Pathogenetic significance of the psychrophilia of Yersinia pseudotuberculosis

The work presents the data indicating that the temperature of Y. pseudotuberculosis cultivation is very important in regulating the activity of pathogenicity factors, necessary for the initiation of the pathogenic process in the cells of the macroorganism. Low temperature (8-10 degrees C), necessary for the growth of Y. pseudotuberculosis, facilitates the activation of invasive and toxic pathogenicity factors. At a growth temperature of 37 degrees C the inhibition of such necessary attributes of virulence as adhesion and invasion into epithelial cells occurs in Y. pseudotuberculosis, which decreases the capacity of these bacteria for inducing the infectious process. The virulence of Y. pseudotuberculosis population, lost as the result of its cultivation in synthetic culture media at a temperature of 37 degrees C, has been found to be restored at low temperature.     

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).     

Effect of blue-green alga (Cyanobacteria) and their exometabolites on formation of resting forms and variability of Yersinia pseudotuberculosis

Research, carried out with the use of bacteriological methods and polymerase chain reaction, revealed that the transformation of Y. pseudotuberculosis, associated with blue-green algae Anabaena variabilis, into resting (noncultivable) forms took shorter time than in soil extract containing no algae. The exometabolites of "old" cultures of these algae sharply accelerated the formation of resting Y. pseudotuberculosis forms. The influence of the algae and the products of their metabolism was manifested far more intensively at 22 degrees C than at 4 degrees C. After passage through infusoria resting Y. pseudotuberculosis forms, preserved in the mucous covering of cyanobacteria, partially reverted into vegetative forms, capable of growing on solid culture media. The revertants essentially differed from the initial vegetative forms by having lower enzymatic activity, agglutinability and cytopathogenicity, as well as by the loss of plasmid p45. The probable role of blue-green algae, widely spread in soils and water reservoirs, in the processes of reversible transformation of Y. pseudotuberculosis vegetative and resting forms, closely connected with seasonal changes of temperature conditions.     

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.     

DNA, RNA, and protein biosynthesis in cells of Yersinia pseudotuberculosis at various cultivation temperatures

Y. pseudotuberculosis cells cultivated at temperatures of 37 degrees C and 8 degrees C were found to be capable of incorporating exogenic precursors into DNA, RNA and protein. The linear growth of thymidine incorporation occurred during 8 hours of cultivation at 37 degrees C, then the amount of the incorporated label decreased. At 8 degrees C the level of thymidine incorporation into DNA gradually increased for 80 hours and longer, but not reaching the level of incorporation observed at 37 degrees C. The incorporation of uridine into RNA of Y. pseudotuberculosis cells reached its maximum after 4 hours of cultivation at 37 degrees C, at a lower temperature of cultivation the incorporation of uridine into bacterial cells was almost linear, though slower, and lasted for 20 hours. The content of radioactive alanine in Y. pseudotuberculosis protein increased during 16 hours of cultivation at a high temperature, while at 8 degrees C the growth of the incorporation level lasted for at least 40 hours. For all precursors under study the incorporation rate into the cell biopolymers at the initial stages of cultivation was higher at 37 degrees C, than at a lower temperature.     

The biochemical mechanisms of energy support in Yersinia pseudotuberculosis cells at a low cultivation temperature

The object of the study was the first stage of biological oxidation: the transfer of hydrogen electrons to the components of the respiratory chain of Y. pseudotuberculosis cells by NAD and NADF, coenzymes of pyridine-dependent dehydrogenases, having labile redox properties. The study revealed that in the low-temperature cultivation of Y. pseudotuberculosis an increase in the content of NAD and NADF was 1.5- to 2.0-fold greater than that observed at 37 degrees C, which was indicative of the fact that at low environmental temperature pyridine-dependent dehydrogenases played a more important role than at high temperature (37 degrees C). This, in combination with other mechanisms, made it possible for bacterial cells to maintain the level of energy metabolism, necessary for their survival, in the environment with low and constantly changing temperature.     

