The human gut microbiome is composed of millions of microbial genes, performing a variety of functions contributing to the host’s homeostasis. The disturbance of normal gut microbiome composition and function is associated with diseases. Dietary components including food additives, i.e., sweeteners, play a pivotal role in shaping the human gut microbiome. Despite many studies pointing out the association between sweeteners consumption and health issues, the mechanisms are still unclear and whether sweeteners can directly change the gut microbiome remains largely unknown. In this study, we investigated the responses of the human gut microbiome to 20 common sweeteners, using an approach combining high-throughput in-vitro microbiome culturing and metaproteomics, which provided both taxonomic and functional profile. Sweeteners that belonged to sugar alcohols and glycosides were revealed to induce larger changes in the microbiome metaproteome, as compared with other non-caloric artificial sweeteners (NAS). Changes in taxa abundance were found to be associated with all tested sweeteners at genus level. Clustering analysis based on functional profiling categorized sweeteners into two major clusters, including one cluster comprising 6 sugar alcohols which induced greater functional responses including reduced transport and metabolism of lipid and amino acids, and promoted translation, ribosomal structure and biogenesis, as compared with the other cluster comprising NAS. Taxon-specific functional analyses showed that microbial enzymes from Lachonospiraceae, Faecalibacterium, Eubacterium, Coprococcus and Roseburia hominis were the major contributors to altered butyrate-producing pathways by sweeteners. This study provides a comprehensive profiling of sweeteners-induced gut microbiome changes, and may serve as a basis to understand sweeteners-relevant health issues from a microbiome point of view.

The human gut microbiome exists as a community of microorganisms in symbiosis with its host. Prebiotics are functional compounds that modulate this microbial community, promoting the growth and activity of bacteria that are beneficial to human health. Resistant starches (RS), a subclass of prebiotics, are compounds linked to a number of host-beneficial effects when included in human diets. Understanding how RS shapes gut flora composition and function is crucial to understanding these effects; however, these effects are clouded by the complexity of the microbiome’s interactions. Comprehensively characterizing microbiome shifts as the result of prebiotics is an intriguing bioanalytical problem. In the thesis project, I hypothesize that: RS changes microbiome biochemical pathway expression community-wide and at different taxonomic levels; that RS forms will affect microbiome bacterial taxonomic distribution; and that a linear programming optimization approach can parsimoniously distribute ambiguous peptide abundances amongst their constituent species, leading to different interpretations of functional and structural characteristics in microbiome metaproteomics data. To address these hypotheses, the thesis project utilizes a combined metaproteomics and bioinformatics approach. The Figeys lab-developed RapidAIM bioanalytical assay is deployed to generate label-free mass spectrometry metaproteomics data, testing for these effects experimentally. Further, Cerberus, a bioinformatics platform for microbiome metaproteomics analyses, was developed to integrate workflows from different software sources into a unified pipeline. Cerberus also implements a novel linear optimization approach addressing the shared-peptide problem. Through experimental data analyses using Cerberus, microbiomes encountering RS showed concerted taxonomic shifts, general and specific functional modulations linked to these taxonomic changes, and a significantly variable pathway expression profile for host-beneficial microbiome processes. The peptide-species linear optimization procedure demonstrates how naïve approaches to the shared-peptide problem greatly skew downstream taxonomic and functional analyses in metaproteomics experiments, marking an important consideration for microbiome studies seeking to resolve taxon-specific alterations.

