[THE EFFECT OF waaL LIGASE GENES DELETION ON MOTILITY AND STRESS ADAPTATION REACTIONS OF YERSINIA ENTEROCOLITICA 6471/76].

The aim of current study was to estimate the influence of waaL(OS) and waaL(PS) genes deletion on lipopolysaccharide (LPS) synthesis, bacterial motility and stress resistance of bacteria Yersinia enterocolitica 6471/76. Single and double waaL mutants were created by replacing the wild-type alleles in bacterial chromosome for mutant ones. The phenotypes of mutants were visualized by DOC-PAGE gels stained with silver and immunoblot with specific to O-polysaccharide and outer core monoclonal antibodies. Bacterial motility was evaluated by the diameter of the migration zone. Wild type bacteria and mutants were analyzed by bacterial growth curves in a hypertonic medium. Participation of WaaL ligases in resistance to osmotic pressure was found only in case of both ligese genes deletion. Also the YeO3_os_ps mutants showed motility decreasing, which recovered after adding a functionally active gene. Thus, deletion of both waaL ligase genes lead to a drastic reduction in bacterial motility and increase their sensitivity to hypertonic medium that can indirectly characterize biological role of WaaL ligases.

Isolation and characterization of Yersinia-specific bacteriophages from pig stools in Finland.

AIMS: Bacteriophages infect bacteria, and they are present everywhere in the world including the intestinal tracts of animals. Yersiniosis is a common foodborne infection caused by Yersinia enterocolitica and Yersinia pseudotuberculosis. As these bacteria are frequently isolated from pigs, we wanted to know whether Yersinia-specific bacteriophages are also present in the pig stools and, if so, whether there is a positive or negative association between the prevalence of the Yersinia phages and the pathogenic Yersinia in the stool samples. METHODS AND RESULTS: Altogether 793 pig stool samples collected between November 2010 and March 2012 from 14 Finnish pig farms were screened for the presence of bacteriophages able to infect Y. enterocolitica serotype O:3, O:5,27 or O:9 strains, or Y. pseudotuberculosis serotype O:1a, O:1b or O:3 strains. Yersinia phages were isolated from 90 samples from eight farms. Yersinia enterocolitica O:3 was infected by 59 phages, 28 phages infected serotypes O:3 and O:5,27, and eight phages infected serotypes O:3, O:5,27 and O:9, and Y. pseudotuberculosis O:1a by eight phages. Many phages originating from pigs in the same farm were identical based on their restriction enzyme digestion patterns; 20 clearly different phages were selected for further characterization. Host ranges of these phages were tested with 94 Yersinia strains. Six of the phages infected eight strains, 13 phages infected three strains, and one phage infected only one strain, indicating that the phages had a relatively narrow host range. CONCLUSIONS: There was a clear association between the presence of the host bacteria and specific phages in the stools. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated bacteriophages may have potential as biocontrol agents for yersiniosis in both humans and pigs in future, and as alternatives or in addition to antibiotics. To our knowledge, this is the first reported isolation of Yersinia-specific phages from pig stool samples.

Identification of distinct lipopolysaccharide patterns among Yersinia enterocolitica and Y. enterocolitica-like bacteria.

The lipopolysaccharide (LPS) of strains representing various serotypes of Yersinia enterocolitica and Y. enterocolitica-like bacteria was studied by deoxycholate-PAGE and silver staining analysis. Four main types of LPS were detected based on the O-polysaccharide (O-PS): (i) LPS with homopolymeric O-PS, (ii) LPS with ladder-forming heteropolymeric O-PS, (iii) LPS with single-length O-PS, and (iv) semi-rough LPS without O-PS. Within the first three types, several subvariants were detected. Selected serotypes representing all above LPS types are sensitive to bacteriophage φR1-37 indicating that they share the phage receptor, a hexasaccharide called outer core in Y. enterocolitica O:3. Whereas phage φR1-37-resistant mutants of homopolymeric O-PS have lost only the outer core, those of ladder-forming or single-length O-PS have lost also the O-PS suggesting that in the latter ones the outer core is bridging between O-PS and lipid A-core. This work forms a basis of further structural, biochemical and genetic studies of these LPSs.

