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Alternative routes of gut microbial methylamine metabolism that may limit trimethylamine N-oxide, a trigger for atherosclerosis.

肠道微生物甲胺代谢的替代途径可能会限制三甲胺 N-氧化物（动脉粥样硬化的触发因素）。

基本信息

批准号：
9908066
负责人：
JOSEPH Adrian KRZYCKI
金额：
$ 39.84万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-01 至 2022-12-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Cardiovascular disease is the leading cause of mortality in the United States. Evidence is accumulating that certain human intestinal microbes contribute to atherosclerosis leading to cardiovascular disease, thereby increasing the risk of heart attack, stroke, and death. Gut microbes convert quaternary amines (QAs), e.g. carnitine, butyrobetaine, choline, and glycine betaine, to trimethylamine (TMA). TMA enters the bloodstream and once converted by liver flavoproteins to trimethylamine-N-oxide (TMAO), can trigger macrophage mediated vascular lipid deposition. Serum TMAO levels accordingly correlate with atherosclerosis and the above catastrophic health events. TMA production by QA lyases or reductases has long been considered the sole route of microbial QA degradation under the anaerobic conditions prevalent in the gut; but recent evidence reveals a more complex microbial ecology of QAs. Intestinal isolates have been shown to catabolically remove the N-methyl groups of QAs during growth; and the demethylated QA products do not generate TMA. The central hypothesis of this work is that QA demethylation might serve to moderate microbial TMA production in the human intestine, providing a mechanism for homeostasis or therapeutic control of TMAO levels; and thereby a means to decrease the risk of cardiovascular disease. There is a critical need for identification of enzymes mediating demethylation of QAs, the organisms in which they operate, and their prevalence in the human gut. The working hypothesis for the mechanism of QA demethylation is that members of the MttB superfamily, one of which is known to demethylate glycine betaine, use a range of QA substrates. QA demethylating microbes contain multiple MttB proteins. An example is the human intestinal symbiont Eubacterium limosum that demethylates all QAs known to serve as TMA precursor in the gut, and produces increased levels of MttB family members during growth by QA demethylation. The specific aims of this project period are independent yet synergistic. Proteomics coupled to biochemical methods will identify and characterize key proteins that mediate catabolic demethylation of QAs by E. limosum. The knowledge gained will fuel ongoing ecological examination of human gut microbiota with focus on QA demethylation. Enrichment cultures for isolation will be made from human fecal samples for novel trophic groups of QA demethylating microbes, as well those consuming QA demethylation products. A metagenomic examination with deep sequencing of gut microbiota DNA from human fecal samples will allow statistical enumeration of genes whose products generate TMA from QAs versus those whose products demethylate QAs and avoid TMA generation. The results will be correlated with urine levels of QA metabolites. The overall outcomes of this project period will uncover a general mechanism by which many QAs are demethylated in the gut, and provide an initial test of the hypothesis that the interaction of QA degrading microbes in the gut may provide a means to control levels of TMA, and thereby the proatherogenic compound TMAO.

描述（由申请人提供）：心血管疾病是美国死亡的主要原因。越来越多的证据表明，某些人类肠道微生物有助于动脉粥样硬化，导致心血管疾病，从而增加心脏病发作，中风和死亡的风险。肠道微生物将季胺（QA），例如肉毒碱、丁酰甜菜碱、胆碱和甘氨酸甜菜碱转化为三甲胺（TMA）。TMA进入血流，一旦被肝脏黄素蛋白转化为三甲胺-N-氧化物（TMAO），可引发巨噬细胞介导的血管脂质沉积。因此，血清TMAO水平与动脉粥样硬化和上述灾难性健康事件相关。长期以来，QA裂解酶或还原酶的TMA生产一直被认为是肠道中普遍存在的厌氧条件下微生物QA降解的唯一途径;但最近的证据揭示了QA的更复杂的微生物生态学。肠道分离株已被证明在生长过程中分解代谢除去QA的N-甲基;脱甲基QA产物不产生TMA。这项工作的中心假设是，QA去甲基化可能有助于缓和人类肠道中微生物TMA的产生，提供了一种稳态或TMAO水平治疗控制的机制;从而降低心血管疾病风险的手段。迫切需要鉴定介导QA去甲基化的酶、它们在其中操作的生物体以及它们在人类肠道中的流行。QA去甲基化机制的工作假设是MttB超家族的成员（其中一个已知使甜菜碱去甲基化）使用一系列QA底物。QA脱甲基微生物含有多种MttB蛋白。一个实例是人肠道共生体淤泥真杆菌（Eubacterium limosum），其使已知在肠道中充当TMA前体的所有QA脱甲基化，并且在生长期间通过QA脱甲基化产生增加水平的MttB家族成员。本项目期间的具体目标是独立的，但又是协同的。蛋白质组学结合生物化学方法将鉴定和表征介导E. limosum。所获得的知识将推动正在进行的人类肠道微生物群的生态学研究，重点是QA去甲基化。用于分离的富集培养物将从人粪便样本中制备，用于QA脱甲基微生物的新营养群以及消耗QA脱甲基产物的微生物。对来自人类粪便样品的肠道微生物群DNA进行深度测序的宏基因组学检查将允许对其产物从QA产生TMA的基因与其产物使QA脱甲基并避免TMA产生的基因进行统计计数。结果将与QA代谢物的尿液水平相关。本项目期间的总体结果将揭示许多QA在肠道中去甲基化的一般机制，并提供假设的初步测试，即肠道中QA降解微生物的相互作用可能提供控制TMA水平的方法，从而控制促动脉粥样硬化化合物TMAO。