The osmotic regulator OmpR is involved in the response of Yersinia enterocolitica O:9 to environmental stresses and survival within macrophages

Various environmental signals control the expression of the virulence factors in pathogenic Yersinia enterocolitica strains. The role of the osmotic regulator OmpR protein in controlling the production of Yop proteins, virulence determinants in Y. enterocolitica O:9 (European type) has been studied. An ompR deletion mutant was constructed via allelic exchange with an ompR gene of Y. enterocolitica mutagenized in vitro by a reverse genetic polymerase chain reaction (PCR)-based strategy. The ompR mutant showed a reduced ability to survive under conditions of various environmental stresses in vitro. In particular, low pH stress resulted in increased cell mortality levels. Under conditions of high osmolarity, the wild strain's Yop protein production was reduced, whereas protein levels from the mutant strain remained constant regardless of osmolarity variance. In J774A.1 macrophage cell culture survival of the ompR mutant was decidedly lower than that of the wild-type strain, suggesting that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis.     

Structure of the O-chain of the phenol-phase soluble cellular lipopolysaccharide of Yersinia enterocolitica serotype O:9

The phenol-phase soluble cellular lipopolysaccharide isolated by the phenol/water extraction method from Yersinia enterocolitica serotype O:9 cells was shown by hydrolytic, periodate oxidation, methylation and nuclear magnetic resonance studies to be an S-type lipopolysaccharide with a linear O-antigenic polysaccharide of 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. The serological cross-reactivity between Y. enterocolitica serotype O:9 and the lipopolysaccharides of Vibrio cholerae and Brucella species can now be related to the presence of N-acylated 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residues in their respective O-antigenic chains.     

Structure of the lipopolysaccharide O-chain of Yersinia enterocolitica serotype O:5,27

The cellular lipopolysaccharide produced by Yersinia enterocolitica serotype O:5,27 was of the S-type and composed of an antigenic O-chain polysaccharide linked through a core oligosaccharide region, which in turn was linked through 3-deoxy-D-manno-octulonosyl units to a lipid A moiety. The O-chain polysaccharide was composed of equal molar amounts of L-rhamnose and D-xylulose. By partial hydrolysis, periodate oxidation, methylation, specific optical rotation, and 13C and 1H nuclear magnetic resonance studies, the structure of the O-chain was established as being a linear backbone of alternating 1,3-linked alpha-L-rhamnopyranosyl and beta-L-rhamnopyranosyl units, to which 2,2-linked beta-D-threo-pent-2-ulofuranoside (D-xylulofuranoside) units were present on every L-rhamnopyranosyl residue, as shown below. (Formula: see text)     

Characterization of the Six Glycosyltransferases Involved in the Biosynthesis of Yersinia enterocolitica Serotype O:3 Lipopolysaccharide Outer Core

Yersinia enterocolitica (Ye) is a Gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage φR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage φR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.     

Characterization and biological role of the O-polysaccharide gene cluster of Yersinia enterocolitica serotype O:9

Yersinia enterocolitica serotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) in Y. enterocolitica O:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer of N-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of the gnd gene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster, galF and galU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.     

Chromosome-mediated resistance of Yersinia enterocolitica serotype O9 to intracellular killing by mouse peritoneal macrophages

Abstract The survival of Yersinia enterocolitica serotype O9 within mouse peritoneal macrophages was investigated. To evaluate the role of the virulence plasmid in the resistance to intracellular killing, an isogenic pair of virulent (plasmid-bearing) and avirulent (plasmid-less) O9 strains was used. The virulent strain was able to express plasmid-encoded outer membrane proteins and to colonize the Peyer's patches of orally infected mice. When mice were infected intraperitoneally, both strains were recovered at similar rates and over the same time from the peritoneal cavity. When in vitro assays were performed, both strains showed similar resistance to intracellular killing by monolayers of resident and inflammatory peritoneal macrophages. Previous opsonization of bacteria did not modify their survival within macrophage monolayers. We concluded that serotype O9 strains display a chromosome-mediated resistance to intracellular killing by mouse peritoneal macrophages. Moreover, macrophage resistance does not seem to be of importance for virulence of serotype O9 strains in mice.     

