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Background The intestinal microbiota plays a crucial role in protecting the host from pathogenic microbes, modulating immunity and regulating metabolic processes. We studied the simplified human intestinal microbiota (SIHUMIx) consisting of eight bacterial species with a particular focus on the discovery of novel small proteins with less than 100 amino acids (= sProteins), some of which may contribute to shape the simplified human intestinal microbiota. Although sProteins carry out a wide range of important functions, they are still often missed in genome annotations, and little is known about their structure and function in individual microbes and especially in microbial communities. Results We created a multi-species integrated proteogenomics search database (iPtgxDB) to enable a comprehensive identification of novel sProteins. Six of the eight SIHUMIx species, for which no complete genomes were available, were sequenced and de novo assembled. Several proteomics approaches including two earlier optimized sProtein enrichment strategies were applied to specifically increase the chances for novel sProtein discovery. The search of tandem mass spectrometry (MS/MS) data against the multi-species iPtgxDB enabled the identification of 31 novel sProteins, of which the expression of 30 was supported by metatranscriptomics data. Using synthetic peptides, we were able to validate the expression of 25 novel sProteins. The comparison of sProtein expression in each single strain versus a multi-species community cultivation showed that six of these sProteins were only identified in the SIHUMIx community indicating a potentially important role of sProteins in the organization of microbial communities. Two of these novel sProteins have a potential antimicrobial function. Metabolic modelling revealed that a third sProtein is located in a genomic region encoding several enzymes relevant for the community metabolism within SIHUMIx. Conclusions We outline an integrated experimental and bioinformatics workflow for the discovery of novel sProteins in a simplified intestinal model system that can be generically applied to other microbial communities. The further analysis of novel sProteins uniquely expressed in the SIHUMIx multi-species community is expected to enable new insights into the role of sProteins on the functionality of bacterial communities such as those of the human intestinal tract. EtVDiT6mSbNC8m7xJtMZ7w Video abstract

Many functions in host–microbiota interactions are potentially influenced by intestinal transit times, but little is known about the effects of altered transition times on the composition and functionality of gut microbiota. To analyze these effects, we cultivated the model community SIHUMIx in bioreactors in order to determine the effects of varying transit times (TT) on the community structure and function. After five days of continuous cultivation, we investigated the influence of different medium TT of 12 h, 24 h, and 48 h. For profiling the microbial community, we applied flow cytometric fingerprinting and revealed changes in the community structure of SIHUMIx during the change of TT, which were not associated with changes in species abundances. For pinpointing metabolic alterations, we applied metaproteomics and metabolomics and found, along with shortening the TT, a slight decrease in glycan biosynthesis, carbohydrate, and amino acid metabolism and, furthermore, a reduction in butyrate, methyl butyrate, isobutyrate, valerate, and isovalerate concentrations. Specifically, B. thetaiotaomicron was identified to be affected in terms of butyrate metabolism. However, communities could recover to the original state afterward. This study shows that SIHUMIx showed high structural stability when TT changed—even four-fold. Resistance values remained high, which suggests that TTs did not interfere with the structure of the community to a certain degree.

The human gut microbiota plays a vital role in maintaining host health by acting as a barrier against pathogens, supporting the immune system, and metabolizing complex carbon sources into beneficial compounds such as short-chain fatty acids. Brilliant blue E-133 (BB), is a common food dye that is not absorbed or metabolized by the body, leading to substantial exposure of the gut microbiota. Despite this, its effects on the microbiota are not well-documented. In this study, we cultivated the Simplified Human Microbiota Model (SIHUMIx) in a three-stage in vitro approach (stabilization, exposure, and recovery). Using metaproteomic and metabolomic approaches, we observed significant shifts in microbial composition, including an increase in the relative abundance of Bacteroides thetaiotaomicron and a decrease in beneficial species such as Bifidobacterium longum and Clostridium butyricum. We observed lower protein abundance in energy metabolism, metabolic end products, and particularly lactate and butyrate. Disturbance in key metabolic pathways related to energy production, stress response, and amino acid metabolism were also observed, with some pathways affected independently of bacterial abundance. These functional changes persisted during the recovery phase, indicating that the microbiota did not fully return to its pre-exposure state. Our findings suggest that BB has a lasting impact on gut microbiota structure and function, raising concerns about its widespread use in the food industry. This study underscores the need for further research into the long-term effects of food colorants on the gut microbiota and their potential health implications.