Characterization of anti-ECA antibodies in rabbit antiserum against rough Yersinia enterocolitica O:3.

Enterobacterial common antigen (ECA) is a characteristic surface component in bacteria belonging to the Enterobacteriaceae family. It is generally integrated in the outer membrane via a linkage to phosphatidylglycerol (ECA(PG)) and at the same time in some special cases via a linkage to lipopolysaccharide (ECA(LPS)); the latter form is immunogenic. Yersinia enterocolitica O:3 expresses both ECA(PG) and ECA(LPS). To study whether ECA-immunogenicity of Y. enterocolitica O:3 is temperature-regulated, rabbits were immunized with ECA-expressing Y. enterocolitica O:3 bacteria grown at 22 and 37°C. To induce minimal amount of anti-LPS antibodies, immunization was performed with YeO3-c-trs8-R, an LPS mutant missing both O-polysaccharide and the outer core hexasaccharide. However, abundant antibodies specific for LPS core were still present in the obtained antisera such that the reactivity of ECA-specific antibodies could not be detected. To obtain "monovalent" anti-ECA antisera, the sera were absorbed with ECA-negative bacteria. Absorption with live bacteria removed efficiently the anti-LPS antibodies, whereas this was not the case with boiled bacteria. Western blotting revealed that the specificity of the monovalent anti-ECA antiserum was different from that of a monoclonal anti-ECA antibody (mAb 898) as it did not react with ECA(PG), and this suggested that in Y. enterocolitica O:3 ECA(LPS) only one or two ECA repeat unit(s) is/are linked to LPS. Both ECA(PG) and ECA(LPS) expression were found to be regulated by temperature and repressed at 37°C.

Characterisation of non-pathogenic Yersinia pseudotuberculosis-like strains isolated from food and environmental samples.

Non-pathogenic Yersinia pseudotuberculosis-like strains were recovered from Finnish food and environmental samples. These strains could not be differentiated from Y. pseudotuberculosis strains using API 20E or other phenotypical tests. However, all of the strains were inv-, and virF-negative with polymerase chain reaction (PCR), while all Y. pseudotuberculosis strains used as controls were inv-positive and fresh Y. pseudotuberculosis strains were also virF-positive, indicating that the Y. pseudotuberculosis-like strains were non-pathogenic. Using pulsed-field gel electrophoresis (PFGE) with NotI enzyme and ribotyping with EcoRI and HindIII enzymes, the Y. pseudotuberculosis-like strains, which grouped genetically together, could be differentiated from true Y. pseudotuberculosis strains and from strains belonging to other sucrose-negative Yersinia species. In addition, the O-antigen gene cluster of one Y. pseudotuberculosis-like strain was characterized, and it differed from those of known Y. pseudotuberculosis serotypes. This study demonstrates that identification of Y. pseudotuberculosis from food and environmental sources using solely biochemical reactions can be incorrect, and when a strain cannot be serotyped to known Y. pseudotuberculosis serotypes, the pathogenic potential of isolates should be determined.

Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis.

This report describes the development of in-house real-time PCR assays using minor groove binding probes for simultaneous detection of the Bacillus anthracis pag and cap genes, the Francisella tularensis 23 KDa gene, as well as the Yersinia pestis pla gene. The sensitivities of these assays were at least 1 fg, except for the assay targeting the Bacillus anthracis cap gene, which showed a sensitivity of 10 fg when total DNA was used as a template in a serial dilution. The clinical value of the Bacillus anthracis- and Francisella tularensis-specific assays was demonstrated by successful amplification of DNA from cases of cow anthrax and hare tularemia, respectively. No cross-reactivity between these species-specific assays or with 39 other bacterial species was noted. These assays may provide a rapid tool for the simultaneous detection and identification of the three category A bacterial species listed as biological threats by the Centers for Disease Control and Prevention.

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.

Validated 5' nuclease PCR assay for rapid identification of the genus Brucella.

