Integrated analysis of microbe-host interactions in Crohn's disease reveals potential mechanisms of microbial proteins on host gene expression

Abstract

Inflammatory responses of the intestinal epithelial barrier in patients with Crohn's disease (CD), a chronic inflammatory bowel disease (IBD), are associated with gut microbial alterations. At a community level, there is scarce mechanistic evidence on the effects of gut microbial alterations on host mucosal barrier responses. We used a computational microbe-host interaction prediction framework based on network diffusion and systems biology to integrate publicly available paired gut microbial and intestinal gene expression datasets. The ileal signaling network potentially modulated by the microbiota was enriched with immune-related pathways such as those associated with IL-4, IL-2, IL-13, NFkB, and toll-like receptors. We identified bacterial proteins eliciting post-translational modifications on host receptors, resulting in the de-repression of pro-inflammatory cytokines via critical hub proteins such as NFkB. The signaling networks were over-represented with CD associated genes and CD drug targets. Using datasets generated from our validation cohorts, we confirmed some of the results.

Keywords: Gastroenterology; Microbiome; Molecular genetics.

Graphical abstract

Figure 1

Graphical description of the workflow used to infer the plausible effect of the altered CD gut microbial proteins on host intestinal gene expression

Figure 2

Overview of the stepwise results leading to the inference of the site-specific signaling networks (A) Schematic displaying the procedure based on multiple evidences to identify bacterial species relevant to (based on enhanced abundance and transcriptional activity) CD. (B) Figure displaying the relationship between the fraction of orthologous groups of proteins (from the 8 CD relevant species) which are capable of binding to human proteins and the number of species with protein membership in the orthologous groups. (C) Hairball representation of the signaling network capturing the potential effect of the microbial proteins on ileal gene expression in CD patients. (D) In-degree distribution of the ileal signaling network. (E and F) The segregation of the ileal signaling network into sequential layers based on their position in the signaling chains mediating the effect of the bacterial proteins on ileal gene expression. The size of the nodes in E denotes the clustering co-efficient, whereas in F it stands for the betweenness centrality. The top three ranked nodes according to the clustering co-efficient are indicated in E while the top three transcription factors (TFs) ranked by the betweenness centrality are shown in (F).

Figure 3

Functional analysis of the microbiota-modulated ileal signaling network (A) Top 10 Reactome pathways enriched (adj.p-value ≤ 0.1) in the ileal signaling network (B) and overlap between them. (C) Expression profiles of the genes belonging to the top 10 Reactome pathways enriched (adj.p-value ≤ 0.1) among the differentially expressed genes in the ileal signaling network. (D) Overlap between the ileal DE-Gs potentially modulated by the altered microbiota in CD patients and DE-Gs measured in an independent cohort of CD patients. (E) Modules (tightly connected networks) detected by MCODE within the ileal signaling network. (F) Over-represented Reactome signaling pathways (adj.p-value ≤ 0.1) in the network modules inferred from the ileal signaling network. (G) Bacterial proteins with post-translational modification functions and inferred to influence the differential expression of genes in the ileum. EC: Adhesive invasive E. coli (AIEC); BF: Bacteroides fragilis; CC: Clostridium clostridioforme; CH: Clostridium hathewayi; CS: Clostridium symbiosum; KP: Klebsiella pneumoniae; RG: Ruminococcus gnavus; VP: Veillonella parvula. ∗ - Number of bacterial species in which a protein member from the ortholog group was detected. Singleton proteins or ortholog groups with just membership from a single species have not been shown in this figure.

Figure 4

Examples of regulatory and mechanistic interactions mediated by microbial proteins followed by the comparison of the test and validation cohorts (A) Heatmap representation of the transcriptional factors (among the top 20 nodes in the ileal signaling network ranked by the betweenness centrality) regulating the DE-Gs in the ileal signaling network. The number of targets for each of the TFs and the involvement of the DE-Gs in immune responses are depicted. The FC TC and FC VC columns stand for the expression level (in log2 scale) of the DE-Gs from the test and validation cohorts respectively. (B and C) Inferred molecular mode of action of the subtilisin-domain containing type S8 peptidase (encoded by four different species with enhanced abundances in CD) on ileal gene expression in CD patients. The potential cleavage of MAP3K7 by the peptidase in turn enhances the activity of transcription factors (SMAD3, NFKB1, NFKBIA) which subsequently results in the upregulation of genes which are involved in inflammation, recruitment and adhesion of immune cells, remodeling of extracellular matrix and encoding enzymes involved in the production of inflammatory precursors. (D) Comparison between the test (TC) and validation cohorts (VC) of the log2 normalized expression fold changes (1.5 ≤ log2FC ≤−1.5, FDR ≤ 0.05) of the transcriptional regulatory targets of SMAD3, NFKB1, NFKBIAwhich are modulated by the subtilisin-domain containing type S8 peptidase. (E) Heatmap representation of the transcriptional factors (among the top 20 nodes in the ileal and rectal signaling networks ranked by the betweenness centrality) regulating the DE-Gs in the ileal and rectal signaling networks. TFs with less than 10 targets each among the ileal and rectal DE-Gs are not shown. The number of targets specific to ileum/rectum and common to both ileum and rectum for each of the TFs is depicted. The FC I TC and FC I VC columns stand for the expression level (in log2 scale) of the ileal DE-Gs from the test and validation cohorts respectively. FC R TC stands for the expression level of the DE-Gs in the rectal signaling network. (F) Summary of the over-represented gene ontological biological processes and Reactome pathways in the microbiome-modulated ileal and rectal signaling network.

Figure 5

Expression signature based matching of drugs to targets in the ileal signaling network (A) Search scores representing the extent to which a drug can induce/reverse the gene expression signatures of the DE-Gs in the ileal signaling network. Only the top 15 perturbation/drugs are shown. (B) The expression profiles of the ileal network DE-Gs in response to various perturbation/drugs. The horizontal red-bars along the rows indicate the extent to which the corresponding ileal DEG is differentially expressed in CD patients. The red-label bars along the columns indicate the search score which is representative of the extent to which the perturbation/drug elicited gene expression can induce/reverse the signature profile of the DEG. The red and blue squares of the matrix indicate the upregulation and downregulation of the DEG in response to the corresponding perturbation/drug. (C) Enriched (adj P-value ≤ 0.05) ontology terms (from the Mammalian Phenotype) of the ileal network DE-Gs which are also overlapping with the Wortmannin elicited gene expression signature.