Biology subjects

Rakoff-Nahoum, S.

Publications and source records attributed to Rakoff-Nahoum, S..

7 recordsLinked to original sources

bioRxiv2026

Identification of the complete pathway for conversion of bilirubin to urobilinogen by human gut bacteria

Bilirubin, the predominant product of heme catabolism in mammals, enters the intestine via the hepatobiliary system and subsequently is metabolized by the gut microbiome. This process consumes bilirubin and generates multiple downstream derivatives, such as urobilinogen and stercobilinogen. Levels of bilirubin and its derivatives are associated with susceptibility to inflammatory and metabolic disorders, but the microbial species and enzymes that metabolize bilirubin have remained largely unknown. Here, demonstrate that metabolism of bilirubin to urobilinogen requires two separate reactions that can occur in either order and identify novel enzymes and pathway intermediates required for conversion. We find that bilirubin reductase (BilR), an enzyme that was recently discovered and proposed to convert bilirubin to urobilinogen, is specific for reducing the methine bridges of bilinoids, converting bilirubin to the novel intermediate divinylurobilinogen and mesobilirubin to urobilinogen. Using transcriptomic profiling, we identify the bilinoid vinyl reductase (BilV) responsible for reducing the vinyl groups of bilirubin and divinylurobilinogen. BilV is a flavin-dependent oxidoreductase of the Old Yellow Enzyme (OYE) superfamily with a broad distribution across human gut bacteria that overlaps with but does not completely mirror the distribution of BilR. These findings establish the complete pathway for bacterial conversion of bilirubin to urobilinogen, enabling defined studies to interrogate how this metabolism contributes to human health and disease.

microbiology↗

bioRxiv2025

Systematic identification and characterization of regulators of aryl hydrocarbon receptor signaling

The human aryl hydrocarbon receptor (AHR) integrates chemical signals derived from the environment, gut microbes, and endogenous sources to regulate processes ranging from intestinal barrier integrity to xenobiotic detoxification. Despite strong evidence that dysregulation of AHR signaling is a causal factor in metabolic and autoimmune disorders, we currently lack a comprehensive understanding of the factors that regulate AHR activity in human cells. Here, we use genome-scale CRISPR screening to systematically identify regulators of AHR signaling in hepatocytes. The resulting datasets recapitulate the core AHR signaling pathway and identify a large network of regulators. Many of these factors have roles beyond AHR signaling, reflecting that AHR signaling is deeply integrated into human cell biology. We further dissect this network to reveal novel modes of regulation of AHR expression, protein levels, and signaling. For example, we find that the E3 ubiquitin ligase UBR5 sustains AHR signaling by counteracting degradation of ligand-bound AHR. Finally, we identify components of the AHR regulatory network that are specific to cell types and ligands as potential nodes to manipulate AHR signaling in a targeted manner for therapeutic benefit. Overall, our results define the regulatory network that underpins AHR activation, with implications for our understanding of host-microbe interactions and integrative chemosensation and the etiology of metabolic and inflammatory disorders.

genomics↗

bioRxiv2025

A glycan atlas of the mammalian intestine through ontogeny and inflammation

The muco-epithelial interface in the mammalian gut is composed of a mucus and epithelial lining fundamental to barrier function, microbe-host interactions, and intestinal homeostasis. This barrier is heavily glycosylated by O-linked sugars covalently linked to mucin glycoproteins, and N-linked sugars that coat epithelial surface proteins. Gut O- and N-glycans are thought to play central roles in barrier function, host defense, nutrition and attachment for commensals and pathogens, immunoregulation and cell-cell interactions. However, the precise nature of the glycans and how glycan composition changes through development, as a function of diet, and during inflammation, remains incompletely understood. Here, we apply O- and N-glycomic platforms to profile glycans on mucus and intestinal epithelium. By mapping individual glycan species spatially and temporally we identify 57 O- and 18 N-glycans in the mouse intestine, and observe that fucosylation and sialylation varies according to intestinal region and developmental stage. We identify a subset of glycans regulated by the gut microbiome, and observe a constriction of the glycan repertoire during inflammation in both mice and humans. Together, these results provide an atlas of individual intestinal glycans and their dynamic range through ontogeny and inflammation, and represent a significant resource for our understanding of the role of intestinal glycans in health and disease and glycan-focused therapies for intestinal inflammation and shaping the gut microbiome. Highlights- Individual glycans vary across gut region and developmental stage - Terminal fucose and sialic acid residues vary across space and time - The microbiome influences gut glycan composition early in life - Gut inflammation in mice and humans converge on a restricted glycan repertoire eTOC blurbMicrobes colonizing the mammalian intestines encounter mucus and an epithelial layer highly decorated by glycans. Siegel et al. use glycomics to map these sugars in high resolution across gut region, microbial colonization, development and inflammation in both humans and mice.