A real-time, genus-specific 5' nuclease PCR assay for amplification of a 322-bp fragment of the per gene was developed for rapid (<2 h) identification of Brucella spp. from agar plates. The assay, including an internal amplification control (116 bp), identified Brucella strains (n = 23) and did not detect non-Brucella strains (n = 174), indicating its usefulness, particularly for laboratories with stringent quality assurance programs.

Proper expression of the O-antigen of lipopolysaccharide is essential for the virulence of Yersinia enterocolitica O:8 in experimental oral infection of rabbits.

The O-antigen of lipopolysaccharide (LPS) is required for virulence in Yersinia enterocolitica serotype O:8. Here we evaluated the importance of controlling the O-antigen biosynthesis using an in vivo rabbit model of infection. Y. enterocolitica O:8 wild-type strain was compared to three mutants differing in the O-antigen phenotype: (i) the rough strain completely devoid of the O-antigen, (ii) the wzy strain that lacks the O-antigen polymerase (Wzy protein) and expresses LPS with only one repeat unit, and (iii) the wzz strain that lacks the O-antigen chain length determinant (Wzz protein) and expresses LPS without modal distribution of O-antigen chain lengths. The most attenuated strain was the wzz mutant. The wzz bacteria were cleared from the tissues by day 30, the blood parameters were least dramatic and histologically only immunomorphological findings were seen. The level of attenuation of the rough and the wzy strain bacteria was between the wild-type and the wzz strain. Wild-type bacteria were highly resistant to killing by polymorphonuclear leukocytes, the wzz strain bacteria were most sensitive and the rough and wzy strain bacteria were intermediate resistant. These results clearly demonstrated that the presence of O-antigen on the bacterial surface is not alone sufficient for full virulence, but also there is a requirement for its controlled chain length.

Expression of heterologous O-antigen in Yersinia pestis KIM does not affect virulence by the intravenous route.

All strains of Yersinia pestis examined have been found to lack an O-antigen. In other members of the Enterobacteriaceae, the rough phenotype often results in attenuation. However, Y. pestis is the aetiological agent of bubonic plague. In evolving from the ancestral enteropathogenic Yersinia pseudotuberculosis, and with the development of an arthropod-vectored systemic pathogenesis, smooth LPS production is not necessary for Y. pestis virulence and the metabolic burden has been alleviated by inactivation of the O-antigen biosynthetic operon. To investigate this, Y. pestis strain KIM D27 was transformed with a plasmid carrying the operon encoding the O-antigen of Yersinia enterocolitica O : 3. Expression of the O-antigen could be detected in silver-stained gels. The receptor for bacteriophage phiYeO3-12 has been shown to be O-antigen, and infection by this bacteriophage results in lysis of Y. enterocolitica O : 3. Expression of the O-antigen in Y. pestis conferred sensitivity to lysis by phiYeO3-12. The O-antigen-expressing clone was shown to be as virulent in mice by the intravenous route of challenge as the rough wild-type. Assays showed no alteration in the ability of Y. pestis to resist lysis by cationic antimicrobial peptides, serum or polymyxin.

Mutations in the genes for synthesis of the outer core region of the lipopolysaccharide of Yersinia enterocolitica O:3.

AIMS: The aim of this study was to construct non-polar frame-shift mutations in some of the individual genes responsible for the biosynthesis of the branching outer core (OC) hexasaccharide of the lipopolysaccharide (LPS) in Yersinia enterocolitica O:3 (YeO:3). METHODS AND RESULTS: Chromosomal segments of YeO:3 containing wbcN, wbcO and wbcQ genes were cloned into a suicide vector. A frame-shift mutation was introduced into each gene by modifying a unique restriction enzyme recognition site. Each recombinant plasmid with a modified OC gene was mobilized into YeO:3 to allow for allelic exchange between the modified gene and the wild type chromosomal gene. The exchange was confirmed by demonstrating the absence of the particular restriction site in the chromosome of each mutant strain. Analysis of LPS by gel electrophoresis showed that the LPS of the mutants was lacking the OC. Therefore, the constructed wbcN, wbcO and wbcQ strains are true mutants with frame-shifts in the corresponding genes. CONCLUSIONS: The products of the wbcN, wbcO and wbcQ genes are putative glycosyltransferases and, based on the present analysis, essential for the biosynthesis of the OC hexasaccharide. The absence of OC in the LPS of these mutants further supports the hypothesis that the OC hexasaccharide is a single O-antigen O-unit that is not polymerized in YeO:3. SIGNIFICANCE AND IMPACT OF THE STUDY: These mutants provide information on the unique nature of the synthesis of OC of YeO:3 LPS. They are valuable for future biochemical studies to establish the roles of the products of individual OC genes.

Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure.

Pathogenic biotypes of Yersinia enterocolitica (serotypes O:3, O:8, O:9, and O:13), but not environmental biotypes (serotypes O:5, O:6, O:7,8, and O:7,8,13,19), increased their permeability to hydrophobic probes when they were grown at pH 5.5 or in EGTA-supplemented (Ca(2+)-restricted) media at 37 degrees C. A similar observation was also made when representative strains of serotypes O:8 and O:5 were tested after brief contact with human monocytes. The increase in permeability was independent of the virulence plasmid. The role of lipopolysaccharide (LPS) in this phenomenon was examined by using Y. enterocolitica serotype O:8. LPS aggregates of bacteria grown in acidic or EGTA-supplemented broth took up more N-phenylnaphthylamine than LPS aggregates of bacteria grown in standard broth and also showed a marked increase in acyl chain fluidity which correlated with permeability, as determined by measurements obtained in the presence of hydrophobic dyes. No significant changes in O-antigen polymerization were observed, but lipid A acylation changed depending on the growth conditions. In standard medium at 37 degrees C, there were hexa-, penta-, and tetraacyl lipid A forms, and the pentaacyl form was dominant. The amount of tetraacyl lipid A increased in EGTA-supplemented and acidic media, and hexaacyl lipid A almost disappeared under the latter conditions. Our results suggest that pathogenic Y. enterocolitica strains modulate lipid A acylation coordinately with expression of virulence proteins, thus reducing LPS packing and increasing outer membrane permeability. The changes in permeability, LPS acyl chain fluidity, and lipid A acylation in pathogenic Y. enterocolitica strains approximate the characteristics in Yersinia pseudotuberculosis and Yersinia pestis and suggest that there is a common outer membrane pattern associated with pathogenicity.

Characterization of the physiological substrate for lipopolysaccharide heptosyltransferases I and II.

L-Glycero-D-manno-heptopyranose is a characteristic compound of many lipopolysaccharide (LPS) core structures of Gram-negative bacteria. In Escherichia coli two heptosyltransferases, namely WaaC and WaaF, are known to transfer L-glycero-D-manno-heptopyranose to Re-LPS and Rd(2)-LPS, respectively. It had been proposed that both reactions involve ADPL-glycero-D-manno-heptose as a sugar donor; however, the structure of this nucleotide sugar had never been completely elucidated. In the present study, ADPL-glycero-D-manno-heptose was isolated from a heptosyltransferase-deficient E. coli mutant, and its structure was determined by nuclear magnetic resonance spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry as ADPL-glycero-beta-D-manno-heptopyranose. This compound represented the sole constituent of the bacterial extract that was accepted as a sugar donor by heptosyltransferases I and II in vitro.

YadA, the multifaceted Yersinia adhesin.

