Microbe-derived uremic solutes enhance thrombosis potential in the host

Abstract

Alterations in gut microbial composition and function have been linked to numerous diseases. Identifying microbial pathways responsible for producing molecules that adversely impact the host is an important first step in the development of therapeutic interventions. Here, we first use large-scale clinical observations to link blood levels of defined microbial products to cardiovascular disease risks. Notably, the previously identified uremic toxins p-cresol sulfate and indoxyl sulfate were shown to predict 5-year mortality risks. After identifying the microbes and microbial enzymes involved in the generation of these uremic toxins, we used bioengineering technologies coupled with colonization of germ-free mice to show that the gut microbial genes that generate p-cresol and indole are sufficient to confer p-cresol sulfate and indoxyl sulfate formation, and a pro-thrombotic phenotype in vivo. The findings and tools developed serve as a critical step in both the study and targeting of these gut microbial pathways in vivo.

Keywords: cardiovascular disease; gut microbes; indoxyl sulfate; mortality; p-cresol sulfate; uremic toxins.

Fig 1

Untargeted metabolomics reveals that p-cresol is associated with overall mortality. (A) Comparison of electron ionization spectra of the compound detected in plasma and an authentic standard of the trimethylsilyl derivative of p-cresol. (B) Relative plasma levels of p-cresol in sequential stable subjects undergoing elective diagnostic cardiac evaluation. Subjects (n = 1,149; discovery cohort) were divided into groups as indicated based on whether or not they died during the 5-year follow-up. In the box-whisker plot, the upper and lower boundaries of the box represent the 25th and 75th percentiles, the median is marked by a horizontal line inside the box, and the whiskers represent 10% and 90% measured values. (C) Kaplan-Meier estimates of the risk of incident death by quartile of relative amounts of p-cresol from the untargeted analysis. (D) Forest plots showing overall mortality within 5 years among test subjects according to the quartiles for the relative level of p-cresol (black), or a multivariable Cox model for hazard ratio that includes adjustments for age, sex, current smoking, high-density lipoprotein, low-density lipoprotein, triglyceride level, systolic blood pressure, diabetes mellitus, and high-sensitivity C-reactive protein (adjusted, red). Symbols represent hazard ratios and the 95% CIs are indicated by the line length.

Fig 2

Stable isotope-dilution LC-MS/MS analyses verify systemic levels of uremic toxins p-cresol sulfate and indoxyl sulfate are associated with incident overall mortality risks. Plasma levels of p-cresol sulfate (A) and indoxyl sulfate (D) in sequential stable subjects undergoing elective diagnostic cardiac evaluation. Subjects (n = 3,954) were divided into groups as indicated based on whether or not they experienced an incident death event within 5 years. In the box-whisker plot, the upper and lower boundaries of the box represent the 25th and 75th percentiles, the median is marked by a horizontal line inside the box, and the whiskers represent 10% and 90% measured values. Kaplan-Meier estimates and the risk of incident overall mortality ranked by quartile of (B) p-cresol sulfate and (E) indoxyl sulfate levels. Forest plots indicate the hazard ratio (95% CI) for incident (5 years) risks of overall mortality for (C) p-cresol sulfate and (F) indoxyl sulfate quartiles. Hazard ratio (unadjusted, black) and multivariable Cox model adjusted [gray; adjusted for age, sex, current smoking, high-density lipoprotein, low-density lipoprotein, triglyceride level, systolic blood pressure, diabetes mellitus, and high-sensitivity C-reactive protein (model 1) and model 1 + kidney function (model 2)]. Symbols represent hazard ratios and the 95% confidence intervals are indicated by line length.

Fig 3

Long-term mortality risk among patient subgroups. Hazard ratio for 5-year overall mortality based on the Cox proportional hazards regression analysis comparing fourth vs first (referent) quartiles (Q). Data points (open circles) in the center indicate HR and 95% CIs are represented by line length. Numbers for each subgroup are indicated by n. eGFR, estimated glomerular filtration rate; LDL-c, low-density lipoprotein cholesterol; HDL-c, high-density lipoprotein cholesterol; TG, triglyceride.

Fig 4

A collaborative pathway for the production of p-cresol. (A) GC-MS chromatogram of p-cresol production from metabolites of Clostridium sp. D5 and B. hydrogenotrophica DSM 10507 in the presence of 4-HPA and tyrosine (Tyr). (B) GC-MS chromatograms from mono-cultures and co-cultures. Bt, B. thetaiotaomicron; Bv, Bacteroides vulgatus; Cs, Clostridium sporogenes; D5, Clostridium sp. D5. (C) Urine p-cresol sulfate (pCS) from germ-free mice were co-colonized with either B. vulgatus and Clostridium D5 or B. thetaiotaomicron and C. sporogenes or B. thetaiotaomicron and Clostridium sp. D5. (D) The production of 4-HPA in B. thetaiotaomicron mutants. Data bars indicate the average LC-MS ion counts (negative mode) of three biological replicates ± SD. (E) Schematic of a collaborative pathway for the production of p-cresol and p-cresol sulfate.

Fig 5

Engineered Bacteroides strains synthesize p-cresol and indole in vitro. (A) Schematic presentation of genes involved in p-cresol and indole production. (B) Representative GC-MS chromatograms of selected ions for p-cresol (red; m/z 107.1 selected ion) and indole (blue; m/z 117.1 selected ion) production in culture from engineered B. thetaiotaomicron strains. Strains with the hpdBCA operon produce p-cresol, whereas strains containing the BT1492 gene produce indole. (C) Amounts of p-cresol (red) and indole (blue) produced by cultured B. thetaiotaomicron mutants (n = 3) measured by GC-MS.

Fig 6

Difference in microbial genes responsible for p-cresol or indole production is associated with increased in vivo thrombosis potential. (A) Scheme illustrating microbial transplant study design. Germ-free (C57B1/6) mice were subjected to gavage with four different engineered B. thetaiotaomicron strains with different capacities for p-cresol and indole production: (ΔBT1942 (white), hpdBCA ΔBT1942 (red), Δtdk (blue), and hpdBCA Δtdk (purple). (B) Levels of pCS and IS in mouse plasma 2 days post gavage and 24 h post folic acid intraperitoneal (IP) injection at time of thrombosis model. (C, D) In vivo thrombosis potential was measured by the FeCl₃-induced carotid artery injury model. Representative vital microscopy images of carotid artery thrombus formation are shown at the indicated time points following arterial injury (C), and time to cessation of blood flow in mice (D) measured in the indicated number of animals (n = 7–9). The bar represents mean time to cessation of blood. Significance was measured with a Kruskal-Wallis (K.W.) test followed with Dunn’s multiple comparisons test.

Fig 7

Elevated levels of tryptophanase and hpdBCA gene homologs are associated with ASCVD. The metagenomics data from Jie at al.’s study (41) were used to determine fecal abundance of tryptophanase and hpdBCA genes in individuals with (n = 218) and without ASCVD (n = 187). (A, C) Box-whisker (5%–95%) plots of tryptophanase (A) and hpdBCA (C) gene abundance in the fecal metagenome of control individuals vs individuals with ASCVD. P-values were calculated using Wilcoxon rank-sum test. (B, D) Forest plots indicating the ASCVD prevalence according to the tertiles of tryptophanase (B) and hpdBCA (D) gene abundance. The multivariable logistic regression model for odds ratio in panels B and D included adjustments for age, sex, and hyperlipidemia. The 95% CI is indicated by line length.