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

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 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.