Burkholderia cepacia under different ecological conditions: the amount and variability of the bacterial population

In a series of prolonged experiments with the use of the bacteriological method and PCR analysis the amount and state of B. cepacia population, associated and not associated with infusoria Tetrahymena pyriformis, were dynamically evaluated under different conditions: in water, brain heart broth, soil extract and at different temperature (4 degrees C and 25 degrees C). In soil extract at 25 degrees C B. cepacia existed in the vegetative state for the period of up to 3 months, while at 4 degrees C, in the absence of protozoa, the transition of these microorganisms into the uncultivable forms occurred in 9 days, and they could be detected only with the use of PCR. Protozoa maintained the existence of the vegetative bacteria for as long as 2 months, and in 3-4 months uncultivable forms of B. cepacia cells were registered. In water at low temperature B. cepacia disappeared in 2 months, evidently, eaten up by infusoria. The population variability of B. cepacia under different conditions of their existence was established: S-R dissociation, a decrease in biochemical activity, growth deceleration. A high level of cytopathogenicity in B. cepacia pigment-forming clones was noted. In the process of transition into the uncultivable state pigment formation in B. cepacia population decreased up. The ecological plasticity and multi-pathogenicity of B. cepacia as phytopathogens and the causative agents of human diseases are discussed.     

Effect of temperature on synthesis of polyphosphates in Yersinia pseudotuberculosis and Listeria monocytogenes under starvation conditions

It was found that at low temperature (6-8 degrees C) in the absence of nitrogen supply and at the presence of phosphate ions in the medium, Yersinia pseudotuberculosis and Listeria monocytogenes are able to actively synthesize reserve substances as polyphosphates. Most of the bacterial polyphosphates are alkali-soluble, especially at the preliminary stage of cell growth (lag-phase). This is proved by electron microscopic studies of ultrastructure of model microorganisms. During a long starvation period under conditions of carbon and energy source deficit, L. monocytogenes and Y. pseudotuberculosis consume this biopolymer for biosynthetic and bioenergetic processes.     

Thermotropic behavior of lipids and the morphology of Yersinia pseudotuberculosis cells with a high content of lysophosphatidylethanolamine

The content of lysophosphatidylethanolamine (LPE) in Y. pseudotuberculosis cells was found to increase during their growth at 8 degrees C under stationary conditions (without stirring the medium) and at 37 degrees C when the medium contained glucose. The maximum level of LPE (up to 45% of the total phospholipids) was observed in cells grown at 8 degrees C under stationary conditions. Such cells showed an enhanced growth rate, a reduced yield of biomass, an altered cell morphology, and an increased cell area. The cells contained unsaturated fatty acids, phosphatidylethanolamine (PE), and total phospholipids in small amounts, whereas neutral lipids and diphosphatidylglycerol were abundant. In addition, the cells contained an amount of methylated PE and phospholipids of unknown structure. Irrespective of whether the temperature for growth was low or high, the LPE-rich cells showed a high value (32-36 degrees C) of the maximum temperature of thermal transition of lipids (Tmax). This finding is indicative of a densification of the membrane lipid matrix of the LPE-rich cells. The suggestion is made that LPE is accumulated in glucose-fermenting bacterial cells in response to stress caused by oxygen deficiency and low pH values of the growth medium. The possible relationship between LPE accumulation and the virulence of Y. pseudotuberculosis cells grown at low temperatures is discussed.     

Population structure and evolution of pathogenicity of Yersinia pseudotuberculosis

Yersinia pseudotuberculosis is an enteric human pathogen but is widespread in the environment. Pathogenicity is determined by a number of virulence factors, including the virulence plasmid pYV, the high-pathogenicity island (HPI), and the Y. pseudotuberculosis-derived mitogen (YPM), a superantigen. The presence of the 3 virulence factors varies among Y. pseudotuberculosis isolates. We developed a multilocus sequence typing (MLST) scheme to address the population structure of Y. pseudotuberculosis and the evolution of its pathogenicity. The seven housekeeping genes selected for MLST were mdh, recA, sucA, fumC, aroC, pgi, and gyrB. An MLST analysis of 83 isolates of Y. pseudotuberculosis, representing 19 different serotypes and six different genetic groups, identified 61 sequence types (STs) and 12 clonal complexes. Out of 26 allelic changes that occurred in the 12 clonal complexes, 13 were mutational events while 13 were recombinational events, indicating that recombination and mutation contributed equally to the diversification of the clonal complexes. The isolates were separated into 2 distinctive clusters, A and B. Cluster A is the major cluster, with 53 STs (including Y. pestis strains), and is distributed worldwide, while cluster B is restricted to the Far East. The YPM gene is widely distributed on the phylogenetic tree, with ypmA in cluster A and ypmB in cluster B. pYV is present in cluster A only but is sporadically absent in some cluster A isolates. In contrast, an HPI is present only in a limited number of lineages and must be gained by lateral transfer. Three STs carry all 3 virulence factors and can be regarded as high-pathogenicity clones. Isolates from the same ST may not carry all 3 virulence factors, indicating frequent gain or loss of these factors. The differences in pathogenicity among Y. pseudotuberculosis strains are likely due to the variable presence and instability of the virulence factors.     

