Molecular mechanisms of Salmonella invasion: the type III secretion system of the pathogenicity island 1

Salmonella spp. are facultative intracellular pathogens which are able to enter into non-phagocytic cells as an essential step in their pathogenic life cycle. The majority of the molecular determinants involved in this entry process are encoded in a pathogenicity island located at the centisome 63 of the bacterial chromosome, and belong to a specialized protein secretion system termed “type III” or “contactdependent”. This secretion system is used by Salmonella spp. and several other bacterial pathogens to translocate bacterial effector proteins into the eukaryotic cell. Thus, a bidirectional biochemical cross-talk with the host cell is initiated, which leads to several responses such as membrane ruffling, bacterial internalization and the activation of various transcription factors

CD8 T-cell induction against vascular endothelial growth factor receptor 2 by Salmonella for vaccination purposes against a murine melanoma.

The Salmonella type III secretion system (T3SS) efficiently translocates heterologous proteins into the cytosol of eukaryotic cells. This leads to an antigen-specific CD8 T-cell induction in mice orally immunized with recombinant Salmonella. Recently, we have used Salmonella's T3SS as a prophylactic and therapeutic intervention against a murine fibrosarcoma. In this study, we constructed a recombinant Salmonella strain translocating the immunogenic H-2D(b)-specific CD8 T-cell epitope VILTNPISM (KDR2) from the murine vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 is a member of the tyrosine protein kinase family and is upregulated on proliferating endothelial cells of the tumor vasculature. After single orogastric vaccination, we detected significant numbers of KDR2-tetramer-positive CD8 T cells in the spleens of immunized mice. The efficacy of these cytotoxic T cells was evaluated in a prophylactic setting to protect mice from challenges with B16F10 melanoma cells in a flank tumor model, and to reduce dissemination of spontaneous pulmonary melanoma metastases. Vaccinated mice revealed a reduction of angiogenesis by 62% in the solid tumor and consequently a significant decrease of tumor growth as compared to non-immunized mice. Moreover, in the lung metastasis model, immunization with recombinant Salmonella resulted in a reduction of the metastatic melanoma burden by approximately 60%

A rapid change in virulence gene expression during the transition from the intestinal lumen into tissue promotes systemic dissemination of Salmonella.

Bacterial pathogens causing systemic disease commonly evolve from organisms associated with localized infections but differ from their close relatives in their ability to overcome mucosal barriers by mechanisms that remain incompletely understood. Here we investigated whether acquisition of a regulatory gene, tviA, contributed to the ability of Salmonella enterica serotype Typhi to disseminate from the intestine to systemic sites of infection during typhoid fever. To study the consequences of acquiring a new regulator by horizontal gene transfer, tviA was introduced into the chromosome of S. enterica serotype Typhimurium, a closely related pathogen causing a localized gastrointestinal infection in immunocompetent individuals. TviA repressed expression of flagellin, a pathogen associated molecular pattern (PAMP), when bacteria were grown at osmotic conditions encountered in tissue, but not at higher osmolarity present in the intestinal lumen. TviA-mediated flagellin repression enabled bacteria to evade sentinel functions of human model epithelia and resulted in increased bacterial dissemination to the spleen in a chicken model. Collectively, our data point to PAMP repression as a novel pathogenic mechanism to overcome the mucosal barrier through innate immune evasion

Vanishing Twist near Focus-Focus Points

We show that near a focus-focus point in a Liouville integrable Hamiltonian system with two degrees of freedom lines of locally constant rotation number in the image of the energy-momentum map are spirals determined by the eigenvalue of the equilibrium. From this representation of the rotation number we derive that the twist condition for the isoenergetic KAM condition vanishes on a curve in the image of the energy-momentum map that is transversal to the line of constant energy. In contrast to this we also show that the frequency map is non-degenerate for every point in a neighborhood of a focus-focus point.Comment: 13 page

Clinical Study The Use of Interferon Gamma Release Assays in the Diagnosis of Active Tuberculosis

