Cytotoxic necrotizing factor-Y boosts Yersinia effector translocation by activating Rac protein.

Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac.

Gut proteases target Yersinia invasin in vivo.

BACKGROUND: Yersinia enterocolitica is a common cause of food borne gastrointestinal disease. After oral uptake, yersiniae invade Peyer's patches of the distal ileum. This is accomplished by the binding of the Yersinia invasin to ß1 integrins on the apical surface of M cells which overlie follicle associated lymphoid tissue. The gut represents a barrier that severely limits yersiniae from reaching deeper tissues such as Peyer's patches. We wondered if gut protease attack on invasion factors could contribute to the low number of yersiniae invading Peyer's patches. FINDINGS: Here we show that invasin is rapidly degraded in vivo by gut proteases in the mouse infection model. In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin. Protease treated yersiniae are shown to be less invasive in a cell culture model. YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases. CONCLUSIONS: We demonstrate that gut proteases target important Yersinia virulence factors such as invasin and YadA in vivo. Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

Destabilization of YopE by the ubiquitin-proteasome pathway fine-tunes Yop delivery into host cells and facilitates systemic spread of Yersinia enterocolitica in host lymphoid tissue.

Pathogenic Yersinia species inject a panel of Yop virulence proteins by type III protein secretion into host cells to modulate cellular defense responses. This enables the survival and dissemination of the bacteria in the host lymphoid tissue. We have previously shown that YopE of the Y. enterocolitica serogroup O8 is degraded in the host cell through the ubiquitin-proteasome pathway. YopE normally manipulates rearrangements of the actin cytoskeleton and triggers phagocytosis resistance. To shed light into the physiological role of YopE inactivation, we mutagenized the lysine polyubiquitin acceptor sites of YopE in the Y. enterocolitica serogroup O8 virulence plasmid. The resulting mutant strain escaped polyubiquitination and degradation of YopE and displayed increased intracellular YopE levels, which was accompanied by a pronounced cytotoxic effect on infected cells. Despite its intensified activity on cultured cells, the Yersinia mutant with stabilized YopE showed reduced dissemination into liver and spleen following enteral infection of mice. Furthermore, the accumulation of degradation-resistant YopE was accompanied by the diminished delivery of YopP and YopH into cultured, Yersinia-infected cells. A role of YopE in the regulation of Yop translocation has already been described. Our results imply that the inactivation of YopE by the proteasome could be a tool to ensure intermediate intracellular YopE levels, which may effectuate optimized Yop injection into host cells. In this regard, Y. enterocolitica O8 appears to exploit the host ubiquitin proteasome system to destabilize YopE and to fine-tune the activities of the Yop virulence arsenal on the infected host organism.

A key role for E-cadherin in intestinal homeostasis and Paneth cell maturation.

BACKGROUND: E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn's disease. METHODS AND FINDINGS: To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen. CONCLUSION: These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.

In vivo analysis of Yersinia enterocolitica infection using luxCDABE.

Yersiniae expressing an L-arabinose-inducible luxCDABE reporter were used to analyze the colonization of mice. Infection of live mice was followed over a period of 6 days. These experiments revealed frequent colonization of cervical lymph nodes after oral, but not intravenous infection. Furthermore, the well-known colonization of the small intestine, Peyer's patches (PPs) of the ileum, the cecal lymph follicle, mesenteric lymph nodes, liver, and spleen was easily detectable. Removal of the intestinal tract of mice revealed that the number of abscessed PPs and other tissues can be easily quantified. Experiments with an invasin mutant expressing luxCDABE revealed a significantly reduced number of abscessed PPs, cecal lymph follicles, and lymph nodes in yersiniae lacking invasin.

Analysis of Yersinia enterocolitica invasin expression in vitro and in vivo using a novel luxCDABE reporter system.

