Mikael Skurnik

The surface‐located YopN protein is involved in calcium signal transduction in Yersinia pseudotuberculosis

The low‐calcium response (Icr) is strongly conserved among the pathogenic Yersinia species and is observed when the pathogen is grown at 37°C in Ca2+‐depleted medium. This response is characterized by a general metabolic downshift and by a specific induction of virulence‐plasmid‐encoded yop genes. Regulation of yop expression is exerted at transcriptional level by a temperature‐regulated activator and by Ca2+‐regulated negative elements. The yopN gene was shown to encode a protein (formerly also designated Yop4b) which is surface‐located when Yersmia is grown at 37°C. yopN was found to be part of an operon that is induced during the low‐calcium response. Insertional inactivation of the yopN gene resulted in derepressed transcription of yop genes. A hybrid plasmid containing the yopN gene under the control of the tac promoter fully restored the wild‐type phenotype of the yopN mutant. Thus the surface‐located YopN somehow senses the calcium concentration and transmits a signal to shut off yop transcription when the calcium concentration is high.

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.

Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O:8.

The Y. enterocolitica O:8 (YeO8) O‐antigen repeat units consist of five sugar residues: N‐acetyl‐d‐galactosamine (GalNAc), d‐galactose (Gal), d‐mannose (Man), l‐fucose (Fuc), and 6‐deoxy‐d‐gulose (6d‐Gul). The nucleotide sequence of the O‐antigen gene cluster of the YeO8 strain 8081‐c was determined. Altogether, 18 open reading frames (ORFs) were identified and shown to be essential for O‐antigen biosynthesis. We previously characterized the 3′‐end of the O‐antigen gene cluster and identified four genes: two for GDP‐Man biosynthesis, one for UDP‐Gal biosynthesis, and one for O‐antigen polymerase. Based on sequence similarity, Tn5‐insertion phenotypes and chemical analysis, the 14 new genes were assigned the following functions: four genes are involved in the biosynthesis of CDP‐6d‐Gul and two in GDP‐Fuc biosynthesis. Five gene products were assigned sugar transferase functions and one gene product was similar to Wzx, the O‐antigen flippase. Two genes remained unassigned. By genetic complementation we also showed that YeO8 O‐antigen biosynthesis was dependent on N‐acetyl‐glucosaminyl:undecaprenylphosphate transferase (GlcNAc transferase), the WecA (formerly known as Rfe) protein. Data obtained from chemical‐composition analysis suggest that in addition to being GlcNAc transferase, WecA may also function as a GalNAc transferase. Using a restriction‐deficient derivative of Y. enterocolitica O:8 strain 8081, a rough mutant, designated 8081‐R2, was isolated. 8081‐R2 was complemented in trans with a cloned O‐antigen gene cluster restoring surface O‐antigen expression. The virulence of the wild‐type strain and that of the complemented strain were significantly higher (approx. 100‐fold) than that of the rough mutant in an orally infected mouse model, showing that YeO8 O‐antigen is a virulence factor.

Analysis of the yopA gene encoding the Yop1 virulence determinants of Yersinia spp.

The Yop proteins of Yersinia are important virulence determinants. The Yop1 protein sequences of Yersinia pestis, Yersinia pseudotuberculosis, and two Yersinia enterocolitica serotypes, O:3 and O:8, deduced from the nucleotide sequences of the corresponding yopA genes, were compared. Most differences were found in the hydrophilic domains of the proteins, whereas the hydrophobic domains were conserved. The amino acid sequences revealed a signal sequence 25 amino acids long. No cysteine residues were present, even though Yop1 forms a polymeric structure.

Lipopolysaccharide O antigen status of Yersinia enterocolitica O:8 is essential for virulence and absence of O antigen affects the expression of other Yersinia virulence factors

