Autoaggregating Yersinia enterocolitica express surface fimbriae with high surface hydrophobicity

For 13 strains of Yersinia enterocolitica , there was a good correlation between the production of the broad-spectrum, mannose-resistant Yersinia haemagglutinin (MR/Y HA), the presence of fimbriae and high surface hydrophobicity. Each of these characters was expressed in cultures grown at low (< 32°C) but not at high (> 35°C) temperatures.     

YscP, a Yersinia protein required for Yop secretion that is surface exposed, and released in low Ca2+

The Yersinia Ysc apparatus is made of more than 20 proteins, 11 of which have homologues in many type III systems. Here, we characterize YscP from Yersinia enterocolitica. This 515-residue protein has a high proline content, a large tandem repetition and a slow migration in SDS-PAGE. Unlike the products of neighbouring genes, it has a counterpart only in Pseudomonas aeruginosa and it varies even between Yersinia Ysc machineries. An yscPDelta97-465 mutant was unable to secrete any Yop, even under conditions overcoming feedback inhibition of Yop synthesis. Interestingly, a cloned yscPDelta57-324 from Yersinia pestis introduced in the yscPDelta97-465 mutant can sustain a significant Yop secretion and thus partially complemented the mutation. This explains the leaky phenotype observed with the yscP mutant of Y. pestis. In accordance with this secretion deficiency, YscP is required for the delivery of Yop effectors into macrophages. Mechanical shearing, immunolabelling and electron microscopy experiments showed that YscP is exposed at the bacterial surface when bacteria are incubated at 37 degrees C in the presence of Ca2+ and thus do not secrete Yops. At 37 degrees C, when Ca2+ ions are chelated, YscP is released like a Yop protein. We conclude that YscP is a part of the Ysc injectisome which is localized at the bacterial surface and is destabilized by Ca2+ chelation.     

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.     

A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25 degrees C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22 degrees C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22 degrees C and plating on CIN agar (48 h at 25 degrees C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

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.     

Production of bacteriocin-like substances byYersinia frederiksenii,Y. kristensenii,andY. intermedia strains

The production of bacteriocin-like substances by strains of Yersinia frederiksenii, Y. kristensenii and Y. intermedia in broth culture was established. These substances showed a selective activity against Y. enterocolitica, Y. frederiksenii, Y. kristensenii and Y. intermedia strains. Electron micrographs revealed the presence of phage tails in culture media. The production of these substances was detected in cultures grown at 25 degrees C but not in those grown at 37 degrees C, while these bacteriocin-like substances were active at 25 and 37 degrees C. Y. enterocolitica serogroups 0:3 and 0:9 were more susceptible to these bacterin-like substances than strains of Yersinia isolated from environmental sources.     

The cultural properties and virulence of Yersinia]

Study of the cultivation properties of 82 enterobacterial strains has revealed that the colonies of virulent Y. enterocolitica (serovars O3, O9) and Y. pseudotuberculosis (serovar I) are temperature-sensitive. This sign, closely connected with the presence and expression of the virulence plasmid with a molecular weight of 44-48 MD, is not characteristic of other strains. Virulent Yersinia grown in nutrient agar for 48 hours at 37 degrees C form colonies which are smaller in diameter than those formed during cultivation at 26 degrees C (with the significance of differences t greater than or equal to 4), their diameter at 37 degrees C not exceeding 1.0 mm. The test for the determination of the temperature-sensitive morphology of Yersinia colonies, along with the tests for other virulence markers, is probably suitable for the detection of the causative agents of yersiniosis or pseudotuberculosis.     

Antigenic relationship and functional properties of Yersinia porins

We have studied the molecular structure and functional properties of major pore-forming proteins isolated as peptidoglycan (PG)-protein complexes from four Yersinia species (Y. intermedia, Y. enterocolitica, Y. kristensenii and Y. frederiksenii) cultured as various temperatures. Despite the close antigenic relationship, Yersinia porins revealed different functional properties. When reconstituted in model membranes, the PG-protein complexes induced conductance which was different for the "cold" (grown at 6-8 degrees C) and "warm" (grown at 37 degrees C) variants of microbial cultures. We conclude that the functional state of Yersinia porins in the outer membrane depends on the cultivation temperature.     