A real-time PCR assay for the specific identification of serotype O:9 of Yersinia enterocolitica

A real-time PCR assay was developed based on a 181-bp fragment of the recently cloned per gene, including an internal amplification control (124 bp), for the detection of Yersinia enterocolitica O:9 (Ye O:9). The validation included 48 Ye O:9, 33 Y. enterocolitica non-O:9 and 35 other closely-related bacterial strains, containing per gene homologies. The assay was specific for the Ye O:9 tested, the detection limit was 1-10 genome copies of purified DNA and amplification efficiency was between 90.5-103%, indicating a linear regression throughout the detection window.     

Membrane filter technique for the isolation of Yersinia enterocolitica.

A membrane filter procedure was developed for the isolation of Yersinia enterocolitica from aquatic environments. Primary differentiation was based on the fermentation of sorbitol, the absence of lysine decarboxylase and arginine decarboxylase-dihydrolase activities, and the production of urease. Sodium deoxycholate was incorporated as an inhibitor of background organisms. The presumptive identification of Y. enterocolitica was accomplished in 50 h, and the rate of identity confirmation of typical colonies was 88%. The mean recovery rate of 15 strains from phosphate buffer suspensions was 91%, and quantitative recovery was demonstrated for low populations of the organism in both laboratory-prepared and naturally occurring mixed cultures. The technique was used to isolate 33 strains of Y. enterocolitica from 15 of 27 river water samples and from prechlorinated sewage effluent. Nine (27%) of the isolates were rhamnose positive, and only five (15%) were serotypable. Two isolates were identified as serotype O:4 (or O:4,32), two were O:17, and the fifth was O:40.     

Production of Outer Membrane Vesicles by the Plague Pathogen Yersinia pestis

Many Gram-negative bacteria produce outer membrane vesicles (OMVs) during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that Yersinia pestis, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by Y. pestis contain catalytically active Pla that is competent for plasminogen activation and α2-antiplasmin degradation. The abundance of OMV-associated proteins released by Y. pestis is significantly elevated at 37°C compared to 26°C and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp). In addition, we show that Y. pestis OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that Y. pestis may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand.     

Structural identification of the lipopolysaccharide O-antigen produced by Yersinia enterocolitica serotype O:28.

The structure of the O-antigenic polysaccharide (O-PS) component of the lipopolysaccharide produced by Yersinia enterocolitica serotype O:28 has been elucidated. From chemical methods involving glycose analysis, periodate oxidation, methylation and the use of one- and two-dimensional NMR spectroscopy, the O-PS was found to be a polymer of repeating branched hexasaccharide units composed of L-rhamnose (four parts), 2-acetamido-2-deoxy-D-glucose (one part), and 2-acetamido-2-deoxy-D-galacturonic acid (one part) having the following structure:     

Structure of the O-specific polysaccharide of the Yersinia enterocolitica serovar O:4.32 lipopolysaccharide. Serologic relations of lipopolysaccharides of Y. enterocolitica O:4.32 and Y. intermedia O:4.33

O-Specific polysaccharide has been isolated on mild acid hydrolysis of the lipopolysaccharide from Yersinia enterocolitica O: 4.32 (strain 96) and shown to consist of yersiniose B (3,6-dideoxy-4-C-(1-hydroxyethyl)-D-xylo-hexose, YerB) acetylated at C1' and 2-acetamido-2-deoxy-D-galactose residues in a molar ratio 1:2. Acid hydrolysis, methylation and 13C NMR studies indicated the polysaccharide to be composed of trisaccharide repeating units of the following structure: (sequence; see text) The data obtained revealed structural and serological interrelation between O-antigens of Y. enterocolitica O:4.32 and Y. intermedia O:4.33.     

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.     

Studies on the interactions of immunostimulated macrophages and Yersinia enterocolitica O:8

