Viewpoint Cite This: Biochemistry XXXX, XXX, XXX−XXX
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The Gut Microbiome Says NO to microRNA-Mediated Gene Silencing Zheng Wei†,‡ and Jason M. Crawford*,‡,§,∥ †
Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut 06520, United States Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States § Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States ∥ Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut 06536, United States
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In humans, AGO2 is a component of a multiprotein assembly including GW182 family proteins. Structural modeling and immunoprecipitation analysis showed that Snitrosylated Cys691 of AGO2 disrupts an essential interaction with GW182. In C. elegans, let-7 miRNA regulates lin-41 silencing during development, and microbiota-dependent Snitrosylation of ALG-1 disrupted let-7 miRNA-mediated gene silencing, leading to derepression of lin-41. This led to developmental effects in the host as anticipated. Specifically, through analyzing survival in a temperature-sensitive let-7 roundworm mutant cocultured with wild-type or NOSdeficient B. subtilis, they found that NO-proficient bacteria promoted vulval rupture and animal death during vulval morphogenesis, relative to control bacteria (Δnos). These results indicate that microbiota can shape the post-translational landscape of the host proteome to regulate miRNA activity and host development. Other related roundworms, such as from the genus Heterorhabditis, have formed a tight symbiosis with a specific microbiota member, in this case, members of the Photorhabdus genus, via specialized fimbriae,3 and these bacteria similarly regulate developmental decisions and the biological fate of their host nematode. While specific developmental mechanisms differ, C. elegans may intriguingly be able to alter their more variable microbiome composition by controlling their depth, and, by extension, the associated oxygen tension and microbial composition in the soil microenvironment. Discovery of microbiota-derived S-nitrosylation as a mechanism of host regulation in the model C. elegans opens new general lines of inquiry in microbiome research. It remains unknown to the extent that reactive NO and NO-associated molecules could permeate the thick mucus layer in mammalian host epithelia and modify host proteins directly. It is also unclear if microbiota derived NO, directly or indirectly, could manipulate endogenous NO synthesis and associated signaling. Indeed, ROS and RNS stimulate NO synthesis in, for example, human macrophages. Nevertheless, the oral microbiota of humans is also thought to represent a potential source of NO that could affect cardiovascular homeostasis.4 Among the ∼1000 candidate S-nitrosylated proteins in C. elegans, numerous hits are involved in immunity and metabolism, suggesting that microbiota-derived NO may intimately participate in immune signaling at the host/bacteria interface. NO plays several established roles in immunity. As a toxic agent, NO restricts infection of pathogens. As a signaling
itric oxide (NO) is a signaling molecule in vasoregulation, neurotransmission, and immunological function in humans.1 Endothelial nitric oxide synthase (eNOS, NOS-3; vasodilation) and neuronal NOS (nNOS, NOS-1) are primarily constitutively expressed in mammals, whereas in macrophages and other immune cells, NOS is induced (iNOS, NOS-2) in response to pathogen infection, where its product NO serves as a defensive cytotoxin. Dysregulation of NO production causes broad pathophysiological responses and diseases. NO is a short-lived, reactive free-radical species and contributes to the spontaneous formation of other reactive nitrogen species (RNS) and reactive oxygen species (ROS), which collectively mediate the immunomodulatory effects of NOS-2-derived NO. Essential indirect NO signaling roles can be mediated by protein post-translational modifications by Snitrosating susceptible thiols of cysteine residues to modulate kinase, transcription factor, and signal transduction pathways. Longer-term storage of NO is achieved through the formation of S-nitrosoglutathione, which is a substrate for transnitrosation reactions with other thiols. While the importance of NO and NO-derived molecules in modulating host responses has been well-recognized,1 the roles of the microbiota, the collection of microbes in and on the host, in NO regulation and host responses, remain largely obscure. Stamler and co-workers tested the hypothesis that gut microbiota-derived NO could serve as an interkingdom signal and regulate host responses via NO-mediated post-translational modifications using the roundworm Caenorhabditis elegans as a model system.2 They found that C. elegans Snitrosylation can be mediated by intestinal bacteria, Bacillus subtilis or Escherichia coli, which produce NO via distinct mechanisms. They isolated total proteins from young adult stage (L4) roundworms cocultured with B. subtilis and enriched S-nitrosylated proteins using a resin-assisted capture technique. Approximately 1000 candidate S-nitrosylated host proteins were identified by mass spectrometry. While widespread microbiota-derived and NO-mediated protein S-nitrosylation in C. elegans likely has a variety of biological effects, the team focused on the microbiotadependent S-nitrosylation of C. elegans Argonaute protein (ALG-1) (see Figure 1) and its homologous mammalian counterpart, Argonaute 2 (AGO2), at conserved cysteine residues. These Argonaute proteins regulate gene expression via microRNA (miRNA)-mediated gene silencing. Through site-directed mutagenesis studies, they found that Cys855 of ALG-1 and Cys691 of AGO2 represent a conserved site of Snitrosylation in Argonaute proteins. © XXXX American Chemical Society
Received: March 25, 2019
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DOI: 10.1021/acs.biochem.9b00262 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. Model describes microbiome-mediated regulation of C. elegans development through S-nitrosylation of ALG-1. S-nitrosylation of ALG-1 disrupts its protein−protein interactions with a GW182-family protein required for miRNA let-7-mediated lin-41 gene silencing. In mammals such as humans and mice, it is unclear how much microbiome-derived NO penetrates the thick mucus layer of the intestine, which contains molecules such as immunoglobulin A (IgA), antimicrobial peptides (AMPs), and glycoproteins among other molecules, before accessing the intestinal epithelial cells. Whether similar effects occur in mammals as they do in C. elegans remain exciting questions in the field. [NarG = nitrate reductase.]
