Analysis of factors which determine the existence of Yersinia pseudotuberculosis in a saprophytic phase

Data are presented on the effects of a variety of abiotic and biotic environmental factors on the existence and changes in the numbers of Y. pseudotuberculosis. Experiments with sterile soil showed that Y. pseudotuberculosis populations were resistant over a wide range of major abiotic factors: temperature (0-30 degrees C), humidity (15-50%), pH (5.9-9.0). Although exerting some effect on the duration of different growth phases, the above abiotic factors did not influence, within the tested range, the general nature of populational dynamics of the microbe. Comparative experiments carried out in sterile and natural soil specimens using an RNA-polymerase mutant warranted the conclusion that the numbers of Y. pseudotuberculosis in soil (water) are largely controlled by the biotic components of ecosystems, including microflora and microfauna. Y. pseudotuberculosis was shown to exist in the environment (vegetable storehouses and substrate of rodent nests) in association with bacteria belonging to the family Enterobacteriaceae as well as the genera Acinetobacter and Pseudomonas. Endosymbiotic relationships are described between Y. pseudotuberculosis and the free-living infusorian Tetrahymena pyriformis which sustains microbial populations in the soil (water).     

Variability of the clonal structure of Yersinia pseudotuberculosis population under different conditions

In a series of experiments the dynamics of the clonal structure of Y. pseudotuberculosis population was evaluated by cytopathogenicity in soil extract, as well as in associations with blue-green algae (cyanobacteria) and infusoria, under different temperature conditions. In all variants of experiments made at low environmental temperature (10 degrees C) a considerable part of Y. pseudotuberculosis clones (25-40%) was found to be cytopathogenic, while at 22 degrees C such clones were absent or had low cytopathogenicity. At the same time experiments made under the same temperature conditions (10 degrees C) showed the variability of the clonal structure of the bacterial population in different associations and sterile soil extract, as well as at different periods of the experiments. At low temperatures Y. pseudotuberculosis virulent (cytopathogenic) clones, in contrast to avirulent ones, were characterized by the presence of virulence plasmid p45, as well as high urease and catalase activity. The results of the experiments are discussed from the viewpoint of the clonal concept of bacterial populations and their pathogenicity.     

Reduced secretion of YopJ by Yersinia limits in vivo cell death but enhances bacterial virulence

Numerous microbial pathogens modulate or interfere with cell death pathways in cultured cells. However, the precise role of host cell death during in vivo infection remains poorly understood. Macrophages infected by pathogenic species of Yersinia typically undergo an apoptotic cell death. This is due to the activity of a Type III secreted effector protein, designated YopJ in Y. pseudotuberculosis and Y. pestis, and YopP in the closely related Y. enterocolitica. It has recently been reported that Y. enterocolitica YopP shows intrinsically greater capacity for being secreted than Y. pestis YopJ, and that this correlates with enhanced cytotoxicity observed for high virulence serotypes of Y. enterocolitica. The enzymatic activity and secretory capacity of YopP from different Y. enterocolitica serotypes have been shown to be variable. However, the underlying basis for differential secretion of YopJ/YopP, and whether reduced secretion of YopJ by Y. pestis plays a role in pathogenesis during in vivo infection, is not currently known. It has also been reported that similar to macrophages, Y. enterocolitica infection of dendritic cells leads to YopP-dependent cell death. We demonstrate here that in contrast to Y. enterocolitica, Y. pseudotuberculosis infection of bone marrow-derived dendritic cells does not lead to increased cell death. However, death of Y. pseudotuberculosis-infected dendritic cells is enhanced by ectopic expression of YopP in place of YopJ. We further show that polymorphisms at the N-terminus of the YopP/YopJ proteins are responsible for their differential secretion, translocation, and consequent cytotoxicity. Mutation of two amino acids in YopJ markedly enhanced both translocation and cytotoxicity. Surprisingly, expression of YopP or a hypersecreted mutant of YopJ in Y. pseudotuberculosis resulted in its attenuation in oral mouse infection. Complete absence of YopJ also resulted in attenuation of virulence, in accordance with previous observations. These findings suggest that control of cytotoxicity is an important virulence property for Y. pseudotuberculosis, and that intermediate levels of YopJ-mediated cytotoxicity are necessary for maximal systemic virulence of this bacterial pathogen.     

