Search

The Yersinia virulence factor YopJ inhibits the host immune response and induces apoptosis by blocking multiple signaling pathways, including the MAPK and NFkappaB pathways in the infected cell. YopJ is a cysteine protease that cleaves a reversible post-translational modification in the form of ubiquitin or a ubiquitin-like protein. Homologues of YopJ are expressed in animal and plant pathogens, as well as a plant symbiont, suggesting a universal mechanism of regulating or modulating a variety of signaling pathways. The ability of YopJ to block the innate immune response, its activity as a ubiquitin-like protein protease and its activity with respect to mammalian signalling pathways are discussed in this review.

Virulent bacteria are able to sense temperature changes and respond by modifying the expression of--among others--genes that code for virulence factors. The chromatin-associated protein H-NS has been shown to play a role in the thermomodulation of virulence factor expression. In addition to H-NS, proteins of the Hha/YmoA family have also been identified in different enterobacteria as participating in the thermoregulation of some virulence factors. For one of these proteins, the Hha protein, it has been shown that it interacts with H-NS, and both proteins form a nucleoid-protein complex responsible for the thermoregulation of, at least, E. coli hemolysin. The presence of genes coding for homologues of both proteins on some conjugative plasmids and their relation to thermoregulation suggests that this complex could also play a role in the regulation of plasmid transfer.

Enteropathogenic species of the genus Yersinia penetrate the intestinal epithelium and then spread to the lymphatic system, where they proliferate extracellularly. At this location, most other bacteria are effectively ingested and destroyed by the resident phagocytes. Yersinia, on the other hand binds to receptors on the external surface of phagocytes, and from this location it blocks the capacity of these cells to exert their phagocytic function via different receptors. The mechanism behind the resistance to phagocytosis involves the essential virulence factor YopH, a protein tyrosine phosphatase that is translocated into interacting target cells via a type III secretion machinery. YopH disrupts peripheral focal complexes of host cells, seen as a rounding up of infected cells. The focal complex proteins that are dephosphorylated by YopH are focal adhesion kinase and Crk-associated substrate, the latter of which is a common substrate in both professional and non-professional phagocytes. In macrophages additional substrates have been found, the Fyn-binding/SLP-76-associated protein and SKAP-HOM. Phagocytosis is a rapid process that is activated when the bacterium interacts with the phagocyte. Consequently, the effect exerted by a microbe to block this process has to be rapid and precise. This review deals with the mechanisms involved in impeding uptake as well as with the role of the YopH substrates and focal complex structures in normal cell function.

Yersinia species utilize a type III secretion system to inject toxins, called Yops (Yersinia outer proteins), into eukaryotic cells. The N-termini of the Yops serve as type III secretion signals, but they do not share a consensus sequence. To simplify the analysis of type III secretion signals, we replaced amino acids 2-8 of the secreted protein YopE with all permutations (27 or 128) of synthetic serine/isoleucine sequences. The results demonstrate that amphipathic N-terminal sequences, containing four or five serine residues, have a much greater probability than hydrophobic or hydrophilic sequences to target YopE for secretion. Multiple linear regression analysis of the synthetic sequences was used to obtain a model for N-terminal secretion signals. The model accurately classifies the N-terminal sequences of native type III substrates as efficient secretion signals.

The type III mechanism of protein secretion is a pathogenic strategy shared by a number of gram-negative pathogens of plants and animals that has evolved in order to inject virulence proteins into the cytosol of target eukaryotic cells. The pathogens of the Yersinia genus represent a model system where much progress has been made in understanding this secretion pathway. Herein, we review what has been recently learned in yersiniae about the various environmental signals that induce type III secretion, how the synthesis of secretion substrates is regulated, and how such a diverse group of proteins is recognized as a substrate for secretion.

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.

Many Gram-negative pathogens utilize a type III secretion system (TTSS) to inject toxins into the cytosol of eukaryotic cells. Previous studies have indicated that exported substrates are targeted to the Yersinia TTSS via the coding regions of their 5' mRNA sequences, as well as by their cognate chaperones. However, recent results from our laboratory have challenged the role of mRNA targeting signals, as we have shown that the amino termini of exported substrates are crucial for type III secretion. Here, we discuss the nature of these amino-terminal secretion signals and propose a model for the secretion of exported substrates by amino-terminal and chaperone-mediated signals. In addition, we discuss the roles of chaperones as regulators of virulence gene expression and present models suggesting that such regulation can occur independently of the delivery of their substrates to the secretion apparatus.