Résumé: La subérine, un polymère lipidique et complexe des plantes est retrouvé dans divers tissus dont le périderme de la pomme de terre. Le processus biologique de sa dégradation reste encore peu connu et est attribué aux champignons. Des échantillons de sol provenant d'un champ de pommes de terre ont été inoculés dans un milieu de culture contenant de la subérine comme source de carbone. Une approche métaprotéomique a été utilisée pour identifier les populations bactériennes qui se développent en présence de la subérine sur une période d'incubation de 60 jours. Le nombre de spectres normalisé (NSpC) des protéines extracellulaires produites par la communauté bactérienne du sol ont considérablement diminué du jour 5 au jour 20, puis ont augmenté lentement, révélant une succession de bactéries, où la population des bactéries du genre Pseudomonas à croissance rapide a diminué et a été remplacée par d’autres espèces bactériennes qui pouvaient se développer en présence de la subérine. La récalcitrance de la subérine a été démontrée par l'émergence de bactéries auxotrophes telles qu’Oscillatoria dans les derniers jours de la culture bactérienne. Néanmoins, l'identification de deux lipases dans le surnageant de la culture suggère qu'au moins certaines espèces bactériennes peuvent dégrader la subérine. Une des lipases (I4WGM2) a été associée à Rhodanobacter thiooxydans. Lorsque cultivée dans un milieu contenant de la subérine, la souche de R. thiooxidans LCS2 a produit trois lipases, dont I4WGM2. R. thiooxidans LCS2 a également produit d'autres protéines liées au métabolisme des lipides, des transporteurs de chaines d’acide gras et les enzymes de la [béta]-oxydation. Ceci suggère que R. thiooxydans pourrait participer à la dégradation de la subérine. / Abstract: Suberin is a complex lipidic plant polymer found in various tissues including potato periderm. The biological degradation process of suberin is poorly characterized and is attributed to fungi. Soil samples from a potato field were used to inoculate a culture medium containing suberin as carbon source and a metaproteomics approach was used to identify bacterial populations that develop in the presence of suberin, over a 60-day incubation period. The normalized spectral counts of predicted extracellular proteins produced by the soil bacterial community drastically decreased from day 5 to day 20 and then slowly increased, revealing a succession of bacteria. The population of fast-growing pseudomonads declined and was replaced by species that could develop in the presence of suberin. The recalcitrance of suberin was demonstrated by the emergence of auxotrophic bacteria such as Oscillatoria in the last days of the assay. Nevertheless, the identification of two putative lipases in the culture supernatants suggests that at least some bacterial species could degrade suberin. One of the lipases (I4WGM2) was associated with Rhodanobacter thiooxydans. When grown in a suberin-containing medium, R. thiooxydans strain LCS2 produced three lipases, including I4WGM2. This strain also produced other proteins linked to lipid metabolism, including fatty acid and lipid transporters and [beta]-oxidation enzymes, suggesting that R. thiooxydans could participate in suberin degradation.

Metaproteomics, as a proteomic approach to analyse environmental samples, is a new and expanding field of research. The field promises new ways of determining the status of the organisms present in a sample, and could provide additional information compared to metagenomics. Being a novel field of research, robust methods and protocols have not yet been established. In this thesis, we examine several methods for a reliable extraction of protein from soil and periphyton samples. The extraction should preferably be fast, compatible with downstream analysis by mass spectrometry and extract proteins in proportion to their presence in the original sample. A variety of methods and buffers were used to extract proteins from soil and periphyton samples. Concentration determinations showed that all of these methods extracted enough protein for further analysis. For purification and digestion of the samples, several methods were used. The purified samples were analysed on three different mass spectrometers, with the Orbitrap Velos Pro delivering the best results. The results were matched against four genomic and metagenomic databases for identification of proteins, of which the UniProt/SwissProt database gave the best result. A maximum of 52 proteins were identified from periphyton samples when searching against UniProt/SwissProt with strict settings, of which the majority were highly conserved proteins. The main limitation for this type of work is currently the lack of proper metagenomic databases.