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram‐negative bacteria. Although much attention has been given to the biological effects of its lipid A portion, a great body of evidence indicates that its O chain polysaccharide (O antigen) portion plays an important role in the bacterium–host interplay. In this work we have studied in‐depth the role of the O antigen in Yersinia enterocolitica serotype O:8 pathogenesis. We made a detailed virulence analysis of three mutants having different O antigen phenotypes: (i) LPS with no O antigen (rough mutant); (ii) LPS with one O unit (semirough mutant) and (iii) LPS with random distribution of O antigen chain lengths. We demonstrated that these LPS O antigen mutants were attenuated in virulence regardless of the infection route used. Co‐infection experiments revealed that the rough and semirough mutants were severely impaired in their ability to colonize the Peyers patches and in contrast to the wild‐type strain they did not colonize spleen and liver. The mutant with random distribution of O antigen chain lengths, however, survived better but started to be cleared from mouse organs after 8u2003days. As an explanation to this attenuation we present here evidence that other Yersinia virulence factors depend on the presence of O antigen for their proper function and/or expression. We demonstrated that in the rough mutant: (i) the YadA function but not its expression was altered; (ii) Ail was not expressed and (iii) inv expression was downregulated. On the other hand, expression of flhDC, the flagellar master regulatory operon, was upregulated in this mutant with a concomitant increase in the production of flagellins. Finally, expression of yplA, encoding for the Yersinia phospholipase A, was also upregulated accompanied by an increased flagellar type III secretion system mediated secretion of YplA to culture medium. Together these findings suggest that the absence of O antigen in the outer membrane of Yersinia either directly or indirectly, for example through a cellular or membrane stress, could act as a regulatory signal.

The lipopolysaccharide outer core of Yersinia enterocolitica serotype O:3 is required for virulence and plays a role in outer membrane integrity

Lipopolysaccharide (LPS) of Yersinia enterocolitica O:3 has an inner core linked to both the O‐antigen and to an outer core hexasaccharide that forms a branch. The biological role of the outer core was studied using polar and non‐polar mutants of the outer core biosynthetic operon. Analysis of O‐antigen‐ and outer core‐deficient strains suggested a critical role for the outer core in outer membrane properties relevant in resistance to antimicrobial peptides and permeability to hydrophobic agents, and indirectly relevant in resistance to killing by normal serum. Wild‐type bacteria but not outer core mutants killed intragastrically infected mice, and the intravenous lethal dose was ≈104‐fold higher for outer core mutants. After intragastric infection, outer core mutants colonized Peyers patches and invaded mesenteric lymph nodes, spleen and liver, and induced protective immunity against wild‐type bacteria. In mice co‐infected intragastrically with an outer core mutant–wild type mixture, both strains colonized Peyers patches similarly during the first 2 days, but the mutant was much less efficient in colonizing deeper organs and was cleared faster from Peyers patches. The results demonstrate that outer core is required for Y. enterocolitica O:3 full virulence, and strongly suggest that it provides resistance against defence mechanisms (most probably those involving bactericidal peptides).

Complete Nucleotide Sequence and Likely Recombinatorial Origin of Bacteriophage T3

We report the complete genome sequence (38,208 bp) of bacteriophage T3 and provide a bioinformatic comparative analysis with other completely sequenced members of the T7 group of phages. This comparison suggests that T3 has evolved from a recombinant between a T7-like coliphage and a yersiniophage. To assess this, recombination between T7 and the Yersinia enterocolitica serotype O:3 phage phiYeO3-12 was accomplished in vivo; coliphage progeny from this cross were selected that had many biological properties of T3. This represents the first experimentally observed recombination between lytic phages whose normal hosts are different bacterial genera.

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.

Molecular genetics and biochemistry of Yersinia lipopolysaccharide

Studies on the molecular genetics of bacterial LPS1 serve at least two main purposes: (i) to help develop an understanding of the biology, biochemistry and genetics of this bacterial surface macro‐molecule, and (ii) to provide a basis for both vaccine development and virulence experiments. Both of these goals have been the driving force in studies of Yersinia LPS carried out during the last decade. Here we will review the progress made in the molecular genetics and biochemistry of Yersinia LPS. A deep understanding has been achieved with respect to Y. enterocolitica serotype 0:3, reaching as far as a detailed analysis of the gene clusters directing the biosynthesis of the outer core oligosaccharide and of the O‐ag2. The O‐ag gene clusters of Y. enterocolitica serotype 0:8 and Y. pseudotuberculosis serotypes 0:2a and 0:5a have also been cloned and partially characterized. LPS biosynthesis of these Yersinia species includes examples of the two major variations recognized in the biosynthesis of this macromolecule: (i) homopolymeric or O‐antigen polymerase‐independent biosynthesis, and (ii) heteropolymeric or O‐antigen polymerase‐dependent biosynthesis.

Pathogenic Yersinia enterocolitica Strains Increase the Outer Membrane Permeability in Response to Environmental Stimuli by Modulating Lipopolysaccharide Fluidity and Lipid A Structure

ABSTRACT 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 (Ca2+-restricted) media at 37°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°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.