Highly pathogenic Yersinia carry a pathogenicity island termed high-pathogenicity island (HPI). The Yersinia HPI comprises genes involved in the synthesis of the siderophore yersiniabactin and can thus be regarded as an iron-uptake island. A unique characteristic of the HPI is its wide distribution among different enterobacteria such as Escherichia coli, Klebsiella, Citrobacter and Salmonella. Other types of iron-uptake systems are also carried by different pathogenicity islands in enterobacteria.

Yersinia spp. inject effector proteins (Yersinia outer proteins, Yops) into target cells via a type III secretion apparatus. The effector YopE was recently shown to possess GAP activity towards the Rho GTPases RhoA, Rac and CDC42 in vitro. To investigate the intracellular, 'in vivo' targets of YopE we generated a Yersinia enterocolitica strain [WA(pYLCR+E)] that injects 'life-like' amounts of YopE as only effector. Primary human umbilical vein endothelial cells (HUVEC) were infected with WA(pYLCR+E) and were then stimulated with: (i) bradykinin to induce actin microspikes followed by ruffles as an assay for CDC42 activity followed by CDC42 stimulated Rac activity; (ii) sphingosine-1-phosphate to form ruffles by direct Rac activation; or (iii) thrombin to generate actin stress fibres through Rho activation. In WA(pYLCR+E)-infected HUVEC microspike formation stimulated with bradykinin remained intact but the subsequent development of ruffles was abolished. Furthermore, ruffle formation after stimulation with sphingosine-1-phosphate or thrombin induced production of stress fibres was unaltered in the infected cells. These data suggest that YopE is able to inhibit Rac- but not Rho- or CDC42-regulated actin structures and, more specifically, that YopE is capable of blocking CDC42Hs dependent Rac activation but not direct Rac activation in HUVEC. This provides evidence for a considerable specificity of YopE towards selective Rac-mediated signalling pathways in primary target cells of Yersinia.

Enteropathogenic Yersinia are gram-negative bacterial species that translocate from the lumen of the intestine and are able to grow within deep tissue sites. During the earliest stages of disease, the organism is able to bind integrin receptors that are presented on the apical surface of M cells in the intestine, which allows its internalization and subsequent translocation into regional lymph nodes. The primary integrin substrate is the outer-membrane protein invasin, which binds with extraordinarily high affinity to at least five different integrins that have the (beta)(1) chain. Bacterial uptake into host cells is modulated by the affinity of receptor-substrate interaction, receptor concentration and the ability of the substrate to aggregate target receptors.

The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.

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.

Many Gram-negative pathogens use a type III secretion machine to translocate protein toxins across the bacterial cell envelope. Pathogenic Yersinia spp. export at least 14 Yop proteins via a type III machine, which recognizes secretion substrates by signals encoded in yop mRNA or chaperones bound to unfolded Yop proteins. During infection, substrate recognition appears to be regulated in a manner that allows the Yersinia type III pathway to direct Yops to the bacterial envelope, the extracellular medium or into the cytosol of host cells.

The Yersinia genus includes human and animal pathogens (plague, enterocolitis). The fine structures of the endotoxin lipids A of seven strains of Yersinia enterocolitica, Yersinia ruckeri and Yersinia pestis were determined and compared using mass spectrometry. These lipids differed in secondary acylation at C-2': this was dodecanoic acid (C(12)) for two strains of Y. enterocolitica and Y. ruckeri, tetradecanoic acid (C(14)) in two other Y. enterocolitica and hexadecenoic acid (C(16:1)) in Y. pestis. The enterocolitica lipids having a mass identical to that of Escherichia coli were found to be structurally different. The results supported the idea of a relation between membrane fluidity and environmental adaptability in Yersinia.

The gram-negative type III secretion pathway translocates bacterial proteins directly into eukaryotic host cells, thus allowing a pathogen to interfere directly with host signalling pathways. Protein and inositol phosphatases and protein kinases have been identified as delivered effectors in three bacterial pathogens, Salmonella, Shigella and Yersinia, and it is expected that several more such type III effectors will be found.

