Elsevier

Journal of Proteomics

Volume 201, 15 June 2019, Pages 93-103
Journal of Proteomics

Metaproteomics of fecal samples of Crohn's disease and Ulcerative Colitis

https://doi.org/10.1016/j.jprot.2019.04.009Get rights and content

Highlights

  • Fecal metaproteome analyses separated healthy controls from CD patients, UC patients and GCA patients.

  • Increase of NETs and IgG and decrease of IgA and transcriptional regulatory protein RprY from B. fragilis while CD and UC.

  • Identification of potential marker metaproteins for CD, UC, IBS and GCA, such as human enzyme sucrose-isomaltase for CD.

Abstract

Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic inflammatory bowel diseases (IBD) of the gastrointestinal tract. This study used non-invasive LC-MS/MS to find disease specific microbial and human proteins which might be used later for an easier diagnosis.

Therefore, 17 healthy controls, 11 CD patients and 14 UC patients but also 13 Irritable Bowel Disease (IBS) patients, 8 Colon Adenoma (CA) patients, and 8 Gastric Carcinoma (GCA) patients were investigated. The proteins were extracted from the fecal samples with liquid phenol in a ball mill. Subsequently, the proteins were digested tryptically to peptides and analyzed by an Orbitrap LC-MS/MS. For protein identification and interpretation of taxonomic and functional results, the MetaProteomeAnalyzer software was used.

Cluster analysis and non-parametric test (analysis of similarities) separated healthy controls from patients with CD and UC as well as from patients with GCA. Among others, CD and UC correlated with an increase of neutrophil extracellular traps and immune globulins G (IgG). In addition, a decrease of human IgA and the transcriptional regulatory protein RprY from Bacillus fragilis was found for CD and UC. A specific marker in feces for CD was an increased amount of the human enzyme sucrose-isomaltase.

Significance

Crohn's Disease and Ulcerative Colitis are chronic inflammatory diseases of the gastrointestinal tract, whose diagnosis required comprehensive medical examinations including colonoscopy. The impact of the microbial communities in the gut on the pathogenesis of these diseases is poorly understood. Therefore, this study investigated the impact of gut microbiome on these diseases by a metaproteome approach, revealing several disease specific marker proteins. Overall, this indicated that fecal metaproteomics has the potential to be useful as non-invasive tool for a better and easier diagnosis of both diseases.

Introduction

Intestinal microbiota participates in food degradation and synthesize several vitamins. Furthermore, the microbiome interacts with the host, directly influencing its immune system [1]. The gastrointestinal tract (GIT) is colonized by about 1013–1014 microorganisms [2,3]. As an accepted concept homeostasis between microbiota and the immune system is a precondition for human health [4]. However, the GIT could be affected by several diseases such as inflammatory bowel disease (IBD) [5,6], which pathophysiology is linked with an overactivation of the immune system.

Crohn's Disease (CD) and Ulcerative Colitis (UC) as the two main types of IBD are chronic, recurrent inflammations with different clinical, morphological and histopathological pattern. Their prevalence surpass 0.3% in western countries with an increase also in industrialized countries whose societies have become more westernized [7]. CD causes transmural, granulomatous inflammations, which can occur discontinuously in the entire GIT. UC causes superficial inflammation, bleeding and mucosa atrophy in the distal rectum and the colon [8]. The recurrent inflammation in both diseases counts as a risk factor for premalignant transformation. Patients with IBD are under increased risk to develop colon adenoma (CA) and colorectal carcinomas [9] diminishing during that last decades in western countries. In contrast to CD and UC, irritable bowel syndrome (IBS) is a chronic functional gastrointestinal disorder dominated by abdominal discomfort and altered bowel habit which is not linked with structural or biochemical changes [10].

Unfortunately, IBD comprise different diseases with multifactorial pathogeneses, which are currently poorly understood. Genetic alterations represent a risk factor for IBD. Additionally dysfunction in the intestinal inflammatory cascade [4] and environmental factors such as smoking and increased hygienic standards are mentioned to be involved in the pathogenesis, too. Furthermore, several studies [4,[11], [12], [13]] showed the correlation of IBD with a dysbiosis in the microbiome. Frank et al. 2007 [11] observed a reduction of short chain fatty acid producing bacteria in patients with IBD but it remains unclear whether the dysbiosis is a risk factor for the disease or a result. In contrast to IBD, for monoinfections caused by bacteria such as Helicobacter pylori it is known that they have the potency to trigger precancerous lesions and thus, the development of Colon Adenoma (CA) or Gastric Carcinoma (GCA) [14].

The diagnosis of IBD is based on endoscopy as well as on clinical presentation, histopathological and laboratory findings. In addition, physicians may support the diagnosis by measuring single protein markers such as calprotectin and lactoferrin as unspecific inflammatory indicators [15]. Unfortunately, these tests focus on surrogate parameters for unspecific inflammation and do not allow to discriminate between bacterial inflammation and IBD.