microbiology↗

bioRxiv2024

Enteric glia regulate Paneth cell secretion and intestinal microbial ecology

Glial cells of the enteric nervous system (ENS) interact closely with the intestinal epithelium and secrete signals that influence epithelial cell proliferation and barrier formation in vitro. Whether these interactions are important in vivo, however, is unclear because previous studies reached conflicting conclusions [1]. To better define the roles of enteric glia in steady state regulation of the intestinal epithelium, we characterized the glia in closest proximity to epithelial cells and found that the majority express PLP1 in both mice and humans. To test their functions using an unbiased approach, we genetically depleted PLP1+ cells in mice and transcriptionally profiled the small and large intestines. Surprisingly, glial loss had minimal effects on transcriptional programs and the few identified changes varied along the gastrointestinal tract. In the ileum, where enteric glia had been considered most essential for epithelial integrity, glial depletion did not drastically alter epithelial gene expression but caused a modest enrichment in signatures of Paneth cells, a secretory cell type important for innate immunity. In the absence of PLP1+ glia, Paneth cell number was intact, but a subset appeared abnormal with irregular and heterogenous cytoplasmic granules, suggesting a secretory deficit. Consistent with this possibility, ileal explants from glial-depleted mice secreted less functional lysozyme than controls with corresponding effects on fecal microbial composition. Collectively, these data suggest that enteric glia do not exert broad effects on the intestinal epithelium but have an essential role in regulating Paneth cell function and gut microbial ecology.

neuroscience↗

bioRxiv2022

Vaginal microbiome-host interactions modeled in a human vagina-on-a-chip

BackgroundA dominance of non-iners Lactobacillus species in the vaginal microbiome is optimal and strongly associated with gynecological and obstetric health, while the presence of diverse obligate or facultative anaerobic bacteria and a paucity in Lactobacillus species, similar to communities found in bacterial vaginosis (BV), is considered non-optimal and associated with adverse health outcomes. Various therapeutic strategies are being explored to modulate the composition of the vaginal microbiome; however, there is no human model that faithfully reproduces the vaginal epithelial microenvironment for preclinical validation of potential therapeutics or testing hypotheses about vaginal epithelium-microbiome interactions. ResultsHere, we describe an organ-on-a-chip (Organ Chip) microfluidic culture model of the human vaginal mucosa (Vagina Chip) that is lined by hormone-sensitive, primary vaginal epithelium interfaced with underlying stromal fibroblasts, which sustains a low physiological oxygen concentration in the epithelial lumen. We show that the Vagina Chip can be used to assess colonization by optimal L. crispatus consortia as well as non-optimal Gardnerella vaginalis-containing consortia, and to measure associated host innate immune responses. Co-culture of the L. crispatus consortia was accompanied by maintenance of epithelial cell viability, accumulation of D- and L-lactic acid, maintenance of a physiologically relevant low pH, and down regulation of proinflammatory cytokines. In contrast, co-culture of G. vaginalis-containing consortia in the Vagina Chip resulted in epithelial cell injury, a rise in pH, and upregulation of proinflammatory cytokines. ConclusionThis study demonstrates the potential of applying human Organ Chip technology to create a preclinical model of the human vaginal mucosa that can be used to better understand interactions between the vaginal microbiome and host tissues, as well as to evaluate the safety and efficacy of live biotherapeutics products.

microbiology↗

bioRxiv2021

Characterization of vaginal microbial enzymes identifies amylopullulanases that support growth of Lactobacillus crispatus on glycogen

The healthy human vaginal microbiota is generally dominated by lactobacilli, and the transition to a more diverse community of anaerobic microbes is associated with health risks. Glycogen released by lysed epithelial cells is believed to be an important nutrient source in this environment. However, the mechanism by which vaginal bacteria metabolize glycogen is unclear, with evidence implicating both microbial and human enzymes. Here, we biochemically characterize six glycogen-degrading enzymes (GDEs) from vaginal bacteria that support the growth of amylase-deficient L. crispatus on glycogen. We reveal variations in the pH tolerance and susceptibility to inhibition between enzymes from different organisms. Analysis of vaginal microbiome datasets show these enzymes are expressed in all Community State Types. Finally, we confirm the presence and activity of bacterial GDEs in cervicovaginal fluid. This work establishes that bacterial GDEs can participate in the breakdown of glycogen, providing insight into metabolism that may shape the vaginal microbiota.

biochemistry↗

bioRxiv2020

Multi-kingdom quantitation reveals distinct ecological drivers of predictable early-life microbiome assembly

The infant gut microbiota develops remarkably predictably1-7, with pioneer species colonizing after birth, followed by an ordered succession of other microbes. This predictable assembly is vital to health8,9, yet the forces underlying it remain unknown. The environment, host and microbe-microbe interactions are all likely to shape microbiota dynamics, but in such a complex ecosystem identifying the specific role of any individual factor has remained a major challenge10-14. Here we use multi-kingdom absolute abundance quantitation, ecological modelling, and experimental validation to overcome this challenge. We quantify the absolute bacterial, fungal, and archaeal dynamics in a longitudinal cohort of 178 preterm infants. We uncover with exquisite precision microbial blooms and extinctions, and reveal an inverse correlation between bacterial and fungal loads the infant gut. We infer computationally then demonstrate experimentally in vitro and in vivo that predictable assembly dynamics are driven by directed, context-dependent interactions between microbes. Mirroring the dynamics of macroscopic ecosystems15-17, a late-arriving member,Klebsiella, exploits the pioneer,Staphylococcus, to gain a foothold within the gut. Remarkably, we find that interactions between kingdoms drive assembly, with a single fungal species,Candida albicans, inhibiting multiple dominant gut bacteria. Our work unveils the centrality of simple microbe-microbe interactions in shaping host-associated microbiota, a critical step towards targeted microbiota engineering.

microbiology↗