Type III secretory genes(Bscl, J, K, L, N and O) have recently been identified in Bordetella bronchiseptica and shown to be under the control of the BvgAS locus. We examined a 35 616 byte DNA sequence amplified from Bordetella pertussis Tohama I for homology with known type III secretory genes in Yersinia spp. and Pseudomonas sppand a total of 20 homologous open reading frames were detected. Putative type III secretion proteins in B. pertussis were designated according to their homology with type III secretion proteins in B. bronchiseptica, Yersinia and Pseudomonas. These ORFs were arranged in two putative operons, which together we have designated as the BpeI locus. The first spans nucleotides 23385-7888 and encodes the putative proteins LcrH1, BopD, BopB, LcfH2, BscI, BscJ, BscK, BscL, BscN, BscO, BscQ, BscR, BscS, BscT, BscU, and BscC, in this order. The second spans nucleotides 23580-29863 and encodes the putative proteins LcrE, LcrD, BscD and BscF, in this order. The homology of these proteins to type III secretory proteins was B. bronchiseptica (73-99%), Yersinia spp. (17-65%), Pseudomonas spp. (18-64%). The B. pertussis proteins were similar to their homologues in B. bronchiseptica, Yersinia and Pseudomonas in terms of length, molecular weight and isoelectric point. Coiled-coil domains were detected in putative translocation proteins, BopB and BopD. BopB and BopD were similar to each other, to the RTX toxin family and to cyaA, cyaB, cyaD and cyaE. The percentage G+C content of the sequence analysed was 66.16%, which is similar to the published percentage G+C (67-70%) for the B. pertussis chromosome., (Copyright 1999 Academic Press.)

Bacterial pathogens of both animals and plants use type III secretion machines to inject virulence proteins into host cells. Although many components of the secretion machinery are conserved among different bacterial species, the substrates for their type III pathways are not. The Yersinia type III machinery recognizes some secretion substrates via a signal that is encoded within the first 15 codons of yop mRNA. These signals can be altered by frameshift mutations without affecting secretion of the encoded polypeptides, suggesting a mechanism whereby translation of yop mRNA is coupled to the translocation of newly synthesized polypeptide. We report that the type III machinery of Erwinia chrysanthemi cloned in Escherichia coli recognizes the secretion signals of yopE and yopQ. Pseudomonas syringae AvrB and AvrPto, two proteins exported by the recombinant Erwinia machine, can also be secreted by the Yersinia type III pathway. Mapping AvrPto sequences sufficient for the secretion of reporter fusions in Yersinia revealed the presence of an mRNA secretion signal. We propose that 11 conserved components of type III secretion machines may recognize signals that couple mRNA translation to polypeptide secretion.

Fundamental to the virulence of microbial pathogens is their capacity for adaptation and survival within variable, and often hostile, environments encountered in the host. We describe a novel, extragenomic mechanism of surface modulation which may amplify the adaptive and pathogenic potential of numerous bacterial species, including Staphylococcus, Yersinia, and pathogenic Neisseria species, as well as Helicobacter pylori and Streptococcus pyogenes. The mechanism involves specific bacterial recruitment of heparin, glycosaminoglycans, or related sulfated polysaccharides, which in turn serve as universal binding sites for a diverse array of mammalian heparin binding proteins, including adhesive glycoproteins (vitronectin and fibronectin), inflammatory (MCP-3, PF-4, and MIP-1alpha) and immunomodulatory (gamma interferon) intermediates, and fibroblast growth factor. This strategy impacts key aspects of microbial pathogenicity as exemplified by increased bacterial invasion of epithelial cells and inhibition of chemokine-induced chemotaxis. Our findings illustrate a previously unrecognized form of parasitism that complements classical virulence strategies encoded within the microbial genome.

Yersinia bercovieri, a recently identified Y. enterocolitica-like species, produces a heat-stable enterotoxin (designated YbST) which has biologic activity in infant mice and increases short circuit current in Ussing chambers. Although YbST has some properties in common with the heat-stable enterotoxins of Y. enterocolitica (YST I and YST II), it appears to be a novel toxin because (i) it was not neutralized by anti-YST I antiserum, (ii) YbST-neutralizing antiserum did not neutralize YST I, and (iii) Y. bercovieri strains did not hybridize with genetic probes for yst I, yst II, and other known enterotoxins.

