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

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 (t1/2) 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.

Targeted Intracellular Demethylation of Methylmercury Enhances Elimination Kinetics and Reduces Developmental Toxicity in Transgenic Drosophila.

Methylmercury (MeHg) persists today as a priority public health concern. Mechanisms influencing MeHg metabolism, kinetics, and toxicity outcomes are therefore essential knowledge for informing exposure risks. Evidence points to different toxic potencies of MeHg and inorganic mercury (Hg2+), highlighting the role for biotransformation (demethylation) in regulating MeHg toxicokinetics/dynamics. Whereas microbial MeHg demethylation in the gut is seen to influence elimination kinetics, the potential for systemic demethylation in tissues and target organs to influence MeHg toxicity remains uncertain. To investigate the consequences of systemic MeHg demethylation across development, we engineered transgenic Drosophila to express the bacterial organomercurial lyase enzyme (merB) in a targeted and tissue-specific manner. With all combinations of merB-induced demethylation, ubiquitously (via an actin promoter) or in a tissue-specific manner (ie, gut, muscle, neurons), we observe a rescue of MeHg-induced eclosion failure at the pupal to adult transition. In MeHg-fed larvae with ubiquitous or targeted (gut and muscle) merB expression, we see a significant decrease in MeHg body burden at the pupal stage relative to control flies. We also observe a significant increase in the MeHg elimination rate with merB demethylation induced in adults (control, t1/2 = 7.2 days; merB flies, t1/2 = 3.1 days). With neuronal-specific merB expression, we observe a rescue of MeHg-induced eclosion failure without a decrease in Hg body burden, but a redistribution of Hg away from the brain. These results demonstrate the previously unidentified potential for intracellular MeHg demethylation to promote transport and elimination of Hg, and reduce developmental MeHg toxicity. Impact Statement: These findings demonstrate the potential for MeHg demethylation in situ to contribute significantly to the MeHg elimination and distribution kinetics of whole animals and thereby affords a means of protection against the toxic insult of MeHg. Therefore, this study reveals important insight into processes that can determine an individual's resistance or susceptibility to MeHg and provides rationale for therapies targeting a novel metabolism-based pathways to alleviate toxicity risk stemming from MeHg exposure.

Organomercurial Lyase (MerB)-Mediated Demethylation Decreases Bacterial Methylmercury Resistance in the Absence of Mercuric Reductase (MerA).

The mer operon encodes enzymes that transform and detoxify methylmercury (MeHg) and/or inorganic mercury [Hg(II)]. Organomercurial lyase (MerB) and mercuric reductase (MerA) can act sequentially to demethylate MeHg to Hg(II) and reduce Hg(II) to volatile elemental mercury (Hg0) that can escape from the cell, conferring resistance to MeHg and Hg(II). Most identified mer operons encode either MerA and MerB in tandem or MerA alone; however, microbial genomes were recently identified that encode only MerB. However, the effects of potentially producing intracellular Hg(II) via demethylation of MeHg by MerB, independent of a mechanism to further detoxify or sequester the metal, are not well understood. Here, we investigated MeHg biotransformation in Escherichia coli strains engineered to express MerA and MerB, together or separately, and characterized cell viability and Hg detoxification kinetics when these strains were grown in the presence of MeHg. Strains expressing only MerB are capable of demethylating MeHg to Hg(II). Compared to strains that express both MerA and MerB, strains expressing only MerB exhibit a lower MIC with MeHg exposure, which parallels a redistribution of Hg from the cell-associated fraction to the culture medium, consistent with cell lysis occurring. The data support a model whereby intracellular production of Hg(II), in the absence of reduction or other forms of demobilization, results in a greater cytotoxicity than the parent MeHg compound. Collectively, these results suggest that in the context of MeHg detoxification, MerB must be accompanied by an additional mechanism(s) to reduce, sequester, or redistribute generated Hg(II). IMPORTANCE Mercury is a globally distributed pollutant that poses a risk to wildlife and human health. The toxicity of mercury is influenced largely by microbially mediated biotransformation between its organic (methylmercury) and inorganic [Hg(II) and Hg0] forms. Here, we show in a relevant cellular context that the organomercurial lyase (MerB) enzyme is capable of MeHg demethylation without subsequent mercuric reductase (MerA)-mediated reduction of Hg(II). Demethylation of MeHg without subsequent Hg(II) reduction results in a greater cytotoxicity and increased cell lysis. Microbes carrying MerB alone have recently been identified but have yet to be characterized. Our results demonstrate that mer operons encoding MerB but not MerA put the cell at a disadvantage in the context of MeHg exposure, unless subsequent mechanisms of reduction or Hg(II) sequestration exist. These findings may help uncover the existence of alternative mechanisms of Hg(II) detoxification in addition to revealing the drivers of mer operon evolution.