Within the last years the development of high throughput methods to identify genes (metagenomics/metatranscriptomics) [16] or proteins (metaproteomics) [[17], [18], [19]] enabled the examination of the taxonomic and functional composition of the microbial communities in the human gut. Thereby, metagenomics/metatranscriptomics focus on the taxonomic and functional inventory of the microbial communities in the human. In contrast metaproteomics studies the expression of the proteins from the microbial communities and from the host. Identification of host proteins provides additional knowledge about the patients' health status, e.g. by monitoring proteins associated to the immune system or secreted enzymes from liver and pancreas, or apoptotic cells from the surface of the digestion system.

Up to now, several alterations within the taxonomic and functional composition of the microbiome of patients with IBD were observed based on metagenome and metaproteome studies [4,[11], [12], [13],18,20,21]. For instance, Casen et al. 2015 [22] correlated CD with a shift from predominantly beneficial bacteria to potentially pathogenic bacteria. However, our understanding of the pathogenesis of IBD is still limited due to the complexity of the diseases and of the microbiome. Most of the studies investigating the microbiome of patients with IBD focused only on one specific disease. This study investigated 71 fecal metaproteomes from healthy individuals and patients with CD, UC, IBS, CA and GCA. The aim of this experimental design was to identify universal marker proteins for the diseases in samples from a representative clinical background and to proof whether non-invasive metaproteome analysis may distinguish between the different diseases.

Section snippets

Methods

Ethics statement

The ethics committees at the Otto-von-Guericke University, Magdeburg, Germany and the Hannover Medical School approved this study by an amendment to previous studies (Number 42/08, Number 47/15 and 2087-2013). The study was performed in accordance with the Declaration of Helsinki. All patients received comprehensive information about the studies and gave their written consents.

Results

All together this study investigated the microbial and human metaproteins in 71 fecal samples from patients with CD, UCa, UCr, IBS, GCA and CA and healthy individuals as controls. Therefore, we searched for disease specific metaprotein patterns as well as certain taxonomic phyla and metaproteins which could be used as diagnostic markers for a specific disease.

Discussion

The main goal of this study was to proof the concept whether metaproteomics may distinguish between patients with different diseases and healthy individuals to support the diagnosis of GIT diseases. Therefore, the microbial and human metaproteins in fecal samples of patients with CD, UC, IBS, CA, GCA and a control group of healthy individuals were examined. In total 2969 metaproteins were identified, revealing interdependencies between diseases and metaprotein profiles. Additionally, identified

Conclusion

The study presented in this paper benefitted from the metaproteomics approach, by enabling the combined identification of multiple human and microbial metaproteins from a single fecal sample. Cluster analysis and specific marker metaproteins, i.e. human sucrose-isomaltase (UniRef50_P14410) and microbial RprY (UniRef50_Q9AE24), could be shown to differentiate between healthy individuals and patients of several GIT diseases.

Consequently, non-invasive metaproteome analysis of fecal samples may

Authors contribution

C.S., B.H.; Sample Collection & Clinical data recording: C.S., A.L., T.S.; Disease Diagnosis: C.S., A.L., L.B., B.H.; Medical support: A.L., L.B., S.S., A.C.; Conceptual design and project managing, T.S., R.H., R. V.-V.; Design & Performance of experiments, T.S., R.H.; MS-Analytics: S.P., R.H.; Computing Support, K.S.; Data Analysis, T.S., R.H., R. V.-V., D.B.; Writing of manuscript, T.S., R.H., R. V.-V., D.B., K.S.; Funding Acquisition, R.H.; Supervision, R.H.

Conflict of interest

All authors declare no conflicts of interest.

Patient consent

Obtained.

Ethical approval

The samples for this study were taken at the Otto-von-Guericke University Magdeburg and the Hannover Medical School while different projects (Magdeburg: Number 42/08, Number 47/15 and Hannover 2087-2013). Amendments to re-use the samples for this study were given by the local Ethics Committees.

Acknowledgements and funding

We thank the physicians for the diagnoses of the disease and for the medical support of this work as well as the patients for their permission to participate in this study. We acknowledge Corina Siewert for the laboratory support. This project was financed by the German Research Society (DFG) under number HE 8077/1-1.

References (67)

  • R. Sender et al.

    Revised estimates for the number of human and Bacteria cells in the body

    PLoS Biol.

    (2016)
  • N. Kaur et al.

    Intestinal dysbiosis in inflammatory bowel disease

    Gut Microbes

    (2011)
  • C. Maaser et al.

    ECCO-ESGAR guideline for diagnostic assessment in IBD part 1: initial diagnosis, monitoring of known IBD, detection of complications

    J. Crohn's Colitis

    (2018)
  • A. Sturm et al.