A novel luxCDABE plasmid for the analysis of promoter elements by site-specific integration into the genome of Yersinia enterocolitica was constructed. The versatility of this reporter system was demonstrated by comparing the activity of the inv promoter in the Y. enterocolitica high-pathogenic serotype O : 8 (strain WA-314) with that of the low pathogenic serotype O : 9 (strain Y127). The luciferase activity of a transcriptional fusion between the inv promoter of serotype O : 8 and luxCDABE was about fourfold lower than the activity of the respective O : 9 promoter. This correlated with lower invasin production by Y. enterocolitica serotype O : 8 compared with serotypes O : 9, O : 3 and O : 5,27. However, Y. enterocolitica of serotype O : 8 revealed higher invasiveness than serotype O : 9. When both invasins were expressed in trans at similar levels in the Y. enterocolitica O : 8 Deltainv background strain, cell invasion assays showed a slightly higher invasiveness of the strain producing Inv(O : 8) than the strain producing Inv(O : 9). We provide experimental evidence that this might be due to a higher binding capacity of Inv(O : 8) for cells expressing beta1 integrins compared with Inv(O:9). The Y. enterocolitica O : 8 strain harbouring the P(inv)((O : 8)) : : luxCDABE fusion was then successfully used to follow inv expression in a mouse infection model. These experiments showed for the first time that the inv promoter is active in infected living mice, especially in Peyer's patches of the ileum, the caecal lymph follicle, and the lymph nodes, liver and spleen. The production of invasin in the spleen was demonstrated by Western blot analysis. In conclusion, the presented reporter system enables stable genomic integration of the luxCDABE operon into the chromosome of Yersinia, facilitates in vitro quantification of promoter activities under different bacterial growth conditions, and enables detection of promoter activities in a mouse model.

Beta1 integrin-dependent engulfment of Yersinia enterocolitica by macrophages is coupled to the activation of autophagy and suppressed by type III protein secretion.

Autophagy is a central lysosomal degradation process that is essential for the maintenance of cellular homeostasis. Autophagy has furthermore emerged as integral part of the host immune response. Autophagic processes promote the separation and degradation of intracellular microorganisms which contributes to the development of innate and adaptive immunity. Some pathogenic microbes have therefore evolved mechanisms to evade or impede autophagy. We analyzed the effects of the enteropathogenic bacterium Yersinia enterocolitica on autophagy in macrophages. Yersiniae use a number of defined adhesins and secreted proteins to manipulate host immune responses. Our results showed that Y. enterocolitica defective in type III protein secretion efficiently activated autophagy in macrophages. Autophagy was mediated by the Yersinia adhesins invasin and YadA and particularly depended on the engagement of beta(1) integrin receptors. Several autophagy-related events followed beta(1) integrin-mediated engulfment of the bacteria including the formation of autophagosomes, processing of the marker protein LC3, redistribution of GFP-LC3 to bacteria-containing vacuoles, and the segregation of intracellular bacteria by autophagosomal compartments. These results provide direct evidence for the linkage of beta(1) integrin-mediated phagocytosis and autophagy induction. Multiple microbes signal through integrin receptors, and our results suggest a general principle by which the sensing of an extracellular microbe triggers autophagy. Owing to the importance of autophagy as host defense response, wild-type Y. enterocolitica suppressed autophagy by mobilizing type III protein secretion. The subversion of autophagy may be part of the Y. enterocolitica virulence strategy that supports bacterial survival when beta(1) integrin-dependent internalization and autophagy activation by macrophages are deleterious for the pathogen.

Cross-talk between type three secretion system and metabolism in Yersinia.