Latent effects of early-life methylmercury exposure on motor function in Drosophila.

The developmental toxicant, methylmercury (MeHg), can elicit motor deficits that last well into adulthood. Recent studies using Drosophila showed that the developing musculature is sensitive to high doses of MeHg, where a larval feeding paradigm resulted in compromised myotendinous junction (MTJ) formation during development, by a mechanism involving the NG2 homologue, kon-tiki (kon). Low-dose exposures to MeHg that do not produce muscle pathology during development, nevertheless result in impaired flight behavior later in adult life. The present study evaluated the potential for relatively low-dose exposure to produce latent adult muscle pathology and motor impairments, as assayed by climbing and flight, as well as to evaluate molecular mechanisms that may contribute to motor deficits. Wildtype larvae were fed 0, 2, 2.5, or 5 µM MeHg laden food until eclosion. The effect of 5 µM MeHg on MTJ-related gene expression during pupal development was assessed via quantitative RT-qPCR analysis. Upon eclosion, adults were transferred to standard food bottles for 4, 11, or 30 days prior to motor testing. Survivorship (%) was determined from a subset of 200 flies per treatment. Average climbing speed (cm/s) was quantified 4-days post-eclosion (PE). Flight ability was assayed 11- or 30-days PE by measuring landing height (cm) of flies dropped into an adhesive-lined vertical column. In parallel, total body mercury was measured to estimate the influence of residual MeHg at the time of motor testing. Muscle morphology was assessed using immuno-fluorescence microscopy. Exposure to 5uM MeHg significantly reduced climbing speed, and flight ability 4 and 11 - days PE, respectively. While age-related flight deficits were seen in each sex, flight deficits due to MeHg persisted to 30-day PE timepoints exclusively in males. Expression of kon was upregulated across the window of pupal development essential to establishing adult MTJ. However, experimentally restricting the induction of comparable levels of kon to muscle during the same periods did not recapitulate the flight deficits, indicating that muscle-specific induction of kon alone is not sufficient to contribute to latent flight impairments. Adult flight muscle morphology of 11-day PE flies treated with 5 µM MeHg was indistinct from controls, implying muscle structure is not grossly perturbed to impair flight. Collectively, the current data suggest that developmental exposure to 5 µM MeHg reduces flight ability in each sex at 11 day-PE and that latent deficits at 30-day PE are male-specific. It remains to be determined whether the developing MTJ of Drosophila is a sensitive target of MeHg, and whether or not kon acts in conjunction with additional MTJ factors to constitute a MeHg target.

Neuroligin-1 Is a Mediator of Methylmercury Neuromuscular Toxicity.

Methylmercury (MeHg) is a developmental toxicant capable of eliciting neurocognitive and neuromuscular deficits in children with in utero exposure. Previous research in Drosophila melanogaster uncovered that developmental MeHg exposure simultaneously targets the developing musculature and innervating motor neuron in the embryo, along with identifying Drosophila neuroligin 1 (nlg1) as a gene associated with developmental MeHg sensitivity. Nlg1 and its transsynaptic partner neurexin 1 (Nrx1) are critical for axonal arborization and NMJ maturation. We investigated the effects of MeHg exposure on indirect flight muscle (IFM) morphogenesis, innervation, and function via flight assays and monitored the expression of NMJ-associated genes to characterize the role of Nlg1 mediating the neuromuscular toxicity of MeHg. Developmental MeHg exposure reduced the innervation of the IFMs, which corresponded with reduced flight ability. In addition, nlg1 expression was selectively reduced during early metamorphosis, whereas a subsequent increase was observed in other NMJ-associated genes, including nrx1, in late metamorphosis. Developmental MeHg exposure also resulted in persistent reduced expression of most nlg and nrx genes during the first 11 days of adulthood. Transgenic modulation of nlg1 and nrx1 revealed that developing muscle is particularly sensitive to nlg1 levels, especially during the 20-36-h window of metamorphosis with reduced nlg1 expression resulting in adult flight deficits. Muscle-specific overexpression of nlg1 partially rescued MeHg-induced deficits in eclosion and flight. We identified Nlg1 as a muscle-specific, NMJ structural component that can mediate MeHg neuromuscular toxicity resulting from early life exposure.