Enhancing effect of low culture temperature on specific antibody productivity of recombinant Chinese hamster ovary cells: clonal variation

To understand the different responses of recombinant Chinese hamster ovary (rCHO) cells to low culture temperature regarding specific productivity (q), 12 parental clones and their corresponding amplified clones producing a humanized antibody were cultivated at 32 and 37 degrees C. The specific growth rate of all clones, including both parental and amplified clones, decreased by 30-63% at 32 degrees C, compared to rates at 37 degrees C. In contrast, their specific antibody productivity (qAb) was significantly enhanced at 32 degrees C. Furthermore, the degree of qAb enhancement at 32 degrees C varied a lot from 4- to 25-fold among the parental clones. At 32 degrees C, most of the amplified clones, regardless of methotrexate (MTX) levels, also showed enhanced qAb but to a lesser extent than their parental clones. However, clone 14 amplified at 0.32 microM MTX (clone 14-0.32) and clone 20 amplified at 1 microM MTX (clone 20-1.00), unlike their parental clones, did not show enhanced qAb at 32 degrees C. Thus, it was found that the enhancing effect of low culture temperature on q of rCHO cells depends on clones. Taken together, the results obtained here emphasize the importance of clonal selection for the successful application of low culture temperature to the enhanced foreign protein production in rCHO cells.     

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 effect of the trophic and temperature conditions of cultivation on lipid synthesis by Yersinia pseudotuberculosis

The comparative study of the synthesis lipids in Y. pseudotuberculosis, depending on the conditions of their cultivation (at different temperatures in mineral media and in media, containing organic compounds), has been carried out. As demonstrated in this study, temperature in the main inducing factor, affecting the synthesis of lipids of definite classes and fatty acids, incorporated into these lipids. During the cultivation of Y. pseudotuberculosis in mineral and organic media under the conditions of low temperature their lipid composition remains unchanged, but at 6 degrees C the synthesis of unsaturated fatty acids prevails, while at 37 degrees C saturated fatty acids are mainly synthesized. On mineral media at 37 degrees C bacteria synthesize mostly nonpolar lipids in the form of reserve substances, represented by triglycerides and free fatty acids.     

Comparison of temperature effects on soil respiration and bacterial and fungal growth rates

Temperature is an important factor regulating microbial activity and shaping the soil microbial community. Little is known, however, on how temperature affects the most important groups of the soil microorganisms, the bacteria and the fungi, in situ. We have therefore measured the instantaneous total activity (respiration rate), bacterial activity (growth rate as thymidine incorporation rate) and fungal activity (growth rate as acetate-in-ergosterol incorporation rate) in soil at different temperatures (0-45 degrees C). Two soils were compared: one was an agricultural soil low in organic matter and with high pH, and the other was a forest humus soil with high organic matter content and low pH. Fungal and bacterial growth rates had optimum temperatures around 25-30 degrees C, while at higher temperatures lower values were found. This decrease was more drastic for fungi than for bacteria, resulting in an increase in the ratio of bacterial to fungal growth rate at higher temperatures. A tendency towards the opposite effect was observed at low temperatures, indicating that fungi were more adapted to low-temperature conditions than bacteria. The temperature dependence of all three activities was well modelled by the square root (Ratkowsky) model below the optimum temperature for fungal and bacterial growth. The respiration rate increased over almost the whole temperature range, showing the highest value at around 45 degrees C. Thus, at temperatures above 30 degrees C there was an uncoupling between the instantaneous respiration rate and bacterial and fungal activity. At these high temperatures, the respiration rate closely followed the Arrhenius temperature relationship.     