Pathogenic yersiniae utilize a type three secretion system (T3SS) to inject Yop proteins into host cells in order to undermine their immune response. YscM1 and YscM2 proteins have been reported to be functionally equivalent regulators of the T3SS in Yersinia enterocolitica. Here, we show by affinity purification, native gel electrophoresis and small angle x-ray scattering that both YscM1 and YscM2 bind to phosphoenolpyruvate carboxylase (PEPC) of Y. enterocolitica. Under in vitro conditions, YscM1, but not YscM2, was found to inhibit PEPC with an apparent IC(50) of 4 mum (K(i) = 1 mum). To analyze the functional roles of PEPC, YscM1, and YscM2 in Yop-producing bacteria, cultures of Y. enterocolitica wild type and mutants defective in the formation of PEPC, YscM1, or YscM2, respectively, were grown under low calcium conditions in the presence of [U-(13)C(6)]glucose. The isotope compositions of secreted Yop proteins and nine amino acids from cellular proteins were analyzed by mass spectrometry. The data indicate that a considerable fraction of oxaloacetate used as precursor for amino acids was derived from [(13)C(3)]phosphoenolpyruvate by the catalytic action of PEPC in the wild-type strain but not in the PEPC(-) mutant. The data imply that PEPC is critically involved in replenishing the oxaloacetate pool in the citrate cycle under virulence conditions. In the YscM1(-) and YscM2(-) mutants, increased rates of pyruvate formation via glycolysis or the Entner-Doudoroff pathway, of oxaloacetate formation via the citrate cycle, and of amino acid biosynthesis suggest that both regulators trigger the central metabolism of Y. enterocolitica. We propose a "load-and-shoot cycle" model to account for the cross-talk between T3SS and metabolism in pathogenic yersiniae.

Yersinia enterocolitica Yop mutants as oral live carrier vaccines.

Attenuated enteropathogenic yersiniae that translocate heterologous antigens into the cytosol of antigen presenting cells via their type three secretion system (TTSS) are considered promising candidates for the development of live oral vaccine carrier strains that induce CD8 T cell responses. Wild type Yersinia enterocolitica of serotype O:8 however efficiently suppresses the immune response of the host by translocating effector proteins called Yersinia outer proteins (Yops) into the cytosol of immune cells. We therefore tested immunogenicity, protective efficacy, and virulence ofyop mutants that translocate the model antigen Listeriolysin (LLO) of Listeria monocytogenes in a mouse model. A deltayopP mutant-based vaccine carrier strain induced the highest numbers of LLO91-99-specific CD8 T cells and effectively protected mice against a lethal challenge with Listeria whereas deltayopPT, deltayopPV(K42Q), and deltayopPO mutants of Y. enterocolitica induced fewer CD8 T cells and conferred only partial protection. The deltayopPH, deltayopPE, deltayopPM, and deltayopPQ mutants induced the weakest CD8 T cell response and did not significantly protect mice against Listeria presumably due to the strong attenuation of these strains in the mouse model. Even though a Y. enterocolitica strain WA-C(pTTSS), which translocated only LLO (but not Yops), induced superior MHC class I-restricted antigen presentation in DC compared to the deltayopP mutants in vitro, this strain was not able to significantly colonize mouse tissue or to induce CD8 T cell responses in vivo. The success in designing a Yersinia oral vaccine carrier is therefore dependent to a great extent on the subtle balance between immunogenicity and attenuation.

Unusual, virulence plasmid-dependent growth behavior of Yersinia enterocolitica in three-dimensional collagen gels.

As a first approach to establishing a three-dimensional culture infection model, we studied the growth behavior of the extracellular pathogen Yersinia enterocolitica in three-dimensional collagen gels (3D-CoG). Surprisingly, we observed that plasmidless Y. enterocolitica was motile in the 3D-CoG in contrast to its growth in traditional motility agar at 37 degrees C. Motility at 37 degrees C was abrogated in the presence of the virulence plasmid pYV or the exclusive expression of the pYV-located Yersinia adhesion gene yadA. YadA-producing yersiniae formed densely packed (dp) microcolonies, whereas pYVDelta yadA-carrying yersiniae formed loosely packed microcolonies at 37 degrees C in 3D-CoG. Furthermore, we demonstrated that the packing density of the microcolonies was dependent on the head domain of YadA. Moreover, dp microcolony formation did not depend on the capacity of YadA to bind to collagen fibers, as demonstrated by the use of yersiniae producing collagen nonbinding YadA. By using a yopE-gfp reporter, we demonstrated Ca(2+)-dependent expression of this pYV-localized virulence gene by yersiniae in 3D-CoG. In conclusion, this study revealed unique plasmid-dependent growth behavior of yersiniae in a three-dimensional matrix environment that resembles the behavior of yersiniae (e.g., formation of microcolonies) in infected mouse tissue. Thus, this 3D-CoG model may be a first step to a more complex level of in vitro infection models that mimic living tissue, enabling us to study the dynamics of pathogen-host cell interactions.

