Tomas de Wouters

Quantifying diet-induced metabolic changes of the human gut microbiome

The human gut microbiome is known to be associated with various human disorders, but a major challenge is to go beyond association studies and elucidate causalities. Mathematical modeling of the human gut microbiome at a genome scale is a useful tool to decipher microbe-microbe, diet-microbe and microbe-host interactions. Here, we describe the CASINO (Community And Systems-level INteractive Optimization) toolbox, a comprehensive computational platform for analysis of microbial communities through metabolic modeling. We first validated the toolbox by simulating and testing the performance of single bacteria and whole communities in vitro. Focusing on metabolic interactions between the diet, gut microbiota, and host metabolism, we demonstrated the predictive power of the toolbox in a diet-intervention study of 45 obese and overweight individuals and validated our predictions by fecal and blood metabolomics data. Thus, modeling could quantitatively describe altered fecal and serum amino acid levels in response to diet intervention.

ANGPTL4 expression induced by butyrate and rosiglitazone in human intestinal epithelial cells utilizes independent pathways.

Short-chain fatty acids (SCFAs), such as butyrate and propionate, are metabolic products of carbohydrate fermentation by the microbiota and constitute the main source of energy for host colonocytes. SCFAs are also important for gastrointestinal health, immunity, and host metabolism. Intestinally produced angiopoietin-like protein 4 (ANGPTL4) is a secreted protein with metabolism-altering properties and may offer a route by which microbiota can regulate host metabolism. Peroxisome proliferator-activated receptor (PPAR)-γ has previously been shown to be involved in microbiota-induced expression of intestinal ANGPTL4, but the role of bacterial metabolites in this process has remained elusive. Here, we show that the SCFA butyrate regulates intestinal ANGPTL4 expression in a PPAR-γ-independent manner. Although PPAR-γ is not required for butyrate-driven intestinal ANGPTL4 expression, costimulating with PPAR-γ ligands and SCFAs leads to additive increases in ANGPTL4 levels. We suggest that PPAR-γ and butyrate rely on two separate regulatory sites, a PPAR-responsive element downstream the transcription start site and a butyrate-responsive element(s) within the promoter region, 0.5 kb upstream of the transcription start site. Furthermore, butyrate gavage and colonization with Clostridium tyrobutyricum, a SCFA producer, can independently induce expression of intestinal ANGPTL4 in germ-free mice. Thus, oral administration of SCFA or use of SCFA-producing bacteria may be additional routes to maintain intestinal ANGPTL4 levels for preventive nutrition or therapeutic purposes.

The Human Gut Microbiome and Its Dysfunctions

The human gastrointestinal tract hosts more than 100 trillion bacteria and archaea, which together make up the gut microbiota. The amount of bacteria in the human gut outnumbers human cells by a factor of 10, but some finely tuned mechanisms allow these microorganisms to colonize and survive within the host in a mutual relationship. The human gut microbiota co-evolved with humans to achieve a symbiotic relationship leading to physiological homeostasis. The microbiota provides crucial functions that human cannot exert themselves while the human host provides a nutrient-rich environment. Chaotic in the early stages of life, the assembly of the human gut microbiota remains globally stable over time in healthy conditions and absence of perturbation. Following perturbation, such as antibiotic treatment, bacteria will recolonize the niches with a composition and diversity similar to the basal level since the ecosystem is highly resilient. Yet, recurrent perturbations lead to a decrease in resilience capacity of the gut microbiome. Shifts in the bacterial composition and diversity of the human gut microbiota have been associated with intestinal dysfunctions such as inflammatory bowel disease and obesity. More than specific bacteria, a general destructuration of the ecosystem seems to be involved in these pathologies. Application of metagenomics to this environment may help in deciphering key functions and correlation networks specifically involved in health maintenance. In term, fecal transplant and synthetic microbiome transplant might be promising therapies for dysbiosis-associated diseases.

