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京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23332号 / 生博第450号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 片山 高嶺, 教授 永尾 雅哉, 教授 上村 匡 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Bisphenols are used in the process of polymerization of polycarbonate plastics and epoxy resins. Bisphenols can easily migrate out of plastic products and enter the gastrointestinal system. By increasing colonic inflammation in mice, disrupting the intestinal bacterial community structure and altering the microbial membrane transport system in zebrafish, bisphenols seem to interfere with the gut microbiome. The highly abundant human commensal bacterium Bacteroides thetaiotaomicron was exposed to bisphenols (Bisphenol A (BPA), Bisphenol F (BPF), Bisphenol S (BPS)), to examine the mode of action, in particular of BPF. All chemicals caused a concentration-dependent growth inhibition and the half-maximal effective concentration (EC50) corresponded to their individual logP values, a measure of their hydrophobicity. B. thetaiotaomicron exposed to BPF decreased membrane fluidity with increasing BPF concentrations. Physiological changes including an increase of acetate concentrations were observed. On the proteome level, a higher abundance of several ATP synthase subunits and multidrug efflux pumps suggested an increased energy demand for adaptive mechanisms after BPF exposure. Defense mechanisms were also implicated by a pathway analysis that identified a higher abundance of members of resistance pathways/strategies to cope with xenobiotics (i.e., antibiotics). Here, we present further insights into the mode of action of bisphenols in a human commensal gut bacterium regarding growth inhibition, and the physiological and functional state of the cell. These results, combined with microbiota-directed effects, could lead to a better understanding of host health disturbances and disease development based on xenobiotic uptake.

The human gut microbiome is home to many hundreds of different microbes which play a crucial role in human physiology. For most of them, little is known about how their genetic diversity translates into functional traits and how they interact with their host, which is to some extent due to the lack of isolate genomes. Cultivation-free metagenomic approaches yield extensive amounts of bacterial genetic data, and recently developed algorithms allow strain-level resolution and reconstruction of bacterial genomes from metagenomes, yet bacterial within-species diversity and transmission dynamics after fecal microbiota transplantation remain largely unexplored over cohorts and using these technological advances. To investigate bacterial within-species diversity I first undertook large-scale exploratory studies to characterize the population-level genomic makeup of the two key human gut microbes Eubacterium rectale and Akkermansia muciniphila , leveraging many hundreds of bacterial draft genomes reconstructed from short-read shotgun metagenomics datasets from all around the planet. For E. rectale , I extended previous observations about clustering of subspecies with geography, which suggested isolation by distance and the putative ancestral loss of four distinct motility operons, rendering a subspecies specifically found in Europe immotile. For A. muciniphila, I found that there are several closely related but undescribed Akkermansia spp. in the human gut that are all likely human-specific but are differentially associated with host body mass index, showcasing metabolic differences and distinct co-abundance patterns with putative cognate phages . For both species, I discovered distinct subspecies-level genetic variation in structural polysaccharide synthesis operons. Next, utilizing a complementary strain-resolved approach to track strains between individuals, I undertook a fecal microbiota transplantation (FMT) meta-analysis integrating 24 distinct clinical metagenomic datasets. I found that patients with an infectious disease or those who underwent antibiotic treatment displayed increased donor strain uptake and that some bacterial clades engraft more consistently than others. Furthermore, I developed a machine-learning framework that allows optimizing microbial parameters - such as bacterial richness - in the recipient after FMT based on donor microbiome features, representing first steps towards making a rational donor choice. Taken together, in my work I extended the strain-level understanding of human gut commensals and showcased that genomes from metagenomes can be suitable to conduct large-scale bacterial population genetics studies on other understudied human gut commensals. I further confirmed that strain-resolved metagenomics allows tracking of strains and thus inference of strain engraftment characteristics in an FMT meta-analysis, revealing important differences in engraftment over cohorts and species and paving the way towards better designed FMTs. I believe that my work is an important contribution to the field of microbiome research, showcasing the power of shotgun metagenomics, modern algorithms and large-scale data analysis to reveal previously unattainable insights about the human gut microbiome.

