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

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Triclosan is widely used as an antimicrobial compound in consumer products like hand soaps, toothpastes, athletic wear, and even children’s toys. It has become a prominent environmental contaminant because of its industrial utilization and subsequent discharge into wastewater. Indeed, triclosan is ubiquitous in the United States – one study found it in the urine of 75% of tested human participants.1 Its presence in most households and throughout the environment indicate that we must have a deeper understanding of it affects long term health.

A previous study from the Zhang group at the University of Massachusetts-Amherst demonstrated that antimicrobial compounds, including triclosan, increased inflammation and tumor formation in the colon.2 Interestingly, this effect was observed only in mice with an intact gut microbiota, not germ-free mice, indicating that the toxicity of triclosan was likely mediated by intestinal microbes. At this point, the Zhang group reached out to the Redinbo group at the University of North Carolina-Chapel Hill to evaluate the enzymes responsible for generating the gut toxicity of triclosan. Together, we hypothesized that triclosan’s mammalian metabolism by glucuronidation and sulfation might be reversed by gut microbial beta-glucuronidases (GUS) and sulfatases, reactivating triclosan in the GI tract. In our Nature Communications study published on January 10 2022, which includes the Zhang, Redinbo, and Cai (Hong Kong Baptist University) groups as well as an international team of investigators, we show that gut microbial GUS enzymes are the culprits that drive the intestinal toxicity of triclosan.

First, to confirm that gut microbial enzymes were acting upon triclosan metabolites, we examined the metabolite profile of triclosan-fed mice in various regions of the GI tract. The small intestine contained mostly sulfate- and glucuronide-conjugated triclosan, consistent with our knowledge of its metabolism. The colon was, however, dominated by free triclosan, indicating that the sulfated and glucuronidated compounds had been hydrolyzed upon reaching the distal GI tract. Similar results were observed in the feces of human subjects who used toothpaste containing triclosan. Furthermore, when antibiotic-treated or germ-free mice were given triclosan, the ratio of triclosan to triclosan-glucuronide (TCS-G) decreased, establishing that the gut microbiota mediates the conversion of TCS-G back to triclosan.

The Redinbo group had previously created a catalog of gut microbial GUS enzymes from the human and mouse intestinal microbiome and grouped them into distinct structural clades.3–5 Here we examined the ability of a panel of representative purified GUS enzymes to process TCS-G, which revealed that two of the structural classes – Loop 1 and FMN-binding GUSs – preferentially process TCS-G. Next, we examined human fecal lysates, which contain enzymes extracted from fecal samples, using a unique activity-based probe-enabled proteomics pipeline.6 The rate of TCS-G processing by each human fecal lysate was measured and then correlated with the composition of GUS enzymes in each sample as determined by our proteomics pipeline following enrichment with a covalent GUS probe. Loop 1 GUS abundance correlated with TCS-G turnover rate, corroborating our in vitro data. We did not see any significant correlation with FMN-binding GUS enzymes, suggesting that Loop 1 GUS enzymes are primarily responsible for TCS-G turnover. Finally, we elucidated several key structural features of Loop 1 and FMN-binding GUS enzymes that enable them to process TCS-G efficiently. The key collective takeaway from these data is that the Loop 1 GUSs drive the conversion of TCS-G to triclosan.

After identifying these gut microbial enzymes, we postulated that triclosan-induced colitis might be alleviated by selectively inhibiting the bacterial proteins responsible. To test this prediction, we employed a GUS inhibitor (GUSi) that effectively targets both Loop 1 and FMN-binding enzymes.7 Mice were treated in four groups, with vehicle or triclosan coadministered with or without GUSi, and all mice were treated with dextran sodium sulfate to induce colitis. As previously reported2, mice who received triclosan had worsened colitis than those receiving vehicle as evidenced by decreased colon length, increased crypt damage, and increased immune cells and expression of pro-inflammatory markers. Here we showed that each of these effects was abolished by GUSi coadministration. These results reaffirm the inflammatory properties of triclosan and support the conclusion that gut microbial GUS enzymes drive triclosan-induced colitis by converting the metabolite TCS-G back to the native triclosan.

Interestingly, the U.S. Food and Drug Administration restricted the usage of triclosan in over-the-counter hand soaps in 2016. However, this act was mainly based on evidence that showed that the addition of triclosan to these soaps offered no additional health benefit over plain soap, not on the potential toxicity of triclosan. Indeed, triclosan is still present in many consumer products including toothpastes. As we show here, the adverse health effects of triclosan are clear, and suggest that the presence of this compound should be re-evaluated.

Please find our complete study linked here: https://www.nature.com/articles/s41467-021-27762-y

References

  1. Calafat, A. M., Ye, X., Wong, L. Y., Reidy, J. A. & Needham, L. L. Urinary concentrations of triclosan in the U.S. population: 2003-2004. Environ. Health Perspect. 116, 303–307 (2008).
  2. Yang, H. et al. A common antimicrobial additive increases colonic inflammation and colitis-associated colon tumorigenesis in mice. Sci. Transl. Med. 10, 4116 (2018).
  3. Pellock, S. J. et al. Discovery and Characterization of FMN-Binding β-Glucuronidases in the Human Gut Microbiome. J. Mol. Biol. 431, 970–980 (2019).
  4. Pollet, R. M. et al. An Atlas of β-Glucuronidases in the Human Intestinal Microbiome. Structure 25, 967-977.e5 (2017).
  5. Creekmore, B. C. et al. Mouse Gut Microbiome-Encoded β-Glucuronidases Identified Using Metagenome Analysis Guided by Protein Structure. mSystems 4, 1–13 (2019).
  6. Jariwala, P. B. et al. Discovering the Microbial Enzymes Driving Drug Toxicity with Activity-Based Protein Profiling. ACS Chem. Biol. acschembio.9b00788 (2019). doi:10.1021/acschembio.9b00788
  7. Pellock, S. J. et al. Gut Microbial β-Glucuronidase Inhibition via Catalytic Cycle Interception. ACS Cent. Sci. 4, 868–879 (2018).

Morgan Walker

Ph.D. Candidate, University of North Carolina - Chapel Hill