The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds.

Virulent Yersinia species (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica) possess a 70 kb virulence plasmid that encodes the Yop virulon. This virulence system allows extracellular bacteria adhering at the surface of eukaryotic cells to secrete and inject bacterial effector proteins, called Yops, into the cytosol of these cells in order to disarm them. These secreted Yop proteins are remarkably conserved among the different species. A Y. enterocolitica O:8 strain was found to secrete a protein antigenically related to YopM but significantly larger. Sequencing of the corresponding gene showed that the protein was a YopM variant with three repeats of one domain. Comparison of the yopM gene of various Yersinia strains by PCR amplification, as well as analysis of the secreted Yop proteins by SDS-PAGE and Western blotting revealed that, unlike the other Yops, the YopM protein shows some heterogeneity., (Copyright 1998 Academic Press)

The hydrophobic probe N-phenyl-1-naphthylamine accumulated less in non-pathogenic Yersinia spp. and non-pathogenic and pathogenic Yersinia enterocolitica than in Yersinia pseudotuberculosis or Yersinia pestis. This was largely due to differences in the activity of efflux systems, but also to differences in outer membrane permeability because uptake of the probe in KCN/arsenate-poisoned cells was slower in the former group than in Y. pseudotuberculosis and Y. pestis. The probe accumulation rate was higher in Y. pseudotuberculosis and Y. pestis grown at 37 degrees C than at 26 degrees C and was always highest in Y. pestis. These yersiniae had LPSs with shorter polysaccharides than Y. enterocolitica, particularly when grown at 37 degrees C. Gel<-->liquid-crystalline phase transitions (Tc 28-31 degrees C) were observed in LPS aggregates of Y. enterocolitica grown at 26 and 37 degrees C, with no differences between non-pathogenic and pathogenic strains. Y. pseudotuberculosis and Y. pestis LPSs showed no phase transitions and, although the fluidity of LPSs of Y. pseudotuberculosis and Y. enterocolitica grown at 26 degrees C were close below the Tc of the latter, they were always in a more fluid state than Y. enterocolitica LPS. Comparison with previous studies of Salmonella choleraesuis subsp. choleraesuis serotype minnesota rough LPS showed that the increased fluidity and absence of transition of Y. pseudotuberculosis and Y. pestis LPSs cannot be explained by their shorter polysaccharides and suggested differences at the lipid A/core level. It is proposed that differences in LPS-LPS interactions and efflux activity explain the above observations and reflect the adaptation of Yersinia spp. to different habitats.

Pathogenic Yersiniae adhere to and kill macrophages by targeting some of their Yop proteins into the eukaryotic cytosol. There is debate about whether YopE targeting proceeds as a direct translocation of polypeptide between cells or in two distinct steps, each requiring specific signals for YopE secretion across the bacterial envelope and for translocation into the eukaryotic cytosol. Here, we used the selective solubilization of the eukaryotic plasma membrane with digitonin to measure Yop targeting during Yersinia infections of HeLa cells. YopE, YopH, YopM and YopN were found in the eukaryotic cytosol but not in the extracellular medium. When bound to SycE chaperone in the Yersinia cytoplasm, YopE residues 1-100 are necessary and sufficient for the targeting of hybrid neomycin phosphotransferase. Electron microscopic analysis failed to detect an extracellular intermediate of YopE targeting, suggesting a one-step translocation mechanism.