Yersinia as oral live carrier vaccine: influence of Yersinia outer proteins (Yops) on the T-cell response.

Attenuated enteropathogenic Yersinia strains are attractive candidates for the development of oral live carrier vaccines. Yersiniae colonize the small intestine and invade lymphoid tissue of the terminal ileum where they replicate extracellularly. Yersiniae can be engineered to secrete or translocate heterologous antigens into the cytosol of antigen-presenting cells by their type 3 secretion system (T3SS). This results in the induction of both cellular and humoral immune responses to heterologous antigens of viral, bacterial and parasitic origin. In this review, we summarize the progress in developing Yersinia-based vaccine carrier strains by mutating the T3SS effector proteins of Yersinia called Yops (Yersinia outer proteins) to both attenuate the strains and to modulate the T-cell response.

Salmonella type III-mediated heterologous antigen delivery: a versatile oral vaccination strategy to induce cellular immunity against infectious agents and tumors.

Salmonella type III secretion system (T3SS)-mediated translocation can be used for efficient delivery of heterologous antigens to the cytosol of antigen-presenting cells leading to prominent CD8 T-cell priming in orally immunized mice. The time point and duration of hybrid protein translocation during the Salmonella infection cycle can be modulated by employing various type III carrier molecules. The p60 protein of Listeria monocytogenes was used as model antigen to construct chimeric SspH2/p60. SspH2 is a "Salmonella pathogenicity island 2" (SPI2) protein that is known to be translocated by Salmonella during intracellular survival and replication in macrophages. This SPI2 carrier molecule is sufficient to induce a concomitant p60-specific CD4 and CD8 T-cell response in Salmonella-vaccinated mice. Moreover, T3SS-mediated antigen delivery results in an efficient priming of central and effector memory CD8 T cells in spleens of these animals. This vaccination strategy can also be employed to efficiently protect mice from an aggressive fibrosarcoma transfected with p60 in a prophylactic setting.

Invasion and dissemination of Yersinia enterocolitica in the mouse infection model.

Yersinia enterocolitica is one of the most common causes of food borne gastrointestinal disease. After oral uptake yersiniae replicate in the small intestine, invade Peyer's patches of the distal ileum and disseminate to spleen and liver. In these tissues and organs yersiniae replicate extracellularly and form exclusively monoclonal microabscesses. Only very few yersiniae invade Peyer's patches and establish just a very few monoclonal microabscesses. This is due to both Yersinia and host specific factors.

The Yersinia enterocolitica type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1.

BACKGROUND: Pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) share a virulence plasmid encoding a type three secretion system (T3SS). This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins), the specific Yop chaperones (Sycs), and the Ysc (Yop secretion) proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study. RESULTS: We have established the large-scale production of recombinant SycO in its outright form. We confirm that Y. enterocolitica SycO forms homodimers which is typical for Syc chaperones. SycO overproduction in Y. enterocolitica decreases secretion of Yops into the culture supernatant suggesting a regulatory role of SycO in type III secretion. We demonstrate that in vitro SycO interacts with YscM1, a negative regulator of Yop expression in Y. enterocolitica. However, the SycO overproduction phenotype was not mediated by YscM1, YscM2, YopO or YopP as revealed by analysis of isogenic deletion mutants. CONCLUSION: We present evidence that SycO is integrated into the regulatory network of the Yersinia T3SS. Our picture of the Yersinia T3SS interactome is supplemented by identification of the SycO/YscM1 interaction. Further, our results suggest that at least one additional interaction partner of SycO has to be identified.

