Fecal Host Transcriptomics for Non-invasive Human Mucosal Immune Profiling: Proof of Concept in Clostridium difficile Infection

Abstract

Background:Host factors play an important role in pathogenesis and disease outcome in Clostridium difficile infection (CDI), and characterization of these responses could uncover potential host biomarkers to complement existing microbe-based diagnostics.Methods:We extracted RNA from fecal samples of patients with CDI and profiled human mRNA using amplicon-based next-generation sequencing (NGS). We compared the fecal host mRNA transcript expression profiles of patients with CDI to controls with non-CDI diarrhea.Results:We found that the ratio of human actin gamma 1 (ACTG1) to 16S ribosomal RNA (rRNA) was highly correlated with NGS quality as measured by percentage of reads on target. Patients with CDI could be differentiated from those with non-CDI diarrhea based on their fecal mRNA expression profiles using principal component analysis. Among the most differentially expressed genes were ones related to immune response (IL23A, IL34) and actin-cytoskeleton function (TNNT1, MYL4, SMTN, MYBPC3, all adjusted P-values < 1 × 10−3).Conclusions:In this proof-of-concept study, we used host fecal transcriptomics for non-invasive profiling of the mucosal immune response in CDI. We identified differentially expressed genes with biological plausibility based on animal and cell culture models. This demonstrates the potential of fecal transcriptomics to uncover host-based biomarkers for enteric infections.

Plakoglobin and High-Mobility Group Box 1 Mediate Intestinal Epithelial Cell Apoptosis Induced by Clostridioides difficile TcdB

“…Mileto et al discovered that TcdB causes severe disruption of β-catenin/E-cadherin in colonic epithelia of mice infected with TcdB-producing C. difficile strains ( 21 ). A study found that the JUP mRNA level is upregulated in host fecal mRNA transcript expression profiling data from patients with CDI ( 53 ). However, no previous study identified a functional role of JUP in CDI.…”

Plakoglobin and HMGB1 mediate intestinal epithelial cell apoptosis induced by Clostridioides difficile TcdB

“…Mileto et al discovered that TcdB causes severe disruption of β-catenin/E-cadherin in colonic epithelia of mice infected with TcdB-producing C. difficile strains (Mileto et al, 2020). A study found that JUP mRNA level is upregulated in host fecal mRNA transcript expression profiling data from patients with CDI (Schlaberg et al, 2018). However, no previous study identified a functional role of JUP in CDI.…”

The Multiomics Analyses of Fecal Matrix and Its Significance to Coeliac Disease Gut Profiling

“…In addition, the detection of these specific biomarkers and pathways could serve as the diagnosis of CeD pre-disease state [ 222 , 228 ]. While there have been few CeD transcriptomics studies involving the use of fecal matter, fecal host transcriptomics can be used to determine the gene expressions of bacterial species involved in pathogenesis; for example, mRNA expression profiles of immune response and actin-cytoskeleton were enriched with Clostridium difficile infection [ 229 ]. Additionally, fecal host, transcriptomics can be used to investigate the active functional profiles of various stages of CeD [ 215 , 229 ].…”

“…While there have been few CeD transcriptomics studies involving the use of fecal matter, fecal host transcriptomics can be used to determine the gene expressions of bacterial species involved in pathogenesis; for example, mRNA expression profiles of immune response and actin-cytoskeleton were enriched with Clostridium difficile infection [ 229 ]. Additionally, fecal host, transcriptomics can be used to investigate the active functional profiles of various stages of CeD [ 215 , 229 ]. The analysis of fecal matter over blood analysis showed an increased representation of energy metabolisms pathways in CeD subjects and, along with computational functional analysis, fecal transcriptomics can predict functional pathways of pre-CeD subjects [ 212 ].…”