Identification of NF-κB Modulation Capabilities within Human Intestinal Commensal Bacteria

The intestinal microbiota plays an important role in modulation of mucosal immune responses. To seek interactions between intestinal epithelial cells (IEC) and commensal bacteria, we screened 49 commensal strains for their capacity to modulate NF-κB. We used HT-29/kb-seap-25 and Caco-2/kb-seap-7 intestinal epithelial cells and monocyte-like THP-1 blue reporter cells to measure effects of commensal bacteria on cellular expression of a reporter system for NF-κB. Bacteria conditioned media (CM) were tested alone or together with an activator of NF-κB to explore its inhibitory potentials. CM from 8 or 10 different commensal species activated NF-κB expression on HT-29 and Caco-2 cells, respectively. On THP-1, CM from all but 5 commensal strains stimulated NF-κB. Upon challenge with TNF-α or IL-1β, some CM prevented induced NF-κB activation, whereas others enhanced it. Interestingly, the enhancing effect of some CM was correlated with the presence of butyrate and propionate. Characterization of the effects of the identified bacteria and their implications in human health awaits further investigations.

Reporter bacteriophage A511::celB transduces a hyperthermostable glycosidase from Pyrococcus furiosus for rapid and simple detection of viable Listeria cells

Reporter bacteriophages for detection of pathogenic bacteria offer fast and sensitive screening for live bacterial targets. We present a novel strategy employing a gene encoding a hyperthermophilic enzyme, permitting the use of various substrates and assay formats. The celB gene from the hyperthermophilic archaeon Pyrococcus furiosus specifying an extremely thermostable β-glycosidase was inserted into the genome of the broad host range, virulent Listeria phage A511 by homologous recombination. It is expressed at the end of the infectious cycle, under control of the strong major capsid gene promoter Pcps. Infection of Listeria with A511::celB results in strong gene expression and synthesis of a fully functional β-glycosidase. The reporter phage was tested for detection of viable Listeria cells with different chromogenic, fluorescent or chemiluminescent substrates. The best signal-to-noise ratio and sufficiently high sensitivity was obtained using the inexpensive substrate 4-Methylumbelliferyl-α-D-Glucopyranoside (MUG). The reporter phage assay is simple to perform and can be completed in about 6 h. Phage infection, as well as the subsequent temperature shift, enzymatic substrate conversion and signal recordings are independent from each other and may be performed separately. The detection limit for viable Listeria monocytogenes in an assay format adapted to 96-well microplates was 7.2 x 102 cells per well, corresponding to 6 x 103 cfu per ml in suspension. Application of the A511::celB protocol to Listeria in spiked chocolate milk and salmon demonstrate the usefulness of the reporter phage for rapid detection of low numbers of the bacteria (10 cfu/g or less) in contaminated foods.

Probiotics tailored to the infant: a window of opportunity

Initial neonatal gut colonization is a crucial stage for developing a healthy physiology, beneficially influenced by breast-feeding. Breast milk has been shown not only to provide nutrients and bioactive immunological compounds, but also commensal bacteria, including gut-associated anaerobic bacteria such as Bifidobacterium species. Infant formulas are increasingly supplemented with probiotic bacteria despite uncertainties regarding their efficacy, and lack of mechanistic understanding. Breast milk may be a valuable source of such bacteria which, upon validation of their mechanism of action, might open a window of opportunity for developing probiotic-supplemented infant formula with proven efficacy.

Does Our Food (Environment) Change Our Gut Microbiome (‘In-Vironment’): A Potential Role for Inflammatory Bowel Disease?

Human biology can only be fully assessed by combining an analysis of both the host and its surrounding environment. As a part of the environment, the human gastrointestinal tract hosts more than 100 trillion bacteria making up the gut microbiota. The human host provides a nutrient-rich environment while the microbiota provides indispensable functions that humans cannot exert themselves. Shifts in the bacterial makeup of the human gut microbiota have been associated with disorders such as inflammatory bowel disease (IBD), irritable bowel syndrome and obesity. However, since most bacteria inhabiting our gut are not cultivable to date, until recently little was known about their individual functions. Metagenomics, i.e. the analysis of the collective genomes present in a defined ecosystem, gives insight into these specific functions. The first extensive catalogue of the intestinal metagenome outnumbers the size of the human genome by a factor of 150. Recently, 3 distinct ‘types’ of gut composition within the human population have been highlighted. These so-called ‘enterotypes’ are characterized by the dominant genera (Bacteroides, Prevotella and Ruminococcus) and their co-occurring phylogenetic groups. In accordance with the previously described impact of nutritional behavior (diet, probiotics and prebiotics) on specific bacterial populations, an association has been observed between long-term dietary habits and enterotypes. This recent discovery, i.e. that belonging to one or the other enterotype might be modulated by the diet opens up new perspectives in the fields of IBD, nutrition and therapeutic strategies.