Serogroup-related escape of Yersinia enterocolitica YopE from degradation by the ubiquitin-proteasome pathway.

Pathogenic Yersinia spp. employ a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell to modify the host immune response. One strategy of the infected host cell to resist the bacterial attack is degradation and inactivation of injected bacterial virulence proteins through the ubiquitin-proteasome pathway. The cytotoxin YopE is a known target protein of this major proteolytic system in eukaryotic cells. Here, we investigated the sensitivity of YopE belonging to different enteropathogenic Yersinia enterocolitica serogroups to ubiquitination and proteasomal degradation. Analysis of the YopE protein levels in proteasome inhibitor-treated versus untreated cells revealed that YopE from the highly pathogenic Y. enterocolitica serotype O8 was subjected to proteasomal destabilization, whereas the YopE isotypes from serogroups O3 and O9 evaded degradation. Accumulation of YopE from serotypes O3 and O9 was accompanied by an enhanced cytotoxic effect. Using Yersinia strains that specifically produced YopE from either Y. enterocolitica O8 or O9, we found that only the YopE protein from serogroup O8 was modified by polyubiquitination, although both YopE isotypes were highly homologous. We determined two unique N-terminal lysines (K62 and K75) in serogroup O8 YopE, not present in serogroup O9 YopE, that served as polyubiquitin acceptor sites. Insertion of either lysine in serotype O9 YopE enabled its ubiquitination and destabilization. These results define a serotype-dependent difference in the stability and activity of the Yersinia effector protein YopE that could influence Y. enterocolitica pathogenesis.

Staphylococcus pettenkoferi sp. nov., a novel coagulase-negative staphylococcal species isolated from human clinical specimens.

Five coagulase-negative, novobiocin-susceptible staphylococcal strains were isolated from human blood cultures in different German and Belgian medical facilities. A novel species, 'Staphylococcus pettenkoferi' was proposed recently to accommodate two of these strains (B3117(T) and A6664), although the name was not validly published. All five strains belonged to the genus Staphylococcus because they were non-motile, Gram-positive, catalase-positive cocci with peptidoglycan type (A3 alpha type L-lys-gly(2-4)-L-Ser-Gly), menaquinone pattern (MK-7, MK-6 and MK-8) and major cellular fatty acids (ai-C(15 : 0), ai-C(17 : 0) and i-C(15 : 0)) that corresponded to those of staphylococci. Phenotypically, the isolates most closely resembled Staphylococcus capitis subsp. capitis and Staphylococcus auricularis, but they could be distinguished from these species by physiological tests and chemotaxonomic investigations. The results of DNA-DNA hybridization, chemotaxonomic investigations and 16S rRNA gene and RNA polymerase B gene (rpoB) sequence analysis enabled strains B3117(T), K6999, 229 and 230 to be differentiated genotypically and phenotypically from known Staphylococcus species, indicating that these isolates are representatives of a novel species. The name Staphylococcus pettenkoferi sp. nov. is proposed for this novel species, with strain B3117(T) (=CIP 107711(T)=CCUG 51270(T)) as the type strain. Due to differences in the results of physiological and chemotaxonomic investigations and DNA-DNA hybridization data, strain A6664 was not included in the description of the novel species.

Yersinia enterocolitica infection of mice reveals clonal invasion and abscess formation.

