Abstract Introduction The growing food allergy epidemic is thought to be related to changing environmental factors, particularly changes in the gut microbiome. While prior work has demonstrated that food allergy can be modulated by gut microbes, little is known about how food allergen-specific CD4+ T cells are affected by gut microbial composition. Here, we report that food allergy severity differs between mice obtained from two different specific pathogen-free vendors (Jackson Labs [Jax] and Taconic Biosciences [Tac]) along with differences in food-allergen specific Treg cells in the small intestine. Methods Mice were sensitized to ovalbumin (OVA) via intraperitoneal injection of OVA with alum. Two weeks later, animals were challenged with 3-7 gavages of OVA. Animals were observed for development of diarrhea within 60 minutes following challenge. Body temperature was tracked before and after gavage. Lymphocytes were isolated from small intestine lamina propria (SI LP) and stained with OVA peptide:MHCII tetramers or for markers of mucosal mast cells (MMC). Intestinal permeability was measured in naive Tac and Jax animals using FITC-dextran uptake assay. In co-housing experiments, mice were co-housed for 4 weeks prior to OVA sensitization. Results Mice from Tac developed diarrhea and anaphylaxis after fewer allergen exposures than mice from Jax. We also found that Tac mice have significantly fewer allergen-specific regulatory T cells in SI LP compared to mice from Jax. In addition, Tac mice had a greater abundance of MMCs and increased intestinal permeability. This increased food allergy severity phenotype was transferable via co-housing, which corresponded to a shift in Jax microbial communities towards those found in Tac mice. Conclusion Our findings demonstrate for the first time that food allergen-specific Treg cells can be modulated by gut microbial community composition, which in turn is correlated to food allergy severity. Altered intestinal permeability may be a mechanism behind this relationship. Funding Source Life Science Research Association Topic Categories Mucosal and Regional Immunology (MUC)

Metabolic diseases including type 2 diabetes and obesity pose a significant global health burden. Plant-based diets, including vegan diets, are linked to favorable metabolic outcomes, yet the underlying mechanisms remain unclear. In a randomized trial involving 21 pairs of identical twins, we investigated the effects of vegan and omnivorous diets on the host metabolome, immune system, and gut microbiome. Vegan diets induced significant shifts in serum and stool metabolomes, cytokine profiles, and gut microbial composition. Despite lower dietary glycine intake, vegan diet subjects exhibited elevated serum glycine levels linked to reduced abundance of the gut pathobiont Bilophila wadsworthia. Functional studies demonstrated that B. wadsworthia metabolizes glycine via the glycine reductase pathway and modulates host glycine availability. Removing B. wadsworthia from a complex microbiota in mice elevated glycine levels and improved metabolic markers. These findings reveal a previously underappreciated mechanism by which diet regulates host metabolic status via the gut microbiota.

Bacteria use invertible genetic elements known as invertons to generate heterogeneity among a population and adapt to new and changing environments. In human gut bacteria, invertons are often found near genes associated with cell surface modifications, suggesting key roles in modulating dynamic processes such as surface adhesion and intestinal colonization. However, comprehensive testing of this hypothesis across complex bacterial communities like the human gut microbiome remains challenging. Metagenomic sequencing holds promise for detecting inversions without isolation and culturing, but ambiguity in read alignment limits the accuracy of the resulting inverton predictions. Here, we developed a customized bioinformatic workflow—PhaseFinderDC—to identify and track invertons in metagenomic data. Applying this method to a defined yet complex gut community (hCom2) across different growth environments over time using both in vitro and in vivo metagenomic samples, we detected invertons in most hCom2 strains. These include invertons whose orientation probabilities change over time and are statistically associated with environmental conditions. We used motif enrichment to identify putative inverton promoters and predict genes regulated by inverton flipping during intestinal colonization and surface adhesion. Analysis of inverton-proximal genes also revealed candidate invertases that may regulate flipping of specific invertons. Collectively, these findings suggest that surface adhesion and intestinal colonization in complex gut communities directly modulate inverton dynamics, offering new insights into the genetic mechanisms underlying these processes.

Dietary plants are molecularly rich but the fates of these compounds post-ingestion and their implications for human health are largely unknown. Here, we systematically characterized the major chemical contributions of widely consumed Solanum species (nightshades) to the human metabolome. Using untargeted metabolomics, we found that a series of steroidal alkaloids resulting from glycoalkaloids tomatine, solanine, and chaconine are dominant diet-derived compounds in systemic circulation following ingestion of tomato and potato. By comparing serum and tissue metabolomes of colonized and microbiome-depleted mice, we determined that the gut microbiota modifies these compounds extensively, altering their absorption and gating host exposure. By screening the metabolic products in human urine and stool samples, we established that steroidal glycoalkaloid metabolism varies inter-individually in a population. Furthermore, using a collection of representative human commensal type strains, we found that a limited set of strains is responsible for steroidal glycoalkaloid metabolism, with the chemical output of a community determined by its strain-level composition. These findings enabled the rational design of complex synthetic microbial communities that controlled host exposure to steroidal alkaloid metabolites in vivo. Importantly, microbial metabolism of Solanum metabolites alters their acetylcholinesterase inhibition in vitro and gut motility in vivo. Our study provides insights into the molecular mechanisms of a diet-microbiome interactions and the effects of dietary metabolites on host physiology.

