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

Cholesterol and host cell surface proteins contribute to cell-cell fusion induced by the <i>Burkholderia</i> type VI secretion system 5

<div><p>Following escape into the cytoplasm of host cells, <i>Burkholderia pseudomallei</i> and the related species <i>Burkholderia thailandensis</i> employ the type VI secretion system 5 (T6SS-5) to induce plasma membrane fusion with an adjacent host cell. This process leads to the formation of multinucleated giant cells and facilitates bacterial access to an uninfected host cell in a direct manner. Despite its importance in virulence, the mechanism of the T6SS-5 and the role of host cell factors in cell-cell fusion remain elusive. To date, the T6SS-5 is the only system of bacterial origin known to induce host-cell fusion. To gain insight into the nature of T6SS-5-stimulated membrane fusion, we investigated the contribution of cholesterol and proteins exposed on the host cell surface, which were shown to be critically involved in virus-mediated giant cell formation. In particular, we analyzed the effect of host cell surface protein and cholesterol depletion on the formation of multinucleated giant cells induced by <i>B</i>. <i>thailandensis</i>. Acute protease treatment of RAW264.7 macrophages during infection with <i>B</i>. <i>thailandensis</i> followed by agarose overlay assays revealed a strong reduction in the number of cell-cell fusions compared with EDTA treated cells. Similarly, proteolytic treatment of specifically infected donor cells or uninfected recipient cells significantly decreased multinucleated giant cell formation. Furthermore, modulating host cell cholesterol content by acute cholesterol depletion from cellular membranes by methyl- β-cyclodextrin treatment or exogenous addition of cholesterol impaired the ability of <i>B</i>. <i>thailandensis</i> to induce cell-cell fusions. The requirement of physiological cholesterol levels suggests that the membrane organization or mechanical properties of the lipid bilayer influence the fusion process. Altogether, our data suggest that membrane fusion induced by <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>thailandensis</i> involves a complex interplay between the T6SS-5 and the host cell.</p></div

Acute depletion of host cell surface proteins from RAW264.7 macrophages during infection reduces T6SS-5 stimulated cell-cell fusions.

<p>A) FACS histograms of RAW264.7 macrophages treated with 0.05% trypsin or 0.05% EDTA for 30 min at 37°C and immediately stained with α integrin β-2 (CD18) antibody conjugated to APC. Numbers in the panel indicate the mean fluorescence intensity (log scale). Representative results from one of two independent experiments are shown. B) Viability of macrophages after EDTA or trypsin treatment for 30 min at 37°C. Viability of detached cells was measured immediately after the treatment. Data are shown as mean + SD of three independent experiments performed in duplicate. *, <i>P</i> = 0.0458 (Welch’s t-test)/effect size: 0.346 (Glass’s Δ). C) MNGC formation of macrophages infected with <i>B</i>. <i>thailandensis</i> at an MOI of 50 for 3 h, at which point 0.05% trypsin or 0.05% EDTA was added for 30 min. Detached cells were collected and quantified and MNGC formation was determined at the indicated time points post seeding using an agarose overlay assay. Data represent the mean + SD of two independent experiments performed in duplicate. ***, <i>P</i> < 0.0001 (t-test)/effect size: 1.609 (Hedges’ <i>g</i>); ns, not significant, <i>P</i> value = 0.468 (Mann Whitney test)/effect size: 0.229 (Mann Whitney <i>r</i>). D) Infection of macrophages with <i>B</i>. <i>thailandensis</i> wild type (wt) and ΔT6SS-5 mutant at MOI 50 for 3 h and subsequent treatment with trypsin for 30 min. MNGC formation was determined of detached cells at the indicated time points using agarose overlay assays. Data are shown as mean + SD of two independent experiment performed in duplicate. ***, <i>P</i> < 0.0001 (Mann Whitney test)/effect size: 0.870 (Mann Whitney <i>r</i>); N/D, not detected.</p

Proteins located specifically on the surface of both donor (infected) and recipient (neighboring uninfected) RAW264.7 macrophages contribute to MNGC formation induced by <i>B</i>. <i>thailandensis</i>.

