Differential regulation of physiological activities by RcsB and OmpR in Yersinia enterocolitica.

A thorough understanding of the mechanisms of Rcs and EnvZ/OmpR phosphorelay systems that allow Yersinia enterocolitica to thrive in various environments is crucial to prevent and control Y. enterocolitica infections. In this study, we showed that RcsB and OmpR have the ability to function differently in modulating a diverse array of physiological processes in Y. enterocolitica. The rcsB mutant stimulated flagella biosynthesis and increased motility, biofilm formation and c-di-GMP production by upregulating flhDC, hmsHFRS and hmsT. However, mutation in ompR exhibited a non-motile phenotype due to the lack of flagella. Biofilm formation was reduced and less c-di-GMP was produced through the downregulation of flhDC, hmsHFRS and hmsT expression when Y. enterocolitica was exposed to low osmolarity conditions. Furthermore, OmpR was identified to be important for Y. enterocolitica to grow in extreme temperature conditions. Importantly, ompR mutations in Y. enterocolitica were more sensitive to polymyxin B and sodium dodecyl sulfate than rcsB mutations. Since motility, biofilm formation and environmental tolerance are critical for bacterial colonization of the host, these findings indicated that OmpR is more critical than RcsB in shaping the pathogenic phenotype of Y. enterocolitica.

Function of pYV Plasmid on Biofilm Formation of Yersinia enterocolitica ERL032123 in the Presence of Ca2.

The effect of the virulence plasmid pYV and calcium ions on biofilm of Yersinia enterocolitica biofilm formation was determined using a microtiter plate assay. Loss of the pYV plasmid prevented biofilm formation and the presence of Ca2+ enhanced biofilm formation in cultures containing the pYV plasmid. Scanning electron microscopy supported the result from the microtiter plate assay showing that in the presence of Ca2+, the wild-type Y. enterocolitica strain formed a strong biofilm on a polycarbonate surface. The results implied that Ca2+ promotes Y. enterocolitica biofilm formation through the function of the pYV plasmid.

Antimicrobial activity and mechanism of the human milk-sourced peptide Casein201.

INTRODUCTION: Casein201 is one of the human milk sourced peptides that differed significantly in preterm and full-term mothers. This study is designed to demonstrate the biological characteristics, antibacterial activity and mechanisms of Casein201 against common pathogens in neonatal infection. METHODOLOGY: The analysis of biological characteristics was done by bioinformatics. Disk diffusion method and flow cytometry were used to detect the antimicrobial activity of Casein201. Killing kinetics of Casein201 was measured using microplate reader. The antimicrobial mechanism of Casein201 was studied by electron microscopy and electrophoresis. RESULTS: Bioinformatics analysis indicates that Casein201 derived from ß-casein and showed significant sequence overlap. Antibacterial assays showed Casein201 inhibited the growth of S taphylococcus aureus and Y ersinia enterocolitica. Ultrastructural analyses revealed that the antibacterial activity of Casein201 is through cytoplasmic structures disintegration and bacterial cell envelope alterations but not combination with DNA. CONCLUSION: We conclude the antimicrobial activity and mechanism of Casein201. Our data demonstrate that Casein201 has potential therapeutic value for the prevention and treatment of pathogens in neonatal infection.

Virulence-related genes, adhesion and invasion of some Yersinia enterocolitica-like strains suggests its pathogenic potential.

Yersina enterocolitica-like species have not been extensively studied regarding its pathogenic potential. This work aimed to assess the pathogenic potential of some Y. enterocolitica-like strains by evaluating the presence of virulence-related genes by PCR and their ability to adhere to and invade Caco-2 and HEp-2 cells. A total of 50 Y. frederiksenii, 55 Y. intermedia and 13 Y. kristensenii strains were studied. The strains contained the following genes: Y. frederiksenii, fepA(44%), fes(44%) and ystB(18%); Y. intermedia, ail(53%), fepA (35%), fepD(2%), fes(97%), hreP(2%), ystB(2%) and tccC(35%); Y. kristensenii, ail(62%), ystB(23%), fepA(77%), fepD(54%), fes(54%) and hreP(77%). Generally, the Y. enterocolitica-like strains had a reduced ability to adhere to and invade mammalian cells compared to the highly pathogenic Y. enterocolitica 8081. However, Y. kristensenii FCF410 and Y. frederiksenii FCF461 presented high invasion potentials in Caco-2 cells after five days of pre-incubation increased by 45- and 7.2-fold compared to Y. enterocolitica 8081, respectively; but, the ail gene was not detected in these strains. The presence of virulence-related genes in some of the Y. enterocolitica-like strains indicated their possible pathogenic potential. Moreover, the results suggest the existence of alternative virulence mechanisms and that the pathogenicity of Y. kristensenii and Y. frederiksenii may be strain-dependent.