The new field of ‘omics’ has spawned the development of metaproteomics, an approach that has the ability to identify and decipher the metabolic functions of a proteome derived from a microbial community that is largely uncultivable. With the development and availabilities of high throughput proteomics, high performance liquid chromatography coupled to mass spectrometry (MS) has been leading the field for metaproteomics. MS-based metaproteomics has been successful in its’ investigations of complex microbial communities from soils to the human body. Like the environment, the human body is host to a multitude of microorganisms that reside within the skin, oral cavity, vagina, and gastrointestinal tract, referred to as the human microbiome. The human microbiome is made up of trillions of bacteria that outnumber human genes by several orders of magnitude. These microbes are essential for human survival with a significant dependence on the microbes to encode and carryout metabolic functions that humans have not evolved on their own. Recently, metaproteomics has emerged as the primary technology to understand the metabolic functional signature of the human microbiome. Using a newly developed integrated approach that combines metagenomics and metaproteomics, we attempted to address the following questions: i) do humans share a core functional microbiome and ii) how do microbial communities change in response to disease. This resulted in a comprehensive identification and characterization of the metaproteome from two healthy human gut microbiomes. These analyses have resulted in an extended application to characterize how Crohn’s disease affects the functional signature of the microbiota. Contrary to measuring highly complex and representative gut metaproteomes is a less complex, controlled human-derived microbial community present in the gut of gnotobiotic mice. This human gut model system enhanced the capability to directly monitor fundamental interactions between two dominant phyla, Bacteroides and Firmicutes, in gut microbiomes colonized with two or more phylotypes. These analyses revealed membership abundance and functional differences between phylotypes when present in either a binary or 12-member consortia. This dissertation aims to characterize host microbial interactions and develop MS-based methods that can provide a better understanding of the human gut microbiota composition and function using both approaches.

The gut microbiome and its metabolic processes are dynamic systems. Surprisingly, our understanding of gut microbiome dynamics is limited. Here we report a metaproteomic workflow that involves protein stable isotope probing (protein-SIP) and identification/quantification of partially labeled peptides. We also developed a package, which we call MetaProfiler, that corrects for false identifications and performs phylogenetic and time series analysis for the study of microbiome dynamics. From the stool sample of five mice that were fed with 15-N hydrolysate from Ralstonia eutropha, we identified 15,297 non-redundant unlabeled peptides of which 10,839 of their heavy counterparts were quantified. These peptides revealed incorporation profiles over time that were different between and within taxa, as well as between and within clusters of orthologous groups (COGs). Our study helps unravel the complex dynamics of protein synthesis and bacterial dynamics in the mouse gut microbiome.

No description available.

Extracellular polymeric substances (EPS) in activated sludge are known to account for the flocculent nature of activated sludge. Extensive studies over the last few decades have attempted to extract and characterize activated sludge EPS, but a lack of agreement between studies has also been quite common. The molecular makeup of EPS has, however, remained nearly unexplored, leaving their identity, function, and fate over various stages in the activated sludge system mainly unknown. In spite of their critical involvement in bioflocculation and long history of related research, our understanding of EPS is still greatly limited and better elucidation of their composition and structure is needed. The hypothesis of this research was that activated sludge floc contains different fractions of EPS that are distinguishable by their association with certain cations and that each fraction behaves differently when subjected to shear, aerobic digestion, anaerobic digestion and other processes. In order to examine this floc hypothesis, the research mainly consisted of three sections: 1) development of EPS extraction methods that target cations of interest (divalent cations, especially calcium and magnesium, iron, and aluminum) from activated sludge; 2) molecular investigations on activated sludge EPS using metaproteomic analyses, comprising sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and protein identification by liquid chromatography tandem mass spectrometry (LC/MS/MS), and hemaagglutination (HA)/HA inhibition assays; and 3) investigating the fate of EPS in sludge digestion using SDS-PAGE. Evaluation of prior research and data from preliminary studies led to the development of the three extraction methods that were used to target specific cations from activated sludge and to release their associated EPS into solution. These methods are the cation exchange resin (CER) procedure for extracting Ca²⁺+Mg²⁺, sulfide extraction for removing Fe, and base treatment (pH 10.5) for dissolving Al. The cation selectivity in the three extraction methods, the composition of EPS (protein/polysaccharide), amino acid composition, and a series of sequential extraction data established initial research evidence that activated sludge EPS that are associated with different cations are not the same. SDS-PAGE was successfully applied to study extracellular proteins from several sources of both full- and bench-scale activated sludges. The three extraction methods led to different SDS-PAGE profiles, providing direct evidence that proteins released by the three methods were indeed different sludge proteins. Another important outcome from this stage of research was finding the similarity and differences of extracellular proteins between different sources of activated sludge. SDS-PAGE data showed that many of CER-extracted proteins were well conserved in all the sludges investigated, indicating that a significant fraction of Ca²⁺ and Mg²⁺-bound proteins are universal in activated sludge. On the other hand, protein profiles resulting from sulfide and base extraction were more diverse for different sludges, indicating that Al and Fe and their associated proteins are quite dynamic in activated sludge systems. Protein bands at high densities were analyzed for identifications by LC/MS/MS and several bacterial proteins and polypeptides originating from influent sewage were identified in this study. This was also thought to be the first account of protein identification work for full-scale activated sludge. The analysis of SDS-PAGE post sludge digestion revealed that CER-extracted proteins remained intact in anaerobic digestion while they were degraded in aerobic digestion. While the fate of sulfide-and base-extracted proteins in aerobic digestion was not as clearly resolved, their changes in anaerobic digestion were well determined in this research. Sulfide-extracted protein bands were reduced by anaerobic digestion, indicating that Fe-bound EPS were degraded under anaerobic conditions. While parts of base-extracted proteins disappeared after anaerobic digestion, others became more extractable along with the extraction of new proteins, indicating that the fate of base-extractable proteins, including Al-bound proteins, is more complex in anaerobic digestion than CER-extracted and sulfide-extracted proteins. These results show that Ca²⁺+Mg²⁺, Fe³⁺, and Al³⁺ play unique roles in floc formation and that each cation-associated EPS fraction imparts unique digestion characteristics to activated sludge. Finally, since a considerably different cation content is quite common for different wastewaters, it is postulated that this variability is one important factor that leads to different characteristics of activated sludge and sludge digestibility across facilities. The incorporation of the impact of cations and EPS on floc properties into an activated sludge model might be challenging but will assure a better engineering application of the activated sludge process. / Ph. D.

