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Gut Parabacteroides merdae protects against cardiovascular damage by enhancing branched-chain amino acid catabolism

Nature Metabolism volume 4, pages 1271–1286 (2022)Cite this article

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A Publisher Correction to this article was published on 18 January 2023

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Abstract

Obesity, dyslipidemia and gut dysbiosis are all linked to cardiovascular diseases. A Ganoderma meroterpene derivative (GMD) has been shown to alleviate obesity and hyperlipidemia through modulating the gut microbiota in obese mice. Here we show that GMD protects against obesity-associated atherosclerosis by increasing the abundance of Parabacteroides merdae in the gut and enhancing branched-chain amino acid (BCAA) catabolism. Administration of live P. merdae to high-fat-diet-fed ApoE-null male mice reduces atherosclerotic lesions and enhances intestinal BCAA degradation. The degradation of BCAAs is mediated by the porA gene expressed in P. merdae. Deletion of porA from P. merdae blunts its capacity to degrade BCAAs and leads to inefficacy in fighting against atherosclerosis. We further show that P. merdae inhibits the mTORC1 pathway in atherosclerotic plaques. In support of our preclinical findings, an in silico analysis of human gut metagenomic studies indicates that P. merdae and porA genes are depleted in the gut microbiomes of individuals with atherosclerosis. Our results provide mechanistic insights into the therapeutic potential of GMD through P. merdae in treating obesity-associated cardiovascular diseases.

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Fig. 1: HFD-fed ApoE−/− mice treated with GMD exhibit reduced atherosclerosis.
Fig. 2: GMD-mediated changes of gut microbiota in HFD-fed ApoE−/− mice.
Fig. 3: Gavage with live P. merdae attenuates the formation of atherosclerotic plaque.
Fig. 4: Identification of branched-chain amino acid degradation pathways in P. merdae.
Fig. 5: Increased BCAA degradation by P. merdae attenuates the formation of atherosclerotic plaque in HFD-fed −/− mice.
Fig. 6: Gut BCAA degradation by P. merdae regulates plaque macrophage mTORC1 signaling.

Data availability

Raw sequence data from all 16S rRNA sequencing experiments are deposited in the National Center for Biotechnology Information GenBank repository (BioProject nos PRJNA870664 and PRJNA870654). For the European cohort, which is about cardiometabolic disease, the raw shotgun-sequencing data have been deposited in the European Nucleotide Archive with accession codes PRJEB37249, PRJEB38742, PRJEB41311 and PRJEB46098 and the serum metabolome data have been uploaded to MassIVE with accession MSV000088042 and MSV000088043. For the Chinese population cohort, which is about atherosclerosis, the metagenomic shotgun-sequencing data have been deposited in the European Bioinformatics Institute database under the accession code ERP023788. Additional information and materials will be made available upon reasonable request. Source data are provided with this paper.

Code availability

Any code used to analysis or plot data in this manuscript is available from the corresponding author upon request.

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Acknowledgements

This work was financially supported by the National Key R&D program of China (2019YFA0905602), the Chinese Academy of Sciences Strategic Priority Research Program (class B) (XDB38020300) and the National Natural Science Foundation of China (81773614).

Author information

Author notes

  1. These authors contributed equally: Shanshan Qiao, Chang Liu.

Authors and Affiliations

  1. State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China

    Shanshan Qiao, Li Sun, Tao Wang, Huanqin Dai, Kai Wang, Li Bao, Hantian Li, Wenzhao Wang & Hongwei Liu

  2. Savaid Medical School, University of Chinese Academy of Sciences, Beijing, PR China

    Shanshan Qiao, Li Sun, Tao Wang, Huanqin Dai, Kai Wang, Li Bao, Hantian Li, Shuang-Jiang Liu & Hongwei Liu

  3. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China

    Chang Liu & Shuang-Jiang Liu

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Contributions

S.S.Q. and H.W.L. were responsible for study conceptualization. S.S.Q., L.S., T.W., H.Q.D., W.Z.W. and H.W.L. were responsible for the methodology. Formal analysis was conducted by S.S.Q., C.L. and K.W. Investigation was carried out by S.S.Q., K.W. and L.B. S.S.Q. and C.L. were responsible for resources. S.S.Q. and H.W.L. wrote the original draft. S.S.Q., H.T.L. and H.W.L. were responsible for writing review & editing. Supervision was carried out by S.J.L. and H.W.L. Funding acquisition was the responsibility of H.W.L.

