<i>Escherichia coli</i> strains and plasmids used in this work.

<p><i>Escherichia coli</i> strains and plasmids used in this work.</p

Determination of cellulose amounts.

<p>Strains MG1655, MG1655<i>ΔcarB::cat</i>, and MG1655<i>ΔpyrC::tet</i> were grown 48 hours at 30°C on either LB1/4 agar (no added uracil, Cellulose extraction and determination was performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031252#pone.0031252-Gualdi2" target="_blank">[35]</a>. Data shown are the average of two independent experiments giving very similar results. For strains MG1655<i>ΔcarB::cat</i> and MG1655<i>ΔpyrC::tet</i> grown on LB1/4 agar no glucose was detectable in the assays. A value of 0.5 nmol glucose, corresponding to the lowest detectable concentration in the assay, as determined by a glucose standard curve, was thus arbitrarily assigned to these strains.</p

Congo red binding by <i>E. coli</i> strains deficient in UMP biosynthesis.

<p>The MG1655 strain and isogenic mutants deficient in UMP biosynthetic genes were spotted on either CR medium or CR(ura) medium (CR medium supplemented with 0.25 mM uracil) and grown for 24 hours at 30°C. Plates were incubated for 48 hours at 4°C to enhance Congo red binding.</p

Congo red binding by <i>E. coli</i> strains deficient in pyrimidine sensing (<i>cytR</i> and <i>rutR</i> mutants) and purine biosynthesis (<i>purH</i> mutant).

<p><b>4A.</b> The MG1655 strain and its isogenic mutants in the <i>purH</i>, <i>cytR</i> and <i>rutR</i> genes were spotted either on CR medium (left panel) or on CR(ura) medium (right panel) and grown for 24 hours at 30°C. Plates were incubated for 48 hours at 4°C to enhance Congo red binding. Determination of transcript levels. <b>4B.</b> Relative expression of either the <i>csgD</i> gene (left panel) or the <i>udp</i> gene (right panel) was determined by Real-Time PCR on RNA extracted from overnight cultures of MG1655 and of its isogenic <i>purH</i> and <i>cytR</i> mutants. 16S RNA transcript was used as reference gene. <i>Δ</i>Ct values between the genes of interest and 16S RNA were set at 1 for MG1655 in LB1/4 medium, and transcript levels in other strains and/or growth conditions are expressed as relative values. Experiments were repeated at least three times, each time in duplicate; standard deviations were always lower than 5%.</p

UMP biosynthetic pathways in <i>Escherichia coli</i>.

<p>Adapted from Ecocyc (<a href="http://ecocyc.org/" target="_blank">http://ecocyc.org/</a>).</p

Determination of curli production by Congo red binding.

<p>Phenotypes on CR medium of MG1655 (wild type strain), AM70 (<i>csgA</i> deletion mutant, unable to produce curli), MG1655<i>carB::Tn5kan</i>, MG1655<i>carB::Tn5kan ΔcsgA::cat</i> and MG1655<i>ΔcarB::cat</i>. Strains were grown either at 30°C (for 24 hours) or at 37°C (for 18 hours). Plates were incubated for 48 hours at 4°C to enhance Congo red binding.</p

Determination of gene expression levels.

<p>Relative expression of the <i>csgD</i>, <i>csgB</i>, <i>adrA</i> and <i>bcsA</i> genes determined by Real-Time PCR on RNA extracted from overnight cultures. 16S RNA transcript was used as reference gene. <i>Δ</i>Ct values between the genes of interest and 16S RNA were set at 100 for MG1655 in LB1/4 medium, and transcript levels in other strains and/or growth conditions are expressed as relative values. Experiments were repeated at least three times, each time in duplicate; standard deviations were always lower than 5%.</p

Image_1_Phagocytosis and Epithelial Cell Invasion by Crohn’s Disease-Associated Adherent-Invasive Escherichia coli Are Inhibited by the Anti-inflammatory Drug 6-Mercaptopurine.pdf

<p>Adherent-invasive Escherichia coli (AIEC) strains are overrepresented in the dysbiotic microbiota of Crohn’s disease (CD) patients, and contribute to the onset of the chronic inflammation typical of the disease. However, the effects of anti-inflammatory drugs used for CD treatment on AIEC virulence have not yet been investigated. In this report, we show that exposure of AIEC LF82 strain to amino-6-mercaptopurine (6-MP) riboside, one of the most widely used anti-inflammatory drugs in CD, impairs its ability to adhere to, and consequently to invade, human epithelial cells. Notably, phagocytosis of LF82 treated with 6-MP by human macrophages is also reduced, suggesting that 6-MP affects AIEC cell surface determinants involved both in interaction with epithelial cells and in uptake by macrophages. Since a main target of 6-MP in bacterial cells is the inhibition of the important signal molecule c-di-GMP, we also tested whether perturbations in cAMP, another major signaling pathway in E. coli, might have similar effects on interactions with human cells. To this aim, we grew LF82 in the presence of glucose, which leads to inhibition of cAMP synthesis. Growth in glucose-supplemented medium resulted in a reduction in AIEC adhesion to epithelial cells and uptake by macrophages. Consistent with these results, both 6-MP and glucose can affect expression of cell adhesion-related genes, such as the csg genes, encoding thin aggregative fimbriae (curli). In addition, glucose strongly inhibits expression of the fim operon, encoding type 1 pili, a known AIEC determinant for adhesion to human cells. To further investigate whether 6-MP can indeed inhibit c-di-GMP signaling in AIEC, we performed biofilm and motility assays and determination of extracellular polysaccharides. 6-MP clearly affected biofilm formation and cellulose production, but also, unexpectedly, reduced cell motility, itself an important virulence factor for AIEC. Our results provide strong evidence that 6-MP can affect AIEC-host cell interaction by acting on the bacterial cell, thus strengthening the hypothesis that mercaptopurines might promote CD remission also by affecting gut microbiota composition and/or physiology, and suggesting that novel drugs targeting bacterial virulence and signaling might be effective in preventing chronic inflammation in CD.</p

<i>In vitro</i> phagocytosis of BtCDC272 grown in different conditions by neutrophils in whole blood.

<p><i>In vitro</i> phagocytosis of BtCDC (10 and 100 M.O.I) by human neutrophils of two donors was determined by FACS analysis at 15 min. Data are expressed as the percentage of control MFI. The mean value ± SEM is shown. *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001; two-tailed Student’s <i>t</i> test. 0. Results for each donor, expressed as MFI, are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093009#pone.0093009.s003" target="_blank">Figure S3</a>.</p

Cytokine production by mouse macrophage cell line RAW264.7 exposed to BtCDC272 grown in different conditions and at different M.O.I.

<p>Confluent monolayers of cells were incubated with bacteria cultured at the indicated conditions. Bacteria were added at MOI of 0.005, 0.05, 0.5 or 5 for 4 hours to confluent monolayers of RAW264.7 cells. LPS (100 ng/ml from <i>E. coli</i> Serotype 055:B5; Sigma-Aldrich) was used as positive control of macrophage activation. Murine CXCL2, TNF-α and IL-6 levels were measured in cell supernatants by ELISA Results are mean± SEM of two independent experiments.</p