Genomic Insights and Its Comparative Analysis with Yersinia enterocolitica Reveals the Potential Virulence Determinants and Further Pathogenicity for Foodborne Outbreaks.

Yersinia enterocolitica is a well-known foodborne pathogen causing gastrointestinal infections worldwide. The strain Y. enterocolitica FORC_002 was isolated from the gill of flatfish (plaice) and its genome was sequenced. The genomic DNA consists of 4,837,317 bp with a GC content of 47.1%, and is predicted to contain 4,221 open reading frames, 81 tRNA genes, and 26 rRNA genes. Interestingly, genomic analysis revealed pathogenesis and host immune evasion-associated genes encoding guanylate cyclase (Yst), invasin (Ail and Inv), outer membrane protein (Yops), autotransporter adhesin A (YadA), RTX-like toxins, and a type III secretion system. In particular, guanylate cyclase is a heat-stable enterotoxin causing Yersinia-associated diarrhea, and RTX-like toxins are responsible for attachment to integrin on the target cell for cytotoxic action. This genome can be used to identify virulence factors that can be applied for the development of novel biomarkers for the rapid detection of this pathogen in foods.

Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome.

Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells. While the structure of the distal "needle complex" is well documented, the composition and role of the functionally important cytosolic complex remain less well understood. Using functional fluorescent fusions, we found that the C-ring, an essential and conserved cytosolic component of the system, is composed of ~22 copies of SctQ (YscQ in Yersinia enterocolitica), which require the presence of YscQC, the product of an internal translation initiation site in yscQ, for their cooperative assembly. Photoactivated localization microscopy (PALM) reveals that in vivo, YscQ is present in both a free-moving cytosolic and a stable injectisome-bound state. Notably, fluorescence recovery after photobleaching (FRAP) shows that YscQ exchanges between the injectisome and the cytosol, with a t½ of 68 ± 8 seconds when injectisomes are secreting. In contrast, the secretin SctC (YscC) and the major export apparatus component SctV (YscV) display minimal exchange. Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion. The stabilization of the C-ring depends on the presence of the functional ATPase SctN (YscN). These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion.

Yersinia enterocolitica type III secretion injectisomes form regularly spaced clusters, which incorporate new machines upon activation.

Bacterial type III secretion systems or injectisomes are multiprotein complexes directly transporting bacterial effector proteins into eukaryotic host cells. To investigate the distribution of injectisomes in the bacterium and the influence of activation of the system on that distribution, we combined in vivo fluorescent imaging and high-resolution in situ visualization of Yersinia enterocolitica injectisomes by cryo-electron tomography. Fluorescence microscopy showed the injectisomes as regularly distributed spots around the bacterial cell. Under secreting conditions (absence of Ca(2+) ), the intensity of single spots significantly increased compared with non-secreting conditions (presence of Ca(2+) ), in line with an overall up-regulation of expression levels of all components. Single injectisomes observed by cryo-electron tomography tended to cluster at distances less than 100 nm, suggesting that the observed fluorescent spots correspond to evenly distributed clusters of injectisomes, rather than single injectisomes. The up-regulation of injectisome components led to an increase in the number of injectisomes per cluster rather than the formation of new clusters. We suggest that injectisome clustering may allow more effective secretion into the host cells.

[Presence of Yersinia enterocolitica in conditions of agro complex].

