Comprehensive biomolecular isolation for integrated Multi-omics

Comprehensive biomolecular isolation protocol

In microbial ecology, high-resolution molecular biology approaches are vital for discovering and characterizing the vast microbial diversity, and understanding the interaction of microbial communities with biotic and abiotic environmental factors. Integrated omics, comprising community genomics, transcriptomics, proteomics and metabolomics, is able to reveal the links between genetic potential and functionality in microbial communities in a truly systematic fashion. However, mixed microbial communities are complex, dynamic and heterogeneous and it is therefore essential that biomolecular fractions obtained for high-throughput omic analyses are representative of single undivided samples to facilitate meaningful data integration, analysis and modelling. We have developed a new methodological framework for the reproducible sequential isolation of high-quality polar and non-polar metabolites polar and non-polar, RNA (optionally split into large and small RNA fractions), DNA and proteins from single undivided mixed microbial community samples. The developed methodological framework lays the foundation for standardized molecular eco-systematic studies on a range of different microbial communities in the future.

Publications:

Roume H., Muller E.E.L., Cordes T., Renaut J., Hiller K., and Wilmes P. (2013) A biomolecular isolation framework for Eco-Systems Biology. The ISME Journal 7:110–121 (doi: 10.1038/ismej.2012.72).

Roume H., Heintz-Buschart A., Muller E.E.L. and Wilmes P.* (2013) Sequential isolation of metabolites, RNA, DNA, and proteins from the same unique sample. Methods in Enzymology 513:219–236. Invited article for a special issue on “Microbial Metagenomics, Metatranscriptomics, and Metaproteomics” edited by Edward F. DeLong (doi: 10.1016/B978-0-12-407863-5.00011-3).

Muller EE, Heintz-Buschart A, Roume H, Wilmes P (2013) The sequential isolation of metabolites, RNA, DNA, and proteins from a single, undivided mixed microbial community sample. Protocol Exchange (doi: 10.1038/protex.2014.051).

Shah P., Muller E.E.L., Lebrun L.A., Wampach L., Wilmes P. (2018) Sequential isolation of DNA, RNA, protein and metabolite fractions from murine organs and intestinal contents for integrated omics of host-microbiota interactions. Methods in Molecular Biology, 1841:279-291 (doi: 10.1007/978-1-4939-8695-8_19).

Funding: FNR ATTRACT programme grant “SysBioNaMA”; SysBioWwTBioEng FNR AFR PHD programme grant; MetaLABpop FNR AFR PDR programme grant; European Union Joint Programme – Neurodegenerative Disease Research grant.

High-throughput comprehensive biomolecular extractions for integrated omic analysis of biological samples

Integrated omic analysis of biological samples aims to resolve the information within each biomolecular fraction from genetic potential of the organisms to their functional capacity. Our comprehensive biomolecular isolation protocol lays the foundation for systematic integrated omics. The protocol itself is time consuming and therefore throughput is mediocre. Together with industrial partner, we have developed three robots which allow the automated isolation of DNA, RNA, protein and metbaolites fractions of sufficient quality and quantity for systematic, downstream multi-omic analyses. This provides platform for standardised integrated omic analyses of different biological samples in the future.

Funding: FNR ATTRACT programme grant “SysBioNaMA”; MetaLABpop FNR AFR PDR programme grant; European Union Joint Programme – Neurodegenerative Disease Research grant  

Contaminant-free sRNA sequencing

Comprehensive analyses of biomolecules from low input samples, for example sRNAseq from human plasma samples, are prone to distortion by different biases. The danger of contamination with biomaterial from lab reagents is an important consideration. In this project, we develop strategies to control the cleanness of lab reagents and to improve the safety of commonly used extraction kits.

Collaboration: David Galas (Pacific Northwest Diabetes Research Institute, Seattle, USA), Qiagen  

Publications:

Heintz-Buschart A., Yusuf D., Kaysen A., Etheridge A., Fritz J.V., May P., de Beaufort C., Upadhyaya B.B., Ghosal A., Galas D., Wilmes P.* (2018) Small RNA profiling of low biomass samples: identification and removal of contaminants. BMC Biology, 16:52 (doi: 10.1186/s12915-018-0522-7).

