Non-lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis

Abstract

Trimethylamine (TMA) N-oxide (TMAO), a gut-microbiota-dependent metabolite, both enhances atherosclerosis in animal models and is associated with cardiovascular risks in clinical studies. Here, we investigate the impact of targeted inhibition of the first step in TMAO generation, commensal microbial TMA production, on diet-induced atherosclerosis. A structural analog of choline, 3,3-dimethyl-1-butanol (DMB), is shown to non-lethally inhibit TMA formation from cultured microbes, to inhibit distinct microbial TMA lyases, and to both inhibit TMA production from physiologic polymicrobial cultures (e.g., intestinal contents, human feces) and reduce TMAO levels in mice fed a high-choline or L-carnitine diet. DMB inhibited choline diet-enhanced endogenous macrophage foam cell formation and atherosclerotic lesion development in apolipoprotein e(-/-) mice without alterations in circulating cholesterol levels. The present studies suggest that targeting gut microbial production of TMA specifically and non-lethal microbial inhibitors in general may serve as a potential therapeutic approach for the treatment of cardiometabolic diseases.

Figure 1. The choline analogue 3,3-dimethyl-1-butanol inhibits microbial choline TMA lyase activity

(A) Effect of the indicated choline analogues on microbial TMA lyase activity (measured as d9-TMA production from 100 μM of the indicated d9-labeled substrate) from lysate of E. coli Top10 cells transformed (pBAD vector) with cutC/D genes (from P. mirabilis). (B) Effect of DMB on choline TMA lyase activity in intact P. mirabilis incubated with the indicated concentrations of d9-choline substrate ± DMB (1 mM) at 37°C. (C) DMB effect on choline uptake. P. mirabilis (OD_{600 nm} = 0.5) were pelleted and then re-suspended in minimal media supplemented with the indicated concentrations of d9-choline ± DMB (2 mM) for 15 minutes at 37°C. Intracellular d9-choline was then quantified as described under Methods. (D) Choline TMA lyase activity from intact E. coli Top10 cells transformed with the indicated constructs in the presence vs. absence of DMB. (E) DMB dose response curves for inhibition of choline TMA lyase activity in intact E. coli Top10 cells transformed with cutC/D genes from either D. alaskensis (pUC57 vector) or P. mirabilis (pBAD vector). (F) TMA lyase activity for the indicated substrates in P. mirabilis lysate ± DMB. Data are presented as mean ± SE from three independent replicates (A–F). See also Figures S1, S2, S6.

Figure 2. Inhibitory effect of DMB on alternative microbial TMA lyases, and both mouse cecal and human fecal microbial TMA lyase activities with multiple substrates

(A) TMA lyase activity for the indicated substrates (375 μM each) in combined lysates from E. coli BL21 cells transformed (pET22 vector) with cntA and cntB genes (from A. baumannii) incubated in the presence or absence of DMB. (B) TMA lyase activity with the indicated substrates (375 μM, ± DMB) in intact E. coli BL21 cells transformed (pET22 vector) with yeaW/X genes (from E. coli DH10B). (C–E) TMA lyase activity with the indicated substrates (± DMB) incubated with (C) mouse cecum lysate (1.3 mg/ml protein) or (D–E) human fecal microbes (equivalent to 100 mg feces/ml). The DMB concentration in all reaction systems was 10 mM (in A, B) or 2 mM (C–E). Data are presented as mean ± SE from three independent replicates (A–E). See also Figures S3 and S6C,D.

Figure 3. DMB serves as a non-lethal inhibitor

(A–C) Cells (A, P. mirabilis; B, P. penneri; C, E. fergusonii) were grown in nutrient broth ± DMB (0.1% v/v) and both the growth rate (OD₆₀₀) and total amount of d9-TMA produced from d9-choline (100 μM) (insert) were monitored. (D–E) Demonstration that DMB decreases plasma TMAO levels in male (D) and female (E) C57BL/6J Apoe^−/− mice placed chronically on either 1.0% choline or 1.0% carnitine-supplemented diets in the presence versus absence of DMB (1%, v/v in drinking water). (F) Three groups of male C57BL/6J Apoe^−/− mice (8 week old) were placed on chemically defined diet equivalent to chow supplemented with choline (1.0% total choline). Two groups of mice were administered 1.2 μmol of DMB daily (provided in corn oil, 150 μl total vol) by either (oral) gastric gavage, or subcutaneous (SC) routes, and the third group of mice received sham (vehicle) gavages daily. After two weeks, plasma TMAO levels were determined by LC/MS/MS. Data are presented as mean ± SE from three (A–C) or the indicated numbers (D–F) of independent replicates. See also Figure S4C and Tables S1 and S2.

Figure 4. DMB attenuates choline enhanced foam cell formation and atherosclerosis

(A) Representative Oil-Red-O/hematoxylin staining of peritoneal macrophages, Scale bar, 50 μm, and (B) foam cell quantification, from 20 week old male C57BL/6J Apoe^−/− mice fed chemically defined chow (0.07% total choline) or 1% choline-supplemented diets from time of weaning (4 weeks). Data are mean ± SE. (C) Representative Oil-Red-O/hematoxylin staining of aortic root sections from 20 week old male C57BL/6J Apoe^−/− mouse fed chemically defined chow (0.07% total choline) or choline-supplemented (1.0% total choline) diets in the presence vs. absence of DMB provided in the drinking water, as described under Experimental Procedures. Scale bar, 200 μm. (D) Aortic root lesion area was quantified in 20 week old male C57BL/6J Apoe^−/− mice from the indicated diet and DMB treatment groups. Data are mean ± SE. p values shown were calculated by ANOVA. See also Figure S4 and S5.

Figure 5. DMB alters gut microbial composition

Unweighted UniFrac distances plotted in PCoA space comparing cecal microbial communities in male (A) or female (C) C57BL/6J Apoe^−/− mice fed chow versus choline-supplemented diet in the presence versus absence of DMB. Each data point represents a sample from a distinct mouse projected onto the first two principal coordinates (percent variation explained by each PCo is shown in parentheses). (B, D, E) Impact of diet and DMB on the proportion of several taxa. Data represent mean ± SE (n ≥ 9 mice per group). See also Tables S3 and S4.

Figure 6. Schema showing use of DMB to inhibit gut microbial trimethylamine production for the treatment of atherosclerosis

DMB (3,3-dimethyl-1-butanol) is a structural analogue of choline, and an inhibitor of microbial TMA production (a choline TMA lyase inhibitor). Provision of DMB in the drinking water of atherosclerosis prone Apoe^−/− mice inhibits choline diet dependent enhancement in TMAO, endogenous macrophage foam cell formation, and atherosclerosis development.