Pascale Lepercq

Bacterial protein signals are associated with Crohn’s disease

Objective No Crohn’s disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls. Design We first developed and validated a workflow—including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS—to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions. Results Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins. Conclusions This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis.

Gnotobiotic rats harboring human intestinal microbiota as a model for studying cholesterol-to-coprostanol conversion

The efficiency of microbial reduction of cholesterol to coprostanol in human gut is highly variable among population and mechanisms remain unexplored. In the present study, we investigated whether microbial communities and their cholesterol metabolism characteristics can be transferred to germ-free rats. Two groups of six, initially germ-free rats were associated with two different human microbiota, exhibiting high and low cholesterol-reducing activities. Four months after inoculation, enumeration of coprostanoligenic bacteria, fecal coprostanol levels and composition of the fecal microbial communities were studied in gnotobiotic rats and compared with those of the human donors. Combination of culture (most probable number enumeration of active bacteria) and biochemical approaches (extraction followed by gas chromatography of sterols) showed that gnotobiotic rats harbored a coprostanoligenic bacterial population level and exhibited coprostanoligenic activities similar to those of the corresponding human donor. On the other hand, molecular approaches (whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes, and temporal temperature gradient gel electrophoresis of bacterial 16S rRNA gene amplicons) demonstrated that gnotobiotic rats reproduced a stable microbial community, close to the human donor microbiota at the group or genus levels but different at the dominant species level. These results suggest that the gnotobiotic rat model can be used to explore the still unknown human intestinal microbiota involved in luminal cholesterol metabolism, including regulation of expression of its activity and impact on health.

Composition and metabolism of the intestinal microbiota in consumers and non-consumers of yogurt

The objective of the present study was to evaluate the impact of a regular consumption of yogurt on the composition and metabolism of the human intestinal microbiota. Adult subjects were selected on the basis of daily food records and divided into two groups: yogurt consumers (at least 200 g yogurt consumed per d, n 30); non-consumers (no yogurt, n 21). Their faecal microbiota was analysed using molecular methods (in situ hybridisation and PCR amplification combined with separation by denaturing gel electrophoresis) and its metabolic characteristics were assessed by measuring glycosidase, P-glucuronidase and reductase activities and profiling SCFA, neutral sterols and bile acids. The yogurt starter Lactobacillus delbrueckii ssp. bulgaricus (identity confirmed by 16S rRNA sequencing) was detected in 73% of faecal samples from fermented milk consumers v. 28% from non-consumers (P=0.003). In yogurt consumers, the level of Enterobacteriaceae was significantly lower (P=0.006) and 13-galactosidase activity was significantly increased (P=0.048). In addition, within this group, 3-galactosidase activity and the Bifidobacterium population were both positively correlated with the amount of fermented milk ingested (r 0.66, P<0.0001 and r 0.43, P=0.018, respectively). Apart from these effects, which can be considered beneficial to the host, no other major differences could be detected regarding the composition and metabolic activity of intestinal microbiota.

Bacteroides sp. Strain D8, the First Cholesterol-Reducing Bacterium Isolated from Human Feces

ABSTRACT The microbial community in the human colon contains bacteria that reduce cholesterol to coprostanol, but the species responsible for this conversion are still unknown. We describe here the first isolation and characterization of a cholesterol-reducing bacterium of human intestinal origin. Strain D8 was isolated from a 10−8 dilution of a fresh stool sample provided by a senior male volunteer with a high capacity to reduce luminal cholesterol to coprostanol. Cholesterol-to-coprostanol conversion by strain D8 started on the third day, while cells were in stationary phase, and was almost complete after 7 days. Intermediate products (4-cholesten-3-one and coprostanone) were occasionally observed, suggesting an indirect pathway for cholesterol-to-coprostanol conversion. Resting-cell assays showed that strain D8 could reduce 1.5 μmol of cholesterol/mg bacterial protein/h. Strain D8 was a gram-negative, non-spore-forming, rod-shaped organism identified as a member of the genus Bacteroides closely related to Bacteroides vulgatus, based on its morphological and biochemical characteristics. The 16S rRNA gene sequence of strain D8 was most similar (>99.5%) to those of two isolates of the recently described species Bacteroides dorei. Phylogenetic tree construction confirmed that Bacteroides sp. strain D8 clustered within an independent clade together with these B. dorei strains. Nevertheless, no cholesterol-reducing activity could be detected in cultures of the B. dorei type strain. Based on Bacteroides group-specific PCR-temporal temperature gradient gel electrophoresis, there was no correlation between the presence of a band comigrating with the band of Bacteroides sp. strain D8 and cholesterol conversion in 11 human fecal samples, indicating that this strain is unlikely to be mainly responsible for cholesterol conversion in the human population.