Influence of nutrient-limited growth on pathogenesis-associated outer membrane proteins of Yersinia enterocolitica

From studies based on batch culture, it has been postulated that the expression of the virulence-associated proteins of Yersinia spp. is controlled by temperature and Ca2+, such that these proteins are synthesized only at the higher temperature (37 degrees C) and calcium-scarce conditions of the intracellular environment. It was found, however, that in Yersinia enterocolitica one of these proteins (140 kDa) is not synthesized at submaximal growth rates under any of the relevant conditions, and that another of the implicated proteins (34 kDa), is synthesized even at 28 degrees C during nutrient-limited growth. Thus, temperature and Ca2+ influence the synthesis of these proteins differently under growth conditions that better approximate the natural environments than do batch cultures.     

Heat resistance of Campylobacter and Yersinia strains by three methods

Two methods of determining the heat resistance of bacteria, a glass cup and a test tube method, were compared with a method using capillary tubes. Three strains of Yersinia enterocolitica, one of Campylobacter jejuni and two of C. coli were tested in physiological saline. The differences between the results obtained by the glass cup method and the reference method were not statistically significant for five strains and were small also for the other, a Yersinia strain. D values obtained by the glass cup method at 58, 60 and 62 degrees C were 1.4-1.8, 0.40-0.51 and 0.15-0.19 min (zeta values 4.00-4.52 degrees C) for the Yersinia strains, and 0.42, 0.13 and 0.07 min (zeta value 5.07 degrees C) for one C. coli strain. For the other Campylobacter strains, D values of 0.71-0.78, 0.24-0.28 and 0.12-0.14 min (zeta values 4.94 and 5.60 degrees C) were recorded at 56, 58 and 60 degrees C. D values obtained at 60 degrees C by the test tube method were 2.7-5.0 min and were considered to be unrealistic.     

Partial purification and characterization of phospholipase C from Yersinia enterocolitica

About 34% of the strains of Yersinia enterocolitica isolated from raw milk were found to produce lecithinase. A selected strain produced phospholipase C at 22 degrees C and 37 degrees C; production was optimum at 37 degrees C in the stationary phase (14-16 h). A decrease in phospholipase C activity at various storage temperatures (-5 degrees C, 4 degrees C, 37 degrees C) was also observed, although the enzyme was active over a wide range of temperature (5-65 degrees C) and pH (3.5-7.5). The phospholipase C was partially purified by ammonium sulphate precipitation and Sephadex column chromatography, and characterized.     

Study of the dependence of gene expression of plague pathogen plasmid 65MD on the host bacterium genome

The conjugative cointegrate containing Yersinia pestis 65 Md plasmid coding for the production of traction I antigen and mouse toxin has been transferred into Yersinia pseudotuberculosis, Yersinia enterocolitica and Escherichia coli cells. Analysis of the transconjugants obtained has confirmed the connection of the genetical determinants for the mentioned bacterial products with the 65 Md plasmid. The similar level of fra and tox-genes expression has been found in Yersinia cells while fraction I was not produced in Escherichia coli CA cells. The data on the increased synthesis of fraction I at 40 degrees C as compared with the one at 37 degrees C has been obtained. In some cases the production remained at the same level characteristic of the 37 degrees C. The possibility of formation of different Yersinia Fra+ recombinants in the mixed natural epizootic foci and their role in epizootic infections are discussed.     

A Yersinia enterocolitica serotype 0:3 lipopolysaccharide-specific monoclonal antibody reacts more strongly with bacteria cultured at room temperature than those cultured at 37 degrees C

It has been suggested that the O-side chains of the lipopolysaccharide (LPS) of serotype 0:3 strains of Yersinia enterocolitica vary quantitatively and qualitatively depending on whether they are cultured at 37 degrees C or 25 degrees C. It is uncertain whether this affects the expression of the serotype-specific antigens that are probably carried on the LPS. We studied this question with a serotype 0:3-specific monoclonal antibody, 2C1. This monoclonal antibody immunoprecipitated a 39K major protein from detergent-solubilized 125I-labeled Yersinia preparation. However, results of Western blot experiments demonstrated that the antigens reactive with 2C1 were not actually the 39K protein but the O-side chains of the LPS. Significantly, reactivity could be detected whether the bacteria were cultured at room temperature or at 37 degrees C. However, absorption experiments confirmed that there were less accessible antigenic determinants on the 37 degrees C-LPS. The LPS preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. These SDS-PAGE profiles showed that less O-side chains were present in the 37 degrees C-LPS. Hence, the most likely explanation for our observation is that the 37 degrees C incubation condition induced a partial smooth to rough transition of the Yersinia LPS with a decrease in the amount of 2C1-reactive antigen.     