Funding: FNR Proof-of-Concept programme grant “MycromeDX”.

Bioinformatic approaches for integrated community omics

Binning of metagenomic sequences using nucleotide signatures and machine learning approaches

Any environmental sample, including those taken from human subjects (e.g. from the gastrointestinal tract) contain a complex mixture of microbial organisms. Due to current limitations of isolated culturing of many of these organisms, community genomics are generally used to study microbial consortia in situ. However, the scrambled genomic information obtainable using metagenomics needs to be related back to the organisms of origin, i.e. binned, so as to be able to study the individual community members separately. The advent of next-generation sequencing allows to create very large data sets, hence further increasing the difficulty of processing these complex mixtures. The goal of this computational biology project is to use in silico approaches for the retrieval of the individual sequences of the constituent organisms in an efficient, precise and automated way using state-of-the-art machine learning approaches. Downstream applications (e.g. genomic assembly and multi-omics integration) will benefit from this step by, among others, reducing the complexity, increasing sensitivity and allowing for parallel workflows.

Publications:

Laczny C., Pinel N., Vlassis N. and Wilmes P. (2014) Alignment-free visualization of metagenomic data by nonlinear dimension reduction. Scientific Reports 4:4516 (doi: 10.1038/srep04516).

Laczny C. and Wilmes P. (2014) Identification of condition-specific microbial populations from human metagenomic data. Nucleic Acids as Molecular Diagnostics. (Keller A. & Meese E. eds.) Wiley-VCH, Weinheim, Germany (doi: 10.1002/9783527672165.ch11). 

Laczny C.C., Sternal T., Plugaru V., Atashpendar A., Margossian H.H., Gawron P., Coronado S., van der Maaten L., Vlassis N., and Wilmes P. (2015) VizBin - an application for reference-independent visualization and human-augmented binning of metagenomic data. Microbiome, 3:1 (doi: 10.1186/s40168-014-0066-1).

Laczny C.C., Muller E.E.L., Heintz-Buschart A., Herold M., Lebrun L.A., Hogan A., May P., De Beaufort C., Wilmes P. (2016) Recovery, refinement, and characterisation of hitherto undescribed population-level genomes from the human gastrointestinal tract. Frontiers in Microbiology 7:884 (doi: 10.3389/fmicb.2016.00884).

Funding: FNR AFR PHD programme grant “DissRNA“.

Integrated omics data analysis pipeline

Molecular Eco-Systems Biology generates massive omics datasets derived from next generation sequencing and high-throughput mass spectrometry. This data has to be filtered, processed and analysed in an integrated fashion. We are developing methods which allow integration of metagenomic, metatranscriptomic, metaproteomic and metabolomic data by combining efficient in-house pipelines together with state-of-the-art publicly available software tools.

References:

Narayanasamy S., Jarosz Y., Muller E.E.L., Heintz-Buschart A., Herold M., Kaysen A., Laczny C.L., Pinel N., May P., Wilmes P. (2016) IMP: a reproducible pipeline for reference-independent integrated metagenomic and metatranscriptomic analyses. Genome Biology 17:260 (doi: 10.1186/s13059-016-1116-8).

Funding: FNR ATTRACT programme grant “SysBioNaMA”; FNR AFR PHD programme grant “PopAntiVir“.