Yersinia enterocolitica is a common cause of food-borne gastrointestinal disease leading to self-limiting diarrhea and mesenteric lymphadenitis. Occasionally, focal abscess formation in the livers and spleens of certain predisposed patients (those with iron overload states such as hemochromatosis) is observed. In the mouse oral infection model, yersiniae produce a similar disease involving the replication of yersiniae in the small intestine, the invasion of Peyer's patches, and dissemination to the liver and spleen. In these tissues and organs, yersiniae are known to replicate predominantly extracellularly and to form microcolonies. By infecting mice orally with a mixture of equal amounts of green- and red-fluorescing yersiniae (yersiniae expressing green or red fluorescent protein), we were able to show for the first time that yersiniae produce exclusively monoclonal microcolonies in Peyer's patches, the liver, and the spleen, indicating that a single bacterium is sufficient to induce microcolony and microabscess formation in vivo. Furthermore, we present evidence for the clonal invasion of Peyer's patches from the small intestine. The finding that only very few yersiniae are required to establish microcolonies in Peyer's patches is due to both Yersinia-specific and host-specific factors. We demonstrate that yersiniae growing in the small intestinal lumen show strongly reduced levels of invasin, the most important factor for the early invasion of Peyer's patches. Furthermore, we show that the host severely restricts sequential microcolony formation in previously infected Peyer's patches.

The proteasome pathway destabilizes Yersinia outer protein E and represses its antihost cell activities.

Pathogenic Yersinia spp. neutralize host defense mechanisms by engaging a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell. Although the modulation of the cellular responses by individual Yops has been intensively studied, little is known about the fate of the translocated Yops inside the cell. In this study, we investigated involvement of the proteasome, the major nonlysosomal proteolytic system in eukaryotic cells, in Yop destabilization and repression. Our data show that inhibition of the proteasome in Yersinia enterocolitica-infected cells selectively stabilized the level of YopE, but not of YopH or YopP. In addition, YopE was found to be modified by ubiquitination. This suggests that the cytotoxin YopE is physiologically subjected to degradation via the ubiquitin-proteasome pathway inside the host cell. Importantly, the increased levels of YopE upon proteasome inhibition were associated with decreased activity of its cellular target Rac. Thus, the GTPase-down-regulating function of YopE is enhanced when the proteasome is inhibited. The stabilization of YopE by proteasome inhibitor treatment furthermore led to aggravation of the cytotoxic YopE effects on the actin cytoskeleton and on host cell morphology. Together, these data show that the host cell proteasome functions to destabilize and inactivate the Yersinia effector protein YopE. This implies the proteasome as integral part of the cellular host immune response against the immunomodulatory activities of a translocated bacterial virulence protein.

Yersinia's stratagem: targeting innate and adaptive immune defense.

In contrast to Salmonella and Shigella, enteropathogenic Yersinia species are extracellular multiplying Gram-negative bacteria. This life style requires a sophisticated anti-host strategy, which is implemented by the Yersinia virulence plasmid. This plasmid encodes the type 3 secretion system (injectisome), at least six microinjected anti-host effector proteins, a trimeric coiled coil outer membrane protein (Yersinia adhesin) with cell adhesin and protective functions against complement and defensins, and the released V antigen, which has Toll-like receptor 2 agonist activity.

Nosocomial urosepsis caused by Enterobacter kobei with aberrant phenotype.

Enterobacter kobei is the species of the Enterobacter cloacae complex, which is phenotypically most closely related to the species E. cloacae. This is the first report of infection caused by a new biotype of E. kobei. A patient with a history of urinary bladder operation developed a urosepsis with an Enterobacter isolate displaying the E. cloacae phenotype. The isolate was classified to the species E. kobei by sequence analysis of the 16S-rDNA, 4 protein-coding genes and enterobacterial repetitive intergenic consensus (ERIC)-cluster analysis. E. kobei was originally described to be Voges-Proskauer (VP) negative. However, the isolates of the present case were VP-positive. After analyzing 120 biochemical tests included in the API20E and the Biotype 100 systems, 4 biochemical tests were identified potentially differentiating this new biotype from E. cloacae.