UNLOCKING THE GUT MICROBIOME’S SECRET: THE ESSENTIAL ROLE OF BILIRUBIN REDUCTASE IN HEALTH
Haem is an iron-containing porphyrin ring system that plays a crucial role in oxygen transport via hemoglobin thereby playing a major role in maintaining cellular homeostasis. It also functions as a prosthetic group in various proteins essential in oxygen transport and storage, cellular and xenobiotic metabolism, cell signaling, and transcriptional regulation. Its level in the blood is highly regulated as high levels of free haem have been associated with pro-inflammatory responses, cytotoxicity, compromised vascular function, sepsis, acute lung injury, etc. so its synthesis, utilization, and degradation are tightly regulated. Degradation of haem is mediated by haem oxygenase to form biliverdin which is then converted to another complex called bilirubin which is mediated by biliverdin reductase enzyme.
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| Conversion of haem to bilirubin |
The bilirubin, along with cholesterol and bile acids is secreted into the gut from the liver in the form of conjugated bilirubin called bilirubin diglucuronide which gets either excreted or reabsorbed in the gut. The deconjugation of bilirubin diglucuronide, mediated by human or bacterial beta-glucuronidases allows for easy reabsorption by the enterohepatic circulation thereby elevating its serum levels which have been associated with serious health problems like jaundice and in extreme cases leading to a severe condition called kernicterus, a type of neurological damage induced by the elevated levels of bilirubin. These conditions are generally prevented by our gut microbiome which is solely responsible for the metabolism of bilirubin diglucuronide into more excretable urobilinogen and stercobilinogen thereby maintaining its serum levels in optimum levels where bilirubin has health benefits, acting as an antioxidant.
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| Haem degradation pathway |
The gut microbiome uses bilirubin as a potential terminal electron acceptor in anaerobic respiration, which happens with the conversion of bilirubin into urobilinogen. Despite knowing this information, the enzyme responsible for this conversion termed candidate bilirubin reductase and the gut bacteria producing it were unknown until recently when researchers used screening of gut bacteria, comparative genomics, biochemical tests, fluorescence assays, and metabolomics to discover this enzyme named as bliR and gut bacteria responsible for it.
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A fluorescence assay was performed on extracts from bacteria grown in media with bilirubin and was used to screen the gut bacteria which showed bilirubin reductase activity. The bilirubin reductase was suspected to be oxidoreductase that acts on carbon-carbon double bonds and converts the bilirubin to unstable products urobilinogen and stercobilinogen which readily oxidizes to more stable urobilin and stercobilin which fluorescent with the addition of iodine. At the same time, non-protein-bound bilirubin will not be fluorescent. Positive control was known as bilirubin reducer Clostridioides difficile CD3. Various bacteria from major phyla of human gut microbiota were screened using this method and nine strains were identified with bilirubin reductase activity with three species which was previously not known to have bilirubin reductase activity including Clostridium symbiosum (strains WAL-14163 and WAL-14673), Clostridium sp. M62/1 and Ruminococcus gnavus CC55_001C. All bilirubin reductase activity-positive species were found to be in class Clostridia of the phylum Firmicutes.
The operon in Ruminococcus gnavus was found to be different from other bilirubin reductase-positive bacteria like Clostridium symbiosum and, Clostridioides difficile where it had two extra C-terminal domains, the N-terminal domain having triose-phosphate isomerase (TIM) barrel, extra flavodoxin-like domain and an NADP(H)-binding domain.
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| Comparison between the operons from different bacterial species |
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| Predicted AlphaFold model of Ruminococcus gnavus bliR |
For the validation of bliR as the bilirubin reductase enzyme, the gene from Clostridium symbiosum, Clostridioides difficile and Ruminococcus gnavus was introduced into E.coli10-beta via pCW-lic vector and E.coliT7 via pET-28a(+) vector and the same fluorescence assay was used along with liquid chromatography and tandem mass spectroscopy (LC-MS\MS) and was observed that the strains transformed from bliR gene from Clostridium symbiosum and Clostridioides difficile showed excessive fluorescence and the presence of urobilin and absence of stercobilin was indicated the LC-MS. The strain transformed by the bliR gene from R.gnavus was found to be capable was reducing bilirubin to urobilinogen.
A large-scale analysis of the human gut metagenomes across 4296 infants suggested that the bilirubin reductase was absent in most of the infants analyzed during the first few months when the risk of neonatal jaundice was the highest. It was found mostly by the end of the first year. These observations suggest a strong connection between gut microbiome and the development of jaundice in infants.
The patients suffering from inflammatory bowel disease (IBD) were also found to lack the bilirubin reductase enzyme thereby having lower levels of urobilin and forming pigmented gallstones containing high levels of bilirubin. A significant fragment of the analyzed patients with Crohn’s disease or ulcerative colitis lacked bilirubin reductase which was very much higher compared to 0.1% of healthy adults lacking the enzyme.
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| Comparison of the presence of bliR in healthy adults, infants, and patients suffering from IBD |
In conclusion, bilirubin reductase represents a fascinating example of the crucial interplay between our gut microbiome and overall health. This enzyme, synthesized exclusively by the gut microbiota, plays a pivotal role in regulating bilirubin levels in the blood—a key aspect of maintaining physiological balance and preventing conditions such as jaundice. By nurturing a healthy microbiome through diet and lifestyle choices, we not only support the synthesis of vital enzymes but also contribute to our body’s ability to maintain essential processes. Ultimately, this underscores the importance of a holistic approach to health that considers both the microbiome and its significant impact on bodily functions.
REFERENCES:
Ryter, S. W. (2020). Significance of Heme and Heme Degradation in the Pathogenesis of Acute Lung and Inflammatory Disorders. International Journal of Molecular Sciences, 22(11), 5509. https://doi.org/10.3390/ijms22115509
Hall, B., Levy, S., Dufault-Thompson, K., Arp, G., Zhong, A., Ndjite, G. M., Weiss, A., Braccia, D., Jenkins, C., Grant, M. R., Abeysinghe, S., Yang, Y., Jermain, M. D., Wu, C. H., Ma, B., & Jiang, X. (2024). BilR is a gut microbial enzyme that reduces bilirubin to urobilinogen. Nature microbiology, 9(1), 173–184. https://doi.org/10.1038/s41564-023-01549-x
IMAGE SOURCES:
News Medical Life Sciences, https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20240304113258/ri/1000/src/images/news/ImageForNews_773422_17095699715843124.jpg
American Heart Association, https://www.ahajournals.org/cms/10.1161/CIRCULATIONAHA.105.598698/asset/d64c2f9b-1dda-4190-9235-0043a46f6be6/assets/graphic/16ff1.jpeg
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