Corresponding authors

Correspondence to Shuang-Jiang Liu or Hongwei Liu.

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Competing interests

H.W.L. has a patent related to this work (CN202110786071.9). All other authors declare no competing interests.

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Nature Metabolism thanks Esther Lutgens, Herbert Tilg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Effects of GMD on food intake, body weight gain, body composition, and hepatic steatosis in the HFD-fed ApoE−/− mice.

a, Cumulative food intake (**P = 0.0021). b, Body weight gain (Mod vs. Con: ***P = 0.0008, ****P = 0.0001, **** P < 0.0001, **** P < 0.0001, **** P < 0.0001 and **** P < 0.0001; Mod vs. Statin: *P = 0.0271, ***P = 0.0003 and **P = 0.0062; Mod vs. GMDL: **P = 0.0014, **P = 0.0041, ***P = 0.0003, **** P < 0.0001, **P = 0.0016, ****P = 0.0001, ****P < 0.0001, ****P < 0.0001 and ****P = 0.0001; Mod vs. GMDH: **P = 0.0023, *P = 0.0170, **P = 0.0097, **P = 0.0037, **P = 0.0044, **P = 0.0010, ***P = 0.0004, ****P < 0.0001 and ****P = 0.0001). c, Lean. d, Fat (**** P < 0.0001, **** P < 0.0001 and **** P < 0.0001). e, Inguinal fat (**** P < 0.0001, **** P < 0.0001 and **** P < 0.0001). f, Mesenteric fat (*P = 0.0271). g, Representative images of liver sections after HE or oil red O staining. Abbreviation: Con, normal diet-fed C57BL/6 J mice; Mod, HFD-fed ApoE−/− mice treated with vehicle; Statin, HFD-fed ApoE−/− mice treated with 10 mg/kg atorvastatin; GMDL, HFD-fed ApoE−/− mice treated with 5 mg/kg GMD; GMDH, HFD-fed ApoE−/− mice treated with 10 mg/kg GMD. n = 8 for each group. Data is presented as the mean ±s.e.m. Statistical analysis was performed using one-way ANOVA followed by the Tukey post hoc test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. The P values in each panel were given from left to right, respectively.

Source data

Extended Data Fig. 2 GMD alters the gut microbiota in the HFD-fed ApoE−/− mice.

a, Species accumulation curve. b, α-diversity of the gut microbiota between ApoE−/− +HFD and ApoE−/− +HFD + GMDH groups, as indicated by the observed species, Shannon and Chao1indices. c, Sankey diagram showing the genus of contribution differently among two groups. d, A heat map of the most abundant top 35 bacteria at the genus level among two groups. Blue denotes a low relative abundance across a taxon (row); red denotes a high relative abundance. The color key for the Z score indicates correspondence between the red-blue coloring and standard deviations from the mean abundance of each taxon. Abbreviation: ApoE−/− +HFD, HFD-fed ApoE−/− mice treated with vehicle; ApoE−/− +HFD + GMDH, HFD-fed ApoE−/− mice treated with 10 mg/kg GMD.

Source data

Extended Data Fig. 3 Relative abundances of P. merdae, porA gene reads, and the level of serum BCAAs across a Chinese gut metagenome or European gut metagenome and metabolome.

a-c, The relative abundance of P. merdae (a, *P = 0.0255) and porA gene (b, ****P < 0.0001) in each gut metagenomic dataset, and the positive correlation (c, R2 = 0.261 and P < 0.0001) between the gut metagenomes of atherosclerosis cohorts (n = 214) and their healthy counterparts (n = 171). d and e, The relative abundance of P. merdae (d, **P = 0.0028, **P = 0.0012 and ****P < 0.0001) and the serum BCAAs levels (e, *** P < 0.0001 for listed all comparisons; The exact concentrations of serum BCAAs in each sample were also shown in Supplementary Table 12 from Nature Medicine, VOL 28, P303–P314, 2022, www.nature.com/naturemedicine) in a European cohort study. The study recruited 647 fecal donors comprising a combined group of patients diagnosed with ischemic heart disease (IHD, n = 372 with different drugs) which included cases with acute coronary syndrome (ACS, n = 112), chronic ischemic heart disease (CIHD, n = 158) and heart failure (HF, n = 102) due to CIHD, and the healthy control (HC, n = 275, health by self-report and no intake of lipid-lowering, anti-diabetic or anti-hypertensive drugs). For panel c, R value refers to the standardized effect size estimated by the linear regression, and the corresponding P value is shown. For the other panels, the P values were obtained from Two-tailed Student’s t-tests, * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. The P values in each panel were given from left to right, respectively.