The NF-κB binding site located in the proximal region of the TSLP promoter is critical for TSLP modulation in human intestinal epithelial cells.

Thymic stromal lymphopoietin (TSLP) is constitutively secreted by intestinal epithelial cells. It regulates gut DCs, therefore, contributing to the maintenance of immune tolerance. In the present report, we describe the regulation of TSLP expression in intestinal epithelial cells and characterize the role of several NF‐κB binding sites present on the TSLP promoter. TSLP expression can be stimulated by different compounds through activation of p38, protein kinase A, and finally the NF‐κB pathway. We describe a new NF‐κB binding element located at position –0.37 kb of the promoter that is crucial for the NF‐κB‐dependent regulation of TSLP. We showed that mutation of this proximal NF‐κB site abrogates the IL‐1β‐mediated transcriptional activation of human TSLP in several epithelial cell lines. We also demonstrated that both p65 and p50 subunits are able to bind this new NF‐κB binding site. The present work provides new insight into epithelial cell‐specific TSLP regulation.

Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells

The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB) in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor), we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health.

Adhesion Potential of Intestinal Microbes Predicted by Physico-Chemical Characterization Methods.

Bacterial adhesion to epithelial surfaces affects retention time in the human gastro-intestinal tract and therefore significantly contributes to interactions between bacteria and their hosts. Bacterial adhesion among other factors is strongly influenced by physico-chemical factors. The accurate quantification of these physico-chemical factors in adhesion is however limited by the available measuring techniques. We evaluated surface charge, interfacial rheology and tensiometry (interfacial tension) as novel approaches to quantify these interactions and evaluated their biological significance via an adhesion assay using intestinal epithelial surface molecules (IESM) for a set of model organisms present in the human gastrointestinal tract. Strain pairs of Lactobacillus plantarum WCFS1 with its sortase knockout mutant Lb. plantarum NZ7114 and Lb. rhamnosus GG with Lb. rhamnosus DSM 20021T were used with Enterococcus faecalis JH2-2 as control organism. Intra-species comparison revealed significantly higher abilities for Lb. plantarum WCSF1 and Lb. rhamnosus GG vs. Lb. plantarum NZ7114 and Lb. rhamnosus DSM 20021T to dynamically increase interfacial elasticity (10−2 vs. 10−3 Pa*m) and reduce interfacial tension (32 vs. 38 mN/m). This further correlated for Lb. plantarum WCSF1 and Lb. rhamnosus GG vs. Lb. plantarum NZ7114 and Lb. rhamnosus DSM 20021T with the decrease of relative hydrophobicity (80–85% vs. 57–63%), Zeta potential (-2.9 to -4.5 mV vs. -8.0 to -13.8 mV) and higher relative adhesion capacity to IESM (3.0–5.0 vs 1.5–2.2). Highest adhesion to the IESM collagen I and fibronectin was found for Lb. plantarum WCFS1 (5.0) and E. faecalis JH2-2 (4.2) whereas Lb. rhamnosus GG showed highest adhesion to type II mucus (3.8). Significantly reduced adhesion (2 fold) to the tested IESM was observed for Lb. plantarum NZ7114 and Lb. rhamnosus DSM 20021T corresponding with lower relative hydrophobicity, Zeta potential and abilities to modify interfacial elasticity and tension. Conclusively, the use of Zeta potential, interfacial elasticity and interfacial tension are proposed as suitable novel descriptive and predictive parameters to study the interactions of intestinal microbes with their hosts.