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Gut Microbiota: Rational Manipulation of Gut Bacterial Metalloenzymes Provides Insights into Dysbiosis and Inflammation Vayu M. Rekdal and Emily P. Balskus* Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
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important nature of molybdenum-dependent respiration during inflammation. Previous work had revealed similar patterns for metabolism of nitrate, an additional inflammation-derived substrate reduced by molybdenum enzymes.3 Altogether, the authors hypothesized that these enzymes help Proteobacteria thrive by enabling the metabolism of nitrate and formate, molecules that are more abundant in the inflamed gut. In the current study, Winter and co-workers sought to test this proposal by inhibiting the activities of these key Proteobacterial molybdenum-dependent enzymes, thus helping to elucidate their role in dysbiosis and gut inflammation. Their chosen strategy for enzyme manipulation leveraged a molecular understanding of metallocofactor biosynthesis. All Proteobacterial molybdoenzymes incorporate molybdenum into a cofactor known as bis-molybdopterin guanine dinucleotide (moco). Moco acquires molybdenum early in its complex biosynthesis by two enzymes, MogA and MoeA, which catalyze ATP-dependent insertion of the oxyanion molybdate into a pyranopterin scaffold. Following molybdate installation, the pyranopterin is modified further, producing a fully mature metallocofactor that is inserted into cognate enzymes (Figure 1A). It has been known since the 1980s that the closely related transition metal tungsten (as the oxyanion tungstate) can be incorporated in place of molybdenum during moco biosynthesis.4 Because of differences in the redox potentials of these two metallocofactors, substitution of tungsten for molybdenum can inhibit the activity of Proteobacterial molybdoenzymes. Thus, tungstate and molybdate have been used as tools in microbiology and biochemistry to probe the metal and redox dependence of specific metabolic pathways. Winter and co-workers first explored if tungstate affected Proteobacterial growth on inflammation-associated substrates in vitro and in vivo. Consistent with their previous work, they observed that tungstate blocked molybdoenzyme-dependent growth of the model Proteobacterium E. coli on nitrate, formate, TMAO, and DMSO in culture. They next found that while a wild-type E. coli strain could outcompete a mocodeficient, isogenic mutant in the inflamed gastrointestinal tracts of dextran sodium sulfate (DSS)-treated mice, tungstate treatment abrogated this fitness advantage. While these findings suggested that tungstate could block molybdoenzyme-dependent growth of E. coli in an inflamed gut, they raised the question of how this transition metal may influence the dynamics of the entire gut microbial community. Remarkably, tungstate treatment prevented the inflammation-induced bloom of Proteobacteria while having no effect on the diversity and gene content of the other members of the microbiota under normal
riginally considered to be a bystander in human biology, the gut microbiota has recently emerged as an important player in health and disease. Numerous sequencing-based studies have revealed strong associations between the overall structure of this complex microbial community and conditions like obesity and inflammatory bowel disease. However, it is often unclear whether the gut microbiota causes disease or merely reflects disturbed host physiology. Moving from a descriptive (“who’s there”) to a mechanistic (“what are they doing”) understanding of this community is challenging in part because of the difficulty of manipulating specific microbial functions in complex assemblages. Traditional genetic approaches can be applied to only a small fraction of gut species and cannot yet be used in complex, native communities. They also cannot readily target functions that are distributed broadly across multiple microbial species. Other approaches involve global manipulation of communities, including antibiotic treatment, dietary interventions, and fecal transplants. While such approaches can illuminate global patterns associated with disease, they fail to identify the specific enzymes and metabolic pathways that cause disturbed host physiology. A recent study by Winter and co-workers1 shows how a molecular understanding of gut microbial enzymes can enable more precise manipulation of metabolic activities in this microbial community and ultimately provide new insights into the role of these organisms in human health and disease. This work specifically examined the shift in gut microbial community composition (dysbiosis) that accompanies gastrointestinal inflammation, a condition that is observed in conditions such as inflammatory bowel disease (IBD), but despite the strong associations between inflammatory diseases and dysbiosis, a causal role for altered gut microbiota community composition in sustaining host inflammation had not yet been established. One of the major hallmarks of inflammation-induced dysbiosis is the exponential expansion of facultative anaerobes from the phylum Proteobacteria. These bacteria, which include the well-known model organism Escherichia coli, can survive under both aerobic and strictly anaerobic conditions and are typically a minor constituent of a healthy gut microbial community (∼0.1%). Previous work from these authors had suggested that the Proteobacterial blooms associated with gut inflammation are concomitant with an increase in metabolism involving molybdenum-dependent respiratory enzymes.2 Specifically, metabolomics and metagenomics experiments revealed that mice suffering from gut inflammation displayed a simultaneous increase in the level of free gastrointestinal formate and gut microbial molybdoenzymes that are responsible for formate oxidation. Formate also boosted the growth of commensal E. coli in culture and in mice, highlighting the © XXXX American Chemical Society
Received: March 21, 2018
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DOI: 10.1021/acs.biochem.8b00340 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. Rational gut microbiota manipulation by altering metalloenzymes. (A) Following incorporation of molybdate or tungstate into molybdopterin (MPT), the organometallic complex is converted to the molybdenum cofactor (moco), which is installed intact into its cognate enzyme. (B) Molybdenum-dependent enzymes help gut Proteobacteria thrive during inflammation, enabling growth on nitrate and formate. When tungsten is substituted for molybdenum, the respiratory enzymes do not function properly, preventing Proteobacterial growth and dampening inflammation.