Obesity is a common disease among all ages that has threatened human health and has become a global concern. Gut microbiota can affect human metabolism and thus may modulate obesity. Certain mixes of gut microbiota can protect the host to be healthy or predispose the host to obesity. Modern next-generation sequencing technique allows accessing huge amount of genetic information underlying microbiota and thus provides new insights into the functionality of these micro-organisms and their interactions with the host. Multiple previous studies have demonstrated that the microbiome might contribute to obesity by increasing dietary energy harvest, promoting fat deposition and triggering systemic inflammation. However, these researches are either based on lab cultivation studies or basic statistical analysis. In order to further explore how gut microbiota affect obesity, this thesis utilize a series of machine learning methods to analyze large amount of metagenomics data from human gut microbiome. The publicly available HMP (Human Microbiome Project) metagenomic sequencing data, contain microbiome data for healthy adults, including overweight and obese individuals, were used for this study. HMP gut data were organized based on two different feature definitions: taxonomic information and metabolic reconstruction information. Several widely used classification algorithms: namely Naive Bayes, Random Forest, SVM and elastic net logistic regression were applied to predict healthy or obese status of the subjects based on the cross-validation accuracy. Furthermore, the corresponding feature selection algorithms were used to identify signature features in each dataset that lead to the differences between healthy and obese samples. The results showed that these algorithms perform poorly on taxonomic data than metabolic pathway data though lots of selected taxa are still supported by literature. Among all the combinations between different algorithms and data, elastic net logistic regression has the best cross-validation performance and thus becomes the best model. In this model, several important features are found and some of these are consistent with the previous studies. Rerunning classifiers by using features selected by elastic net logistic regression again further improved the performance of the classifiers. On the other hand, this study uncovered some new features that haven't been supported by previous studies. The new features could also be the potential target to distinguish obese and healthy subjects. The present thesis work compares the strengths and weaknesses of different machine learning techniques with different types of features originating from the same metagenomics data. The features selected by these models could provide a deep understanding of the metabolic mechanisms of micro-organisms. It is therefore worth to comprehensively understand the differences of gut microbiota between healthy and obese subjects, and particularly how gut microbiome affects obesity.

Using a novel, adipose-liver, two-organ, human-on-a-chip system, the metabolic disease non-alcoholic fatty liver disease was modeled. This model was then used to test the effects of the gut microbiome on NAFLD progression. Two products of the gut microbiome, Trimethylamine-n-oxide and butyrate, were selected as representatives of potentially harmful and potentially beneficial compounds. A dose response, adipocyte and hepatocyte monocultures controls, and HoaC systems were run for 14 days. Through this experimentation, it was found that a dysbiosis of the gut microbiome could be influencing NAFLD progression. Additionally, further development and discovery regarding adipose-liver systems was added to the ongoing conversation of HoaC systems and their usages.

Metagenomics is a cultivation-independent approach for obtaining the genomic composition of microbial communities. Microbial communities are ubiquitous in nature. Microbes which are associated with the human body play important roles in human health and disease. These roles span from protecting us against infections from other bacteria, to being the causes of these diseases. A deeper understanding of these communities and how they function inside our bodies allows for advancements in treatments and preventions for these diseases. Recent developments in metagenomics have been driven by the emergence of Next-Generation Sequencing technologies and Third-Generation Sequencing technologies that have enabled cost-effective DNA sequencing and the generation of large volumes of genomic data. These technologies have allowed for the introduction of hybrid DNA assembly techniques to recover the genomes of the constituent microbes. While Next-Generation Sequencing technologies use paired-end sequencing reads from DNA fragments into short reads and have a relatively lower error rate, Third-Generation Sequencing technologies use much longer DNA fragments to generate longer reads, bringing contigs together for larger scaffolds with a higher error rate. Hybrid assemblers leverage both short and long read sequencing technologies and can be a critical step in the advancements of metagenomics, combining these technologies to allow for longer assemblies of DNA with lower error rates. We evaluate the strengths and weaknesses of the hybrid assembly framework using several state-of-the-art assemblers and simulated human microbiome datasets. Our work provides insights into metagenomic assembly and genome recovery, an important step towards a deeper understanding of the microbial communities that influence our well-being.

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