Host-microbe relationships in inflammatory bowel disease detected by bacterial and metaproteomic analysis of the mucosal-luminal interface

Abstract

Background: Host-microbe interactions at the intestinal mucosal-luminal interface (MLI) are critical factors in the biology of inflammatory bowel disease (IBD).

Methods: To address this issue, we performed a series of investigations integrating analysis of the bacteria and metaproteome at the MLI of Crohn's disease, ulcerative colitis, and healthy human subjects. After quantifying these variables in mucosal specimens from a first sample set, we searched for bacteria exhibiting strong correlations with host proteins. This assessment identified a small subset of bacterial phylotypes possessing this host interaction property. Using a second and independent sample set, we tested the association of disease state with levels of these 14 "host interaction" bacterial phylotypes.

Results: A high frequency of these bacteria (35%) significantly differentiated human subjects by disease type. Analysis of the MLI metaproteomes also yielded disease classification with exceptional confidence levels. Examination of the relationships between the bacteria and proteins, using regularized canonical correlation analysis (RCCA), sorted most subjects by disease type, supporting the concept that host-microbe interactions are involved in the biology underlying IBD. Moreover, this correlation analysis identified bacteria and proteins that were undetected by standard means-based methods such as analysis of variance, and identified associations of specific bacterial phylotypes with particular protein features of the innate immune response, some of which have been documented in model systems.

Conclusions: These findings suggest that computational mining of mucosa-associated bacteria for host interaction provides an unsupervised strategy to uncover networks of bacterial taxa and host processes relevant to normal and disease states. (Inflamm Bowel Dis 2012;).

Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.

Figures

FIGURE 1

Self-organizing map (SOM) cluster analysis of the relationships between the amounts of the bacterial phylotypes and proteins in MLI samples from the first cohort of IBD and healthy subjects. Bacterial rRNA gene and metaproteome composition were examined by OFRG and SELDI-MS analyses, respectively. Pearson correlation coefficients for each phylotype-protein pair are depicted by the color in each cell; stronger correlations have brighter colors (see scale bar). Bacterial phylotypes are on the horizontal axis while the proteins are on the vertical axis (see Supporting Information for details). The heat map contains a subset of the bacterial phylotypes; for details on how this subset was selected, see Identifying the Index Bacterial Phylotypes in Materials and Methods.

FIGURE 2

Classification of healthy and IBD subjects by nearest shrunken centroids analyses of the amounts of the index bacterial phylotypes or proteins from intestinal MLI samples. Values are posterior probabilities: healthy (circles) and IBD (triangles). Means are solid horizontal lines and standard deviations are dashed lines (only the lower value is shown). Only second cohort subjects sampled in both cecum (CE) and sigmoid colon (SIG) were used in this analysis (n = 32), and both regions were analyzed separately. A: Bacterial population density values were generated using 15 qPCR assays: 14 index phylotypes (Table S2 and Figure S1) and one targeting all bacterial rRNA genes; the optimal classification solution (threshold = 0.175, error = 4/32) included all bacteria-region variables except Eubacterium 2766 from the cecum and Clostridium 12 from the cecum and sigmoid colon (27/30 phylotype-region variables). B and C: Proteins were enumerated by MALDI-MS. B: Classification from 25 protein-region variables (threshold = 1.788; error = 5/32) produced similar posterior probabilities as those from the bacterial analysis (A); C: The optimal classification solution (threshold = 0.670; error = 3/32) included 490 protein-variables (see Supporting Information for protein lists).

FIGURE 3

Regularized canonical correlation analysis of bacteria and proteins from MLI samples of IBD and healthy subjects. Canonical variates 1 and 2, based on correlation analysis of the levels of bacteria and proteins, are plotted for samples from the cecum and sigmoid colon. CD, HC and UC indicate Crohn’s disease, healthy controls and ulcerative colitis. n = 13 (CD-SIG), 7 (CD-CE), 12 (HC-SIG), 13 (UC-HE), 14 (UC-SIG) and 14 (UC-CE).

FIGURE 4

Self-organizing map (SOM) cluster analysis of the correlations between the levels of the 8 bacteria and 43 proteins that were strongest contributors to the interrelationships identified by the RCCA. Bacterial rRNA gene and metaproteome composition were examined by sequence-selective qPCR and MALDI-MS analyses, respectively. Pearson correlation coefficients for each phylotype-protein pair are depicted by the color in each cell; stronger correlations have brighter colors (see scale bar). Bacterial phylotypes are on the horizontal axis while the proteins are on the vertical axis. The selected bacteria and proteins had the largest coefficients for canonical variates 1 and 2.

FIGURE 5

Network analysis of the relationships between the amounts of selected bacterial phylotypes and proteins in MLI samples from the second cohort of IBD and healthy subjects. The variables examined were the 8 bacteria and 43 proteins depicted in Figure 4. Phylotypes and proteins are depicted as yellow and blue circles, respectively. Only correlations > 0.4 or http://cran.rproject.org/web/packages/sna/index.html).