Growth temperature-dependent variation in the bacteriophage-inactivating capacity and antigenicity of Yersinia enterocolitica lipopolysaccharide

Growth temperature affected the structure of Yersinia enterocolitica Ye 3827 lipopolysaccharide (LPS). Although Y. enterocolitica Ye 3827 synthesized smooth LPS when grown at a low temperature (25 degrees C), partial smooth-rough transition occurred when the bacteria were grown at the physiological temperature (37 degrees C). The structural alteration was detected by bacteriophage-inactivation assay and chemical and immunological analyses. LPS prepared from bacteria grown at 25 degrees C inactivated a number of bacteriophages that recognize the O-antigenic polysaccharide portion of LPS, whereas more than 3000 times the amount of LPS from bacteria grown at 37 degrees C was required for the same degree of inactivation. The antigenic determinant(s) responsible for the major reaction between 25 degrees C-LPS and anti-25 degree C-bacteria was located on the O-antigenic polysaccharide portion of LPS, but those responsible for the major reaction between 37 degrees C-LPS and anti-37 degrees C-bacteria were located on the R-core or inner portion of LPS.     

Role of RpoS in survival of Yersinia enterocolitica to a variety of environmental stresses.

rpoS, a gene that encodes an alternative sigma factor (also known as katF), is critical for the ability of Yersinia enterocolitica grown at 37 degrees C, but not at 26 degrees C, to survive diverse environmental insults such as high temperature, hydrogen peroxide, osmolarity, and low pH. However, a Y. enterocolitica rpoS mutant was not affected in expression of inv or ail, invasion of tissue culture cells, or virulence in mice.     

The effect of the bacterial genome on the expression and behavior of the plasmid determining the Ca2+-dependence of the plague pathogen]

The conjugative cointegrate containing the 47 Md plasmid of Yersinia pestis has been transferred into the strains of the different Yersinia (Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica) and Escherichia coli CA. There appeared in the populations of recombinant Yersinia under the conditions of Ca2+ deficit at 37 degrees C the cells coming into the stasis stage or dying. It was shown on the model of Yersinia enterocolitica that bacterial lethality might be prevented by exclusion of the sheep blood from Ca2+ deficient medium. Ca(2+)-dependence was not expressed in Escherichia coli cells in which the cointegrates were prone to deletions although the cad-genes were preserved intact. The latter conclusion is based on the positive reciprocal transfer of the Cad(+)-marker into Yersinia pestis cells.     

Inhibition of Yersinia enterocolitica serotype O3 by natural microflora of pork

Yersinia enterocolitica serotype O3 did not grow but did survive in inoculated raw ground pork kept at 6 and 25 degrees C. The antagonistic effect of microbial flora, especially Hafnia alvei and environmental Yersinia organisms, on the growth of Y. enterocolitica serotype O3 in raw ground pork was evident. These results were supported by evidence of the inhibition of growth of Y. enterocolitica serotype O3 by Enterobacteriaceae, especially H. alvei and environmental Yersinia organisms, in mixed cultures at 6 and 25 degrees C. We suggest that naturally contaminated pork is a source of human infection, since Y. enterocolitica serotype O3 was capable of surviving in the raw pork for a long time.     

Use of poly-beta-oxybutyric acid by Yersinia pseudotuberculosis and Listeria monocytegenes bacteria at various temperatures

A comparative investigation of the intracellular content of poly-beta-hydroxybutyric acid showed that Yersinia pseudotuberculosis strains accumulated, on the average, lower amounts of this reserve substance than Listeria monocytogenes strains. The intracellular pool of poly-beta-hydroxybutyric acid was responsible for the growth of the bacteria at low temperatures (4-6 degrees C) in the absence of any exogenous carbon and energy source.