Human immune systems are highly variable, with most variation attributable to non-genetic sources. The gut microbiome crucially shapes the immune system; however, its relationship with the baseline immune states of healthy humans remains incompletely understood. Therefore, we performed multi-omic profiling of 110 healthy participants through the ImmunoMicrobiome study. A factor-based integrative approach identified coordinated variation, revealing that the tonic interferon response was amongst the most variable immune features in healthy participants. Microbiome composition, pathways, and stool metabolites varied concomitantly with interferon response pathways. Distinct transcriptional programs involving inflammation and TGF-β in SIGLEC-1highmonocytes and CD69highactivated MAIT and NK cells were representative of these programs. Our study provides extensive data to examine the relationship between the immune states and microbiomes of healthy individuals at steady state, which paves the way for delineating inter-individual differences relevant for disease susceptibility and responses to therapy.

The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype.

Microbial community function depends on both taxonomic composition and spatial organization. While composition of the human gut microbiome has been deeply characterized, less is known about the organization of microbes between regions such as lumen and mucosa and the microbial genes regulating this organization. Using a defined 117 strain community for which we generate high-quality genome assemblies, we model mucosa/lumen organization with in vitro cultures incorporating mucin hydrogel carriers as surfaces for bacterial attachment. Metagenomic tracking of carrier cultures reveals increased diversity and strain-specific spatial organization, with distinct strains enriched on carriers versus liquid supernatant, mirroring mucosa/lumen enrichment in vivo. A comprehensive search for microbial genes associated with this spatial organization identifies candidates with known adhesion-related functions, as well as novel links. These findings demonstrate that carrier cultures of defined communities effectively recapitulate fundamental aspects of gut spatial organization, enabling identification of key microbial strains and genes.

Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1–5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected. Germ-free mice were colonized with complex defined communities to show T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies.

Approximately 15% of US adults have circulating levels of uric acid above its solubility limit, which is causally linked to the disease gout. In most mammals, uric acid elimination is facilitated by the enzyme uricase. However, human uricase is a pseudogene, having been inactivated early in hominid evolution. Though it has long been known that uric acid is eliminated in the gut, the role of the gut microbiota in hyperuricemia has not been studied. Here, we identify a widely distributed bacterial gene cluster that encodes a pathway for uric acid degradation. Stable isotope tracing demonstrates that gut bacteria metabolize uric acid to xanthine or short chain fatty acids. Ablation of the microbiota in uricase-deficient mice causes severe hyperuricemia, and anaerobe-targeted antibiotics increase the risk of gout in humans. These data reveal a role for the gut micro biota in uric acid excretion and highlight the potential for microbiome-targeted therapeutics in hyperuricemia.

Using high-depth whole genome sequencing of F0 mating pairs and multiple individual F1 offspring, we estimated the nuclear mutation rate per generation in the malaria vectors Anopheles coluzzii and Anopheles stephensi by detecting de novo genetic mutations. A purpose-built computer program was employed to filter actual mutations from a deep background of superficially similar artifacts resulting from read misalignment. Performance of filtering parameters was determined using software-simulated mutations, and the resulting estimate of false negative rate was used to correct final mutation rate estimates. Spontaneous mutation rates by base substitution were estimated at 1.00 × 10 −9 (95% confidence interval, 2.06 × 10 −10 —2.91 × 10 −9 ) and 1.36 × 10 −9 (95% confidence interval, 4.42 × 10 −10 —3.18 × 10 −9 ) per site per generation in A. coluzzii and A. stephensi respectively. Although similar studies have been performed on other insect species including dipterans, this is the first study to empirically measure mutation rates in the important genus Anopheles , and thus provides an estimate of µ that will be of utility for comparative evolutionary genomics, as well as for population genetic analysis of malaria vector mosquito species.