Source data

Extended Data Fig. 4 Parabacteroides merdae alters the gut microbiota and reduce hepatic steatosis in the HFD-fed ApoE−/− mice.

a, α-diversity of the gut microbiota between ApoE−/− and PM groups, as indicated by the observed species, Chao1, Shannon, Pielou_e, and Simpson indices. b, Principal coordinate analysis (PCoA) of all samples at the genus level among two groups. c, PCoA of all samples at the species level among two groups. d, A heat map of the most abundant top 30 bacteria at the genus level among two groups and the importance of species to the model decreased from top to bottom, which could be considered as indicators of inter-group differences. e, A heat map of the most abundant top 30 bacteria at the species level among two groups. Blue denotes a low relative abundance across a taxon (row); red denotes a high relative abundance. The color key for the Z score indicates correspondence between the red-blue coloring and standard deviations from the mean abundance of each taxon. f. Representative images of liver sections after HE or oil red O staining. Abbreviation: ApoE−/−, HFD-fed ApoE−/− mice treated with vehicle; PM, HFD-fed ApoE−/− mice treated with P. merdae (2 × 108 CFU/day). n = 10 for each group.

Source data

Extended Data Fig. 5 Gavage with the live P. merdae attenuates hyperlipidemia, hyperglycemia, and atherosclerosis biomarkers in Abx pre-treatment ApoE−/− mice.

a, Experimental design showing groups and durations. b, Body weight change (**P = 0.0025, **P = 0.0031, **P = 0.0047, **P = 0.0018, *P = 0.0109, *P = 0.0446, *P = 0.0155, **P = 0.0018, **P = 0.0018 and *P = 0.0242). c, White fat (P = 0.0073). d, Levels of plasma T-C (*P = 0.0142), T-TG (**P = 0.0086), LDL-C, and HDL-C. e, Plasma ox-LDL (*P = 0.0309). f, Plasma LPS (***P = 0.0004). g, Plasma MCP-1 (**P = 0.0013). h, Plasma TNF-α (*P = 0.0213). i, Plasma IL-1β (**P = 0.0088). j, Plasma hs-CRP. k, Plasma HbA1c (**P = 0.0058). l, Plasma sINS. m, OGTT test (*P = 0.0300). n, AUC of OGTT (*P = 0.0790). o, AOC of OGTT (*P = 0.0228). p, ITT test (*P = 0.0146, *P = 0.0127 and *P = 0.0194). q, AUC of ITT (**P = 0.004). r, AOC of ITT. n = 8 for each group. Abbreviation: ApoE, Abx pre-treatment ApoE−/− mice treated with vehicle; P. merdae, Abx pre-treatment ApoE−/− mice treated with treated with P. merdae (2 × 108 CFU/day). Data is presented as mean ± s.e.m. Statistical analysis was performed using one-way ANOVA followed by the Tukey post hoc test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. AVNM: antibiotics (ampicillin, vancomycin, neomycin, metronidazole). The P values in each panel were given from left to right, respectively.

Source data

Extended Data Fig. 6 Analysis of BCAAs catabolism in the P. merdae and construction of porA-deficient strain of P. merdae.

a, PorA gene cluster for BCAAs metabolism predicted by gutSMASH in P. merdae genome. b, PorA gene cluster in P. merdae genome and its homologs-MGC00000015_c1 in Clostridium sporogenes. c, Schematic layout of mutant construction. d, Validation of pGERM::porA insertion into porA gene of P. merdae genome. e, Growth of strain PMΔporA and PMWT on Gifu anaerobic medium, mid log phases are pointed by gray arrows and late log phases by black arrows. n = 3 for each group.

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Extended Data Fig. 7 The transcription levels of BCAA catabolic enzymes, the level of blood glucose, and insulin sensitivity in the high-fat diet-fed ApoE−/− mice treated with the wild-type or mutant strain of P. merdae.