Influence of Camembert consumption on the composition and metabolism of intestinal microbiota: a study in human microbiota-associated rats

The objective of the present study was to evaluate the consequence of Camembert consumption on the composition and metabolism of human intestinal microbiota. Camembert cheese was compared with milk fermented by yoghurt starters and Lactobacillus casei as a probiotic reference. The experimental model was the human microbiota-associated (HM) rat. HM rats were fed a basal diet (HMB group), a diet containing Camembert made from pasteurised milk (HMCp group) or a diet containing fermented milk (HMfm group). The level of micro-organisms from dairy products was measured in faeces using cultures on a specific medium and PCR-temporal temperature gradient gel electrophoresis. The metabolic characteristics of the caecal microbiota were also studied: SCFA, NH3, glycosidase and reductase activities, and bile acid degradations. The results showed that micro-organisms from cheese comprised 10(5)-10(8) bacteria/g faecal sample in the HMCp group. Lactobacillus species from fermented milk were detected in HMfm rats. Consumption of cheese and fermented milk led to similar changes in bacterial metabolism: a decrease in azoreductase activity and NH3 concentration and an increase in mucolytic activities. However, specific changes were observed: in HMCp rats, the proportion of ursodeoxycholic resulting from chenodeoxycholic epimerisation was higher; in HMfm rats, alpha and beta-galactosidases were higher than in other groups and both azoreductases and nitrate reductases were lower. The results show that, as for fermented milk, Camembert consumption did not greatly modify the microbiota profile or its major metabolic activities. Ingested micro-organisms were able to survive in part during intestinal transit. These dairy products exert a potentially beneficial influence on intestinal metabolism.

Bifidobacterium animalis strain DN-173 010 hydrolyses Bile salts in the gastrointestinal tract of pigs

Background: Bile salt hydrolase (BSH) activity is widespread among ingested bifidobacteria and lactobacilli. It is sometimes considered to be beneficial because of its putative lowering effect on cholesterol absorption and sometimes considered to be deleterious because it may compromise normal fat absorption and even promote the formation of secondary cytotoxic bile acids by the resident intestinal flora. However, the true hydrolysis of bile salts in vivo by ingested living bacteria remains unexplored. The aim of the study was to examine whether or not Bifidobacterium animalis DN-173 010 (used in fermented milks), which demonstrates a BSH activity in vitro, was also active in vivo during its transit in the intestine of pigs. Methods: Direct measurement of total and unconjugated bile acids reabsorbed into the portal vein was done, before and after the pigs had been treated for 2 weeks with two daily doses of≈ 3.5·10[Formula: See Text] colony-forming units of living (6 pigs) or inactivated B. animalis (6 pigs). Results: None of the treatments modified the portal serum concentration of total bile acids over a 6-h postprandial period. Unconjugated bile acids represented up to 44% and 53% of total bile acids after 1 and 2 weeks of treatment with living bacteria, respectively, compared with only 25% (P < 0.05) before treatment or after 1 or 2 weeks of treatment with inactivated bacteria. Conclusions: Living B. animalis DN-173 010 exhibited a BSH activity in the gastrointestinal tract of pigs, most probably in the small bowel. There was no sign of increased formation of secondary bile acids beyond the hydrolysis reaction.

Metatranscriptomics Reveals the Active Bacterial and Eukaryotic Fibrolytic Communities in the Rumen of Dairy Cow Fed a Mixed Diet

Ruminants have a unique ability to derive energy from the degradation of plant polysaccharides through the activity of the rumen microbiota. Although this process is well studied in vitro, knowledge gaps remain regarding the relative contribution of the microbiota members and enzymes in vivo. The present study used RNA-sequencing to reveal both the expression of genes encoding carbohydrate-active enzymes (CAZymes) by the rumen microbiota of a lactating dairy cow and the microorganisms forming the fiber-degrading community. Functional analysis identified 12,237 CAZymes, accounting for 1% of the transcripts. The CAZyme profile was dominated by families GH94 (cellobiose-phosphorylase), GH13 (amylase), GH43 and GH10 (hemicellulases), GH9 and GH48 (cellulases), PL11 (pectinase) as well as GH2 and GH3 (oligosaccharidases). Our data support the pivotal role of the most characterized fibrolytic bacteria (Prevotella, Ruminocccus and Fibrobacter), and highlight a substantial, although most probably underestimated, contribution of fungi and ciliate protozoa to polysaccharide degradation. Particularly these results may motivate further exploration of the role and the functions of protozoa in the rumen. Moreover, an important part of the fibrolytic bacterial community remains to be characterized since one third of the CAZyme transcripts originated from distantly related strains. These findings are used to highlight limitations of current metatranscriptomics approaches to understand the functional rumen microbial community and opportunities to circumvent them.