AIM: Detection of duration of existence of Yersinia enterocolitica in substrates ofagro complex and formation of biofilms by causative agent during artificial semination of forage and meat products. MATERIALS AND METHODS: Y. enterocolitica 09 strain and its rifampicin-resistant (Rmr) mutant were used. Microbial landscape of samples was studied by seeding on selective media (HiMedia), biochemical properties of isolates were controlled on API test-systems (Bio-Merieux). The presence ofyopA gene localized on virulence plasmid pCad was determined in PCR. Vital staining of biofilms was carried out by Live/Dead stain (Invitrogen, USA). Visualization of the data was registered by using GMS-510 (USA) microscope with digital camera and Skope Photo software (USA). Formation ofyersinia bacterial biofilms was confirmed by using scanning electron microscope (SEM) JSM6380 (Japan). RESULTS: Prolonged duration of existence of Y. enterocolitica in substrates of agro complex with conservation of pCad virulence plasmid by causative agent was detected. SEM demonstrated stages of biofilm formation during artificial semination of animal forage, meat products and materials of food equipment in a wide range of temperatures from 10 to 30 degrees C, and vital stain detected viable yersinia in mature biofilms. CONCLUSION: Agro complexes are a variant oftechnogenic foci where ecological conditions for prolonged existence of sapronosis are formed.

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.

Function and molecular architecture of the Yersinia injectisome tip complex.

By quantitative immunoblot analyses and scanning transmission electron microscopy (STEM), we determined that the needle of the Yersinia enterocolitica E40 injectisome consists of 139 +/- 19 YscF subunits and that the tip complex is formed by three to five LcrV monomers. A pentamer represented the best fit for an atomic model of this complex. The N-terminal globular domain of LcrV forms the base of the tip complex, while the central globular domain forms the head. Hybrids between LcrV and its orthologues PcrV (Pseudomonas aeruginosa) or AcrV (Aeromonas salmonicida) were engineered and recombinant Y. enterocolitica expressing the different hybrids were tested for their capacity to form the translocation pore by a haemolysis assay. There was a good correlation between haemolysis, insertion of YopB into erythrocyte membranes and interaction between YopB and the N-terminal globular domain of the tip complex subunit. Hence, the base of the tip complex appears to be critical for the functional insertion of YopB into the host cell membrane.

The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles.

Many pathogenic bacteria use injectisomes to deliver effector proteins into host cells through type III secretion. Injectisomes consist of a basal body embedded in the bacterial membranes and a needle. In Yersinia, translocation of effectors requires the YopB and YopD proteins, which form a pore in the target cell membrane, and the LcrV protein, which assists the assembly of the pore. Here we report that LcrV forms a distinct structure at the tip of the needle, the tip complex. This unique localization of LcrV may explain its crucial role in the translocation process and its efficacy as the main protective antigen against plague.

The needle length of bacterial injectisomes is determined by a molecular ruler.

Size determination represents a fundamental requirement for multicomponent biological structures. Some pathogenic bacteria possess a weapon derived from the flagellum. Like the flagellum, this type-III secretion apparatus, called the injectisome, has a transmembrane basal body, but the external component is a needle-like structure instead of a hook and a filament. Here, we provide evidence that the length of this needle is determined by the size of a protein, YscP, acting as a molecular ruler.

Genetic analysis of the formation of the Ysc-Yop translocation pore in macrophages by Yersinia enterocolitica: role of LcrV, YscF and YopN.