The adhesion protein YadA is encoded by the yadA gene located in the 70-kb virulence plasmid of Yersinia (pYV) that is common to the pathogenic Yersinia species (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica). YadA is a virulence factor of Y. enterocolitica, however, YadA seems to be dispensable for the virulence of Y. pseudotuberculosis, and in wild-type Y. pestis the yadA gene has a frameshift mutation silencing the gene. Expression of the Y. pseudotuberculosis YadA in Y. pestis reduces its virulence. YadA is a homotrimer of ca. 45-kDa subunits that are anchored to the outer membrane via their C-termini, while their N-termini form a globular head on top of a stalk; the 'lollipop'-shaped YadA structure covers the entire bacterial surface giving it hydrophobic properties. The yadA gene expression is induced at 37 degrees C by the temperature-dependent transcriptional activator LcrF. YadA is a multifaceted protein as revealed by its different biological properties. YadA+ bacteria bind to collagens, laminin, fibronectin, intestinal submucosa, mucus, and to hydrophobic surfaces like polystyrene. YadA+ bacteria autoagglutinate in stationary culture and also specifically agglutinate guinea pig red blood cells. YadA is also a potent serum resistance factor as it inhibits the classical pathway of complement. As invasin, it mediates low rate invasion to tissue culture cells. In a rat model of reactive arthritis YadA and specifically YadA-mediated collagen binding is necessary for Y. enterocolitica to induce the disease. Despite of this wealth of information or perhaps because of it, the in vivo role of YadA during infection remains still largely unresolved.

Isolation and structural characterization of an R-form lipopolysaccharide from Yersinia enterocolitica serotype O:8.

The lipopolysaccharide (LPS) of strain 8081-c-R2, a spontaneous R-mutant of Yersinia enterocolitica serotype O:8, was isolated using extraction with phenol/chloroform/light petroleum. Its compositional analysis indicated the presence of D-GlcN, D-Glc, L-glycero-D-manno- and D-glycero-D-manno-heptose, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and phosphate. From deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide, three oligosaccharides (1-3) were isolated using high-performance anion-exchange chromatography, the structures of which were determined by compositional analysis and one- and two-dimensional NMR spectroscopy as [carbohydrate structure see text] in which all sugars are pyranoses, and R and R' represent beta-D-Glc (in 1 and 2) and beta-D-GlcN (in 1 only), respectively. D-alpha-D-Hep is D-glycero-alpha-D-manno-heptose, L-alpha-D-Hep is L-glycero-alpha-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, and P is phosphate. The liberated lipid A was analyzed by compositional analyses and MALDI-TOF MS. Its beta-D-GlcN4P-(1-->6)-alpha-D-GlcN-1-->P backbone is mainly tetra-acylated with two amide- and one ester-linked (at O3 of the reducing GlcN) (R)-3-hydroxytetradecanoic acid residues, and one tetradecanoic acid that is attached to the 3-OH group of the amide-linked (R)-3-hydroxytetradecanoic acid of the nonreducing GlcN. Additionally, small amounts of tri- and hexa-acylated lipid A species occur.

Complete genomic sequence of the lytic bacteriophage phiYeO3-12 of Yersinia enterocolitica serotype O:3.

phiYeO3-12 is a T3-related lytic bacteriophage of Yersinia enterocolitica serotype O:3. The nucleotide sequence of the 39,600-bp linear double-stranded DNA (dsDNA) genome was determined. The phage genome has direct terminal repeats of 232 bp, a GC content of 50.6%, and 54 putative genes, which are all transcribed from the same DNA strand. Functions were assigned to 30 genes based on the similarity of the predicted products to known proteins. A striking feature of the phiYeO3-12 genome is its extensive similarity to the coliphage T3 and T7 genomes; most of the predicted phiYeO3-12 gene products were >70% identical to those of T3, and the overall organizations of the genomes were similar. In addition to an identical promoter specificity, phiYeO3-12 shares several common features with T3, nonsubjectibility to F exclusion and growth on Shigella sonnei D(2)371-48 (M. Pajunen, S. Kiljunen, and M. Skurnik, J. Bacteriol. 182:5114-5120, 2000). These findings indicate that phiYeO3-12 is a T3-like phage that has adapted to Y. enterocolitica O:3 or vice versa. This is the first dsDNA yersiniophage genome sequence to be reported.

Bacterial PCR in the diagnosis of joint infection.

