Altered infant human gut microbiome composition and metabolic activity are implicated in childhood atopy and asthma1. We hypothesized that compositionally distinct neonatal human gut microbiota exist and are differentially related to relative–risk (RR) of childhood atopy and asthma. Using stool samples (n = 298; aged 1–11 months) from a US birth cohort and 16S rRNA sequencing, neonates (median age 35 days) were divisible into three microbiota–composition states (NGM1–3). Each incurred significantly different RR for multi–sensitized atopy at age–two years and doctor–diagnosed asthma at age–four years. The highest risk group, NGM3, showed lower relative abundance of certain bacteria (e.g. Bifidobacterium, Akkermansia and Faecalibacterium), higher relative abundance of particular fungi (Candida and Rhodotorula), and a distinct fecal metabolome enriched for pro-inflammatory metabolites. Ex vivo culture of adult human peripheral T–cells with sterile fecal water from NGM3 subjects increased the proportion of CD4+ cells producing interleukin–4 and reduced the relative abundance of Foxp3+CD25+CD4+ cells. 12,13 DiHOME which discriminated NGM3 from lower–risk NGMs, recapitulated the effect of NGM3 fecal water on Foxp3+CD25+CD4+ cell relative abundance. These findings suggest that neonatal gut microbiome dysbiosis drives CD4+ T–cell dysfunction associated with childhood atopy.

Author contributions S.R.L. designed the study, performed immune assays, animal models, metagenomic analysis, biochemical assays, mass spectrometry and statistical analyses, and developed the manuscript. K.A.S. assisted with animal models, performed all microscopy analysis and contributed to the manuscript. D.L.L. assisted with animal models and human immune assays. A.R.P. assisted with animal models and manuscript editing. K.E.F. and K.M. assisted with metagenomic and statistical analysis. E.F. assisted with microscopy. D.R.O., E.M.Z. and C.C.J. provided WHEALS cohort samples and data. M.M. and M.D.C. provided TIPS cohort samples and data. H.A.B. contributed to manuscript development. S.V.L. designed and supervised the study and developed the manuscript. Competing interests S.V.L. is co-founder of Siolta Therapeutics Inc., and serves as both a consultant and a member of its Board of Directors. Furthermore, the Regents of the University of California, UCSF have filed a provisional patent application (Application number 62/637,175) on behalf of S.V.L. and S.R.L. relating to the methods and compositions of EH genes.

Significant gut microbiota heterogeneity exists among ulcerative colitis (UC) patients, though the clinical implications of this variance are unknown. We hypothesized that ethnically distinct UC patients exhibit discrete gut microbiotas with unique metabolic programming that differentially influence immune activity and clinical status. Using parallel 16S rRNA and internal transcribed spacer 2 sequencing of fecal samples (UC, 30; healthy, 13), we corroborated previous observations of UC-associated bacterial diversity depletion and demonstrated significant Saccharomycetales expansion as characteristic of UC gut dysbiosis. Furthermore, we identified four distinct microbial community states (MCSs) within our cohort, confirmed their existence in an independent UC cohort, and demonstrated their coassociation with both patient ethnicity and disease severity. Each MCS was uniquely enriched for specific amino acid, carbohydrate, and lipid metabolism pathways and exhibited significant luminal enrichment of the metabolic products of these pathways. Using a novel ex vivo human dendritic cell and T-cell coculture assay, we showed that exposure to fecal water from UC patients caused significant Th2 skewing in CD4+ T-cell populations compared to that of healthy participants. In addition, fecal water from patients in whom their MCS was associated with the highest level of disease severity induced the most dramatic Th2 skewing. Combined with future investigations, these observations could lead to the identification of highly resolved UC subsets based on defined microbial gradients or discrete microbial features that may be exploited for the development of novel, more effective therapies.

Pathogens selectively target host cells using adhesion molecules and secreted virulence factors that may utilize protein, lipid, or carbohydrate ligands on the cell surface. The human intestinal pathogen Vibrio cholerae secretes a pore-forming toxin, Vibrio cholerae cytolysin (VCC), which contains two domains that are structurally similar to known carbohydrate-binding proteins. These tandem domains are attached to the carboxy-terminus of the cytolytic domain and contain a β-trefoil fold and a β-prism fold. VCC has been shown to bind glycosylated proteins, and removal of the β-prism domain leads to a large decrease in lytic activity against rabbit erythrocytes. Despite these clues, the identity of the glycan receptors of VCC and the role of glycan binding in toxin activity remains unknown. To better understand this specificity, we used a combination of structural and functional approaches to characterize the carbohydrate-binding activity of the VCC toxin. We first probed the monosaccharide-binding activity of VCC and demonstrated that the toxin exhibits millimolar affinity for aldohexoses. To understand this specificity, we solved the crystal structure of the VCC β-prism domain bound to methyl-α-mannose. Next, we utilized a mammalian glycan screen to determine that the β-prism domain preferentially binds complex N-glycans with a heptasaccharide GlcNAc4 Man3 core (NGA2). Fluorescence anisotropy and surface plasmon resonance indicated an approximately 100-nanomolar affinity of the β-prism domain for the heptasaccharide core. Our results suggest that carbohydrate-binding domains on the VCC toxin facilitate high-affinity targeting of mammalian cell membranes, which may contribute to the ability of VCC to lyse cells at picomolar concentrations.

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