植食性哺乳動物腸道微生物體之多體學系統生物研究

Multi-Omics Systems Biology Studies of the Gut Microbiome of Herbivorous Mammals

劉勃佑(Po-Yu Liu)

指導教授 ： 于宏燦

國立臺灣大學/生命科學院/基因體與系統生物學學位學程/博士(2019年)

https://doi.org/10.6342/NTU201900102

摘要

腸道微生物與哺乳動物宿主共演化和共適應以反映宿主在演化歷史上的生理狀態（如體溫調節）以及外在環境（如食物來源）的改變。小型哺乳動物因體表面積比例大，而具有高質量特異代謝率（mass-specific metabolic rate），意即小型哺乳動物必須有效率地獲取能量以因應快速散失的體熱。然而植食性哺乳動物因缺乏纖維素水解酵素，得仰賴腸道共生微生物進行養分的汲取。此外，腸道微生物相的組成與變化主要受到食物來源的影響，例如季節性物候更替，植物性食物組成也隨之改變，同時植食性哺乳動物透過微生物協助代謝食物中的防禦性植化物，因此動植物交互作用亦與腸道微生物共演化。食葉性飛鼠為最小型之樹棲植食性哺乳動物，佔有特殊的演化與生態棲位，然而對於野生食葉性哺乳動物與腸道微生物的交互作用至今所知仍有限。本研究以野生食葉性飛鼠為研究模式，並根據分子生物學中心法則（the central dogma）整合多體學（multi-omics）方法對飛鼠腸道微生物相進行多源基因體（metagenome）、多源轉錄體（metatranscriptome）和代謝體（metabolome）分析。首先，根據克萊伯定律（Kleiber's law），本研究比較四種不同體型大小的食葉性飛鼠的腸道微生物相與宿主質量特異代謝率和微生物醣類代謝能力之相關性（詳見第二章）。其二，探討食葉性飛鼠（西伯利亞小鼯鼠）腸道微生物的季節性變異與森林地景物候學之動態關係（詳見第三章）。其三，以白面鼯鼠為模式，並利用多源基因體、多源轉錄體和代謝體探討腸道微生物養分汲取與毒性植化物降解之功能（詳見第四章）。此外，為瞭解腸道微生物群聚中微生物個體之交互作用，本研究又以大鼠盲腸分離之纖維素分解細菌群落（consortium）為模式，探討微生物群聚之代謝與生態共生機制（詳見第五章）。四種飛鼠腸道微生物相分析結果顯示，後壁菌門（Firmicutes）是飛鼠消化道中的最優勢菌群，而擬桿菌門（Bacteroidetes）為中小型飛鼠的第二優勢腸道微生物群，但大型飛鼠消化道中不具擬桿菌門細菌；多源基因體預測分析顯示，小型飛鼠的腸道微生物具有最高的醣類代謝潛能（以每單位體重校正）。而西伯利亞小鼯鼠腸道微生物相隨季節性的溫度、地表植被變異；環境溫度於攝氏20度是腸道微生物相組成變化的轉折點，低於此溫度者則有相對多樣的腸道微生物相。白面鼯鼠消化道的代謝體全貌(metabolomic profile)顯示了消化道的化學環境循著消化過程變化，其中包含超過六百種的植物化合物，依消化順序逐漸降解。多源基因體與多源轉錄體同步分析顯示鞭毛、化學趨向和ABC運輸蛋白（ATP-binding cassette transporters）系統為飛鼠盲腸微生物進行養分汲取與毒性植化物降解的關鍵。纖維素分解細菌群落模式透過多源基因體分析顯示共生群落具有代謝互補性，而時間序列分析與競爭模型模擬亦顯示微生物群落共生的穩定平衡。

關鍵字

腸道微生物相 ； 食葉性飛鼠 ； 多體學 ； 微生物群落 ； 共演化

並列摘要

The gut microbiota coevolve and coadapt with their mammalian hosts in response to changes in hosts’ physiological status (e.g. thermal regulation) and external environments (e.g. food sources) over the evolutionary history. In small herbivorous mammals, the gut microbiota helps in efficiently acquiring energy in response to the rapid loss of body heat. Additionally, the gut microbiota of wild mammals is altered according to the food availability per season. Leaf-eating (folivorous) flying squirrels are among the smallest arboreal herbivorous mammals, which occupying the specialized evolutionary and ecological niches; however, little is known about the interactions of the wild folivorous flying squirrels and their gut microbiota. Therefore, in this study, we used the wild folivorous flying squirrels as study models and integrate multi-omics approaches (metagenome, metatranscriptome, and metabolome) based on the central dogma of molecular biology to investigate the gut microbiome of flying squirrels. Chapter 2 was conducted a comparison for the gut microbial compositions and carbohydrate metabolic potentials of four species folivorous flying squirrels with different body sizes and mass-specific metabolic rates based on Kleiber’s law. Chapter 3 described the seasonal dynamics of flying squirrels’ (Siberian flying squirrels) gut microbiota and the landscape phenology of their forest habitats. Chapter 4 provided functional profiles (nutrient extraction and phytotoxin degradation) of the white-faced flying squirrels’ cecal microbiota by using multi-omics tools. In addition, in order to realize the microbial individual interactions within the community, we isolated a cellulolytic microbial consortium from rat cecum to be a model community. Chapter 5 aimed to decipher the metabolically and ecologically sympatric mechanisms among consortium members. The gut microbiota comparison of four flying squirrels revealed that the Firmicutes was the predominant taxa of all the flying squirrel gut microbiota, while the Bacteroidetes was the secondary predominant gut microbial taxa of the small and medium flying squirrels. However, the Bacteroidetes was absent in large flying squirrels. In addition, metagenome prediction demonstrated that small flying squirrels harbored the most carbohydrate metabolic potentials (adjusted by body mass). The gut microbiota of the Siberian flying squirrels were altered along with seasonal temperature and vegetation changes. The 20°C was the turning point of gut microbiota shifting; more diverse gut microbial compositions were found at the lower temperature. The gut metabolomics profile of white-faced flying squirrels demonstrated that the transits of chemical environments along with the digestive process, including more than 600 phytochemicals degraded with this path. Parallel metagenomic and metatranscriptomic analyses revealed that systems of flagella, chemotaxis, and ABC (ATP-binding cassette) transporters were the keys for nutrient extraction and phytotoxin degradation of the cecal microbiota. Metagenomic analysis for cellulolytic consortium revealed that metabolic complementarity occurred within the sympatric consortium. The time-serial analysis and competition model provided the evidence for the steady state of consortium mutualism.

並列關鍵字

gut microbiota ； folivorous flying squirrel ； multi-omics ； microbial consortium ； coevolution

參考文獻

Chapter 1

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