OBJECTIVES: To evaluate the value of broad range bacterial PCR in the diagnosis of joint infection and to find out if there are bacteria causing arthritis which are not cultivable by the present methods. METHODS: Polymerase chain reaction (PCR) with broad range bacterial primers and DNA sequencing (bacterial PCR) was used to analyse 154 synovial fluid (SF) samples from patients with different arthritic diseases. RESULTS: Bacterial DNA was detected in 18 SF samples, including samples from six patients with culture proven purulent arthritis, and from three patients with possible purulent arthritis. Three samples from patients with culture confirmed purulent arthritis remained negative in bacterial PCR. CONCLUSIONS: The results indicate that in the usual diagnostic laboratory setting bacterial PCR does not offer any obvious advantage over bacterial culture in the microbiological diagnosis of joint infection.

Bacteriophage phiYeO3-12, specific for Yersinia enterocolitica serotype O:3, is related to coliphages T3 and T7.

Bacteriophage phiYeO3-12 is a lytic phage of Yersinia enterocolitica serotype O:3. The phage receptor is the lipopolysaccharide O chain of this serotype that consists of the rare sugar 6-deoxy-L-altropyranose. A one-step growth curve of phiYeO3-12 revealed eclipse and latent periods of 15 and 25 min, respectively, with a burst size of about 120 PFU per infected cell. In electron microscopy phiYeO3-12 virions showed pentagonal outlines, indicating their icosahedral nature. The phage capsid was shown to be composed of at least 10 structural proteins, of which a protein of 43 kDa was predominant. N-terminal sequences of three structural proteins were determined, two of them showing strong homology to structural proteins of coliphages T3 and T7. The phage genome was found to consist of a double-stranded DNA molecule of 40 kb without cohesive ends. A physical map of the phage DNA was constructed using five restriction enzymes. The phage infection could be effectively neutralized using serum from a rabbit immunized with whole phiYeO3-12 particles. The antiserum also neutralized T3 infection, although not as efficiently as that of phiYeO3-12. phiYeO3-12 was found to share, in addition to the N-terminal sequence homology, several common features with T3, including morphology and nonsubjectibility to F exclusion. The evidence conclusively indicated that phiYeO3-12 is the first close relative of phage T3 to be described.

Temperature-regulated efflux pump/potassium antiporter system mediates resistance to cationic antimicrobial peptides in Yersinia.

Most bacterial pathogens are resistant to cationic antimicrobial peptides (CAMPs) that are key components of the innate immunity of both vertebrates and invertebrates. In Gram-negative bacteria, the known CAMPs resistance mechanisms involve outer membrane (OM) modifications and specifically those in the lipopolysaccharide (LPS) molecule. Here we report, the characterization of a novel CAMPs resistance mechanism present in Yersinia that is dependent on an efflux pump/potassium antiporter system formed by the RosA and RosB proteins. The RosA/RosB system is activated by a temperature shift to 37 degrees C, but is also induced by the presence of the CAMPs, such as polymyxin B. This is the first report of a CAMPs resistance system that is induced by the presence of CAMPs. It is proposed that the RosA/RosB system protects the bacteria by both acidifying the cytoplasm to prevent the CAMPs action and pumping the CAMPs out of the cell.

Functional mapping of the Yersinia enterocolitica adhesin YadA. Identification Of eight NSVAIG - S motifs in the amino-terminal half of the protein involved in collagen binding.

The virulence plasmid-encoded YadA of Yersinia enterocolitica serotype O:3 is a 430-amino-acid outer membrane protein, synthesized with a 25-amino-acid signal peptide. YadA forms homotrimeric surface structures that function as adhesin between bacteria and collagen as well as other host proteins. The structure-function relationships of YadA were studied, and the collagen-binding determinants of YadA were located to its amino-terminal half. Collagen did not bind to any of the overlapping 16-mer YadA peptides, indicating that the collagen binding site of YadA is conformational. Epitope mapping of YadA identified 12 linear antigenic epitopes altogether. Seven epitopes were uniquely recognized by an anti-YadA antiserum able to inhibit collagen binding. Four of these epitopes shared a motif NSVAIG-S that is repeated eight times within the N-terminal half of YadA. Site-directed mutagenesis showed that these motifs are absolutely required for YadA-mediated collagen binding, revealing a novel type of collagen-binding mechanism.