Bile acids are crucial for the uptake of dietary lipids and can shape the gut-microbiome composition. This latter function is associated with the toxicity of bile acids and can be modulated by bile acid modifying bacteria such as Eggerthella lenta, but the molecular details of the interaction of bacteria depending on bile acid modifications are not well understood. In order to unravel the molecular response to bile acids and their metabolites, we cultivated eight strains from a human intestinal microbiome model alone and in co-culture with Eggerthella lenta in the presence of cholic acid (CA) and deoxycholic acid (DCA). We observed growth inhibition of particularly gram-positive strains such as Clostridium ramosum and the gram-variable Anaerostipes cacae by CA and DCA stress. C. ramosum was alleviated through co-culturing with Eggerthella lenta. We approached effects on the membrane by zeta potential and genotoxic and metabolic effects by (meta)proteomic and metabolomic analyses. Co-culturing with Eggerthella lenta decreased both CA and DCA by the formation of oxidized and epimerized bile acids. Eggerthella lenta also produces microbial bile salt conjugates in a co-cultured species-specific manner. This study highlights how the interaction with other bacteria can influence the functionality of bacteria.

Pelagic aggregates function as biological carbon pumps for transporting fixed organic carbon to sediments. In iron-rich (ferruginous) lakes, photoferrotrophic and chemolithoautotrophic bacteria contribute to CO2 fixation by oxidizing reduced iron, leading to the formation of iron-rich pelagic aggregates (iron snow). The significance of iron oxidizers in carbon fixation, their general role in iron snow functioning and the flow of carbon within iron snow is still unclear. Here, we combined a two-year metatranscriptome analysis of iron snow collected from an acidic lake with protein-based stable isotope probing to determine general metabolic activities and to trace 13CO2 incorporation in iron snow over time under oxic and anoxic conditions. mRNA-derived metatranscriptome of iron snow identified four key players (Leptospirillum, Ferrovum, Acidithrix, Acidiphilium) with relative abundances (59.6–85.7%) encoding ecologically relevant pathways, including carbon fixation and polysaccharide biosynthesis. No transcriptional activity for carbon fixation from archaea or eukaryotes was detected. 13CO2 incorporation studies identified active chemolithoautotroph Ferrovum under both conditions. Only 1.0–5.3% relative 13C abundances were found in heterotrophic Acidiphilium and Acidocella under oxic conditions. These data show that iron oxidizers play an important role in CO2 fixation, but the majority of fixed C will be directly transported to the sediment without feeding heterotrophs in the water column in acidic ferruginous lakes.