Diet posttranslationally modifies the mouse gut microbial proteome to modulate renal function

Abstract

Associations between chronic kidney disease (CKD) and the gut microbiota have been postulated, yet questions remain about the underlying mechanisms. In humans, dietary protein increases gut bacterial production of hydrogen sulfide (H₂S), indole, and indoxyl sulfate. The latter are uremic toxins, and H₂S has diverse physiological functions, some of which are mediated by posttranslational modification. In a mouse model of CKD, we found that a high sulfur amino acid-containing diet resulted in posttranslationally modified microbial tryptophanase activity. This reduced uremic toxin-producing activity and ameliorated progression to CKD in the mice. Thus, diet can tune microbiota function to support healthy host physiology through posttranslational modification without altering microbial community composition.

Fig. 1.. Dietary Saa and the gut microbiota modulate kidney injury severity in a mouse CKD model.

(A) Serum creatinine (Cre) levels of SPF and GF mice on low- versus high-Saa+Ade diet. (B and C) Representative hematoxylin and eosin (H&E) staining (B) and representative trichrome staining (C) of kidneys from mice in (A). (D) Histology-based renal injury scores. (E and F) SPF (E) and GF (F) mouse cecal sulfide levels detected in the lead acetate or methylene blue assay. (G) Normalized E. coli mean gene abundance in CKD patient samples compared with non-CKD controls (PTRI whole-genome shotgun sequencing dataset). (H) Serum Cre levels from ASF or ASF^{E. coli} mice on low- versus high-Saa+Ade diets. (I and J) Representative H&E staining (I) and trichrome staining (J) of kidneys from mice in (H). (K) Histology-based renal injury scores. (L and M) ASF and ASF^{E. coli} cecal sulfide levels detected by lead acetate (L) or methylene blue assay (M). Data represent two [(L) and (M)], three [(A), (D), (H), and (K)], or four [(E) and (F)] independent experiments. Symbols represent individual mice. Bars represent means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Two-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for (A), (D), (E), (F), (H), and (K); the Mann-Whitney test for was used for (L) and (M). Scale bar, 1 mm for the 40× magnification in (B) and 200 mm for the 200× magnification in (B) and (C).

Fig. 2.. Characterization of E. coli S-sulfhydrome reveals that TnaA is a highly S-sulfhydrated protein.

(A) E. coli sulfide production, determined with lead acetate. (B) E. coli sulfide production, determined with methylene blue. OD₆₇₀, optical density at 670 nm. (C) Schematic of S-sulfhydrated protein pulldown method. The S-sulfhydrated protein is highlighted in blue, to distinguish it from the native protein in red. (D) Silver staining of E. coli lysates subjected to S-sulfhydration pulldown and eluted either with or without DTT. m.w., molecular weight. (E) Silver staining of lysates from WT or ΔdecR E. coli lysates subjected to S-sulfhydration pulldown. (F) Heat map of the relative quantity of the 212 S-sulfhydrated proteins by TMT LC-MS³ analysis from S-sulfhydration pulldown fractions from WT E. coli samples eluted with or without DTT and DdecR mutant samples eluted with DTT. Proteins ordered based on q score for enrichment in the DTT-eluted versus non-DTT–eluted samples. Data represent two (E), three [(D) and (F)], four (A), or six (B) independent experiments. Bars represent means ± SEM. **P < 0.01. Statistical significance was determined with a linear model test (A), two-way Kruskal-Wallis test with Dunn’s post hoc test (B), or two-way ANOVA with Tukey’s post hoc test (F).

Fig. 3.. S-Sulfhydration inhibits the indole-producing enzymatic activity of E. coli TnaA.

(A) Representative Western blot analysis of TnaA-His from WT and ΔdecR E. coli lysates subjected to S-sulfhydration pulldown. Loading controls show RpoD in the flowthrough. (B) Same method as in (A) but with E. coli lysates treated with NaCl, H₂O₂, or NaHS. (C) LC-MS/MS analysis of indoles in WT E. coli cultures with cysteine or NaHS. (D and E) Kovac’s assay for indole production in WT E. coli cultures with cysteine or NaHS (D) or with purified TnaA enzyme supplemented with NaCl, Na₂S₄, or DTT (E). Data represent three [(A) and (E)], four [(B) and (D)], or five (C) independent experiments. Bars represent means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Data were analyzed with the Mann-Whitney test (A), two-way ANOVA with Tukey’s post hoc test [(B) and (E)], or two-way Kruskal-Wallis test with Dunn’s post hoc test [(C) and (D)].

Fig. 4.. Dietary Saa modulate cecal indole levels, serum indoxyl sulfate levels, and kidney function in a mouse CKD model.

(A and B) Western blot analysis of TnaA of S-sulfhydration pulldown and flowthrough samples from cecal contents (A) and Kovac’s assay measurement of indole levels in cecal contents (B) from ASF^{E. coli} mice on Saa diets. (C) LC-MS/MS analysis of indole levels in cecal contents from ASF^{E. coli} mice on Saa diets. Left, spectra representative of an experiment with three mice per group and indole standard. (D) LC-MS measurements of serum indoxyl sulfate in ASF mice on the low-Saa+Ade diet and colonized with E. coli strains. (E) Serum creatinine (Cre) levels in ASF mice colonized with E. coli strains and on low-Saa+Ade diets. (F and G) Representative H&E staining (F) and representative trichrome staining (F) of kidneys from mice in (E). (H) Histology-based renal injury scores. Data in (A), (B), (C), (D), (E), and (H) represent three independent experiments. Symbols represent individual mice. Bars represent means ± SEM. *P < 0.05; **P < 0.01. Data were analyzed with the Mann-Whitney test (A to D) or two-way ANOVA with Tukey’s post hoc test [(E) and (H)]. Scale bar, 1 mm [40× magnifications in (F)] or 200 mm [200× magnification in (F) and (G)].