Expression Profiling of Starchy Endosperm Metabolic Proteins at 21 Stages of Wheat Grain Development

Proteomic analysis of albumins and globulins (alg) present in starchy endosperm of wheat (<i>Triticum aestivum</i> cv Récital), at 21 stages of grain development, led to the identification of 487 proteins. Four main developmental phases of these metabolic proteins, with three subphases in phase three and two in phase four, were shown. Hierarchical cluster analysis revealed nine major expression profiles throughout grain development. Classification of identified proteins in 17 different biochemical functions provided a uniform picture of temporal coordination among cellular processes. Proteins involved in cell division, transcription/translation, ATP interconversion, protein synthesis, protein transport, along with amino acid, lipid, carbohydrate and nucleotide metabolisms were highly expressed in early and early mid stages of development. Protein folding, cytoskeleton, and storage proteins peaked during the middle of grain development, while in later stages stress/defense, folic acid metabolism, and protein turn over were the abundant functional categories. Detailed analysis of stress/defense enzymes revealed three different evolutionary profiles. A global map with their predicted subcellular localizations and placement in grain developmental scale was constructed. The present study of complete grain development enriched our knowledge on proteome expression of alg, successively from endosperm cell division and differentiation to programmed cell death

Subproteomic signature comparison of <i>in vitro</i> selected fluoroquinolone resistance and ciprofloxacin stress in <i>Salmonella</i> Typhimurium DT104B

<p><b>Background</b>: Fluoroquinolone resistance in nontyphoidal <i>Salmonella</i> is a situation of serious and international concern, particularly in <i>S.</i> Typhimurium DT104B multiresistant strains. Although known to be multifactorial, fluoroquinolone resistance is still far from a complete understanding.</p> <p><b>Methods</b>: Subproteome changes between an experimentally selected fluoroquinolone-resistant strain (Se6-M) and its parent strain (Se6), and also in Se6-M under ciprofloxacin (CIP) stress, were evaluated in order to give new insights into the mechanisms involved. Proteomes were compared at the intracellular and membrane levels by a 2-DE~LC-MS/MS and a shotgun LC-MS/MS approach, respectively.</p> <p><b>Results</b>: In total, 35 differentially abundant proteins were identified when comparing Se6 with Se6-M (25 more abundant in Se6 and 10 more abundant in Se6-M) and 82 were identified between Se6-M and Se6-M+CIP (51 more abundant in Se6-M and 31 more abundant under ciprofloxacin stress).</p> <p><b>Conclusion</b>: Several proteins with known and possible roles in quinolone resistance were identified which provide important information about mechanism-related differential protein expression, supporting the current knowledge and also leading to new testable hypotheses on the mechanism of action of fluoroquinolone drugs.</p

Proteolytic activity of meprins α and β on AIEC LF82 outer membrane proteins and flagellin.

<p>Total protein extracts from untreated or meprin-treated (100 µg/mL) whole bacteria were immunoblotted for OmpA and OmpC/F (A) or Flagellin (B). The inner membrane protein Lep was used as internal control. Amounts of proteins were quantified by using <i>Image J</i> software. Results are expressed as protein amount relative to Lep. Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05.</p

Intestinal meprin α and meprin β mRNA.

<p>A and B, Mep1A (A) and Mep1B (B) mRNA levels were determined by TaqMan quantitative real time PCR and are displayed as amounts relative to the intestinal epithelial marker villin-1. Healthy controls (hc) were compared with ulcerative colitis (UC) and Crohn's disease (CD) patients. CD patient biopsies were separated into groups with normal appearance or with macroscopic inflammation, as determined by an experienced endoscopist. Statistics were performed using GraphPad Prism 5.0 Software, and <i>P</i> values were calculated with the non-parametric Mann-Whitney test. C and D, Mep1A (C) and Mep1B (D) mRNA levels in C57Bl/6J mouse ileum and colon uninfected or infected with AIEC LF82 bacteria. The effect of AIEC LF82 infection on meprin expression was determined in mouse ileum and colon by quantitative real time PCR. Data are displayed as meprin amounts relative to the housekeeping TATA box binding protein (TBP) gene. Statistics were performed using GraphPad Prism 5.0 Software, and <i>P</i> values were calculated with the one-way ANOVA test. Dot plots show individual samples with relative mRNA (cDNA) amounts on a linear scale. Horizontal bars represent the median.</p

Meprin treatment affect mannose residue recognition by AIEC and AIEC-induced IL-8 secretion by T84 cells.

<p>A, ability of type 1 pili to bind D-mannose residues as determined by a yeast aggregation test. AIEC LF82 bacteria were treated with 100 µg/ml of meprin α or β at 37°C for 120 min. A fixed amount of inactivated yeast cells (<i>Saccharomyces cerevisiae</i>) suspension and decreasing concentrations of treated and untreated bacteria were mixed, and the loss of the ability to form homogenous aggregation was used as the read-out for impaired type 1 pili-yeast interaction. B, Amount of IL-8 secreted by uninfected or AIEC LF82- or type 1 pili negative mutant LF82-Δ<i>fimA</i>-infected T84 cells, at 24 h post-infection. AIEC LF82 and LF82-Δ<i>fimA</i> bacteria were treated with 100 µg/ml of meprins. Il-8 secretion was determined by ELISA. Data are expressed as fold increase in the amount of secreted IL-8 ± SEM by T84 cells infected with untreated or treated bacteria relative to non infected cells. Student's <i>t</i>-test, * <i>P</i><0.05 for comparison between IL-8 secretion induced by untreated versus meprin-treated AIEC LF82 or LF82-Δ<i>fimA</i> bacteria. C, LF82-Δ<i>fimA</i> bacteria were pretreated with exogenous meprin α or meprin β at 100 µg/ml and undifferentiated T84 cells were infected at a MOI of 10. The number of associated bacteria was determined. Results are expressed as the percentage of cell-associated bacteria pretreated with exogenous meprins relative to untreated bacteria, defined as 100%.D, effect of meprins on recombinant human IL-8. Recombinant human IL-8 (110 ng/ml) was treated with meprin α or β (100 µg/ml), electroblotted and detected with mouse anti-human IL-8.</p