Human-microbe co-culture models

Design, fabrication and implementation of a microfluidics-based human-microbial in vitro co-culture device

In the natural world, individual cell populations are typically not found in isolation but are in direct contact with other cell types or organisms. Co-culture systems have been developed for addressing a number of fundamental biological questions relating to interactions between different cell populations but are typically limited in scope. This project is focussed on the development of a modular microfluidics-based co-culture device, termed HuMiX, which allows proximal co-culture of human epithelial cells and microbes. The HuMiX device mimics physiologically relevant spatial dimensions and establishes extracellular matrix conditions, which allow the establishment of stable growth conditions for both cell contingents. We have developed such devices from design to implementation via state-of-the-art micro fabrication techniques. The devices include integrated sensors for online monitoring of physicochemical parameters like concentration of oxygen and pH. As a proof-of-concept, we have recently demonstrated long-term co-culture of human epithelial cell lines (Caco2) with LGG and/or B. caccae,, which forms the basis for developing a microfluidics-based model of the entire human gastrointestinal tract.

Collaborator: Frederic Zenhausern (University of Arizona, Phoenix, USA)

Publications:

Fritz J.V., Desai M.S., Shah P., Schneider J.G and Wilmes P.* (2013) From meta-omics to causality: experimental models for human microbiome research. Microbiome 1:14 (doi: 10.1186/2049-2618-1-14).

Shah P., Fritz J.V., Glaab E., Desai M., Greenhalgh K., Frachet A., Niegowska M., Estes M., Jäger C., Seguin-Devaux C., Zenhausern F., Wilmes P.* (2016) A microfluidics-based in vitro model of the gastrointestinal human-microbe interface. Nature Communications 7:11535 (doi: 10.1038/ncomms11535).

Wilmes P.*, Marta C., van de Wiele T. (2018) Resolving host-microbe interactions in the gut: the promise of in vitro models to complement in vivo research. Current Opinion in Microbiology 44:28-33 (doi: 10.1016/j.mib.2018.07.001).

Funding: FNR CORE programme grant ”HuMiX”; Proof-of-concept FNR programme grant “HuMix2.0” and “iHuMiX”.

Co-culture of human primary cells with gastrointestinal microorganisms

A human individual’s microbiome consists of around 100 trillion cells, which represents at least ten times as many cells as human cells constitute the body. Beneficial effects of the presence of microbial communities on human physiology range from immune cell development and homeostasis, food digestion via the fermentation of non-digestible dietary components in the large intestine to balancing the host’s metabolis and promoting angiogenesis. Negative consequences for the host linked to the human microbiome include for example chronic inflammation and infection. Indeed, shifts in microbial community structure and function (dysbiosis) have been linked to numerous human diseases, including inflammatory bowel disease, diabetes mellitus, obesity, cardiovascular disease and cancer. The largest microbial reservoir of the human body is the gastrointestinal tract (GIT) and, thus, it is also the most studied and important from a biomedical perspective.

Therefore, it seems of great importance to understand and control the interplay between GIT microorganisms and human immune cells located in the gastrointestinal-associated lymphoid tissue (GALT), which constitutes the largest immune compartment in the human body. It is estimated that T cells associated with the small intestinal epithelium alone account for more than 60% of the total body lymphocytes. Currently, it is difficult to study the crosstalk between human immune cells and GI microorganisms in vivo in humans for obvious ethical reasons. Moreover, a systematic manipulation of variables to test the impact of a specific subtype of immune cells on the microbiota as well as the inflammatory effect of specific microbial species on human immune cells is not possible in vivo. In this respect, we plan to develop a microfluidics-based in vitro co-culture system, which allows the culture and modulation of different subtypes of primary immune cells in presence and absence of human gut microorganisms. Bacterial and human immune cells are separated by an epithelial cell monolayer in order to closely mimic the GIT.

Collaborators: RIKEN Center for Integrative Medical Sciences, Laboratory for Gut Homeostasis, Team Leader: Kenya Honda

Publications:

Fritz J.V., Desai M.S., Shah P., Schneider J.G., and Wilmes P. (2013) From meta-omics to causality: experimental models for human microbiome research. Microbiome 1:14 (doi: 10.1186/2049-2618-1-14).

Shah P., Fritz J.V., Glaab E., Desai M., Greenhalgh K., Frachet A., Niegowska M., Estes M., Jäger C., Seguin-Devaux C., Zenhausern F., Wilmes P. (2016) A microfluidics-based in vitro model of the gastrointestinal human-microbe interface. Nature Communications 7:11535 (doi: 10.1038/ncomms11535).