Yersiniae are equipped with the Yop virulon, an apparatus that allows extracellular bacteria to deliver toxic Yop proteins inside the host cell cytosol in order to sabotage the communication networks of the host cell or even to cause cell death. LcrG is a component of the Yop virulon involved in the regulation of secretion of the Yops. In this paper, we show that LcrG can bind HeLa cells, and we analyse the role of proteoglycans in this phenomenon. Treatment of the HeLa cells with heparinase I, but not chondroitinase ABC, led to inhibition of binding. Competition assays indicated that heparin and dextran sulphate strongly inhibited binding, but that other glycosaminoglycans did not. This demonstrated that binding of HeLa cells to purified LcrG is caused by heparan sulphate proteoglycans. LcrG could bind directly to heparin-agarose beads and, in agreement with these results, analysis of the protein sequence of Yersinia enterocolitica LcrG revealed heparin-binding motifs. In vitro production and secretion by Y. enterocolitica of the Yops was unaffected by the addition of heparin. However, the addition of exogenous heparin decreased the level of YopE-Cya translocation into HeLa cells. A similar decrease was seen with dextran sulphate, whereas the other glycosaminoglycans tested had no significant effect. Translocation was also decreased by treatment of HeLa cells with heparinitase, but not with chondroitinase. Thus, heparan sulphate proteoglycans have an important role to play in translocation. The interaction between LcrG and heparan sulphate anchored at the surface of HeLa cells could be a signal triggering deployment of the Yop translocation machinery. This is the first report of a eukaryotic receptor interacting with the type III secretion and associated translocation machinery of Yersinia or of other bacteria.

The resistance to divalent metal ions, antibiotics and H2O2 was investigated in Yersinia kristensenii strains 13, 15, 18 by performing subcultivations with CdSO4 (20 and 100 mg/L) in nutrient agar (NA) and M9 medium with thiamine. Metal resistance of all three strains in NA was the same and decreased in the following sequence: Ni > Zn = Co > Cd. The chloramphenicol (Cmp) resistance ranged between 32 and 256 mg/L and the H2O2 sensitivity was very low or even zero. In the presence of thiamine the metal resistance sequence changed to Zn = Cd > Ni, Co, Ni and Co tolerance being 10-20 mg/L. Cmp resistance of all strains increased to 256 mg/L and H2O2 sensitivity also rose. In Cd-treated cultures, the ratio of glucose to thiamine in culture medium affected Cd resistance. At normal content of glucose and thiamine (5 g/L and 5 mg/L), Cd resistance markedly decreased coincident with thiamine exhaustion in these slowly-growing cultures. The Cmp resistance decreased to 16 mg/L, Ni and Co intolerance and H2O2 hypersensitivity appeared. At lowered glucose or thiamine levels (5 g/L and 2.5 mg/L or 2.5 g/L and 5 mg/L) a marginal decrease of Cd resistance took place in response to limited glucose uptake. Low thiamine or low-glucose cultures were resistant to H2O2, and exhibited a small decrease in Cmp resistance and a low Ni, Co tolerance. The adaptation of strain 15 to Cd induced only a small decrease of Cd resistance. Lowered glucose-to-thiamine ratio in culture medium probably induced in Cd-treated cultures a response triggering Cd resistance.

Five clinical strains of Yersinia isolated in Japan and identified as atypical biotype 3 or biotype 3B, serotype O:3, phage type II (3*/O:3/II) Yersinia enterocolitica were characterised since the biochemical reactions of these strains indicate they might also belong to the species Yersinia bercovieri. Biochemical tests, characterisation of the beta-lactamases and DNA-DNA hybridization studies provided strong evidence indicating that these strains should be classified as Yersinia enterocolitica. A simple scheme combining a disc diffusion test and four biochemical tests was devised for identification of these atypical strains.

Studies on the molecular genetics of bacterial LPS serve at least two main purposes: (i) to help develop an understanding of the biology, biochemistry and genetics of this bacterial surface macromolecule, 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 O:3, reaching as far as a detailed analysis of the gene clusters directing the biosynthesis of the outer core oligosaccharide and of the O-ag. The O-ag gene clusters of Y. enterocolitica serotype O:8 and Y. pseudotuberculosis serotypes O:2a and O: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.

Yersinia adhering at the surface of eukaryotic cells secrete a set of proteins called Yops. This secretion which occurs via a type III secretion pathway is immediately followed by the injection of some Yops into the cytosol of eukaryotic cells. Translocation of YopE and YopH across the eukaryotic cell membranes requires the presence of the translocators YopB and YopD. YopE and YopH are modular proteins composed of an N-terminal secretion signal, an internalization domain, and an effector domain. Secretion of YopE and YopH requires the presence of the specific cytosolic chaperones SycE and SycH, respectively. In this work, we have mapped the regions of YopE and YopH that are involved in binding of their cognate chaperone. There is only one Syc-binding domain in YopE (residues 15-50) and YopH (residues 20-70). This domain is localized immediately after the secretion signal and it corresponds to the internalization domain. Removal of this bifunctional domain did not affect secretion of YopE and YopH and even suppressed the need for the chaperone in the secretion process. Thus SycE and SycH are not secretion pilots. Instead, we propose that they prevent intrabacterial interaction of YopE and YopH with proteins involved in translocation of these Yops across eukaryotic cell membranes.