The impact of abiotic factors (temperature and glucose) on physicochemical properties of lipids from Yersinia pseudotuberculosis

The impact of the availability of glucose in nutrition medium and growth temperature on the composition and thermotropic behavior of lipids from Yersinia pseudotuberculosis (Enterobacteriaceae) was studied. Y. pseudotuberculosis was grown in nutrition broth (NB) with/without glucose at 8 and 37 degrees C, corresponding to the temperatures of saprophytic and parasitic phases of this bacterium life. The decrease of phosphatidylethanolamine, phosphatidylglycerol and unsaturated fatty acids and the parallel increase of lysophosphatidylethanolamine and diphosphatidylglycerol and saturated and cyclopropane acids were the most significant changes with temperature in bacterial phospholipid (PL) classes and fatty acids, respectively. Glucose did not effect the direction of temperature-induced changes in the contents of PLs, fatty acids, however it enhanced (for PLs) or diminished (for fatty acids) intensity of these changes. The thermally induced transitions of lipids were studied by differential scanning calorimetry (DSC). It was revealed that the addition of glucose to NB induced a sharp shift of DSC thermograms to lower temperatures in the "warm" variants of bacteria. The peak maximum temperature (Tmax) of thermal transitions dropped from 50 to 26 degrees C that is the optimal growth temperature of Y. pseudotuberculosis. Tmax of total lipids of the cells grown at 8 degrees C without glucose in NB was equal to growth temperature that corresponded to the classical mechanism of homeoviscous adaptation of bacteria. An addition of glucose to NB at this growth temperature caused the subsequent reduction of Tmax to -8 degrees C, while the temperature ranges of thermograms were not substantially changed. So, not only the temperature growth of bacteria, but also the presence of glucose in NB can modify the physical state of lipids from Y. pseudotuberculosis. In this case, both factors affect additively. It is suggested that glucose influences some membrane-associated proteins and then the fluidity of lipid matrix through temperature-inducible genes.     

The entry portals and routes of Yersinia pseudotuberculosis penetration into the warm-blooded organism

This work presents data indicating that Y. pseudotuberculosis population, cultivated at lower temperature (6-8 degrees C), has a high potential of cellular and tissue invasiveness. Pseudotuberculosis has been experimentally shown to start as generalized infection due to the rapid (during 10-15 minutes) penetration of the infective agent through the epithelium of the mouth cavity, the small intestine, the urinary bladder, conjunctiva, pulmonary alveoli and vascular walls into the blood and then into internal organs. The data, obtained in this study, on the penetration of Y. pseudotuberculosis through any mucous surfaces in different species of warm-blooded animals are indicative, to a considerable extent, of the fact that this process occurs due to the action of nonspecific mechanisms.     

Fatty-acid composition of cells and lipopolysaccharides in different Yersinia species under the conditions of growth at low temperature

Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii and Y. ruckeri grown at 4 degrees C were characterized by fatty acid composition with a high content of C16:1 and C18:1, as well as the proportion of saturated to nonsaturated fatty acids equal to, on the average, 2.0. In Yersinia lipopolysaccharides a relatively high level of C16:1 and C12:0 was observed with the prevalence of 3-OH-C14:0. In the fatty-acid spectra of both cells and lipopolysaccharides no essential difference was noted. Thus, during growth at low temperature differences, earlier detected in the studied Yersinia species grown at 37 degrees C and making it possible to divide 7 Yersinia species into 2 groupes, were completely leveled. These results confirmed the close phylogenetic relationship between the Yersinia species under study and were indicative of more pronounced biological community of Yersinia under the conditions of growth at low temperature.     

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.     

Variation in lipid A structure in the pathogenic yersiniae

Important pathogens in the genus Yersinia include the plague bacillus Yersinia pestis and two enteropathogenic species, Yersinia pseudotuberculosis and Yersinia enterocolitica. A shift in growth temperature induced changes in the number and type of acyl groups on the lipid A of all three species. After growth at 37 degrees C, Y. pestis lipopolysaccharide (LPS) contained the tetra-acylated lipid IV(A) and smaller amounts of lipid IV(A) modified with C10 or C12 acyl groups, Y. pseudotuberculosis contained the same forms as part of a more heterogeneous population in which lipid IV(A) modified with C16:0 predominated, and Y. enterocolitica produced a unique tetra-acylated lipid A. When grown at 21 degrees C, however, the three yersiniae synthesized LPS containing predominantly hexa-acylated lipid A. This more complex lipid A stimulated human monocytes to secrete tumour necrosis factor-alpha, whereas the lipid A synthesized by the three species at 37 degrees C did not. The Y. pestis phoP gene was required for aminoarabinose modification of lipid A, but not for the temperature-dependent acylation changes. The results suggest that the production of a less immunostimulatory form of LPS upon entry into the mammalian host is a conserved pathogenesis mechanism in the genus Yersinia, and that species-specific lipid A forms may be important for life cycle and pathogenicity differences.