Microbial enzymes induce colitis by reactivating triclosan in the mouse gastrointestinal tract

Abstract

Emerging research supports that triclosan (TCS), an antimicrobial agent found in thousands of consumer products, exacerbates colitis and colitis-associated colorectal tumorigenesis in animal models. While the intestinal toxicities of TCS require the presence of gut microbiota, the molecular mechanisms involved have not been defined. Here we show that intestinal commensal microbes mediate metabolic activation of TCS in the colon and drive its gut toxicology. Using a range of in vitro, ex vivo, and in vivo approaches, we identify specific microbial β-glucuronidase (GUS) enzymes involved and pinpoint molecular motifs required to metabolically activate TCS in the gut. Finally, we show that targeted inhibition of bacterial GUS enzymes abolishes the colitis-promoting effects of TCS, supporting an essential role of specific microbial proteins in TCS toxicity. Together, our results define a mechanism by which intestinal microbes contribute to the metabolic activation and gut toxicity of TCS, and highlight the importance of considering the contributions of the gut microbiota in evaluating the toxic potential of environmental chemicals.

Metagenomics Combined with Activity-Based Proteomics Point to Gut Bacterial Enzymes that Reactivate Mycophenolate

“…This approach has been employed previously to examine drug and toxin reactivation rates by fecal lysates and to correlate these values with meta-proteomic GUS abundances. 21,23 Here we found MPA reactivation rates between 3 nM/sec and 114 nM/sec for MMF-treated transplant recipients' samples, and between 6 and 18 nM/sec for the healthy individual samples (Figure 4B). Thus, a range of values were observed across all samples, and the highest rates recorded were from MMFtreated transplant recipient samples.…”

“…21, 23 The proteomics pipeline outlined in Figure 4A was followed to output peptide fragments that were then used to identify individual GUS proteins. 21, 23 Importantly, though, the current study uses the protein sequences derived from shotgun metagenomics collected from these exact fecal samples (Figures 2 and 3) as the reference database to identify GUS proteins, rather than a standard reference metagenome like the Integrated Gene Catalog (IGC). 28 In doing so, we found that GUS proteome percent peptide coverage was significantly improved as determined by a by a Wilcoxon matched-pairs signed rank test (P = 0.031; Supplemental Figure 11) using the cohort-specific metagenomics data compared to the IGC as a reference database.…”

“…In all prior studies that apply activity-based proteomics towards discovering structural classes of GUS responsible for drug reactivation, “Loop 1” GUS enzymes were most strongly correlated with increased rate of reactivation. 21, 34 Some classes of GUS enzymes were sparsely represented across our cohort as a whole (Figure 4; Supplemental Figure 11), with no “Loop 2” GUS enzymes being detected and with “Loop 1” GUS enzymes only being detected in four of nine samples. The increased abundance of FMN-binding GUS enzymes in MMF recipients relative to healthy individuals supports our conclusion that FMN-binding GUS promote MPA reactivation in the gut, which may in turn contribute to GI toxicity and systemic recirculation of active MPA.…”

“…Human fecal samples were processed as previously described (21). 5 -10 g of thawed fecal material collected from each donor was resuspended in 25 mL cold extraction buffer (25 mM HEPES pH 6.5, 25 mM NaCl, one Roche Complete EDTA-free protease inhibitor tablet in 50 mL buffer) and 500 mg autoclaved garnet beads then vortexed.…”

Lactational delivery of Triclosan promotes non-alcoholic fatty liver disease in newborn mice

“…With considerable evidence that TCS alters biological responses 21 , several key studies in mice have confirmed that long term exposure has detrimental effects on both the intestinal tract and liver. Chronic exposure to TCS has been shown to increase colonic inflammation and colitis-associated colon tumorgenesis 47 , which has recently been linked to reactivation of TCS from its glucuronide metabolite by specific intestinal microbial β-glucuronidase enzymes 48 . Our previous studies have shown TCS can function as a liver tumor promoter stimulating liver tumorigenesis, due in part to induction of oxidative stress and fibrosis 21,22 .…”