Interferon gamma release assays (IGRAs) are in vitro immunologic diagnostic tests used to identify Mycobacterium tuberculosis infection. They cannot differentiate between latent and active infections. The cutoff suggested by the manufacturer is 0.35 IU/mL for latent tuberculosis. As IGRA tests were recently approved for the differential diagnosis of active tuberculosis, we assessed the diagnostic accuracy of the latest generation IGRA for detection of active tuberculosis in a low-incidence area in Germany. Our consecutive case series includes 61 HIV negative, Mycobacterium tuberculosis culture positive patients, as well as 234 control patients. The retrospective analysis was performed over a period of two years. In 11/61 patients with active tuberculosis (18.0%) the test result was <0.35 IU/mL, resulting in a sensitivity of 0.82. We recommend establishing a new cut-off value for the differential diagnosis of active tuberculosis assessed by prospective clinical studies and in various regions with high and low prevalence of tuberculosis

Concomitant Cytosolic Delivery of Two Immunodominant Listerial Antigens by Salmonella enterica Serovar Typhimurium Confers Superior Protection against Murine Listeriosis

During its interaction with host cells, Salmonella enterica serovar Typhimurium employs a type III secretion system for cytosolic targeting of virulence factors. This protein translocation mechanism is a useful tool for heterologous antigen delivery by attenuated Salmonella vaccine carrier strains. In the present study, we used the Yersinia outer protein E (YopE) as a carrier molecule for Salmonella type III-dependent cytosolic delivery of the immunodominant CD8 T-cell antigens listeriolysin O (LLO) and p60 of Listeria monocytogenes. It is shown that concomitant translocation of hybrid YopE/LLO and YopE/p60 proteins by Salmonella led to antigen presentation and CD8 T-cell priming efficacies comparable to those of translocation of single listerial antigens. However, simultaneous translocation of LLO and p60 significantly surpassed single cytosolic antigen delivery in the ability to protect against Listeria. For the first time, this study demonstrates that concomitant expression of two independent antigens via the same recombinant plasmid leads to superior protection against a challenge with an intracellular bacterial pathogen. In conclusion, these findings emphasize the versatility of Salmonella type III-mediated heterologous antigen delivery for the induction of cytotoxic T-lymphocyte-mediated immunity

Oral Vaccination with Recombinant Yersinia enterocolitica Expressing Hybrid Type III Proteins Protects Gerbils from Amebic Liver Abscess

Protection against invasive amebiasis was achieved in the gerbil model for amebic liver abscess by oral immunization with live attenuated Yersinia enterocolitica expressing the Entamoeba histolytica galactose-inhibitable lectin that has been fused to the Yersinia outer protein E (YopE). Protection was dependent on the presence of the YopE translocation domain but was independent from the antibody response to the ameba lectin

Evaluation of the Novel Helicobacter pylori ClariRes Real-Time PCR Assay for Detection and Clarithromycin Susceptibility Testing of H. pylori in Stool Specimens from Symptomatic Children▿

The aim of the present study was to evaluate the Helicobacter pylori ClariRes assay (Ingenetix, Vienna, Austria) for the detection of H. pylori infection and the simultaneous clarithromycin susceptibility testing of the H. pylori isolates in stool samples from 100 symptomatic children. The results obtained by this novel biprobe real-time PCR method were directly compared with the results obtained from histological examination of gastric biopsy specimens, culturing, the [13C]urea breath test, and a monoclonal antibody-based stool antigen enzyme immunoassay (EIA). Fecal specimens from all 54 children who were shown to be noninfected by “gold standard” tests gave true-negative PCR results (specificity, 100%). Of the remaining 46 individuals with a positive H. pylori status, 29 were found to be positive by real-time PCR (sensitivity, 63%). For these 29 cases, the H. pylori ClariRes assay confirmed all results from phenotypic clarithromycin susceptibility testing by Etest. In summary, this investigation demonstrates that detection of Helicobacter DNA in stool samples by real-time PCR is a difficult task and that this method cannot replace the stool antigen EIA (sensitivity, 95.7%) for the accurate diagnosis of H. pylori infection in children