Pathogenic yersiniae secrete about a dozen anti-host proteins, the Yops, by a pathway which does not involve cleavage of a classical signal peptide. The Yop secretory apparatus, called Ysc, for Yop secretion, is the archetype of type III secretion systems (which serve for the secretion of virulence proteins by several animal and plant pathogens) and is related to the flagellar assembly apparatus. The Yop secretion signal is N-terminal but has not been defined to date. Apart from the Ysc machinery, secretion of at least four Yops requires cytoplasmic proteins called Syc (for specific Yop chaperone). Each Syc protein binds to its cognate Yop. Unlike most cytoplasmic chaperones, these proteins do not have an ATP-binding domain, and are presumably devoid of ATPase activity. They share a few common properties: an acidic pl, a size in the range of 15-20 kDa, and a putative amphipathic alpha-helix in the C-terminal portion. They were recently shown to have counterparts in other pathogenic bacteria, where they appear to have a similar function.

The purpose of the study was the determination of biochemical features of strains belonging to Yersinia genus isolated from clinical material and other sources, and an assessment of the usefulness of certain biochemical tests for the detection of potentially pathogenic Yersinia strains. In all, 110 strains were studied, including 48 from the archives of the National Institute of Hygiene, 38 isolated from food of animal or plant origin, and 24 isolated from blood and faeces of patients. On the ground of the biochemical features the isolated strains were recognized as belonging to 5 species: Y. enterocolitica(83 strains), Y. pseudotuberculosis(12 strains), Y. frederiksenii(7 strains), Y. kristensenii(2 strains) and Y. intermedia(4 strains). Two strains differed in their features from the typical species reveale as yet in Yersinia genus. All strains isolated from clinical material were recognized as Y. enterocolitica, while those isolated from food included also Y. frederiksenii, Y. intermedia and Y. kristensenii. The isolated strains grew well on CIN medium forming characteristic violet-pink colonies with irregular outlines after 24-48 hours of incubation at 25 degrees C or 37 degrees C. The potential pathogenicity was assessed on the basis of the presence of autoagglutination (AA) and absent ability of breaking down of salicin, aesculin and pyrazinamide. Only two strains of Y. enterocolitica 03 isolated from faeces and 5 strains of Y. pseudotuberculosis from the archives had AA ability. Low frequency of AA was explained with possible loss of plasmids conveying virulence as a result of multiple passages of the strains, and the fact that many of them were present in the R phase. All strains of Y. enterocolitica 03 and both Y. enterocolitica 09 strains from the archives could be assumed to be potentially pathogenic for man and animals, while no strain isolated from food showed the set of features which could suggest its possible pathogenicity.

A raw milk bacterial isolate, identified as Yersinia kristensenii was found to produce a bacteriocin which was inhibitory to Yersinia enterocolitica but not to other selected species of Yersinia or Gram-negative bacteria. Maximum production of bacteriocin was obtained when the organism was grown in shake culture at 28 degrees C. Mitomycin C at a concentration of 0.5 micrograms ml-1 induced bacteriocin production. The bacteriocin was partially purified and characterized by ammonium sulphate fractionation and gel filtration. The bacteriocin was completely inactivated when treated with proteolytic enzymes (trypsin and chymotrypsin). Bacteriocin activity was heat-resistant and it retained some of its activity after 5 min at boiling temperature. A total of 15 bacteriocin sensitive-suspected food isolates were further identified biochemically as Yersinia enterocolitica and a non-sensitive isolate was identified as Yersinia intermedia.

