarica , and ulegeica ) isolated in 38 natural plague foci of Russia and neighboring states. Revealed is the fact that all the strains of Yersinia pestis main subspecies manifest equal dependence in growth on three amino-acids such as methionine, phenylalanine, and threonine, while strains isolated from certain natural foci have an additional demand for cysteine, leucine, and arginine. Strains of non-main subspecies differ in their nutrient requirements both from the strains of the main subspecies and among themselves. Strains of subspecies caucasica stand in need of thiamine and such amino-acids as phenylalanine, tyrosine and arginine; subspecies altaica – phenylalanine, arginine and leucine; subspecies hissarica – phenylalanine, leucine and methionine; ulegeica – phenylalanine. Detected is a number of mutations that lead to auxotrophy in Yersinia pestis strains of different subspecies

Phylogenetic Analysis of Yersinia pestis Strains of Medieval Biovar, Isolated in Precaspian North-Western Steppe Plague Focus in the XX Century

Objective of the study – comparative phylogenetic analysis of Yersinia pestis strains, isolated in Precaspian North-Western steppe focus in 1924–1926, 1972, and 1986–1990 to understand the causes of focal reactivation during different time periods of the XX century.Materials and methods. The work included 30 strains of Yersinia pestis from Precaspian North-Western steppe natural focus and adjacent plague foci. Whole genome sequencing of eight Y. pestis strains from the former was carried out. Also whole-genome sequences of 16 strains from neighboring natural foci were used. Whole-genome sequencing of Y. pestis strains was conducted in Ion PGM system (Life technologies). SNPs search across the core genome was performed using software package Wombac 2.0. Tree diagram Maximum Likelihood, HKU85 model, was constructed to analyze phylogenetic relations.Results and discussion. It is established that in early XX century (1924–1926), strains of phylogenetic branches 2.MED4 and 2.MED1, belonging to medieval biovar, main subspecies, circulated on Ergenin Upland in the Precaspian North-Western steppe natural focus. Later on they became extinct in the territory. It is shown that the strains, isolated on Ergenin Upland in 1972, constituted a common subcluster on the dendrogram with the strains from low-mountain and piedmont plague foci of Caucasus and Transcaucasia, dated the same time period. It was inferred that epizootic manifestations on Ergenin upland in 1972, after a long recess since 1938, were caused by importation of Y. pestis strains from low-mountain natural plague foci of Caucasus and Transcaucasia. It was noted that expansion of Caucasian strains was of short-term character, and plague infected animals have not been found on Ergenin Upland since 1974 (including modern period). It is established that Y. pestis strains isolated in the eastern part of Precaspian North-Western steppe focus between 1986 and 1990, do not have close genetic relation to the strains that circulated on Ergenin Upland in 1924–1926 and 1972. It is determined that each epizootic period (1913–1938 and 1972–1973) in Precaspian North-Western steppe natural focus culminated in the elimination of the circulating Y. pestis strains and rehabilitation of the focal territory

Construction of the Reagent Panel “GenPest-subspecies/Altai-RGF”

The aim of the study was to develop a test system that allows for detecting plague pathogen DNA in clinical and biological samples, environmental objects with the simultaneous determination of its appurtenance to the main and non-main subspecies, differentiation of the altai biovar central asiatica subspecies separately. Materials and methods. Primer sets for specific genetic markers have been selected using the VectorNTI 10 software, optimal conditions for PCR were determined for RotorGene devices. To assess the specificity and sensitivity of the developed set of reagents, 44 strains of microorganisms were used, of which 19 were Yersinia pestis strains and 25 strains of heterologous microorganisms. The diagnostic sensitivity of “GenPest-subspecies/Altai-RGF” is 98.6 % with a confidence level of probability of 91 %. The diagnostic specificity of “GenPest-subspecies/Altai-RGF” is ≥ 99 % with a confidence level of 91 %. Results and discussion. A medical product “A set of reagents for the detection and differentiation of plague pathogen strains of the main and non-main subspecies (altai biovar, subspecies central asiatica exclusively) by the polymerase chain reaction with hybridization-fluorescent registration of results in real-time mode (GenPest-subspecies/Altai-RGF)” has been developed. The set of reagents passed the state registration in accordance with the established procedure. The use of the developed set of reagents is relevant for the Gorno-Altai high-mountain plague focus of the Russian Federation and the adjacent part of Mongolia

Phylogenetic Analysis of <i>Yersinia pestis</i> Strains of Medieval Biovar from Natural Plague Foci of the Russian Federation and Bordering Countries

Objective of the study is to conduct phylogenetic investigation of Yersinia pestis strains (medieval biovar) from plague foci of Russia and bordering countries, using SNP-analysis of the genome-wide sequences of these strains. Materials and methods. Carried out has been sequencing of 14 Y. pestis strains, medieval biovar, from 13 natural plague foci of Russia and neighboring states, as well as their comparison to 9 strains of the same biovar, contained in the NCBI GenBank database. Using software products - Wombac 2.0 and Bionumerics 7.1, revealed is the presence of 1875 core SNPs, on the basis of which a dendrogram of phylogenetic relations between medieval strains is constructed. Results and conclusions. In consequence of genome-wide SNP-analysis, it is established that Y. pestis strains, medieval biovar, from plague foci of Russia and bordering states are assigned to 2.MED1 phylogenetic line and fall under two major evolutionary branches, the first one of which includes strains from the Caucasus and Caspian-Sea regions, and the second one - from Central Asia and China. The data obtained can be used for the development of molecular-genetic methods for differentiation of Y. pestis strains, medieval biovar