Functional dissection of translocon proteins of the Salmonella Pathogenicity Island 2-encoded type III secretion system

<p>Abstract</p> <p>Background</p> <p>Type III secretion systems (T3SS) are essential virulence factors of most Gram-negative bacterial pathogens. T3SS deliver effector proteins directly into the cytoplasm of eukaryotic target cells and for this function, the insertion of a subset of T3SS proteins into the target cell membrane is important. These proteins form hetero-oligomeric pores acting as translocon for the delivery of effector proteins. <it>Salmonella enterica </it>is a facultative intracellular pathogen that uses the <it>Salmonella </it>Pathogenicity Island 2 (SPI2)-encoded T3SS to manipulate host cells in order to survive and proliferate within the <it>Salmonella</it>-containing vacuole of host cells. Previous work showed that SPI2-encoded SseB, SseC and SseD act to form the translocon of the SPI2-T3SS.</p> <p>Results</p> <p>Here we investigated the structural requirements of SseB and SseD to form a functional translocon. Based on bioinformatic predictions, deletional analyses of SseB and SseD were performed and the effect on secretion by the T3SS, formation of a translocon, translocation of effector proteins and intracellular replication was investigated. Our data showed that both SseB and SseD are very sensitive towards alterations of the primary structure of the proteins. Although proteins encoded by mutant alleles were still secreted, we observed that all mutations resulted in a loss of function of the SPI2-T3SS.</p> <p>Conclusion</p> <p>These observations indicate that translocon proteins of the SPI2-T3SS are highly evolved towards the formation of multi-subunit complex in the host cell membrane. Structural alterations are not tolerated and abrogate translocon function.</p

Intersecting Knowledge Fields and Integrating Data-Driven Computational Design en Route to Performance-Oriented and Intensely Local Architectures

This paper discusses research by design efforts in architectural education, focused on developing concepts and methods for the design of performance-oriented and intensely local architectures. The pursued notion of performance foregrounds the interaction between a given architecture and its local setting, with consequences not only for the design product but also for the related processes by which it is generated. Integrated approaches to data-driven computational design serve to generate such designs. The outlined approach shifts the focus of design attention away from the delivery of finite architectural objects and towards an expanded range of architecture-environment interactions that are registered, instrumentalised and modulated over time. This paper examines ongoing efforts in integrating specific architectural goals and approaches, computational data-driven design methods and generative design processes, based on a range of context-specific and often real-time data sets. The work discussed is produced in the context of the Research Centre for Architecture and Tectonics (RCAT) and the Advanced Computational Design Laboratory (ACDL) at the Oslo School of Architecture and Design

Divergent Roles of Salmonella Pathogenicity Island 2 and Metabolic Traits during Interaction of S. enterica Serovar Typhimurium with Host Cells

The molecular mechanisms of virulence of the gastrointestinal pathogen Salmonella enterica are commonly studied using cell culture models of infection. In this work, we performed a direct comparison of the interaction of S. enterica serovar Typhimurium (S. Typhimurium) with the non-polarized epithelial cell line HeLa, the polarized cell lines CaCo2, T84 and MDCK, and macrophage-like RAW264.7 cells. The ability of S. Typhimurium wild-type and previously characterized auxotrophic mutant strains to enter host cells, survive and proliferate within mammalian cells and deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) was quantified. We found that the entry of S. Typhimurium into polarized cells was much more efficient than entry into non-polarized cells or phagocytic uptake. While SPI2-T3SS dependent intracellular proliferation was observed in HeLa and RAW cells, the intracellular replication in polarized cells was highly restricted and not affected by defective SPI2-T3SS. The contribution of aromatic amino acid metabolism and purine biosynthesis to intracellular proliferation was distinct in the various cell lines investigated. These observations indicate that the virulence phenotypes of S. Typhimurium are significantly affected by the cell culture model applied

Measurement of spin correlation in ttbar production using a matrix element approach

correlation, assuming that the spin of the top quark is either correlated with the spin of the anti-top quark as predicted by the standard model or is uncorrelated. For the first time we use a matrix-element-based approach to study ttbar spin correlation. We use {ttbar -> W+bW-bbar ->l+nubl-nub} final states produced in ppbar collisions at a center of mass energy sqrt(s)=1.96 TeV, where l denotes an electron or a muon. The data correspond to an integrated luminosity of 5.4 fb-1 and were collected with the dzero detector at the Fermilab Tevatron collider. The result agrees with the standard model prediction. We exclude the hypothesis that the spins of the ttbar are uncorrelated at the 97.7% C.L.Comment: 7 pages, 3 figures, submitted to Phys. Rev. Let

Structural Characterization of a Novel Chlamydia pneumoniae Type III Secretion-Associated Protein, Cpn0803

Type III secretion (T3S) is an essential virulence factor used by Gram-negative pathogenic bacteria to deliver effector proteins into the host cell to establish and maintain an intracellular infection. Chlamydia is known to use T3S to facilitate invasion of host cells but many proteins in the system remain uncharacterized. The C. trachomatis protein CT584 has previously been implicated in T3S. Thus, we analyzed the CT584 ortholog in C. pneumoniae (Cpn0803) and found that it associates with known T3S proteins including the needle-filament protein (CdsF), the ATPase (CdsN), and the C-ring protein (CdsQ). Using membrane lipid strips, Cpn0803 interacted with phosphatidic acid and phosphatidylinositol, suggesting that Cpn0803 may associate with host cells. Crystallographic analysis revealed a unique structure of Cpn0803 with a hydrophobic pocket buried within the dimerization interface that may be important for binding small molecules. Also, the binding domains on Cpn0803 for CdsN, CdsQ, and CdsF were identified using Pepscan epitope mapping. Collectively, these data suggest that Cpn0803 plays a role in T3S