Presence of muramic acid, a bacterial cell wall component, was analysed by gas chromatography-mass spectrometry in synovial fluid (SF) of 40 patients with acute inflammatory arthritis. SF muramic acid was observed in 4/14 patients with acute, culture negative inflammatory arthritis of unclear origin. Each of these four patients had a history of a recent bacterial disease (pansinuitis, purulent leg ulcer, erysipelas, unexplained fever with suspicion of cholecystitis and urinary tract infection). In the bacterial arthritis, SF muramic acid was detected in 6/12 patients (in 2/6 culture negative cases). In the reactive arthritis due to Salmonella or Yersinia, the rate of positivity was 2/14. Nineteen samples of traumatic SF effusion were muramic acid negative. These findings indicate that several cases of undefined acute inflammatory arthritis are of bacterial origin.

Pathogenic bacteria of the species Yersinia, including Yersinia pestis, block phagocytosis by macrophages. This process involves the YopE protein, which induces disruption of the host cell actin microfilament structure. Here, we show that the contact between the pathogen and the mammalian cell induces expression and then polarized transfer of YopE into the eukaryotic cell. While the bacteria remain at the surface of the target cell, the YopE cytotoxin is transferred through the host cell plasma membrane and YopE is only recovered within the cytosol of the target cell. The results suggest that the pathogen senses cell structures and focuses the transfer of YopE to occur solely at the interaction zone between the bacterium and the eukaryotic cell. The regulation of this process is shown to involve surface-located YopN sensor protein of the bacterium.

Yersinia enterocolitica strains of serotypes lethal to mice have been reported previously to produce an endogenous siderophore. In this study, an ethyl acetate-extractable siderophore was characterized and given the name yersiniophore. Yersiniophore was produced by 16 of 16 human isolates of serotypes O:4, O:4,32, O:8, O:21, and one nonhuman isolate of serotype O:21. It was not produced by isolates of serotype O:3, O:5, or O:9. One strain of Yersinia pseudotuberculosis produced yersiniophore, but strains of Yersinia kristensenii, Yersinia frederiksenii, and Yersinia intermedia did not produce or utilize yersiniophore. Food and water isolates of Y. enterocolitica produced a water-soluble siderophore but not yersiniophore. Sixty-two strains of Y. enterocolitica including 42 isolates from human infections, 2 animal isolates, and 18 water and food isolates were examined for utilization of yersiniophore, the water-soluble siderophore, and ferrioxamine. Yersiniophore promoted growth rate, iron binding, and uptake in 17 of 62 strains, all of which produced yersiniophore. Ten of 17 food and water isolates and one human isolate were capable of utilizing the water-soluble siderophore. Utilization studies suggest that at least one additional water-soluble siderophore may be produced. Ferrioxamine promoted the growth of 60 of 62 strains examined; however, only the 17 strains which produced yersiniophore actively accumulated [59Fe]ferrioxamine. Yersiniophore production and utilization may be important in clinical infections since all human strains belonging to serotype O:8 produced yersinophore. The water-soluble siderophore was not detected in human isolates.

Pathogenic bacteria frequently express surface proteins with affinity for components of the mammalian extracellular matrix, i.e. collagens, laminin, fibronectin or proteoglycans. This review summarizes our current knowledge on the mechanisms of bacterial adherence to extracellular matrices and on the biological significance of these interactions. The best-characterized bacterial proteins active in these interactions are the mycobacterial fibronectin-binding proteins, the fibronectin- and the collagen-binding proteins of staphylococci and streptococci, specific enterobacterial fimbrial types, as well as the polymeric surface proteins YadA of yersinias and the A-protein of Aeromonas. Some of these bacterial proteins are highly specific for an extracellular matrix protein, some are multifunctional and express binding activities towards a number of target proteins. The interactions can be based on a protein-protein or on a protein-carbohydrate interaction, or on a bridging mechanism mediated by a bivalent soluble target protein. Many of the interactions have also been demonstrated on tissue sections or in vivo, and adherence to the extracellular matrix has been shown to promote bacterial colonization of damaged tissues.

Pathogenic Yersinia express a complex array of iron-regulated functions and possess mechanisms for inorganic iron and hemin acquisition. These include a unique temperature-regulated hemin storage system, high-affinity transport processes for hemin and inorganic iron, and an iron-responsive regulatory system controlling gene expression. The genetic organization of these systems illuminates several aspects of iron metabolism in the yersiniae.