Genes encoding specific nickel transport systems flank the chromosomal urease locus of pathogenic yersiniae

The transition metal nickel is an essential cofactor for a number of bacterial enzymes, one of which is urease. Prior to its incorporation into metalloenzyme active sites, nickel must be imported into the cell. Here, we report identification of two loci corresponding to nickel-specific transport systems in the gram-negative, ureolytic bacterium Yersinia pseudotuberculosis. The loci are located on each side of the chromosomal urease gene cluster ureABCEFGD and have the same orientation as the latter. The yntABCDE locus upstream of the ure genes encodes five predicted products with sequence homology to ATP-binding cassette nickel permeases present in several gram-negative bacteria. The ureH gene, located downstream of ure, encodes a single-component carrier which displays homology to polypeptides of the nickel-cobalt transporter family. Transporters with homology to these two classes are also present (again in proximity to the urease locus) in the other two pathogenic yersiniae, Y. pestis and Y. enterocolitica. An Escherichia coli nikA insertion mutant recovered nickel uptake ability following heterologous complementation with either the ynt or the ureH plasmid-borne gene of Y. pseudotuberculosis, demonstrating that each carrier is necessary and sufficient for nickel transport. Deletion of ynt in Y. pseudotuberculosis almost completely abolished bacterial urease activity, whereas deletion of ureH had no effect. Nevertheless, rates of nickel transport were significantly altered in both ynt and ureH mutants. Furthermore, the ynt ureH double mutant was totally devoid of nickel uptake ability, thus indicating that Ynt and UreH constitute the only routes for nickel entry. Both Ynt and UreH show selectivity for Ni(2+) ions. This is the first reported identification of genes coding for both kinds of nickel-specific permeases situated adjacent to the urease gene cluster in the genome of a microorganism.     

Action of sodium chloride on Yersinia pseudotuberculosis and Yersinia enterocolitica

The bacteriostatic and bactericidal action of sodium chloride on 60 Y. pseudotuberculosis strains, 75 Y. enterocolitica strains and 158 urine-fermenting strains has been studied. A new specific feature of Y. pseudotuberculosis has been revealed: high sensitivity to sodium chloride. The suitability of the sodium chloride test has been shown for the identification of Yersinia and the differentiation of Y. pseudotuberculosis and Y. enterocolitica.     

Analysis of insect toxin complex gene variability of Yersinia pestis and Yersinia pseudotuberculosis strains

The nucleotide sequences of the Tc's insect toxin complex genes have been analyzed in 18 natural strains of the main and non-main subspecies of Yersinia pestis isolated in different natural foci in the Russian Federation, as well as neighboring and more remote countries, as compared to the data on Y. pestis and Y. pseudotuberculosis strains stored in the NCBI GenBank database. The nucleotide sequences of these genes in plague agent strains have been found to be highly conserved, in contrast to those of the pseudotuberculosis agent. The sequences of two genes, tcaC and tccC2, have been found to be almost identical in Y. pestis strains, whereas other three genes (tcaA, tcaB, and tccC1) contain a few mutations, which, however, are not common for all strains of the plague agent. Exceptions are only strains of the Y. pestis biovar orientalis, whose tcaB gene is in a nonfunctional state due to a nucleotide deletion. The results suggest that the formation of the species Y. pestis as an agent of a natural focal infection with a transmissive mechanism has not resulted in degradation of the Tc's complex genes. Instead, these genes are likely to have been altered as the plague agent have been adapting to the new environment.     

Population structure of the Yersinia pseudotuberculosis complex according to multilocus sequence typing

Multilocus sequence analysis of 417 strains of Yersinia pseudotuberculosis revealed that it is a complex of four populations, three of which have been previously assigned species status [Y.?pseudotuberculosis sensu stricto (s.s.), Yersinia pestis and Yersinia similis] and a fourth population, which we refer to as the Korean group, which may be in the process of speciation. We detected clear signs of recombination within Y.?pseudotuberculosis s.s. as well as imports from Y.?similis and the Korean group. The sources of genetic diversification within Y.?pseudotuberculosis s.s. were approximately equally divided between recombination and mutation, whereas recombination has not yet been demonstrated in Y.?pestis, which is also much more genetically monomorphic than is Y.?pseudotuberculosis s.s. Most Y.?pseudotuberculosis s.s. belong to a diffuse group of sequence types lacking clear population structure, although this species contains a melibiose-negative clade that is present globally in domesticated animals. Yersinia similis corresponds to the previously identified Y.?pseudotuberculosis genetic type G4, which is probably not pathogenic because it lacks the virulence factors that are typical for Y.?pseudotuberculosis s.s. In contrast, Y.?pseudotuberculosis s.s., the Korean group and Y.?pestis can all cause disease in humans.     

Fermentative mechanisms of the psychrophilicity of Yersinia tuberculosis

The specific activity of urease, nitrogenase, hialuronidase and neuraminidase in Y. pseudotuberculosis grown in different culture media and at different temperature has been studied. These enzymes have been found capable of functioning at both relatively low (2-8 degrees C) and high (37 degrees C) temperatures. The thermoadaptive properties of Y. pseudotuberculosis within a wide range of temperatures are ensured by the constant presence of isoenzymes, functioning only at low temperatures or only at high temperatures, in the microbial cells. Low temperature in combination with a definite culture medium triggers the activity of certain enzymatic systems, which explains, to some extent, the biochemical mechanisms of the psychrophilic properties of Y. pseudotuberculosis.     