O-Antigen Delays Lipopolysaccharide Recognition and Impairs Antibacterial Host Defense in Murine Intestinal Epithelial Cells

Although Toll-like receptor (TLR) 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs). Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS) recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S.) Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella

Coordinated Regulation of Virulence during Systemic Infection of Salmonella enterica Serovar Typhimurium

To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM) virulence and tested them in three virulence assays (intraperitoneal [i.p.], and intragastric [i.g.] infection in BALB/c mice, and persistence in 129X1/SvJ mice). Overall, 35 regulators were identified whose absence attenuated virulence in at least one assay, and of those, 14 regulators were required for systemic mouse infection, the most stringent virulence assay. As a first step towards understanding the interplay between a pathogen and its host from a systems biology standpoint, we focused on these 14 genes. Transcriptional profiles were obtained for deletions of each of these 14 regulators grown under four different environmental conditions. These results, as well as publicly available transcriptional profiles, were analyzed using both network inference and cluster analysis algorithms. The analysis predicts a regulatory network in which all 14 regulators control the same set of genes necessary for Salmonella to cause systemic infection. We tested the regulatory model by expressing a subset of the regulators in trans and monitoring transcription of 7 known virulence factors located within Salmonella pathogenicity island 2 (SPI-2). These experiments validated the regulatory model and showed that the response regulator SsrB and the MarR type regulator, SlyA, are the terminal regulators in a cascade that integrates multiple signals. Furthermore, experiments to demonstrate epistatic relationships showed that SsrB can replace SlyA and, in some cases, SlyA can replace SsrB for expression of SPI-2 encoded virulence factors

Measurement of the direct CP violating charge asymmetry in B-+/- -> mu(+/-)nu D-mu(0) decays

We present the first measurement of the CP violating charge asymmetry in B-+/- -> mu(+/-)nu D-mu(0) decays using the full Run II integrated luminosity of 10.4 fb(-1) in proton-antiproton collisions collected with the D0 detector at the Fermilab Tevatron Collider. We measure a difference in the yield of B- and B+ mesons in these decays by fitting the reconstructed invariant mass distributions. This results in an asymmetry of A(mu D0) = [-0.14 +/- 0.20] %, which is consistent with standard model predictions.Department of Energy (United States of America); National Science Foundation (United States of America); Alternative Energies and Atomic Energy Commission (France); National Center for Scientific Research/National Institute of Nuclear and Particle Physics (France); Ministry of Education and Science of the Russian Federation (Russia); National Research Center "Kurchatov Institute" of the Russian Federation (Russia); Russian Foundation for Basic Research (Russia); National Council for the Development of Science and Technology (Brazil); Carlos Chagas Filho Foundation for the Support of Research in the State of Rio de Janeiro (Brazil); Department of Science and Technology (India); Department of Atomic Energy (India); Administrative Department of Science, Technology and Innovation (Colombia); National Council of Science and Technology (Mexico); National Research Foundation of Korea (Korea); Foundation for Fundamental Research on Matter (The Netherlands); Science and Technology Facilities Council (United Kingdom); Royal Society (United Kingdom); Ministry of Education, Youth and Sports (Czech Republic); Bundesministerium fur Bildung und Forschung (Federal Ministry of Education and Research) (Germany); Deutsche Forschungsgemeinschaft (German Research Foundation) (Germany); Science Foundation Ireland (Ireland); Swedish Research Council (Sweden); China Academy of Sciences (China); National Natural Science Foundation of China (China); Ministry of Education and Science of Ukraine (Ukraine)This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Search for a fourth generation t' quark in ppbar collisions at sqrt{s}=1.96 TeV

We present a search for pair production of a fourth generation t' quark and its antiparticle, followed by their decays to a W boson and a jet, based on an integrated luminosity of 5.3/fb of proton-antiproton collisions at sqrt{s}=1.96 TeV collected by the D0 Collaboration at the Fermilab Tevatron Collider. We set upper limits on the t't'bar production cross section that exclude at the 95% C.L. a t' quark that decays exclusively to W+jet with a mass below 285 GeV. We observe a small excess in the muon+jets channel which reduces the mass range excluded compared to the expected limit of 320 GeV in the absence of a signal.Comment: submitted to Phys. Rev. Letter

Search for diphoton events with large missing transverse energy in 6.3 fb-1 of ppbar collisions at sqrt(s)=1.96 TeV

We report a search for diphoton events with large missing transverse energy produced in ppbar collisions at sqrt(s)=1.96 TeV. The data were collected with the D0 detector at the Fermilab Tevatron Collider, and correspond to 6.3 fb-1 of integrated luminosity. The observed missing transverse energy distribution is well described by the standard model prediction, and 95% C.L. limits are derived on two realizations of theories beyond the standard model. In a gauge mediated supersymmetry breaking scenario, the breaking scale Lambda is excluded for Lambda < 124 TeV. In a universal extra dimension model including gravitational decays, the compactification radius R_c is excluded for R_c-1 < 477 GeV.Comment: Submitted to Phys. Rev. Let