MOESM1 of Examination of the effects of Campylobacter concisus zonula occludens toxin on intestinal epithelial cells and macrophages

Additional file 1. SDS-PAGE and Western blot images of EP and EP-ZotP14UCO-S1 proteins. EP and EP-ZotP14UCO-S1 proteins were purified from E. coli transformed with pETBlue-2 vector or pETBlue-2-zot 808T using Ni–NTA columns as per manufacturer’s instruction. The flow through, column wash and eluates were collected and subjected to SDS-PAGE to reveal proteins. Eluates were also subjected to Western blot to detect Zot using anti-histidine antibodies. A and B purification of EP, A showing SDS-PAGE and B showing Western blot. C and D purification of EP-ZotP14UCO-S1, C showing SDS-PAGE and D showing Western blot. The Zot protein (44 kD) was detected in EP-P14UCO-S1 not in EP. FT (Flow through), W1 (first wash), W2 (second wash), El-E4 (elutions 1-4). E1 was used for conducting experiments in this study

Detection of <i>C. concisus</i> in biopsies collected from four intestinal anatomic sites of patients with IBD (CD and UC) and controls^@.

<p>@ Identification of C. concisus was based on Campylobacter genus-PCR and sequencing of the positive PCR products, except for three biopsy samples. The three biopsy samples showed mixed sequences by Campylobacter genus-PCR, therefore were further subjected to C. concisus PCR to examine the presence of C. concisus.</p><p>Biopsies collected from four intestinal anatomic sites were examined; ileal biopsies were not available from two patients with CD, caecal biopsy was not available from one patient with CD and rectal biopsy was not available from one patient with CD.</p><p>*Significantly higher in patients with IBD as compared with the controls (<i>P</i><0.05).</p

<i>Campylobacter</i> genus-PCR positivity in patients with IBD (CD and UC) and controls.

<p>Four biopsies collected from each individual were examined. A <i>Campylobacter</i> genus-PCR positive individual is an individual who had at least one intestinal biopsy positive for <i>Campylobacter</i> genus-PCR. *Significantly different as compared with the controls (<i>P</i><0.05).</p

Percentage of <i>Campylobacter</i> species positivity in patients with IBD (CD and UC) and controls^@.

<p>@A specific Campylobacter species positive individual is an individual who has at least one biopsy positive for the Campylobacter species listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025417#pone-0025417-t003" target="_blank">Table 3</a>, detected by Campylobacter genus-PCR and sequencing. *Significantly higher as compared with the controls (P<0.05).</p

Montreal classification of patients with IBD

<p>Montreal classification of patients with IBD</p

Detection of <i>Campylobacter</i> species in biopsies collected from four intestinal anatomic sites of individuals (n = 33) with normal intestinal histology by <i>Campylobacter</i> genus-PCR and sequencing^*.

<p>*Four biopsies, one each from ileum, caecum, descending colon and rectum of each individual, were examined. Identification of <i>Campylobacter</i> species was based on 97–100% similarity of the sequences of PCR products (503–766 bp) to the sequences of known <i>Campylobacter</i> species.</p

Prevalence of <i>zot</i>-positive <i>C</i><i>. concisus</i> strains in the oral cavity of patients with IBD and controls.

<p>The prevalence of <i>zot</i>-positive <i>C</i><i>. concisus</i> strains in active IBD and healthy controls was not statistically different. <i>zot</i>-positive <i>C</i><i>. concisus</i> strains were not detected in patients in remission who received antibiotics treatment for IBD.</p

Electron Microscopic image of an oral EICC isolate, an oral non-EICC isolate and an enteric EICC isolate.

<p>All isolates were cultured from Patient No. 1.</p

<i>C. concisus</i> isolates used in this study.

#<p>A total of 70 <i>C. concisus</i> isolates were examined in this study.</p>*<p><i>C. concisus</i> isolated from intestinal biopsies and luminal-washout fluid was referred as enteric <i>C. concisus</i> and <i>C. concisus</i> isolated from saliva was referred as oral <i>C. concisus</i>.</p

PCR primers used for amplification of MLST genes in this study.

<p>PCR primers were designed based on the whole genome sequenced <i>C. concisus</i> strain 13826 (Accession No: CP000792.1).</p