Bacterial protein signals are associated with Crohn's disease

Abstract

Objective: No Crohn's disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls.

Design: We first developed and validated a workflow-including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS-to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions.

Results: Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins.

Conclusions: This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis.

Keywords: Crohn's Disease; Enteric Bacterial Microflora; Inflammatory Bowel Disease.

Figure 1

Structure of all crude samples and half the corresponding extracted microbial pellets profiled by 16S rRNA gene pyrosequencing. Hierarchical clustering of the 16S rRNA gene pyrosequencing dataset (at genus and OTU level) showed a high similarity between population structure of crude samples and those of the corresponding microbial extracts, but did not allow distinguishing between clinical status, CD or HC. HC.1 to HC.6 and CD.1 to CD.6 denote HC and CD patients, respectively; suffix letters, F and a, denote native faeces and bacterial cell extracts, respectively. CD, Crohn's disease; HC, healthy controls.

Figure 2

Cluster tree based on the pairwise distances between 2D-DIGE profiles. Similarities between patterns (normalised volumes of 2007 spots) were assessed by unsupervised hierarchical clustering. HC.1 to HC.6 and CD.1 to CD.6 denote HC and CD patients, respectively; g01–12 denote gel numbers. Microbial fractions prepared in duplicate from the same stool specimen always clustered together, reflecting good technical reproducibility of our method, and pairs of duplicates tended to cluster by clinical status, CD or HC, indicating a clear clinically relevant signal in the proteomics data. CD, Crohn's disease; HC, healthy controls; 2D-DIGE, two-dimensional difference gel electrophoresis.

Figure 3

Cluster heat map constructed from the normalised volumes of spots with significant different intensities between HC and CD patients, and that could be robustly identified. Spot numbers and meaningful names for the associated functions are in the right margin. Similarities between patterns are visualised by unsupervised hierarchical clustering. HC.1 to HC.6 and CD.1 to CD.6 denote HC and CD patients, respectively; g01–12 denote gel numbers. Since all spot variables were centred at the mean (the mean has been subtracted to each value), the new mean for each spot variable is now at 0, making half the values negative as indicated in the colour key. Blue and red tones therefore signify under-represented and over-represented, respectively. Proteins highlighted in yellow in the right margin are those that were chosen for SRM validation. As different forms of the same protein (typically TonB-dependent receptors of Bacteroides) may occur in different spots, the number of highlighted spots exceeds 13. Proteins annoted ‘surface’, ‘TonB’, ‘OMP’ and ‘lipoprotein’ in the right margin, may be grouped into ‘cell envelope proteins’ in the text when several categories are concerned, including proteins of unknown function that have specific features known to be characteristic of cell envelope localisation. CD, Crohn's disease; HC, healthy controls; SRM, selected reaction monitoring.

Figure 4

Volcano plot representing results of the LC-SRM assays on the 13 targeted proteins. The logarithmic fold changes (CD vs HC) are plotted against negative logarithmic adjusted p values calculated with the R package MSstats and performed from triplicate injections. All targeted proteins were found to be either upregulated or downregulated in CD patients compared with controls, and the results validated all candidates identified in the discovery experiments. CD, Crohn's disease; HC, healthy controls; SRM, selected reaction monitoring.

Figure 5

Box plots of the relative abundances of faecal bacterial populations found by 454 pyrosequencing. Differences between Crohn's patients (□) and HCs (□) at the different phylogenetic levels were considered *significant for p≤0.05, and ⁽*⁾tendencies were reported up to p≤0.10 (‘glm’ with the ‘quasibinomial family’). Specific traits of CD microbiota were significantly increased abundances of members in the lineage Betaproteobacteria-Burkhoderiales-Alcaligenaceae, a tendency towards increased abundances of Bacteroidaceae-Bacteroides and Blautia, significantly lower numbers of Roseburia, and a tendency towards lower numbers of Alphaproteobacteria, Prevotellaceae-Prevotella and Oscillospira. On the other hand, inter-individual variability was higher in CD patients, which is in agreement with heterogeneity of CD. CD, Crohn's disease; HCs, healthy controls.

Figure 6

Heat map of the correlation matrix between abundance of the varying protein groups and the abundance of the related varying OTUs (left panel). Red and hotter orange tones indicate a positive correlation between abundance of a protein group and abundance of the corresponding OTUs; green and yellow tones indicate absence of correlation as for example, TonB proteins and other uncharacterised surface proteins of Bacteroides in spots 0082, 0480 and 0009, and DnaK of Enterobacteriaceae in spot 1835 (right middle part of the image). Activities that were found to be increased and decreased in CD patients fell in the upper and lower halves of the image, respectively. OTUs that were found to be increased and decreased in abundance in CD patients fell into the right and left halves of the image, respectively. Examples of positive correlations are detailed on right panel and highlighted in yellow on panel A. CD, Crohn's disease.