<p>A) and B) Schematic depicting acute protease treatment of donor cells and recipient cells, respectively. Green shapes indicate bacteria and black bars represent surface proteins. C) Macrophages infected with <i>B</i>. <i>thailandensis</i> at MOI 33 for approximately 6 h were treated with trypsin or EDTA for 30 min. Detached cells were mixed at equal ratios with uninfected and untreated macrophages and seeded at 6×10⁵ cells per well in a 24 well plate using agarose overlay assay. After incubating the cells for 3–4 h MNGC formation was quantified. Data represent the mean + SD of two independent experiments performed in duplicate. ***, <i>P</i> < 0.0001 (t-test)/effect size: 3.226 (Hedges’ <i>g</i>). D) Macrophages were infected with <i>B</i>. <i>thailandensis</i> at MOI 33 for approximately 6 h and mixed with trypsin or EDTA treated uninfected cells at equal ratios. The cells were seeded in a 24 well plate at 6×10⁵ cells per well and brought into contact through agarose overlay. Cell-cell fusions were calculated at 3–4 h post seeding. Data represent the mean + SD of three independent experiments performed in duplicate. *, <i>P</i> = 0.0463 (t-test)/effect size: 0.688 (Hedges’ <i>g</i>).</p

Acute treatment of RAW264.7 macrophages with the cholesterol extracting agent MβCD during infection decreases <i>B</i>. <i>thailandensis</i> mediated host cell fusions.

<p>A) Measurement of total cholesterol content of untreated (control) macrophages and cells treated with 10 mM MβCD for 1 h (MβCD) normalized to total protein content. Data are shown as mean + SD of two independent experiments performed in duplicate. ***, <i>P</i> < 0.0001 (t-test)/effect size: 4.327 (Hedges’ <i>g</i>). B) Viability of macrophages immediately after treatment with 10 mM MβCD for 1 h compared to untreated cells. Data represent mean + SD of two independent experiments performed in duplicate. ns, not significant <i>P</i> = 0.0438 (t-test)/effect size: 0.323 (Hedges’ <i>g</i>). C) MNGC formation of macrophages infected with <i>B</i>. <i>thailandensis</i> at an MOI of 17 and treated with 10 mM MβCD for 1 h shortly before the onset of MNGC formation. Shown are mean values + SD of four independent experiments performed in duplicate. **, <i>P</i> = 0.0027 (t-test)/effect size: 0.916 (Hedges’ <i>g</i>). D) MNGC formation induced by <i>B</i>. <i>thailandensis</i> wild type (wt) and ΔT6SS-5 mutant of macrophages infected and treated as described in C. Data shown are mean values + SD of two independent experiments performed in duplicate. ***, <i>P</i> < 0.0001 (Welch’s t-test)/effect size: 1.454 (Glass’s Δ). E) Schematic illustrating MβCD-based cholesterol extraction from infected donor cells. Green shape represents bacteria, yellow circles indicate cholesterol and the thin black lines denote ring-shaped heptamers formed by MβCD. F) MNGC formation of macrophages infected with <i>B</i>. <i>thailandensis</i> at MOI 33 for approximately 6 h and followed by MβCD (10 mM) treatment for 1 h. To these cells untreated and uninfected macrophages were added at equal ratios and MNGC formation was determined at 2–3 h post seeding. Data represent mean + SD of two independent experiments performed in duplicate. ***, <i>P</i> < 0.0001 (t-test)/effect size: 2.542 (Hedges’ <i>g</i>).</p

Role of Rho GTPases and MAP kinases for GILZ expression.