Funding: FNR: AFR - Development and establishment of a microfluidics-based in vitro culture model to study the impact of HIV infection on the gastrointestinal mucosal barrier - grant to Joëlle Fritz; FNR CORE programme grant, Proof-of-concept FNR programme grant

A study of the molecular mechanisms underlying the response of human colorectal adenocarcinoma enterocytes to prebiotics/probiotics

The majority of the microorganisms constituting the human microbiome inhabit the gastrointestinal tract (GIT) where they play essential roles in governing human health. A variety of diseases including colorectal cancer (CRC) are associated with dysbiosis, a pathological imbalance in the intestinal microbiota. Apart from endogenous microbial consortia, diets supplemented with prebiotics, are thought to have a major effect on GIT microbiota and are inversely correlated with the risk of developing CRC. Furthermore, the use of probiotics including Lactobacillus rhamnosus GG has been found to exhibit anti-cancer effects. Here, we study the synergistic effects of probiotic bacterial strains, dietary components and colorectal adenocarcinoma enterocytes of the human GIT using the microfluidics-based GIT co-culture model (HuMiX). More specifically we are interested in the phenotypical characteristics of the enterocytes using specific cell invasion, migration and proliferation assays. We are also studying the gene expression changes in human enterocytes after different pre-and probiotic treatments within the HuMiX model.

A thorough mechanistic understanding of the interplay between dietary habits, bacterial metabolism and human physiology is required to understand the role of pre-and probiotics and apply them appropriately in CRC therapies and prevention. In this context, this project will likely results in recommendations for dietary and probiotic-based interventions to modulate the microbiota-host relationship in order to reduce the expression of pro-carcinogenic genes and reduce pro-inflammatory responses that have been found to play a pivotal role in CRC.

Collaborators: Serge Haan and Elisabeth Letellier (Life Sciences Research Unit, Luxembourg)

Publication:

Greenhalgh K., Ramiro-Garcia J., Heinken A., Ullmann P., Bintener T., Pacheco M.P., Baginska J., Frachet A., Halder R., Fritz J.V., Sauter T., Thiele I., Haan S., Letellier E., Wilmes P. (2019) Integrated in vitro and in silico modelling delineates the molecular effects of a synbiotic regimen on colorectal cancer-derived cells. Cell Reports 27:1621-1632.e9 (doi: 10.1016/j.celrep.2019.04.001).

Funding: Internal Research Project of the University of Luxembourg “MiDiCa”; FNR AFR PhD grant “NutriHuMiX“; Pump-prime grant from the Luxembourg Personalised Medicine Consortium

Study of human−microbial molecular interactions using a model microbial community representative of the gastrointestinal tract

The microbial communities that inhabit the human gastrointestinal tract play roles in both health and disease. Different species of gut bacteria (~10^3) possess wider metabolic capabilities, including those not encoded in the human genome e.g., degradation of complex carbohydrates present in our diets. Changes in the relative proportions of distinct functional groups of gut bacteria, termed dysbiosis, have been implicated in several intestinal disorders, including diabetes, inflammatory bowel disease (IBD) and colon cancer. Little is known about the mechanisms behind dysbiosis; most studies have taken case-control comparative analytical approaches to identify species with altered abundance during disease. However, most studies have not had the statistical power to infer causal relationships. Therefore, in vivo or in vitro experiments are necessary to examine the effects of different bacterial groups. To carry out such experiments, it is necessary to compile representative artificial gut microbial communities with which hypotheses can be tested. Therefore, we have designed a synthetic (simplified) human intestinal microbiota that contains species with diverse metabolic potentials. This artificial microbiota is being used to study the interaction of gut microbes with the host, using in vivo (gnotobiotic mice) and in vitro (microfluidics-based device; see the section on HuMiX) approaches.

Funding: HuMiX FNR CORE programme grant