1016720), the Camille and Henry Dreyfus Foundation (No. TC-17-011), the Yale Comprehensive Cancer Center, and Yale University.
molecule, NO regulates apoptosis and other types of cell death. In multiple immune cells, high concentrations of NO generated by NOS-2 are rapidly oxidized under aerobic conditions to RNS/ROS, inducing host cell cytotoxicity. While it has been shown that NO and RNS/ROS are involved in apoptosis, it remains unclear whether and how these reactive molecules are directly involved in inflammasome assembly and pyroptosis. Pyroptosis is a common inflammatory form of programmed cell death in intestinal epithelial cells, in which pathogens are recognized by intracellular receptors, resulting in the assembly of inflammasomes,5 promoting the maturation and secretion of pro-inflammatory cytokines IL-1β and IL-18. These cytokines then attract other immune cells for defense and contribute to inflammation. The connections among NO, inflammation, and the microbiota are intriguing, as diseases including obesity, cancer, and inflammatory bowel diseases all share an intimate relationship to microbiome composition. Microbiota-derived NO production and its potential effects on host immune responses in higher organisms adds yet another layer of complexity. Elucidation of NO-associated signaling pathways at the host/bacteria interface will help to eliminate gaps in our knowledge regarding their molecular mechanisms in immune response, cell metabolism, growth, and death. Such an understanding may unearth new strategies to control microbiome-associated inflammatory diseases.
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Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Coleman, J. W. (2001) Nitric oxide in immunity and inflammation. Int. Immunopharmacol. 1, 1397−1406. (2) Seth, P., Hsieh, P. N., Jamal, S., Wang, L., Gygi, S. P., Jain, M. K., Coller, J., and Stamler, J. S. (2019) Regulation of microRNA machinery and development by interspecies S-nitrosylation. Cell 176, 1014−1025. (3) Somvanshi, V. S., Sloup, R. E., Crawford, J. M., Martin, A. R., Heidt, A. J., Kim, K.-s., Clardy, J., and Ciche, T. A. (2012) A single promoter inversion switches Photorhabdus between pathogenic and mutualistic states. Science 337, 88−93. (4) Pinheiro, L. C., Ferreira, G. C., Amaral, J. H., Portella, R. L., de O. C. Tella, S., Passos, M. A., and Tanus-Santos, J. E. (2016) Oral nitrite circumvents antiseptic mouthwash-induced disruption of enterosalivary circuit of nitrate and promotes nitrosation and blood pressure lowering effect. Free Radical Biol. Med. 101, 226−235. (5) Zhu, S., Ding, S., Wang, P., Wei, Z., Pan, W., Palm, N. W., Yang, Y., Yu, H., Li, H. B., Wang, G., Lei, X., de Zoete, M. R., Zhao, J., Zheng, Y., Chen, H., Zhao, Y., Jurado, K. A., Feng, N., Shan, L., Kluger, Y., Lu, J., Abraham, C., Fikrig, E., Greenberg, H. B., and Flavell, R. A. (2017) Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells. Nature 546, 667−670.
AUTHOR INFORMATION
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*E-mail:
[email protected]. ORCID
Jason M. Crawford: 0000-0002-7583-1242 Funding
Our work on the discovery and characterization of metabolites from the human microbiota has been supported by the National Institutes of Health (Nos. 1DP2-CA186575 and R01CA215553), the Burroughs Wellcome Foundation (No. B
DOI: 10.1021/acs.biochem.9b00262 Biochemistry XXXX, XXX, XXX−XXX
