The human intestinal tract is colonized by microbial communities that show a subject-specific composition and a high-level temporal stability in healthy adults. To determine whether this is reflected at the functional level, we compared the faecal metaproteomes of healthy subjects over time using a novel high-throughput approach based on denaturing polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry. The developed robust metaproteomics workflow and identification pipeline was used to study the composition and temporal stability of the intestinal metaproteome using faecal samples collected from 3 healthy subjects over a period of six to twelve months. The same samples were also subjected to DNA extraction and analysed for their microbial composition and diversity using the Human Intestinal Tract Chip, a validated phylogenetic microarray. Using metagenome and single genome sequence data out of the thousands of mass spectra generated per sample, approximately 1,000 peptides per sample were identified. Our results indicate that the faecal metaproteome is subject-specific and stable during a one-year period. A stable common core of approximately 1,000 proteins could be recognized in each of the subjects, indicating a common functional core that is mainly involved in carbohydrate transport and degradation. Additionally, a variety of surface proteins could be identified, including potential microbes-host interacting components such as flagellins and pili. Altogether, we observed a highly comparable subject-specific clustering of the metaproteomic and phylogenetic profiles, indicating that the distinct microbial activity is reflected by the individual composition.

BackgroundInfants born by caesarean section or receiving antibiotics are at increased risk of developing metabolic, inflammatory and immunological diseases, potentially due to disruption of normal gut microbiota at a critical developmental time window. We investigated whether probiotic supplementation could ameliorate the effects of antibiotic use or caesarean birth on infant microbiota in a double blind, placebo-controlled randomized clinical trial. Mothers were given a multispecies probiotic, consisting of Bifidobacterium breve Bb99 (Bp99 2 × 108 cfu) Propionibacterium freundenreichii subsp. shermanii JS (2 × 109cfu), Lactobacillus rhamnosus Lc705 (5 × 109 cfu) and Lactobacillus rhamnosus GG (5 × 109 cfu) (N = 168 breastfed and 31 formula-fed), or placebo supplement (N = 201 breastfed and 22 formula-fed) during pregnancy, and the infants were given the same supplement. Faecal samples of the infants were collected at 3 months and analyzed using taxonomic, metagenomic and metaproteomic approaches.ResultsThe probiotic supplement had a strong overall impact on the microbiota composition, but the effect depended on the infant’s diet. Only breastfed infants showed the expected increase in bifidobacteria and reduction in Proteobacteria and Clostridia. In the placebo group, both birth mode and antibiotic use were significantly associated with altered microbiota composition and function, particularly reduced Bifidobacterium abundance. In the probiotic group, the effects of antibiotics and birth mode were either completely eliminated or reduced.ConclusionsThe results indicate that it is possible to correct undesired changes in microbiota composition and function caused by antibiotic treatments or caesarean birth by supplementing infants with a probiotic mixture together with at least partial breastfeeding.Trial registrationclinicaltrials.gov NCT00298337. Registered March 2, 2006.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0567-4) contains supplementary material, which is available to authorized users.

Metaproteomic research involves various computational challenges during the identification of fragmentation spectra acquired from the proteome of a complex microbiome. These issues are manifold and range from the construction of customized sequence databases, the optimal setting of search parameters to limitations in the identification search algorithms themselves. In order to assess the importance of these individual factors, we studied the effect of strategies to combine different search algorithms, explored the influence of chosen database search settings, and investigated the impact of the size of the protein sequence database used for identification. Furthermore, we applied de novo sequencing as a complementary approach to classic database searching. All evaluations were performed on a human intestinal metaproteome dataset. Pyrococcus furiosus proteome data were used to contrast database searching of metaproteomic data to a classic proteomic experiment. Searching against subsets of metaproteome databases and the use of multiple search engines increased the number of identifications. The integration of P. furiosus sequences in a metaproteomic sequence database showcased the limitation of the target-decoy-controlled false discovery rate approach in combination with large sequence databases. The selection of varying search engine parameters and the application of de novo sequencing represented useful methods to increase the reliability of the results. Based on our findings, we provide recommendations for the data analysis that help researchers to establish or improve analysis workflows in metaproteomics.