The Ysc-Yop type III secretion (TTS) system allows extracellular Yersinia bacteria, adhering to eukaryotic target cells, to inject Yop effector proteins in the cytosol of these cells. The secretion apparatus, called the injectisome, ends up with a needle-like structure made of YscF. YopN, one of the proteins secreted by the injectisome is thought to act as a plug. YopB, YopD and LcrV, three other proteins secreted by the injectisome and called 'translocators' form a pore allowing translocation of the Yop effectors across the target cell plasma membrane. Here, we tested the role of LcrV, YscF and YopN in the formation of this pore in macrophages by monitoring the release of the low-molecular-weight fluorescent dye BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, 623Da) and of the high-molecular-weight lactate dehydrogenase (LDH, 135 kDa). BCECF is released through the translocation pore itself provided no Yop effector is trafficking through the channel. In contrast, LDH is released by the osmotic lysis of the target cell that occurs after pore formation. This release is reduced by the GAP activity of YopE. In order to study the role of LcrV, one has to circumvent the regulatory effect of LcrV on the synthesis of YopB and YopD. We observed here that this regulatory role of LcrV is lost in a yopQ mutant and hence we studied the role of LcrV in a yopQ mutant background. A lcrV, yopQ double mutant was deficient in pore formation while able to produce YopB and YopD. Pore formation was restored by the introduction of lcrV(+) but not yopQ(+) confirming that LcrV itself is directly required for pore formation. Bacteria secreting only YopB, YopD and LcrV could form pores, showing that YopB, YopD and LcrV are sufficient for pore formation provided they are secreted by the same bacterium. LcrV is not involved in secretion of YopB and YopD as suggested previously. Bacteria producing normal Ysc injectisomes, including the YscF needle but no translocators did not form pores, indicating that the needle is not sufficient by itself for pore formation, as was also suggested. yopN mutant bacteria formed needles and released BCECF even if they secreted the effectors. This observation suggests that many translocation pores are not filled in the absence of YopN and thus that YopN might form a link between the needle and the pore, guiding the effectors.

Experimental mixed infection of rabbits with Yersinia enterocolitica and Listeria monocytogenes.

Experimental mixed infection was reproduced in rabbits after per os infection with Yersinia enterocolitica serotype 0:3 cells. Four days later some of animals were re-infected orally with Listeria monocytogenes serotype 4b cells. A third group of healthy rabbits was also infected per os with Listeria monocytogenes. The infectious process was followed dynamically from days 1-28. The experimental animals were examined for clinical, paraclinical and morphological findings. Augmentation of body temperature and alveolar macrophage number, a decreased number of peritoneal macrophages, leucopenia as well as purulent meningoencephalitis, catarrhal pneumonia, lienitis, lymphadenitis and enteritis were detected after experimental mixed infection. Both types of macrophages demonstrated a weak bactericidal activity against Yersinia enterocolitica and a highly expressed killing effect against Listeria monocytogenes. Yersinia and Listeria cells were isolated from the viscera and brain. Both species of bacteria were established intracellularly in the macrophages by electron-microscopic examination. The data received showed that mixed Yersinia enterocolitica 0:3 and Listeria monocytogenes 4b infection of rabbits runs with transitory hyperthermia as a generalized infection and is similar to the Listeria mono-infection. The immunosuppressive effect induced by oral Yersinia enterocolitica infection of rabbits promotes the expression of listerious agents.

Infection of synovial fibroblasts in culture by Yersinia enterocolitica and Salmonella enterica serovar Enteritidis: ultrastructural investigation with respect to the pathogenesis of reactive arthritis.

Synovial fibroblasts were infected with Yersinia enterocolitica or Salmonella enterica serovar Enteritidis and analyzed by electron microscopy and fluorescence in situ hybridization. Intracellular bacterial replication was followed by degradation leading to "ghosts" possessing lipopolysaccharides but not DNA. However, single bacteria survived for more than 2 weeks. Therefore, transient intra-articular infection might be the missing link between initial intestinal infection and late synovial inflammation in the pathogenesis of reactive arthritis.

Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells.

A number of pathogenic, Gram-negative bacteria are able to secrete specific proteins across three membranes: the inner and outer bacterial membrane and the eukaryotic plasma membrane. In the pathogen Yersinia enterocolitica, the primary structure of the secreted proteins as well as of the components of the secretion machinery, both plasmid-encoded, is known. However, the mechanism of protein translocation is largely unknown. Here we show that Y. enterocolitica polymerizes a 6-kDa protein of the secretion machinery into needles that are able to puncture the eukaryotic plasma membrane. These needles form a conduit for the transport of specific proteins from the bacterial to the eukaryotic cytoplasm, where they exert their cytotoxic activity. In negatively stained electron micrographs, the isolated needles were 60-80 nm long and 6-7 nm wide and contained a hollow center of about 2 nm. Our data indicate that it is the polymerization of the 6-kDa protein into these needles that provides the force to perforate the eukaryotic plasma membrane.

