Assessing the role of the gut microbiome in methylmercury demethylation and elimination in humans and gnotobiotic mice

Abstract

The risk of methylmercury (MeHg) toxicity following ingestion of contaminated foodstuffs (e.g., fish) is directly related to the kinetics of MeHg elimination among individuals. Yet, the factors driving the wide range of inter-individual variability in MeHg elimination within a population are poorly understood. Here, we investigated the relationship between MeHg elimination, gut microbiome demethylation activity, and gut microbiome composition using a coordinated human clinical trial and gnotobiotic mouse modeling approach together with metagenomic sequence analysis. We first observed MeHg elimination half-lives (t_1/2) ranging from 28 to 90 days across 27 volunteers. Subsequently, we found that ingestion of a prebiotic induced changes in the gut microbiome and mixed effects (increased, decrease, and no effect) on elimination in these same individuals. Nonetheless, elimination rates were found to correlate with MeHg demethylation activity in cultured stool samples. In mice, attempts to remove the microbiome via generation of germ-free (GF) animals or through antibiotic (Abx) treatment both diminished MeHg demethylation to a similar extent. While both conditions substantially slowed elimination, Abx treatment resulted in significantly slower elimination than the GF condition, indicating an additional role for host-derived factors in supporting elimination. Human fecal microbiomes transplanted to GF mice restored elimination rates to that seen in control mice. Metagenomic sequence analysis of human fecal DNA did not identify genes encoding proteins typically involved in demethylation (e.g., merB, organomercury lyase). However, the abundance of several anaerobic taxa, notably Alistipes onderdonkii, were positively correlated with MeHg elimination. Surprisingly, mono-colonization of GF free mice with A. onderdonkii did not restore MeHg elimination to control levels. Collectively, our findings indicate the human gut microbiome uses a non-conventional pathway of demethylation to increase MeHg elimination that relies on yet to be resolved functions encoded by the gut microbes and the hostClinical Trial NCT04060212, prospectively registered 10/1/2019.

Keywords: Demethylation; Elimination rate; Gut microbiome; Half-life; Methylmercury; Prebiotic.

Fig 1.. MerMES clinical study design.

A. Scheduled fish meal consumption 7 days apart and schematic of sampling and analysis of hair and stool. B. The timeline of Trial 1 and 2 fish meal ingestion, elimination period, sampling and prebiotic intervention (Trial 2).

Fig 2.. In vivo MeHg elimination rates and in vitro demethylation.

A,B. Longitudinal quantification of total Hg (Hg:S) in hair of subjects S13 and S9 over the course of the Trial 1. C. Loss of total mercury due to demethylation, reduction and volatilization in human stool cultures from subjects S9 and S13. D. Correlation and Pearson regression statistics of elimination rate (half-life) and percent Hg remaining in in vitro cultures after 96 hours

Fig 3.. Changes in human MeHg elimination rate with prebiotic exposure.

MeHg elimination rate (t_1/2) of 21 participants following the MerMES protocol (Fig 1) are shown superimposed on the percent composition of indicated taxa (phyla). “A” designates Trial 1 and “B” designates Trial 2 results for each participant. Community composition is denoted at the phyla level, with the exception of Firmicutes, which is subdivided into four monophyletic clades, following the organization of the genome taxonomy database (Version 07-RS207).

Fig 4.. MeHg elimination in mice with various manipulations of the gut microbiome.

Elimination t_1/2 (log transformed) of MeHg in conventional (CV), Antibiotic-treated Conventional (Abx), Germ-Free (GF), Alistipes onderdonkii gnotobiotic mice (A.o.) and mice humanized with two different human gut microbiomes (S27, S36). Bars indicate mean and std. dev. (absolute t_1/2 value in days above the data points). Multiple comparison testing showing significant differences between CV and all other groups are shown, * = p<0.05, ** = p<0.005. Additional comparisons are in Table S2B.

Fig 5.. Total %Hg as Hg(II) in stools of mice with various manipulations of the gut microbiome.

Total Hg as Hg(II) in stools, representing demethylated MeHg, in conventional (CV), Antibiotic-treated conventional (Abx), Germ-Free (GF), Alistipes onderdonkii gnotobiotic mice and mice humanized with two different human gut microbiomes (S27, S36). Bars indicate mean and std. dev. Multiple comparison testing showing significant differences between CV and all other groups are shown, * = p<0.05, ** = p<0.005. Additional comparisons are in Table S3B.

Fig 6.. Principal component ordination (PCO) representing abundance weighted Bray-Curtis dissimilarity of OTUs across 27 metagenomes.

OTUs were generated at 95% ANI (species level) and abundance weighted. Each point represents the taxonomy of a single metagenomic community via the abundance of its OTUs. Greater distance between points indicates greater dissimilarity between metagenome taxonomy. Points are colored based on standard deviations (S.D.) away from mean elimination rate: Purple: >1 S.D. below mean, Blue: < 1 S.D. below mean, Yellow: < 1 S.D. above mean, Red: >1 S.D. above mean. The correlation between MPD (mean pairwise distance) and participant MeHg elimination rate did not reach significance (p-value is 0.689, and the pearson correlation is −0.081)

Fig 7.. OTU abundance, co-occurrence, and correlation to MeHg elimination in Trial 1 metagenomes.

Nodes represent OTUs present in at least 10 of 27 metagenomes surveyed. Size of nodes depicts correlation to MeHg elimination rate where yellow indicates positive and blue indicates negative correlation. A. onderdonkii node is outlined in green. Edges represent significant Pearson correlation between OTU co-occurrence and abundance (p < 0.05). Distance is inverse of correlation (shorter distance = stronger correlation), red lines represent significant negative correlations.