Unraveling the pectinolytic function of Bacteroides xylanisolvens using a RNA-seq approach and mutagenesis

BackgroundDiet and particularly dietary fibres have an impact on the gut microbiome and play an important role in human health and disease. Pectin is a highly consumed dietary fibre found in fruits and vegetables and is also a widely used additive in the food industry. Yet there is no information on the effect of pectin on the human gut microbiome. Likewise, little is known on gut pectinolytic bacteria and their enzyme systems. This study was undertaken to investigate the mechanisms of pectin degradation by the prominent human gut symbiont Bacteroides xylanisolvens.ResultsTranscriptomic analyses of B. xylanisolvens XB1A grown on citrus and apple pectins at mid- and late-log phases highlighted six polysaccharide utilization loci (PUL) that were overexpressed on pectin relative to glucose. The PUL numbers used in this report are those given by Terrapon et al. (Bioinformatics 31(5):647-55, 2015) and found in the PUL database: http://www.cazy.org/PULDB/. Based on their CAZyme composition, we propose that PUL 49 and 50, the most overexpressed PULs on both pectins and at both growth phases, are involved in homogalacturonan (HG) and type I rhamnogalacturonan (RGI) degradation, respectively. PUL 13 and PUL 2 could be involved in the degradation of arabinose-containing side chains and of type II rhamnogalacturonan (RGII), respectively. Considering that HG is the most abundant moiety (>70 %) within pectin, the importance of PUL 49 was further investigated by insertion mutagenesis into the susC-like gene. The insertion blocked transcription of the susC-like and the two downstream genes (susD-like/FnIII). The mutant showed strong growth reduction, thus confirming that PUL 49 plays a major role in pectin degradation.ConclusionThis study shows the existence of six PULs devoted to pectin degradation by B. xylanisolvens, one of them being particularly important in this function. Hence, this species deploys a very complex enzymatic machinery that probably reflects the structural complexity of pectin. Our findings also highlight the metabolic plasticity of B. xylanisolvens towards dietary fibres that contributes to its competitive fitness within the human gut ecosystem. Wider functional and ecological studies are needed to understand how dietary fibers and especially plant cell wall polysaccharides drive the composition and metabolism of the fibrolytic and non-fibrolytic community within the gut microbial ecosystem.

Xylan degradation by the human gut Bacteroides xylanisolvens XB1A(T) involves two distinct gene clusters that are linked at the transcriptional level.

BackgroundPlant cell wall (PCW) polysaccharides and especially xylans constitute an important part of human diet. Xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. Xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. In order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont Bacteroides xylanisolvens XB1AT.ResultsTranscriptomic analyses of B. xylanisolvens XB1AT grown on insoluble oat-spelt xylan (OSX) at mid- and late-log phases highlighted genes in a polysaccharide utilization locus (PUL), hereafter called PUL 43, and genes in a fragmentary remnant of another PUL, hereafter referred to as rPUL 70, which were highly overexpressed on OSX relative to glucose. Proteomic analyses supported the up-regulation of several genes belonging to PUL 43 and showed the important over-production of a CBM4-containing GH10 endo-xylanase. We also show that PUL 43 is organized in two operons and that the knockout of the PUL 43 sensor/regulator HTCS gene blocked the growth of the mutant on insoluble OSX and soluble wheat arabinoxylan (WAX). The mutation not only repressed gene expression in the PUL 43 operons but also repressed gene expression in rPUL 70.ConclusionThis study shows that xylan degradation by B. xylanisolvens XB1AT is orchestrated by one PUL and one PUL remnant that are linked at the transcriptional level. Coupled to studies on other xylanolytic Bacteroides species, our data emphasize the importance of one peculiar CBM4-containing GH10 endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. Our results also suggest that B. xylanisolvens XB1AT has specialized in the degradation of xylans of low complexity. This functional feature may provide a niche to all xylanolytic bacteria harboring similar PULs. Further functional and ecological studies on fibrolytic Bacteroides species are needed to better understand their role in dietary fiber degradation and their impact on intestinal health.

Increasing ursodeoxycholic acid in the enterohepatic circulation of pigs through the administration of living bacteria.

We investigated the feasibility of increasing ursodeoxycholic acid (UDCA) in the enterohepatic circulation of pigs by administering living bacteria capable of epimerising endogenous amidated chenodeoxycholic acid (CDCA) to UDCA. We first demonstrated that combining Bifidobacterium animalis DN-173 010, as a bile salt-hydrolysing bacterium, and Clostridium absonum ATCC 27555, as a CDCA to UDCA epimerising bacterium, led to the efficient epimerisation of glyco- and tauro-CDCA in vitro, with respective UDCA yields of 55.8 (SE 2.8) and 36.6 (SE 1.5)%. This strain combination was then administered to hypercholesterolaemic pigs over a 3-week period, as two daily preprandial doses of either viable (six experimental pigs) or heat-inactivated bacteria (six controls). The main effects of treatment were on unconjugated bile acids (P=0.035) and UDCA (P<0.0001) absorbed into the portal vein, which increased 1.6-1.7- and 3.5-7.5-fold, respectively, under administration of living compared with inactivated bacteria. In bile, UDCA did not increase significantly, but the increase in biliary lithocholic acid with time in the controls was not observed in the experimental pigs (P=0.007), and the same trend was observed in faeces. All other variables (biliary lipid equilibrium, plasma lipid levels and partition of cholesterol between the different lipoprotein classes) remained unaffected by treatment throughout the duration of the experiment. In conclusion, it is feasible to increase the bioavailability of UDCA to the intestine and the liver by administering active bacteria. This may represent an interesting new probiotic activity, provided that in future it could be expressed by a safe food micro-organism.