The lytic activity of Yersinia pestis phage P 3d serovar

The lytic activity of plague phage II, serovar 3, with respect to 1,800 bacterial strains has been studied: 760 Yersinia pestis strains, 262 Y. pseudotuberculosis strains, 252 Y. enterocolitica strains, 166 Escherichia coli strains, 90 Shigella strains and 270 strains of other species. The phage has been found to lyse 81.8% of Y. pestis strains, 1 Y. pseudotuberculosis strain and 1 Y. enterocolitica strain. The representatives of other 19 bacterial species have proved to be resistant to the phage. Though having a wide range of action within Y. pestis, the phage does not lyse most of the strains of the causative agent of plague, isolated in certain natural foci. This fact offers promise for using the phage for the differentiation of Y. pestis.     

Enterotoxic activity of the pseudotuberculosis microbe

For the first time the activity of Y. pseudotuberculosis, its extracellular, surface antigen and Westphal's lipolysaccharide has been studied with the use of biological models (the ligated loop of the small intestine of a rabbit, the pulmonary model, the intradermal tests). This microorganism has been found capable of inducing the accumulation of exudate in the ligated loop of the small intestine of a rabbit and interfering with the permeability of the vascular walls. The toxic action of Y. pseudotuberculosis is neutralized by immunization.     

Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells

Bacterial strains harboring the Yersinia pseudotuberculosis inv locus were analyzed in order to investigate the mechanism of host cell penetration by an invasive pathogen. The inv locus was found to be necessary for Y. pseudotuberculosis to enter HEp-2 cells and sufficient to convert E. coli into a microorganism able to penetrate cultured cells. Both E. coli and Y. pseudotuberculosis strains harboring inv mutations were defective for entry into HEp-2 cells. Furthermore, molecular clones containing inv, and little additional DNA, converted E. coli into a microorganism that was indistinguishable from the parental Yersinia strain with regard to the entry of cultured cells. Data from in vitro protein synthesis indicated that a 103 kd protein was synthesized from inv, saturating the coding capacity of the locus. The nucleotide sequence shows an open reading frame corresponding to a protein of similar size. This protein, called invasin, is necessary for the microorganisms to penetrate HEp-2 cells, and is compartmentalized on the outer surface of the bacterium.     

Biofilm development on Caenorhabditis elegans by Yersinia is facilitated by quorum sensing-dependent repression of type III secretion

Yersinia pseudotuberculosis forms biofilms on Caenorhabditis elegans which block nematode feeding. This genetically amenable host-pathogen model has important implications for biofilm development on living, motile surfaces. Here we show that Y. pseudotuberculosis biofilm development on C. elegans is governed by N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) since (i) AHLs are produced in nematode associated biofilms and (ii) Y. pseudotuberculosis strains expressing an AHL-degrading enzyme or in which the AHL synthase (ypsI and ytbI) or response regulator (ypsR and ytbR) genes have been mutated, are attenuated. Although biofilm formation is also attenuated in Y. pseudotuberculosis strains carrying mutations in the QS-controlled motility regulator genes, flhDC and fliA, and the flagellin export gene, flhA, flagella are not required since fliC mutants form normal biofilms. However, in contrast to the parent and fliC mutant, Yop virulon proteins are up-regulated in flhDC, fliA and flhA mutants in a temperature and calcium independent manner. Similar observations were found for the Y. pseudotuberculosis QS mutants, indicating that the Yop virulon is repressed by QS via the master motility regulator, flhDC. By curing the pYV virulence plasmid from the ypsI/ytbI mutant, by growing YpIII under conditions permissive for type III needle formation but not Yop secretion and by mutating the type III secretion apparatus gene, yscJ, we show that biofilm formation can be restored in flhDC and ypsI/ytbI mutants. These data demonstrate that type III secretion blocks biofilm formation and is reciprocally regulated with motility via QS.     

Pathogenic properties of Yersinia pseudotuberculosis in different biological models

The study of the action of Y. pseudotuberculosis introduced into the cavity of the ligated intestinal loop of a rabbit, into the stomach of 2- to 4-day-old suckling mice or injected intradermally has made it possible to establish the importance of the invasive and toxic activity of this microbe in the pathogenesis of pseudotuberculosis infection. The lesion of the small intestine develops due to the penetration of the bacteria into the mucous membrane and the combined action of the microbial cells inhabiting the stroma, and secreting into the environment of cytotoxins, toxic substances with an enterotropic action or enterotoxins and factors increasing the permeability of the vessels. Y. pseudotuberculosis strains with high and low invasiveness have been isolated.     