Immunological and electron microscopy investigations of the phagocytic and killing activities of peritoneal macrophages from rats and mice against Yersinia enterocolitica serotype O:8 cells were performed. The effect of in vivo application of cytoplasmic membranes (CM) from the stable Escherichia coli WF+ L-form on macrophage activity was also studied. It was established that rat macrophages more actively phagocytosed the plasmidless pYV(-) Y. enterocolitica cells, compared to the plasmid-bearing pYV(+) Y. enterocolitica cells. The killing ability against both variants of the Y. enterocolitica strain was significantly enhanced in macrophages from CM-treated rats after 2 h, 4 h, and 24 h incubation. The CM treatment enhanced the phagocytic activity of the macrophages. The in vitro interaction of normal and immunostimulated rat macrophages with both pYV(+) and pYV(-) variants of Y. enterocolitica did not lead to any additional apoptotic and necrotic changes in macrophages compared to control macrophages, which were cultivated without Y. enterocolitica. Electron-microscopic investigation showed that mouse macrophages eliminated Y. enterocolitica pYV(+) cells in vivo after 24 h. No engulfed or digested bacterial cells were observed. Activation of cell surfaces and vacuolization of macrophage cytoplasm, both of CM-treated non-infected and infected mice, were observed. The experimental results showed that Y. enterocolitica pYV(+) cells could be eliminated by peritoneal macrophages.     

Immunochemical studies on R mutants of Yersinia enterocolitica O:3.

Three mutants of Yersinia enterocolitica O:3, namely: YeO3-R1, YeO3-RfbR7 and YeO3-c-trs8-R were classified on the basis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) profile of isolated lipopolysaccharides (LPS) as belonging to the Ra- (the first) and the Rc-type (the other two mutants). Methylation analysis, in addition to 13C and 1H NMR studies of purified core oligosaccharides revealed structures similar to those established previously for the full core of Y. enterocolitica O:3 in the case of the Ra mutant, and identical to that reported for the Rc mutant Ye75R, in the case of the two other mutants. The O-specific sugar, 6d-L-altrose, which forms a homopolymeric O-chain, was present in small amounts in all three LPS preparations, as well as in the core oligosaccha ride preparations along with the Ra and the Rc sugars, characteristic of the Y. enterocolitica O:3 core. This result is in line with genetic data, indicating that it is the inner core region which is the receptor for the O-specific chain in Y. enterocolitica O:3. This region seems likewise to be the anchoring region for the enterobacterial common antigen (ECA), as shown by SDS/PAGE/Western blot analysis with monoclonal antibodies against ECA. In addition, we also demonstrated that the Ye75R mutant Rc and its parental strain Ye75S, both were ECA-immunogenic strains. So far, ECA-immunogenic strains, i.e. those with LPS-linked ECA, were only identified in E. coli mutants of the R1, R4 and K-12 serotype.     

Model experiments to establish behaviour of Yersinia enterocolitica O:9 strains in various types of fresh dry sausage

In model experiments different kinds of raw sausages were inoculated with liquid cultures of virulent-plasmid-carrying clinical Yersinia (Y.) enterocolitica (e.) strains of the O:9 serotype, doses being between 10⁴ and 10⁵ cfu g^-1. The sausage samples were stored at 3–5° and 13–16°C. During the first 10 d of storage the Y.e. plate count was detected with Desoxycholate-Citrate-Lactose-Sucrose Agar every day, later on in addition to it with phosphate buffer-enrichment and with enrichment according to Schiemann (1982) in intervals of several days' duration. The pH and a _w values, the contents of salt and water were detected. The multitude of complexly acting factors and substances prevents obviously the proliferation of Y.e. in fresh dry sausages. Decay dynamics of Y.e. were found to be considerably affected by storage temperature. Cold storage, basically, had a conservation effect and thus delayed the dying process of model strains. Yersinia enterocolitica -contaminated fresh dry sausage may cause potential danger to consumers, because of relatively extended survival periods of the pathogen. Therefore, manufacturers are expected to observe most stringent hygienic rules of Good Manufacturing Practice.     

A new branched-chain monosaccharide from the Yersinia enterocolitica serotype O:4.32 lipopolysaccharide

A component of the Yersinia enterocolitica O:4,32 lipopolysaccharide has been shown to be a second representative of the new class of monosaccharides and possess the structure of 3,6-dideoxy-4C-(1-hydroxyethyl)-D-xylo-hexose (yersiniose).     

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 the phenol fraction of Brucella for isolating a specific serum against Yersinia enterocolitica serovar O9

A phenol fraction, capable of adsorbing cross-reacting antibodies from Y. enterocolitica O9 antiserum without affecting its serological activity against specific antigens, has been obtained from a Brucella strain. The physicochemical and antigenic characteristics of the preparation are presented. It is recommended that the proposed method be tested in differentiation of intestinal yersiniosis caused by Y. enterocolitica O9 from Brucella infection. This work has resulted in obtaining highly specific antiserum to Y. enterocolitica, serovar O9.