<p>(A) Overexpression of RhoA or RhoB lowers basal GILZ levels. HeLa cells were co-transfected with the p2088 <i>GILZ</i> promoter luciferase reporter and pHM6 based plasmid for overexpression of the indicated Rho GTPases. Means + SD of 3 independent experiments normalized to untreated. Significant differences compared to control vector transfection are indicated by asterisks (p<0.05). In a control experiment, HeLa cells were transfected in the same setting and cell lysates were used for immunoblots to detect RhoA, RhoB, Cdc42 and Rac1 expression. (B) HeLa cells were transfected for 48 h with indicated concentrations of siRNA. Immunoblots were performed from cell lysates for RhoA and RhoB and from cytosolic extracts for GILZ. (C) Toxin B treatment leads to fast and transient MAPK phosphorylation. After treatment of HeLa cells with toxin B for the indicated time spans, levels of phosphorylated as well as total ERK and p38 were assayed by immunoblot. (C) Toxin B induced GILZ expression is mediated by both ERK and p38 MAPK. Cells were pretreated with MAPK phosphorylation inhibitors SB 202190 (p38) or PD 98059 (ERK) 2 h prior to toxin B stimulation and GILZ protein was detected by immunoblot analysis at 6 h or 24 h after stimulation.</p

GILZ is expressed upon stimulation with C3 exotoxin or toxin B.

<p>(A) HeLa cells were incubated with toxin B (50ng/ml) for indicated the time spans and immunoblots were performed from whole cell lysates using anti-Rac1 mAb102 recognizing only non-glucosylated Rac-1, and an anti-Rac1 mAb antibody recognizing total Rac1 as well as antibodies recognizing RhoB, actin or GILZ. (B) Immunoblot of GILZ and actin expression upon stimulation of HeLa cells with C2IN-C3lim (100 ng/mL) + C2IIa (200 ng/mL) for indicated time spans. (C) To explore the expression of additional GILZ isoforms, HeLa cells were transfected with 7.5 nM siRNA specific for GILZ or control siRNA for 48 h and subsequently either left untreated or stimulated with <i>C. difficile</i> toxin B (50 ng/ml) or 100 µM DEX for 4 h. Arrows mark three GILZ isoforms which were inhibited by the used GILZ siRNA. Note that only isoform 1 was induced by the used stimuli. (D) Cells were stimulated with toxin B for 2 or 4 h to determine <i>GILZ</i> mRNA expression by real-time RT-PCR. Mean + SD of 2 independent experiments normalized to untreated. (E) To assay transcriptional activity of the GILZ promoter cells were transfected with a luciferase reporter under control of a 2088 bp <i>GILZ</i> promoter and co-transfected with pCMV-ß-gal (for standardization) 24 h before infection with a <i>Y. enterocolitica</i> pYV⁺ and various mutant strains or treatment with DEX or toxin B. Means + SD of 4 independent experiments normalized to untreated. Significant differences compared to untreated are indicated by asterisks (p<0.05).</p

<i>Yersinia enterocolitica</i> induces GILZ expression.

<p>HeLa cells were infected with a strain harboring the <i>Yersinia</i> virulence plasmid (pYV⁺) or plasmid cured strain (pYV⁻) with MOI 20 or stimulated with 100 µM DEX for indicated time intervals. The amount of GILZ in cytosolic proteins lysates of HeLa cells was detected by immunoblot at different time points. Actin was used as an internal standard. A representative experiment and quantification means + SEM normalized to untreated of at least 3 experiments are shown.</p

Role of myc-1 E-box in TF binding.

<p>Electromobility shift analyses were performed using a double-stranded oligonucleotide probe representing the <i>GILZ</i> promoter sequence −63 to −37 and for some experiments probes with mutations of the E-box cis-elements and the flanking cAMP response (Cre) elements as depicted in (A). HeLa cells were stimulated/infected for 0.5 h or indicated time intervals with toxin B (B, D, E) or <i>Y. enterocolitica</i> (C) and nuclear extracts of these cells were incubated with P³²-labeled GILZ−63/−37 probe. Subsequently band shift analyses were performed. (D) Nuclear extracts were pretreated with a 100-fold excess of indicated cold probes. (E) Nuclear extracts were pretreated with indicated antibodies. Anti-p65 antibody was used as a negative control.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p