The behavior of microbial communities depends on both taxonomic composition and physical structure. Metagenomic sequencing of fecal samples has revealed the composition of human gut microbiomes, but we remain less familiar with the spatial organization of microbes between regions such as lumen and mucosa, as well as the microbial genes that regulate this organization. To discover the determinants of spatial organization in the gut, we simulate mucosal colonization over time using an in vitro culture approach incorporating mucin hydrogel microcosms with a complex yet defined community of 123 human strains for which we generated high-quality genome assemblies. Tracking strain abundance longitudinally using shotgun metagenomic measurements, we observe distinct and strain-specific spatial organization in our cultures with strains enriched on mucin microcosms versus in supernatant, reminiscent of mucosa versus lumen enrichment in vivo . Our high taxonomic resolution data enables a comprehensive search for microbial genes that underlie this spatial organization. We identify gene families positively associated with microcosm-enrichment, including several known for biofilm and adhesion functions such as efflux pumps, gene expression regulation, and membrane proteases, as well as a novel link between a coenzyme F420 hydrogenase gene family and lipo/exopolysaccharide biosynthesis. Our strain-resolved abundance measurements also demonstrate that incorporation of microcosms yields a more diverse community than liquid-only culture by allowing co-existence of closely related strains. Altogether these findings demonstrate that microcosm culture with synthetic communities can effectively simulate lumen versus mucosal regions in the gut, providing measurements of microbial organization with high taxonomic resolution to enable identification of specific bacterial genes and functions associated with spatial structure.

Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex, but not both, limiting their utility. Here, we construct and characterize in vitro a defined community of 104 bacterial species composed of the most common taxa from the human gut microbiota (hCom1). We then used an iterative experimental process to fill open niches: germ-free mice were colonized with hCom1 and then challenged with a human fecal sample. We identified new species that engrafted following fecal challenge and added them to hCom1, yielding hCom2. In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by either hCom2 or a human fecal community are phenotypically similar, suggesting that this consortium will enable a mechanistic interrogation of species and genes on microbiome-associated phenotypes.

Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enabled the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms enabled E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof-of-concept for the extension to other phage-bacterial pairs of interest.

ABSTRACTEfforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex but not both, limiting their utility. In accompanying work, we constructed a complex synthetic community (104 strains, hCom1) containing the most common taxa in the human gut microbiome. Here, we used an iterative experimental process to improve hCom1 by filling open metabolic and/or anatomical niches. When we colonized germ-free mice with hCom1 and then challenged it with a human fecal sample, the consortium exhibited surprising stability; 89% of the cells and 58% of the taxa derive from the original community, and the pre- and post-challenge communities share a similar overall structure. We used these data to construct a second version of the community, adding 22 strains that engrafted following fecal challenge and omitting 7 that dropped out (119 strains, hCom2). In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by hCom2 versus human feces are similar in terms of microbiota-derived metabolites, immune cell profile, and bacterial density in the gut, suggesting that this consortium is a prototype of a model system for the human gut microbiome.

ABSTRACTEfforts to model the gut microbiome have yielded important insights into the mechanisms of interspecies interactions, the impact of priority effects on ecosystem dynamics, and the role of diet and nutrient availability in determining community composition. However, the model communities studied to date have been defined or complex but not both, limiting their utility. Here, we construct a defined community of 104 bacterial strains composed of the most common taxa from the human gut microbiota. By propagating this community in growth media missing one amino acid at a time, we show that branched-chain amino acids have an outsize impact on community structure and identify a pathway in Clostridium sporogenes for generating ATP from arginine. We constructed and propagated the complete set of single-strain dropout communities, revealing a sparse network of strain-strain interactions including a novel interaction between C. sporogenes and Lactococcus lactis driven by metabolism. This work forms a foundation for studying strain-strain and strain-nutrient interactions in highly complex defined communities, and it provides a starting point for interrogating the rules of synthetic ecology at the 100+ strain scale.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

We report the complete mitogenome (Mt) sequences of Aedes (Howardina) busckii and Aedes (Ochlerotatus) taeniorhynchus. The sequences were extracted from one individual per species from the Dutch Leeward Island of Saba. The length of the Ae. busckii Mt was 16,794 bp with 80.6% AT content. The Ae. taeniorhynchus Mt was 16,216 bp long with 79.8% AT content. These are the first full Mt sequences available for these species.

Sample storage for downstream RNA analysis can be challenging in some field settings, especially where access to cryogenic materials or refrigeration/freezer facilities are limited. This has limited RNA-based studies on African malaria vectors collected in the field. We evaluated RNA quality after storing mosquito samples in three different sample preservation media over a 4-week period. Storing mosquito specimens in cold (4°C) media significantly improved yields of intact RNA. Our results indicate commercially available products perform well in keeping RNA integrity as advertised. Moreover, absolute ethanol may be an economical alternative for sample preservation that can be utilized in some resource-limited settings.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S -transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Female Aedes aegypti mosquitoes infect hundreds of millions of people each year with dangerous viral pathogens including dengue, yellow fever, Zika, and chikungunya. Progress in understanding the biology of this insect, and developing tools to fight it, has been slowed by the lack of a high-quality genome assembly. Here we combine diverse genome technologies to produce AaegL5, a dramatically improved and annotated assembly, and demonstrate how it accelerates mosquito science and control. We anchored the physical and cytogenetic maps, resolved the size and composition of the elusive sex-determining “M locus”, significantly increased the known members of the glutathione-S-transferase genes important for insecticide resistance, and doubled the number of chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites. Using high-resolution QTL and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. We predict that AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly arboviral vector.