    ECCO-ESGAR guideline for diagnostic assessment in IBD part 2: IBD scores and general principles and technical aspects

    J. Crohn's Colitis

    (2018)
  • C. Mowat et al.

    Guidelines for the management of inflammatory bowel disease in adults

    Gut

    (2011)
  • R. Francescone et al.

    Microbiome, inflammation, and cancer

    Cancer J.

    (2014)
  • F. Mearin et al.

    Bowel disorders

    Gastroenterology

    (2016 Feb 18)
  • D.N. Frank et al.

    Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases

    Proc. Natl. Acad. Sci. U. S. A.

    (2007)
  • H. Sokol et al.

    Specificities of the fecal microbiota in inflammatory bowel disease

    Inflamm. Bowel Dis.

    (2006)
  • P. Correa et al.

    Helicobacter pylori infection and gastric adenocarcinoma

    US Gastroenterol. Hepatol. Rev.

    (2011)
  • R. Caccaro et al.

    Clinical utility of calprotectin and lactoferrin in patients with inflammatory bowel disease: is there something new from the literature?

    Expert. Rev. Clin. Immunol.

    (2012)
  • Human Microbiome Project, C

    Structure, function and diversity of the healthy human microbiome

    Nature

    (2012)
  • A.R. Erickson et al.

    Integrated metagenomics/metaproteomics reveals human host-microbiota signatures of Crohn's disease

    PLoS One

    (2012)
  • A. Heintz-Buschart et al.

    Integrated multi-omics of the human gut microbiome in a case study of familial type 1 diabetes

    Nat. Microbiol.

    (2016)
  • C. Manichanh et al.

    Reduced diversity of faecal microbiota in Crohn's disease revealed by a metagenomic approach

    Gut

    (2006)
  • C. Casen et al.

    Deviations in human gut microbiota: a novel diagnostic test for determining dysbiosis in patients with IBS or IBD

    Aliment. Pharmacol. Ther.

    (2015)
  • C. Schulz et al.

    Validation of two Calprotectin rapid tests in daily routine

    Clin. Lab.

    (2016)
  • R. Heyer et al.

    Proteotyping of biogas plant microbiomes separates biogas plants according to process temperature and reactor type

    Biotechnol. Biofuels

    (2016)
  • N. Popov et al.

    Eine Störungsfreie Mikromethode zur Bestimmung des Proteingehaltes in Gewebehomogenaten

    Acta biologica et medica Germanica

    (1975)
  • A. Shevchenko et al.

    Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels

    Anal. Chem.

    (1996)
  • T. Muth et al.

    The MetaProteomeAnalyzer: a powerful open-source software suite for metaproteomics data analysis and interpretation

    J. Proteome Res.

    (2015)
  • R. Craig et al.

    TANDEM: matching proteins with tandem mass spectra

    Bioinformatics

    (2004)
  • L.Y. Geer et al.

    Open mass spectrometry search algorithm

    J. Proteome Res.

    (2004)
  • Cited by (38)

    • Multi-omics in Crohn's disease: New insights from inside

      2023, Computational and Structural Biotechnology Journal
    • Neutrophil extracellular traps in inflammatory bowel diseases: Implications in pathogenesis and therapeutic targets

      2021, Pharmacological Research
      Citation Excerpt :

      Dinallo et al. [13] also noted that MPO, NE, PAD4 and histone H3 citrullinated (citH3) were overexpressed in the inflamed colon of patients with UC when compared to patients with CD. However, the proteins that make up NETs were also observed through metaproteomic analysis of fecal samples from patients with CD and UC [34]. In addition, Gottlieb et al. [31] investigated the presence of NETs in biopsy sections of pediatric patients affected by IBD and demonstrated the presence of NETs in samples from patients with both pediatric CD and UC, in which extracellular DNA, MPO, NE and histones were detected in the small intestine and colon.

    • Neutrophil Extracellular Traps in Inflammatory Bowel Disease: Pathogenic Mechanisms and Clinical Translation

      2021, Cellular and Molecular Gastroenterology and Hepatology
      Citation Excerpt :

      To date, 8 studies (Table 1) have demonstrated an increased presence of NETs in the inflamed gut mucosa, stool, or blood in IBD, 4 of which stipulate that NET abundance is positively correlated with active disease.64,66,69,70 Liquid chromatography-mass spectrometry–based IBD proteomics studies reveal an increase in key NET proteins such as myeloperoxidase (MPO) and NE, both of which are highly specific to neutrophils and involved in chromatin decondensation during NET formation,71 as well as increased calprotectin and cathepsin G, both of which have been found in NETs4 in both intestinal biopsies72 and fecal samples.73 Increased levels of NET-associated proteins in IBD have also been demonstrated by immunofluorescence, immunohistochemistry, or Western blot in intestinal biopsies60,65,66,69 as well as in the colonic mucosa of experimental colitis in mice.70

    View all citing articles on Scopus
    View full text