Products of gut microbial Toll/interleukin-1 receptor domain NADase activities in gnotobiotic mice and Bangladeshi children with malnutrition

James S Weagley, Mark Zaydman, Siddarth Venkatesh, Yo Sasaki, Neha Damaraju, Alex Yenkin, William Buchser, Dmitry A Rodionov, Andrei Osterman, Tahmeed Ahmed, Michael J Barratt, Aaron DiAntonio, Jeffrey Milbrandt, Jeffrey I Gordon, James S Weagley, Mark Zaydman, Siddarth Venkatesh, Yo Sasaki, Neha Damaraju, Alex Yenkin, William Buchser, Dmitry A Rodionov, Andrei Osterman, Tahmeed Ahmed, Michael J Barratt, Aaron DiAntonio, Jeffrey Milbrandt, Jeffrey I Gordon

Abstract

Perturbed gut microbiome development has been linked to childhood malnutrition. Here, we characterize bacterial Toll/interleukin-1 receptor (TIR) protein domains that metabolize nicotinamide adenine dinucleotide (NAD), a co-enzyme with far-reaching effects on human physiology. A consortium of 26 human gut bacterial strains, representing the diversity of TIRs observed in the microbiome and the NAD hydrolase (NADase) activities of a subset of 152 bacterial TIRs assayed in vitro, was introduced into germ-free mice. Integrating mass spectrometry and microbial RNA sequencing (RNA-seq) with consortium membership manipulation disclosed that a variant of cyclic-ADPR (v-cADPR-x) is a specific product of TIR NADase activity and a prominent, colonization-discriminatory, taxon-specific metabolite. Guided by bioinformatic analyses of biochemically validated TIRs, we find that acute malnutrition is associated with decreased fecal levels of genes encoding TIRs known or predicted to generate v-cADPR-x, as well as decreased levels of the metabolite itself. These results underscore the need to consider microbiome TIR NADases when evaluating NAD metabolism in the human holobiont.

Trial registration: ClinicalTrials.gov NCT01889329.

Keywords: CP: Microbiology; NAD metabolism; TIR domain structure/activity relationships; childhood malnutrition; defined microbial communities; gnotobiotic mice; human gut microbiome development/functional profiling.

Conflict of interest statement

Declaration of interests A.O. and D.A.R. are co-founders of Phenobiome, Inc., a company pursuing development and biomedical applications of computational tools for predictive phenotype profiling of microbial communities. A.D. and J.M. are co-founders of, and Y.S. served as a consultant to, Disarm Therapeutics, a company dedicated to developing therapeutics for neurodegenerative conditions that is now part of Eli Lilly and Company.

Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
In vitro assays of NADase activities associated with TIR domains (A) Overview of NAD metabolism in humans and bacteria. The predominant pathways catalyzed by bacteria are shown with a solid blue line, while the pathways predominantly utilized by humans are denoted with a solid black line. Reactions that are known, but not widely distributed in bacteria, are indicated with blue dashed lines. (B) Summary of the number of TIR domains assigned to groups defined by Bayesian partitioning with pattern selections (BPPS). The left to right order of BPPS groups is based on the number of TIRs in that group that were identified in 278 fecal microbiome samples obtained from 30 healthy Bangladeshi children and 14 children with acute malnutrition. Subsequent rows indicate the number of TIRs in a given BPPS group that were assayed for NADase activity in vitro after expression in E. coli and the products that they generated from NAD (ADPR, cADPR, v-cADPR-x, or v-cADPR-y). The last row describes the number of TIRs identified in the proteomes of the 26 human gut bacterial strains that were introduced into gnotobiotic mice. (C) Examples of results of HPLC-based assays of NADase activity in recombinant TIRs produced by the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible E. coli expression system. Peaks corresponding to cADPR and its variants are labeled. The inset in the top panel shows production of ADPR and Nam from the purified C. bolteae CLOBOL_01188 protein. (D) Structure of cADPR (Lee et al., 1994). The proposed cyclization sites of its two variant forms, v-cADPR-x and v-cADPR-y, are denoted by dashed lines. (E) MS/MS fragmentation pattern of cADPR, v-cADPR-x, and v-cADPR-y, with the peak area (absorbance units; a.u.) of each fragment normalized to the adenine moiety (m/z 136), which had the largest peak area.
Figure 2
Figure 2
Characterizing the products of NADase-positive TIR domains in the cecal contents of gnotobiotic mice colonized with a 26-member-defined consortium of cultured human gut bacterial taxa and fed NAD precursor-sufficient or deficient diets (A) Design of gnotobiotic mouse experiment. (B–D) Levels of (B) nicotinic acid (NA), (C) NAD, and (D) v-cADPR-x in the cecal contents of germ-free and colonized mice fed the NAD-sufficient or deficient diets. Mean values ± SD are shown. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001 (two-way ANOVA; Tukey’s multiple comparisons test; n = 8 animals/group).
Figure 3
Figure 3
Production of v-cADPR-x in gnotobiotic mice colonized with Bacteroides xylanisolvens and/or Bacteroides thetaiotaomicron (A) Design of gnotobiotic mouse experiment. (B) Absolute abundance of B. xylanisolvens XB1A and B. thetaiotaomicron 7330 in cecal contents as a function of diet and community context. Each dot refers to the abundance of the strain within an individual animal. Mean values ± SD are shown. n = 6 mice per treatment group. ∗p < 0.05; ∗∗∗∗p < 0.0001 (two-way ANOVA; Tukey’s multiple comparisons test). (C) Expression of TIR-domain-encoding genes in B. xylanisolvens XB1A and B. thetaiotaomicron 7330 as a function of diet and community context (n = 6 mice per treatment group). Mean values ± SD are shown. ∗p < 0.05 (DESeq2; Wald test with FDR correction). (D–I) Liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ-MS) of cecal NAD metabolites in germ-free (GF) or mono-colonized animals (D–H) or in animals that had been gavaged with both B. thetaiotaomicron 7330 and B. xylanisolvens XB1A or the entire 26-member consortium (I). Mean values ± SD are shown. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 (two-way ANOVA; Tukey’s post hoc test for comparison of diet and microbial community [D–H] or one-way ANOVA with Tukey’s post hoc test for comparison of community type in mice fed the NAD precursor deficient diet [I]).
Figure 4
Figure 4
Identification and characterization of v-cADPR-x-producing TIR domains in the fecal microbiomes of healthy Bangladeshi infants and children and those with malnutrition (A) Total number of TIR domains encoded by the fecal microbiomes of healthy Bangladeshi infants and children during the first 3 years of postnatal life. (B) Number of TIR domains identified in the fecal microbiomes of Bangladeshi infants and children (healthy and malnourished) predicted to metabolize NAD to ADPR, cADPR, v-cADPR-x, or v-cADPR-y. (C–F) Violin plots showing the total number of reads, generated from the fecal microbiomes of age-matched infants and children with healthy growth phenotypes or with acute malnutrition (weight-for-length Z score [WLZ] < −2), that mapped to all detected TIR-domain-containing genes (C) and genes encoding TIR domains predicted to produce v-cADPR-x (D), cADPR (E), or ADPR (F). Data are normalized for gene length and sequencing depth (n = 82 healthy and 66 SAM donor samples). ∗p < 0.05; ∗∗ <0.01 (Mann-Whitney U test). (G–I) Levels of v-cADPR-x, nicotinic acid (NA), and NAD were quantified by LC-QqQ-MS of feces collected from members of a healthy birth cohort, or children with acute malnutrition. Violin shape illustrates the distribution of values; the inset box plots indicate median values and interquartile range. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (Mann-Whitney U test; n = 10 samples/group).

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