O-specific polysaccharide has been isolated on mild hydrolysis of lipopolysaccharide from Yersinia aldovae and shown to consist of 2-acetamido-2-deoxy-D-glucose, D-glucose, 2-acetamido-2-deoxy-D-galactose, and 3,6-dideoxy-3- [(R)-3-hydroxybutyramido]-D-galactose in molar ratio 2:2:1:1. Acid hydrolysis, methylation, solvolysis with anhydrous hydrogen fluoride, 1H and 13C NMR studies indicated the polysaccharide to be composed of hexasaccharide repeating units of the following structure: [formula see text].

This work represents the first evidence of the presence of an iron uptake system siderophore mediated in the bacterial fish pathogen Yersinia ruckeri. A group of 20 strains representative of this species, with different serotype and origin were examined. All of them were able to grow at high concentrations (from 0.7 to 1.1 mM) of the iron chelator EDDA. Although the Y. ruckeri isolates failed to cross-feed the indicator strains for enterobactin and aerobactin production, the chemical tests revealed the presence in the culture supernatants of phenolate siderophores. At least three outer membrane proteins were induced in iron limiting conditions. All the strains showed a similar pattern of induced membrane proteins regardless their serotype or origin, which suggests a similarity in the iron uptake system. This system could have an important role in the pathogeneicity of Y. ruckeri for fish.

Tissue-binding specificity of the type-3 fimbriae of pathogenic enteric bacteria was determined using frozen sections of human kidney. A wild-type Klebsiella sp. strain and the recombinant strain Escherichia coli HB101(pFK12), both expressing type-3 fimbriae, as well as the purified type-3 fimbriae effectively bound to sites at or adjacent to tubular basement membranes, Bowman's capsule, arterial walls, and the interstitial connective tissue. Bacterial adherence to kidney was decreased after collagenase treatment of the tissue sections. Recombinant strains expressing type-3 fimbriae specifically adhered to type V collagen immobilized on glass slides, whereas other collagens, fibronectin or laminin did not support bacterial adherence. In accordance with these findings, specific binding of purified type-3 fimbriae to immobilized type V collagen was demonstrated. Specific adhesion to type V collagen was also seen with the recombinant strain HB101(pFK52/pDC17), which expresses the mrkD gene of the type-3 fimbrial gene cluster in association with the pap-encoded fimbrial filament of E. coli, showing that the observed binding was mediated by the minor lectin (MrkD) protein of the type-3 fimbrial filament. The interaction is highly dependent on the conformation of type V collagen molecules since type V collagen in solution did not react with the fimbriae. Specific binding to type V collagen was also exhibited by type-3 fimbriate strains of Yersinia and Salmonella, showing that the ability to use type V collagen as tissue target is widespread among enteric bacteria.

Pathogenic bacteria of the genus Yersinia (Y. enterocolitica, Y. pseudotuberculosis and Y. pestis) are pathogens of the invasive type. Although they provoke diseases as different as plague and enterocolitis, the molecular bases of their virulence appear to be surprisingly similar: a 70 kb plasmid confers a degree of resistance to the primary immune response and the presence or absence of chromosome encoded functions modulates virulence. The knowledge of the virulence functions that now emerges suggests the conversion of Y. enterocolitica as a life carrier.

Yersinia strains either urease-positive or urease-negative were examined for precipitation of struvite in human urine at 25 and 37 degrees C. All urease-positive strains and 8 out of 10 urease-negative strains showed the ability to produce these crystals. Incubation time required for precipitation was longer for urease-negative strains and quantity of struvite formed was higher in urease-positive ones. Regarding incubation temperature, no significant influence has been observed.

The presence of hexokinase, aldolase, glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase has been detected in Y. enterocolitica, which indicates the glycolytic and pentosophosphate pathways of glucose catabolism. Y. enterocolitica can be classified as an opportunistic anaerobic microorganism on the basis of the whole complex of its characteristics (growth in both aerobic and anaerobic conditions, the reduction of nitrates into nitrites, the presence of higly active glycolytic enzymes).

The ability of Yersinia to digest polypectate may be of some value in differentiating Y. enterocolitica and Y. pseudotuberculosis from some of the other fermenting gram-negative bacilli, such as Enterobacter agglomerans, with which they can be confused. Pectolytic activity in Yersinia may also have some teleologic or taxonomic significance about which we do not care to speculate.