Real-time RT-PCR result of indicated genes using mRNA from (a) liver (b) muscle (c) adipose of PMΔporA and PMWT; n = 6 for each group. d, Plasma 3-HIB (3-hydroxyisobutyrate) levels, n = 8 for each group. e, Free diet blood glucose (C57 vs. ApoE−/−: **P = 0.0031, ****P < 0.0001, **P = 0.0044, **P = 0.0016 and ***P = 0.0003; PMWT vs. ApoE−/−: *P = 0.0334 and *P = 0.0246); n = 10 for C57 and n = 12 for the other three groups. f, ITT Test (C57 vs. ApoE−/−: ***P = 0.0005, ***P = 0.0002, ****P < 0.0001 and ****P < 0.0001; PMWT vs. ApoE−/−: ***P = 0.0003, ****P < 0.0001 ****P = 0.0001 and ****P = 0.0001; PMΔPorA vs. ApoE−/−: *P = 0.0232); n = 8 for C57 and n = 10 for the other three groups. g, AOC of ITT; n = 8 for C57 and n = 10 for the other three groups. h, AUC of ITT (****P < 0.0001 for all three comparisons); n = 9 for ApoE−/− and n = 10 for the other three groups. Abbreviation: C57: normal-diet-fed C57BL/6 J mice; ApoE−/−, HFD-fed ApoE−/− mice; PMΔPorA, HFD-fed ApoE−/− mice treated with porA-deficient P. merdae mutant; PMWT, HFD-fed ApoE−/− mice treated with wild-type P. merdae. Data is mean ± s.e.m. Statistical analysis was performed using one-way ANOVA followed by the Tukey post hoc test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. The P values in each panel were given from left to right, respectively.

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Extended Data Fig. 8 IgA levels in atherosclerotic plaques and plasma of ApoE−/− mice treated with the wild-type or mutant strain of P. merdae.

a, Immunostaining of IgA (red) levels and the macrophage marker CD68 (green) in atherosclerotic plaques; Representative images are shown on the left and quantification on the above; n = 5 for each group; AU, arbitrary units; Scale bars, 100 μm (left), 50 μm (right). b, Plasma secreted IgA (*P = 0.0359); n = 12 for each group. Abbreviation: PMΔPorA, ApoE−/− mice treated with porA-deficient P. merdae mutant; PMWT, ApoE−/− mice treated with wild-type P. merdae. Data is presented as mean ± s.e.m. Statistical analysis was performed using Unpaired two-sided Student’s t-test (Gaussian model). * P < 0.05.

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Extended Data Fig. 9 Efficacy comparison of GMD and P. merdae on high cholesterol diet (HCD) -fed ApoE−/− mice or high-fat diet (HFD) -fed ApoE−/− mice.

a, Experimental design showing groups and durations. b, Body weight (HFD vs. HFD + GMD: *P = 0.0432, *P = 0.0207, **P = 0.0028, *P = 0.0150, **P = 0.0030 and **P = 0.0011); n = 6 for each group. c, Plasma cholesterol parameters CHO (*P = 0.0216 and ****P = 0.0001); LDL-C (*P = 0.0129 and ****P = 0.0001) and HDL-C (****P = 0.0001); n = 5 for HCD and HCD + GMD, n = 6 for HFD and HFD + GMD. d, Plasma TG (**P = 0.0010); n = 5 for HCD and n = 6 for the other three groups. e, Free diet glucose (**P = 0.0015). f, Plasma hs-CRP (**P = 0.0011); n = 6 for HFD + GMD and n = 5 for the other three groups. g, Plasma ox-LDL (**P = 0.0049); n = 5 for HCD and HCD + GMD, n = 6 for HFD and HFD + GMD. h, Representative H&E and Masson of cross-sections of blue-blackaortic roots and the quantitation of plaque area (*P = 0.0352, ***P = 0.0006 and ****P < 0.0001); n = 6 for HFD + GMD and n = 5 for the other three groups; Masson staining of a cross-section of one of the aortic roots shown in (h), cardiomyocytes (red), nucleus (blue-black), collagen (blue); scale bar = 400 μm. i, Experimental design. j, Plasma TG; n = 6 for PMWT and n = 5 for the other two groups. k, Plasma cholesterol parameters; n = 6 for PMWT and n = 5 for the other two groups. l, Plasma ox-LDL; n = 5 for HCD and n = 6 for the other two groups. m, Plasma hs-CRP; n = 6 for PMWT and n = 5 for the other two groups. n, Representative H&E and Masson of cross-sections of aortic roots and the quantitation of plaque area; n = 5 for each group; Masson staining of a cross-section of one of the aortic roots shown in (n), cardiomyocytes (red), nucleus (blue-black), collagen (blue); scale bar = 300 μm. Abbreviation: HCD, high cholesterol diet (HCD)-fed ApoE−/− mice; HCD-GMD, HCD-fed ApoE−/− mice treated with 10 mg/kg GMD; HFD, HFD-fed ApoE−/− mice; HFD-GMD, HFD-fed ApoE−/− mice treated with 10 mg/kg GMD; PMΔPorA, HCD-fed ApoE−/− mice treated with porA-deficient P. merdae mutant; PMWT, HCD-fed ApoE−/− mice treated with wild-type P. merdae. Statistical analysis was performed using one-way ANOVA followed by the Tukey post hoc test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. The P values in each panel were given from left to right, respectively.

