Expression of JHP0290 homologues in different <i>H</i>. <i>pylori</i> strains.

<p>Whole cell lysate <b>(A)</b> and culture broth (Supernatant) from equal number of cells <b>(B)</b> of various <i>H</i>. <i>pylori</i> strains as indicated in figure legends were immunoblotted with anti-JHP0290 antibody. Blot shown is representative of results obtained in more than five independent experiments. The graph shows western blot band intensities quantified by the ImageJ software. <b>(C)</b> Sequence homology analysis of JHP0290 homologues in <i>H</i>. <i>pylori</i> strains J99 (JHP0290), 26695 (HP0305), P12 (HPP12_HP0304) and HPAG1 (HPAG1_HP0307) using Clustal Omega software (EMBL-EBI). N-terminal 1–17 amino acids were identified as signal peptide by the SignalP 4.1 software (ExpPASy Bioinformatics Resource Portal). Conserved Cysteine at position 162 is marked by an arrow.</p

Primers used in this study.

<p>Primers used in this study.</p

rJHP0290 activates ERK MAPK in gastric epithelial cells.

<p>AGS <b>(A)</b> and MKN45 <b>(B)</b> cells were treated with rJHP0290 for various time points (min) as indicated in the figure legends. Cell lysates were prepared and immunoblotted with anti-phospho-ERK antibody followed by reprobing with anti-ERK and anti-actin antibody to confirm equal loading. Blot shown is representative of results obtained in five independent experiments. The graph shows the western blot band intensities normalized to the actin control in five experiments. Statistically significant differences are indicated by * (p < 0.05).</p

Effect of rJHP0290 on gastric epithelial cell proliferation.

<p>AGS <b>(A)</b>, MKN45 <b>(B)</b> and PHSEC <b>(C)</b> cells were incubated either with protein storage buffer (Ctrl) or different concentrations of rJHP0290 as indicated in the figure legends for 48 h and 72 h, followed by MTT assay to assess proliferation. Values indicate mean ± SD of six independent experiments performed at least in triplicate. <b>(D)</b> AGS and MKN45 cells were treated for 48h with buffer (ctrl) or rJHP0290 Wt (100 ng/ml) or rJHP0290 C162A (100 ng/ml) or an irrelevant His-tagged protein (Irr-His, 100 ng/ml). rJHP0290 was subjected to boiling for 30 min (heat) or was treated with polymyxin B (PMB) for 1 h before treatment of cells. The relative BrdU incorporation in cells was measured. Values indicate mean ± SD of four independent experiments performed in triplicate. Statistically significant differences are indicated by * (p < 0.05).</p

rJHP0290 modulates cell cycle progression in gastric epithelial cells.

<p>AGS cells were incubated either with protein storage buffer (Ctrl) or rJHP0290 (100 ng/ml) for 24 h. Cells were washed with PBS, fixed in ice-cold 70% ethanol at 4°C and stained with PI and RNAse A in PBS for 30 min. The DNA profile was generated by FACS. One representative profile (A) and mean ± SD of six independent experiments (B) are shown. Statistically significant differences are indicated by * (p < 0.05).</p

<i>Helicobacter pylori</i> Protein JHP0290 Exhibits Proliferative and Anti-Apoptotic Effects in Gastric Epithelial Cells

<div><p>The influence of <i>Helicobacter pylori</i> infection on gastric epithelial cell proliferation, apoptosis and signaling pathways contributes to the development of infection-associated diseases. Here we report that JHP0290, which is a poorly functionally characterized protein from <i>H</i>. <i>pylori</i>, regulates multiple responses in human gastric epithelial cells. The differential expression and release of JHP0290 homologues was observed among <i>H</i>. <i>pylori</i> strains. JHP0290 existed in monomeric and dimeric forms in <i>H</i>. <i>pylori</i> cell extracts and culture broth. Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form. The dimeric form of the protein was found to bind more efficiently to gastric epithelial cells than the monomeric form. The exposure of gastric epithelial cells to rJHP0290 induced proliferation in a dose-dependent manner. Faster progression into the cell cycle was observed in rJHP0290-challenged gastric epithelial cells. Furthermore, we detected an anti-apoptotic effect of rJHP0290 in gastric epithelial cells when the cells were treated with rJHP0290 in combination with Camptothecin (CPT), which is an inducer of apoptosis. CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290. In addition, the activation of ERK MAPK and the transcription factor NFκB was observed in rJHP0290-challenged gastric epithelial cells lines. Our results suggest that JHP0290 may affect <i>H</i>. <i>pylori</i>-induced gastric diseases via the regulation of gastric epithelial cell proliferation and anti-apoptotic pathways.</p></div

Fungal Infection Induces Sex-Specific Transcriptional Changes and Alters Sexual Dimorphism in the Dioecious Plant <i>Silene latifolia</i>

<div><p>Sexual dimorphism, including differences in morphology, behavior and physiology between females and males, is widespread in animals and plants and is shaped by gene expression differences between the sexes. Such expression differences may also underlie sex-specific responses of hosts to pathogen infections, most notably when pathogens induce partial sex reversal in infected hosts. The genetic changes associated with sex-specific responses to pathogen infections on the one hand, and sexual dimorphism on the other hand, remain poorly understood. The dioecious White Campion (<i>Silene latifolia</i>) displays sexual dimorphism in floral traits and infection with the smut fungus <i>Micobrotryum lychnidis-dioicae</i> induces a partial sex reversal in females. We find strong sex-specific responses to pathogen infection and reduced sexual dimorphism in infected <i>S</i>. <i>latifolia</i>. This provides a direct link between pathogen-mediated changes in sex-biased gene expression and altered sexual dimorphism in the host. Expression changes following infection affected mainly genes with male-biased expression in healthy plants. In females, these genes were up-regulated, leading to a masculinization of the transcriptome. In contrast, infection in males was associated with down-regulation of these genes, leading to a demasculinization of the transcriptome. To a lesser extent, genes with female-biased expression in healthy plants were also affected in opposite directions in the two sexes. These genes were overall down-regulated in females and up-regulated in males, causing, respectively, a defeminization in infected females and a feminization of the transcriptome in infected males. Our results reveal strong sex-specific responses to pathogen infection in a dioecious plant and provide a link between pathogen-induced changes in sex-biased gene expression and sexual dimorphism.</p></div

Effects of infection, host sex and their interaction on gene expression in <i>S</i>. <i>latifolia</i>.

<p>Numbers of non sex-linked (86, 824) and sex-linked (1, 564) contigs (and percentages) that are differentially expressed as a consequence of infection with <i>M</i>. <i>lychnidis-dioicae</i>, host sex and the interaction between infection and host sex. The model with two factors (infection and sex) and their interaction was calculated.</p

Gene expression changes upon infection in female and male <i>S</i>. <i>latifolia</i>.

<p>Numbers and percentages of differentially expressed contigs in infected females and males for non sex-linked (86, 824) and sex-linked (1, 564) contigs only. Significantly more changes were found in sex-linked than in non sex-linked contigs (χ²-tests tests, P-values ≤0.001).</p

Heat maps and clustering of gene expression in non-infected and infected female and male <i>S</i>. <i>latifolia</i>.

<p>Heat maps and hierarchical clustering analyses of expression patterns for contigs that are significantly differentially expressed between healthy and infected females (a, c) and males (b, d) for non sex-linked (a, b) and sex-linked (c, d) contigs. Dark green colour indicates low expression and white high gene expression intensity.</p