Type III secretion: The bacteria-eukaryotic cell express

Type III secretion (T3S) is an export pathway used by Gram-negative pathogenic bacteria to inject bacterial proteins into the cytosol of eukaryotic host cells. This pathway is characterized by (i) a secretion nanomachine related to the bacterial flagellum, but usually topped by a stiff needle-like structure; (ii) the assembly in the eukaryotic cell membrane of a translocation pore formed by T3S substrates; (iii) a non-cleavable N-terminal secretion signal; (iv) T3S chaperones, assisting the secretion of some substrates; (v) a control mechanism ensuring protein delivery at the right place and time. Here, we review these different aspects focusing in open questions that promise exciting findings in the near futur

Structural basis for selective targeting of Rac subfamily GTPases by a bacterial effector protein

Ras-homology (Rho) family GTPases are conserved molecular switches controlling fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, molecular mechanisms of target selectivity have remained elusive. Here, we report a bacterial effector protein that targets all four Rac subfamily members of Rho family GTPases, but none of the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 facilitate modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural paradigm for target selectivity towards Rac subfamily GTPases and provides a highly selective tool for their functional analysis

Деякі проблеми використання тимчасово зайнятих земель

<div><p>Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype <em>Yersinia enterocolitica</em> which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different <em>Yersinia</em> mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, <em>Clostridium difficile</em> toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal <em>GILZ</em> promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the <em>GILZ</em> promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of <em>GILZ</em> promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.</p> </div

Soft windowing application to improve analysis of high-throughput phenotyping data.

MOTIVATION: High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors. RESULTS: Here we introduce \u27soft windowing\u27, a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources. AVAILABILITY AND IMPLEMENTATION: The method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

Cooperation, competition and antibiotic resistance in bacterial colonies

Bacteria commonly live in dense and genetically diverse communities associated with surfaces. In these communities, competition for resources and space is intense, and yet we understand little of how this affects the spread of antibiotic- resistant strains. Here, we study interactions between antibiotic-resistant and susceptible strains using in vitro competition experiments in the opportunistic pathogen Pseudomonas aeruginosa and in silico simulations. Selection for intracellular resistance to streptomycin is very strong in colonies, such that resistance is favoured at very low antibiotic doses. In contrast, selection for extracellular resistance to carbenicillin is weak in colonies, and high doses of antibiotic are required to select for resistance. Manipulating the density and spatial structure of colonies reveals that this difference is partly explained by the fact that the local degradation of carbenicillin by β-lactamase-secreting cells protects neighbouring sensitive cells from carbenicillin. In addition, we discover a second unexpected effect: the inducible elongation of cells in response to carbenicillin allows sensitive cells to better compete for the rapidly growing colony edge. These combined effects mean that antibiotic treatment can select against antibiotic-resistant strains, raising the possibility of treatment regimes that suppress sensitive strains while limiting the rise of antibiotic resistance. We argue that the detailed study of bacterial interactions will be fundamental to understanding and overcoming antibiotic resistance

Structural basis for selective AMPylation of Rac-subfamily GTPases by Bartonella effector protein 1 (Bep1)

Small GTPases of the Ras-homology (Rho) family are conserved molecular switches that control fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, the molecular mechanism of target selectivity has remained largely elusive. Here we report a bacterial effector protein that selectively targets members of the Rac subfamily in the Rho family of small GTPases but none in the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from; Bartonella rochalimae; is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding G4 motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 enable modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural understanding of target selectivity toward Rac-subfamily GTPases and provides a highly selective tool for their functional analysis

Type III secretion:the bacteria-eukaryotic cell express

Type III secretion (T3S) is an export pathway used by Gram-negative pathogenic bacteria to inject bacterial proteins into the cytosol of eukaryotic host cells. This pathway is characterized by (i) a secretion nanomachine related to the bacterial flagellum, but usually topped by a stiff needle-like structure; (ii) the assembly in the eukaryotic cell membrane of a translocation pore formed by T3S substrates; (iii) a non-cleavable N-terminal secretion signal; (iv) T3S chaperones, assisting the secretion of some substrates; (v) a control mechanism ensuring protein delivery at the right place and time. Here, we review these different aspects focusing in open questions that promise exciting findings in the near future

A Bartonella effector acts as signaling hub for intrinsic STAT3 activation to trigger anti-inflammatory responses

Chronically infecting pathogens avoid clearance by the innate immune system by promoting premature transition from an initial pro-inflammatory response toward an anti-inflammatory tissue-repair response. STAT3, a central regulator of inflammation, controls this transition and thus is targeted by numerous chronic pathogens. Here, we show that BepD, an effector of the chronic bacterial pathogen Bartonella henselae targeted to infected host cells, establishes an exceptional pathway for canonical STAT3 activation, thereby impairing secretion of pro-inflammatory TNF-α and stimulating secretion of anti-inflammatory IL-10. Tyrosine phosphorylation of EPIYA-related motifs in BepD facilitates STAT3 binding and activation via c-Abl-dependent phosphorylation of Y705. The tyrosine-phosphorylated scaffold of BepD thus represents a signaling hub for intrinsic STAT3 activation that is independent from canonical STAT3 activation via transmembrane receptor-associated Janus kinases. We anticipate that our findings on a molecular shortcut to STAT3 activation will inspire new treatment options for chronic infections and inflammatory diseases

CHC regulates NF-κB activation independently of endocytosis and CLCa.

<p>(<b>A</b>) Uptake of Alexa 594-transferrin (Alexa 594-Tf) in cells transfected with control (left panels), AP2M1 (middle panels) or CHC (right panels) siRNAs; Scale bars, 10 µm. (<b>B</b>) Quantification of transferrin uptake by automated image analysis (results are expressed as the mean ± SD of 12 images; graph representative of 2 independent experiments). (<b>C</b>) AP2M1 knockdown fails to enhance IκBα degradation. Cell lysates from control, AP2M1 or CHC siRNA-transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as a loading control. (<b>D</b>) Densitometric quantification of the levels of IκBα shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017158#pone-0017158-g004" target="_blank">Figure 4C</a> (graph representative of 2 independent experiments). (<b>E</b>) AP2M1 knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, AP2M1 or CHC siRNAs for 72 hours. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (<b>F</b>) Inhibition of transferrin uptake after dynasore and PAO treatment. HeLa cells were left untreated (Ctrl) or treated with dynasore (80 µM) (Dyn) or PAO (5 µM) 10 minutes before and during the transferrin uptake assay (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (<b>G</b>) Dynasore and PAO fail to enhance basal degradation of IκBα. HeLa cells were pretreated for 10 minutes with dynasore (80 µM) or PAO (5 µM) and analyzed by western immunoblotting using an IκBα antibody. Actin is shown as a loading control (results representative of 2 independent experiments). (<b>H</b>) Long-term inhibition of endocytosis in dynasore-treated HeLa cells. Transferrin uptake in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (<b>I</b>) Long-term inhibition of endocytosis fails to enhance the basal degradation of IκBα. Basal degradation of IκBα in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours. As positive control of the degradation of IκBα, cells were stimulated for 20 minutes with TNFα (results representative of 2 independent experiments). (<b>J</b>) CLCa knockdown fails to enhance IκBα degradation. Cell lysates from control, CLCa or CHC siRNA transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as loading control. (<b>K</b>) Densitometric quantification of IκBα levels shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017158#pone-0017158-g004" target="_blank">Figure 4F</a> (Graph representative of 2 independent experiments). (<b>L</b>) CLCa knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, CLCa or CHC siRNAs for 72 hours and supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).</p