tungstate itself is likely of limited utility because it modulates the activity of all molybdoenzymes. These enzymes are conserved from bacteria to humans, and genetic disorders that affect the production of moco-dependent enzymes like sulfite oxidase or xanthine oxidase can have fatal consequences.4 It is also likely that this treatment alters additional functions of the gut microbiota beyond Proteobacterial metabolism. Molybdoenzymes are present in most gut bacterial phyla, but many of their functions are unknown. However, these enzymes may be important under normal, homeostatic conditions, as two of the three sequenced bacterial genomes encoding the largest number of moco enzymes (>40 each) are human gut bacteria.5 Understanding whether tungstate influences moco enzyme function in other gut microbes and in the host is required to completely elucidate the effects of this treatment. Despite these limitations, Winter and co-workers provide an intriguing proof of concept for rational chemical modulation of specific enzymatic activities in complex microbial communities. DNA sequencing has illuminated that the gut microbiota houses trillions of microorganisms and encodes millions of unique genes, but pinpointing which organisms and enzymes are relevant for disease is a major challenge. Together with efforts to target gut microbial enzymes with small molecule inhibitors, this work the lays groundwork for future studies integrating chemical tools and knowledge to illuminate molecular links between the gut microbiota and human biology.
physiological conditions (Figure 1B). Taken together, these data supported the idea that tungstate could selectively modulate bacterial pathways that contribute to Proteobacterial blooms and overall dysbiosis during inflammation. Having established tungstate treatment as a viable strategy for gut microbiota manipulation in vivo, Winter and co-workers addressed the long-standing question of how the expansion of Proteobacteria affects host physiology. The authors first assessed how tungstate influenced the host inflammatory response in both DSS-treated mice and a genetic model of inflammation (IL10-knockout). In both models, tungstate administration significantly reduced the level of host inflammatory markers and pathological histology in the intestinal mucosa while simultaneously preventing weight loss (Figure 1B). This effect was gut microbiota-dependent, as this tungstate treatment did not change inflammatory responses in germ-free mice, which lack an endogenous microbiota. Importantly, the effects of tungstate were not unique to a mouse microbiota. When fecal communities were transplanted from patients with active IBD into DSS-treated germ-free mice, tungstate could prevent the inflammation-dependent bloom of Proteobacteria. Altogether, these findings confirm that that blooms of Proteobacteria can exacerbate host inflammation and highlight a critical role for moco-dependent enzymes in this process. Nonetheless, a few open questions remain. For example, while this study establishes a role for Proteobacteria in exacerbating inflammation, it does not address whether changes in the microbiota contribute to inflammatory disease initiation. Manipulation of specific microbial metabolic activities could be a potentially exciting therapeutic strategy. However,
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. B
DOI: 10.1021/acs.biochem.8b00340 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry ORCID
Emily P. Balskus: 0000-0001-5985-5714 Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Zhu, W. H., Winter, M. G., Byndloss, M. X., Spiga, L., Duerkop, B. A., Hughes, E. R., Buttner, L., de Lima Romão, E., Behrendt, C. L., Lopez, C. A., Sifuentes-Dominguez, L., Huff-Hardy, K., Wilson, R. P., Gillis, C. C., Tukel, C., Koh, A. Y., Burstein, E., Hooper, L. V., Baumler, A. J., and Winter, S. E. (2018) Precision editing of the gut microbiota ameliorates colitis. Nature 553, 208−211. (2) Hughes, E. R., Winter, M. G., Duerkop, B. A., Spiga, L., Furtado de Carvalho, T., Zhu, W. H., Gillis, C. C., Buttner, L., Smoot, M. P., Behrendt, C. L., Cherry, S., Santos, R. L., Hooper, L. V., and Winter, S. E. (2017) Microbial Respiration and Formate Oxidation as Metabolic Signatures of Inflammation-Associated Dysbiosis. Cell Host Microbe 21, 208−219. (3) Winter, S. E., Winter, M. G., Xavier, M. N., Thiennimitr, P., Poon, V., Keestra, A. M., Laughlin, R. C., Gomez, G., Wu, J., Lawhon, S. D., Popova, I. E., Parikh, S. J., Adams, L. G., Tsolis, R. M., Stewart, V. J., and Baumler, A. J. (2013) Host-derived nitrate boosts growth of E. coli in the inflamed gut. Science 339, 708−711. (4) Hille, R., Hall, J., and Basu, P. (2014) The Mononuclear Molybdenum Enzymes. Chem. Rev. 114, 3963−4038. (5) Rothery, R. A., and Weiner, J. H. (2015) Shifting the metallocentric molybdoenzyme paradigm: the importance of pyranopterin coordination. JBIC, J. Biol. Inorg. Chem. 20, 349−372.
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DOI: 10.1021/acs.biochem.8b00340 Biochemistry XXXX, XXX, XXX−XXX