Effect of meprins on the ability of AIEC strains and <i>Salmonella</i> Typhimurium strain LT2 to adhere to and to invade intestinal epithelial cells.

<p>Bacteria were pretreated with exogenous meprin α or meprin β at 10 µg/ml. A and B, undifferentiated Intestine-407 (I407), Caco-2 and T84 cells infected with AIEC LF82 at a MOI of 10. The number of associated (A) and internalized (B) bacteria was determined. Results are expressed as the percentage of cell-associated (A) or intracellular bacteria (B) relative to initial inoculum. C and D, undifferentiated T84 cells were infected at a MOI of 10 with <i>Salmonella</i> Typhimurium and AIEC strains LF82, LF9, LF15 and LF31. The number of associated (C) and internalized (D) bacteria was determined. Results are expressed as the percentage of cell-associated (C) or intracellular bacteria (D) relative to untreated bacteria, defined as 100% (bar, C and D). Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05.</p

Proteolytic activity of meprins α and β on AIEC LF82 type 1 pili.

<p>A, proteolytic effect of various concentrations (from 1 µg/ml to 100 µg/ml) of meprins on purified AIEC LF82 type 1 pili. Proteins were loaded on 15% polyacrylamid gel for SDS-PAGE and stained with Coomassie brilliant blue. Results are expressed as FimA protein amount for meprin-treated type 1 pili relative to that of untreated type 1 pili. B, proteolytic effect of meprins on type 1 pili expressed at the surface of whole AIEC LF82 bacteria. AIEC bacteria were untreated or treated with active or heat-inactivated meprins at 100 µg/mL. Total proteins were immunoblotted with rabbit antiserum raised against purified type 1 pili and the inner membrane protein Lep, as an internal control. Results are expressed as FimA amount relative to Lep. Amounts of proteins were quantified by using <i>Image J</i> software. Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05. C and D, MALDI-TOF MS profile of purified AIEC LF82 type 1 pili treated with active or heat-inactivated meprin α or meprin β. Spectra were acquired in a mass spectrometer MALDI-TOF from LF82 type 1 pili treated with 100 µg/ml of meprin α (C) or with meprin β (D).</p

Meprins impair AIEC LF82 ability to adhere to and to invade differentiated intestinal epithelial cells.

<p>AIEC LF82 bacteria were treated before infection with increased concentrations of exogenous meprins α and β (from 0.1 µg/ml to 100 µg/ml, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021199#s4" target="_blank">Materials and Methods</a> section). Differentiated T84 cells were infected at a MOI of 10 with untreated or meprin pretreated bacteria. A and D, the number of cell-associated bacteria was determined after a 3 h infection period. B and E, the number of internalized bacteria was determined after a 3 h infection period followed by gentamicin treatment for 1 h. Results are expressed as percentage of cell-associated (adherent + intracellular) (A and D), or intracellular (B and E) bacteria relative to initial inoculum. C and F, effect of meprin treatment on AIEC bacteria viability. Equal amounts of bacteria were exposed or not with a dose of 100 µg/ml of meprin α or β for 120 min. Thereafter, the number of viable bacteria was determined by plating on agar plate. Results are expressed as colony forming units (CFU) per ml (C and F). Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05.</p

Additional file 1: of Xylan degradation by the human gut Bacteroides xylanisolvens XB1AT involves two distinct gene clusters that are linked at the transcriptional level

Table S1. RNA-seq mapping assessment. Table S2. Xylanase specific activity of B xylanisolvens XB1AT. Table S3. Proteins identified by MALDI-TOF MS or LC-ESI-MS/MS over-produced upon growth of B. xylanisolvens XB1AT on OSX relative to xylose. Table S4. Composition of the commercial oat-spelt xylan (SERVA, France) used in this study. Table S5. Primers used for RT-PCR (to amplify the intergenic regions between two consecutive ORFs within PUL 43). Table S6. Primers used for relative RT-qPCR. Table S7. Primers used for insertion mutagenesis into PUL 43 HTCS gene (BXY_29350). Figure S1. Growth of B. xylanisolvens XB1AT (Wt) and PUL 43 HTCS (BXY_29350) mutant on glucose, xylose, wheat arabinoxylan (WAX) and oat-spelt xylan (OSX). Figure S2. B. xylanisolvens XB1AT gene expression in response to oat-spelt xylan (OSX) and xylose relative to glucose obtained from RNA-seq analysis. Figure S3. B. xylanisolvens XB1AT PUL expression in response to xylose relative to glucose at late-log phase obtained from RNA-seq analysis. Figure S4. Schematic layout of the mutant construction and validation of pGERM:HTCS insertion into PUL 43 HTCS gene (BXY_29350) of B. xylanisolvens XB1A genome. (XLSX 454 kb