Interaction of Yersinia enterocolitica and Y. pseudotuberculosis with platelets.

Yersinia enterocolitica is a bacterium capable of growth at 4 degrees C in donated blood and has been responsible for many deaths following transfusion. Interaction of Y. enterocolitica with blood cells is of interest in understanding the mechanisms of survival and growth in blood. The closely related organism Y. pseudotuberculosis is known to invade platelets and cause platelet aggregation by a mechanism that involves expression of the chromosomal inv gene. Yersinia isolates were made to express green fluorescent protein (GFP) and their interaction with platelets was studied by flow cytometry, enterocolitica did not cause platelet aggregation or activation, not even when grown at 22 degrees C to maximise inv expression. Attachment of Y. enterocolitica O:9 to platelets occurred with virulence plasmid-bearing (pYV+) strains grown at 37 degrees C but not with pYV- strains nor with strains grown at 22 degrees C. Y. pseudotuberculosis containing inv did cause platelet activation and aggregation when grown at 22 degrees C, as has been shown before, but also showed enhanced attachment to platelets when grown at 37 degrees C. Electron microscopy studies confirmed that inv-expressing Y. pseudotuberculosis invaded platelets but Y. enterocolitica attached only to the outer surface of platelets. Interaction of Y. enterocolitica O:9 with platelets provided a modest protection against bacterial killing by human serum. Interaction of Y. enterocolitica O:9 with platelets does not lead to platelet invasion or activation, and is mediated through plasmid-coded factors, not inv.

Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins.

The non-fimbrial adhesins, YadA of enteropathogenic Yersinia species, and UspA1 and UspA2 of Moraxella catarrhalis, are established pathogenicity factors. In electron micrographs, both surface proteins appear as distinct 'lollipop'-shaped structures forming a novel type of surface projection on the outer membranes. These structures, amino acid sequence analysis of these molecules and yadA gene manipulation suggest a tripartite organization: an N-terminal oval head domain is followed by a putative coiled-coil rod and terminated by a C-terminal membrane anchor domain. In YadA, the head domain is involved in autoagglutination and binding to host cells and collagen. Analysis of the coiled-coil segment of YadA revealed unusual pentadecad repeats with a periodicity of 3.75, which differs significantly from the 3.5 periodicity found in the Moraxella UspAs and other canonical coiled coils. These findings predict that the surface projections are formed by oligomers containing right- (Yersinia) or left-handed (Moraxella) coiled coils. Strikingly, sequence comparison revealed that related proteins are found in many proteobacteria, both human pathogenic and environmental species, suggesting a common role in adaptation to specific ecological niches.

Infection of human enterocyte-like cells with rotavirus enhances invasiveness of Yersinia enterocolitica and Y. pseudotuberculosis.

Mixed infection with rotavirus and either Yersinia enterocolitica or Y. pseudotuberculosis was analysed in Caco-2 cells, an enterocyte-like cell line highly susceptible to these pathogens. Results showed an increase of bacterial adhesion and internalisation in rotavirus-infected cells. Increased internalisation was also seen with Escherichia coli strain HB101 (pRI203), harbouring the inv gene from Y. pseudotuberculosis, which is involved in the invasion process of host cells. In contrast, the superinfection with bacteria of Caco-2 cells pre-infected with rotavirus resulted in decreased viral antigen synthesis. Transmission electron microscopy confirmed the dual infection of enterocytes. These data suggest that rotavirus infection enhances the early interaction between host cell surfaces and enteroinvasive Yersinia spp.

The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility.

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen Yersinia enterocolitica can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as flgEF, flgMN, flhBA, and fliA, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by Y. enterocolitica to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon, flhDC, was cloned. Mutations within flhDC completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of flhDC indicated that it is required for the production of flagella. When flhDC was expressed from an inducible ptac promoter, flagellin production was shown to be dependent on levels of flhDC expression. Phenotypically, induction of the ptac-flhDC fusion also corresponded to increased levels of both swimming and swarming motility.