BackgroundPeptide spectrum matching (PSM) is the standard method in shotgun proteomics data analysis. It relies on the availability of an accurate and complete sample proteome that is used to make interpretation of the spectra feasible. Although this procedure has proven to be effective in many proteomics studies, the approach has limitations when applied on complex samples of microbial communities, such as those found in the human intestinal tract. Metagenome studies have indicated that the human intestinal microbiome contains over 100 times more genes than the human genome and it has been estimated that this ecosystem contains over 5000 bacterial species. The genomes of the vast majority of these species have not yet been sequenced and hence their proteomes remain unknown. To enable data analysis of shotgun proteomics data using PSM, and circumvent the lack of a defined matched metaproteome, an iterative workflow was developed that is based on a synthetic metaproteome and the developing metagenomic databases that are both representative for but not necessarily originating from the sample of interest.ResultsTwo human fecal samples for which metagenomic data had been collected, were analyzed for their metaproteome using liquid chromatography-mass spectrometry and used to benchmark the developed iterative workflow to other methods. The results show that the developed method is able to detect over 3,000 peptides per fecal sample from the spectral data by circumventing the lack of a defined proteome without naive translation of matched metagenomes and cross-species peptide identification.ConclusionsThe developed iterative workflow achieved an approximate two-fold increase in the amount of identified spectra at a false discovery rate of 1% and can be applied in metaproteomic studies of the human intestinal tract or other complex ecosystems.

Recent metagenomic studies have demonstrated that the overall functional potential of the intestinal microbiome is rather conserved between healthy individuals. Here we assessed the biological processes undertaken in-vivo by microbes and the host in the intestinal tract by conducting a metaproteome analysis from a total of 48 faecal samples of 16 healthy adults participating in a placebo-controlled probiotic intervention trial. Half of the subjects received placebo and the other half consumed Lactobacillus rhamnosus GG for three weeks (1010 cfu per day). Faecal samples were collected just before and at the end of the consumption phase as well as after a three-week follow-up period, and were processed for microbial composition and metaproteome analysis. A common core of shared microbial protein functions could be identified in all subjects. Furthermore, we observed marked differences in expressed proteins between subjects that resulted in the definition of a stable and personalized microbiome both at the mass-spectrometry-based proteome level and the functional level based on the KEGG pathway analysis. No significant changes in the metaproteome were attributable to the probiotic intervention. A detailed taxonomic assignment of peptides and comparison to phylogenetic microarray data made it possible to evaluate the activity of the main phyla as well as key species, including Faecalibacterium prausnitzii. Several correlations were identified between human and bacterial proteins. Proteins of the human host accounted for approximately 14% of the identified metaproteome and displayed variations both between and within individuals. The individually different human intestinal proteomes point to personalized host-microbiota interactions. Our findings indicate that analysis of the intestinal metaproteome can complement gene-based analysis and contributes to a thorough understanding of the activities of the microbiome and the relevant pathways in health and disease.

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Insulin/IGF-like signaling (IIS) and nutrient sensing are among the most potent regulators of health status and aging. Here, a global view of the metabolic changes in C. elegans with impaired function of IIS represented by daf-2 and daf-16 and the intestinal di- and tripeptide transport pept-1 was generated using (1)H nuclear magnetic resonance spectroscopic analysis of worm extracts and spent culture media. We showed that specific metabolic profiles were significantly associated with each type of mutant. On the basis of the metabonomics data, selected underlying processes were further investigated using proteomic and transcriptomic approaches. The observed changes suggest a decreased activity of the one carbon metabolism in pept-1(lg601) mutants. Higher concentration of branched-chain amino acids (BCAA) and altered transcript levels of genes involved in BCAA metabolism were observed in long-living strains daf-2(e1370) and daf-2(e1370);pept-1(lg601) when compared to wild types and daf-16(m26);daf-2(e1370);pept-1(lg601) C. elegans, suggesting a DAF-16-dependent mechanism.

Obesity is associated with the intestinal microbiota in humans but the underlying mechanisms are yet to be fully understood. Our previous phylogenetic study showed that the faecal microbiota profiles of nonobese versus obese and morbidly obese individuals differed. Here, we have extended this analysis with a characterization of the faecal metaproteome, in order to detect differences at a functional level. Proteins were extracted from crude faecal samples of 29 subjects, separated by 1D gel electrophoresis and characterized using RP LC-MS/MS. The peptide data were analyzed in database searches with two complementary algorithms, OMSSA and X!Tandem, to increase the number of identifications. Evolutionary genealogy of genes: nonsupervised orthologous groups (EggNOG) database searches resulted in the functional annotation of over 90% of the identified microbial and human proteins. Based on both bacterial and human proteins, a clear clustering of obese and nonobese samples was obtained that exceeded the phylogenetic separation in dimension. Moreover, integration of the metaproteomics and phylogenetic datasets revealed notably that the phylum Bacteroidetes was metabolically more active in the obese than nonobese subjects. Finally, significant correlations between clinical measurements and bacterial gene functions were identified. This study emphasizes the importance of integrating data of the host and microbiota to understand their interactions.