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Extended Data Fig. 10 Influence of HCD and HFD on circulating BCAAs, insulin sensitivity, and plaque macrophage mTORC1 signaling in ApoE−/−mice.

a, Experimental design. b, Body weight (CON vs. HCD: *P = 0.0301, **P = 0.0075, **P = 0.0060, *P = 0.0157, **P = 0.0011, **P = 0.0090, ***P = 0.0008 and ***P = 0.0007; CON vs. HFD: *P = 0.0485, *P = 0.0240, **P = 0.0032, **P = 0.0014, **P = 0.0031, **P = 0.0016 and ***P = 0.0004; HCD vs. HFD: *P = 0.0225, **P = 0.0084, **P = 0.0043, ***P = 0.0002, ****P = 0.0001, ***P = 0.0005, ***P = 0.0002 and ****P = 0.0001). c, Individual body weight after 14 weeks of treatment (****P < 0.0001 and ****P < 0.0001). d, Plasma lipid parameter CHO (****P < 0.0001 and *P = 0.0155); LDL-C (****P < 0.0001 and *P = 0.0429); HDL-C (**P = 0.0038) and TG (****P < 0.0001). e, Free diet blood glucose (CON vs. HCD: *P = 0.0151, *P = 0.0380, ****P < 0.0001 and *P = 0.0261; CON vs. HFD: ***P = 0.0007, ****P < 0.0001, ****P = 0.0001, ****P = 0.0001, ***P = 0.0002 and ****P < 0.0001; HCD vs. HFD: ***P = 0.0007, ****P < 0.0001, ****P = 0.0001, ****P = 0.0001, ***P = 0.0002 and ****P < 0.0001). f, OGTT test (CON vs HCD: P = 0.0431; CON vs. HFD: ****P = 0.0001 and **P = 0.0016). g, AOC of OGTT (*P = 0.0165 and *P = 0.0265). h, AUC of OGTT (****P < 0.0001 and ****P < 0.0001). i, Plasma insulin (***P = 0.0003 and **P = 0.0019). j, Plasma ox-LDL (****P = 0.0001 and *P = 0.0104). k, Plasma hs-CRP (*P = 0.0269 and *P = 0.0115). l, Plasma BCAAs levels (*P = 0.0433, **P = 0.0016, **P = 0.0096, ***P = 0.0005, *P = 0.0112 and ***P = 0.0009). m, Fecal BCAAs levels (*P = 0.0151, **P = 0.0022 and *P = 0.0249). n, Representative H&E and Masson of cross-sections of aortic roots and quantitative data; n = 5 for each group; scale bar = 300 μm; Masson staining of a cross-section of one of the aortic roots shown in (n), cardiomyocytes (red), nucleus (blue-black), collagen (blue). o, Immunostaining of pS6 levels, colocalization of pS6 (red) with the macrophage marker CD68 (green) in atherosclerotic plaques, and the quantifications of CD68 aera, average pS6 intensity (**P = 0.0035) and CD68 with pS6 colocalization (*P = 0.0268); Representative images are shown on the top and quantification on the bottom; n = 3 for each group; AU, arbitrary units; Scale bar = 200 μm. Abbreviation: CON, Normal diet-fed C57BL/6 J mice; HCD, high cholesterol diet-fed ApoE−/− mice; HFD, high-fat diet-fed ApoE−/− mice. n = 6 for each group unless otherwise indicated. Data is presented as mean ± s.e.m. Statistical analysis was performed using unpaired two-sided Student’s t-test (Gaussian model). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The P values in each panel were given from left to right, respectively.

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Qiao, S., Liu, C., Sun, L. et al. Gut Parabacteroides merdae protects against cardiovascular damage by enhancing branched-chain amino acid catabolism. Nat Metab 4, 1271–1286 (2022). https://doi.org/10.1038/s42255-022-00649-y

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