Chemotaxis of Yersinia pseudotuberculosis as a mechanism in its search for target tissues of the host organism

Y. pseudotuberculosis cells grown at biologically low temperature have been shown capable of chemotaxis with respect to carbohydrates and amino acids. During cultivation at 36-37 degrees C Y. pseudotuberculosis cells retain this property for 10-15 hours and then lose it. The mechanism of chemotaxis makes it possible for Y. pseudotuberculosis to "find" human and animal tissues and can facilitate the realization of the pathogenicity potential of these bacteria. When administered orally to mice motile bacteria, i. e. those grown at 6-8 degrees C, have been more virulent for the animals than nonmotile ones cultivated at 36-37 degrees C.     

Adaptive ultrastructural changes in soil-resident Yersinia pseudotuberculosis bacteria

After the prolonged residence (from 1 month to 2 years) in flow soil columns at 6 - 8 degrees C and 18 - 20 degrees C a complex of ultrastructural changes was detected in Y. pseudotuberculosis bacteria, depending on temperature, the duration of residence in the soil and, to a definite extent, on the strain. They were manifested in the form of cell-wall changes, the formation of the capsule and intercellular slime, changes in the ribosomal saturation of cytoplasm and the conformation state of DNA in the nucleoid zone. As the result of adaptation to nutritional deficit storage substances were accumulated in the form of electron-dense inclusions (polyphosphates) and pseudovacuoles (poly-beta-oxibutyric acid). Temperature influenced the process of mitotic division and the state of chromatin in bacteria. The described ultrastructural changes Y. pseudotuberculosis may be regarded as natural adaptive reaction to the altered conditions of their residence.     

The response regulator PhoP negatively regulates Yersinia pseudotuberculosis and Yersinia pestis biofilms

A few Yersinia pseudotuberculosis strains form biofilms on the head of the nematode Caenorhabditis elegans , but numerous others do not. We show that a widely used Y. pseudotuberculosis strain, YPIII, is biofilm positive because of a mutation in phoP , which encodes the response regulator of a two-component system. For two wild-type Y. pseudotuberculosis that do not make biofilms on C. elegans , deletion of phoP was sufficient to produce robust biofilms. In Yersinia pestis , a phoP mutant made more extensive biofilms in vitro than did the wild type. Expression of HmsT, a diguanylate cyclase that positively regulates biofilms, is diminished in Y. pseudotuberculosis strains with functional PhoP.     

Associations of the soil amoeba Hartmannella rhysodes with the bacterial causative agents of plague and pseudotuberculosis in an experiment]

Some aspects of relationships between soil ameba and the causative agents of plague and pseudotuberculosis, capable of forming natural associations, are considered. Ameba can phagocytose bacteria causing plague and pseudotuberculosis. Cases of the preservation of individual bacterial cells at the stationary phase and in precysts of amebae have been registered.     

Molecular analysis of the 3,6-dideoxyhexose pathway genes of Yersinia pseudotuberculosis serogroup IIA.

Salmonella enterica and Yersinia pseudotuberculosis are the only examples in nature known to use a variety of 3,6-dideoxyhexose derivatives as O antigen constituents. To allow a comparison of the responsible biosynthetic genes of the two organisms, we have sequenced a section of the Y. pseudotuberculosis serogroup IIA rfb region that contained the genes for the abequose biosynthetic pathway. Comparison of the identified genes with the rfb region of S. enterica LT2 showed that the two dideoxyhexose pathway gene clusters are related. The arrangement of the genes was largely conserved, and the G + C compositions of the two DNA regions were strikingly similar; however, the degree of conservation of nucleotide and protein sequences suggested that the two gene clusters have been evolving independently for considerable time. Hybridization experiments showed that the dideoxyhexose pathway genes are widespread throughout the various serogroups of Y. pseudotuberculosis.     

An experimental study of Yersinia in plants]

Y. enterocolitica and Y. pseudotuberculosis have been experimentally found to penetrate into plants (cabbage, salad, pea, oat) from infected soil and water and to remain in different plant organs (roots, seeds, leaves) up to 30 days (the term of observation). The processes of S-R dissociation in Yersinia cultures isolated from plants, as well as changes in some enzymatic properties, have been established. In the test on Infusoria the cytotoxic action of strains isolated from plants has been noted. The possible role of plants in carrying pathogenic bacteria from the soil to the surface ecological systems and the subsequent infection of animals and humans is discussed.     

Population dynamics of Yersinia pseudotuberculosis in association with the infusorian Tetrahymena pyriformis

The decisive role of biotic factors in the preservation of the viability of Y. pseudotuberculosis is shown. The results of experiments suggest that protozoa may serve as reservoir in the spread of Y. pseudotuberculosis infection.