A host-microbiota interactome reveals extensive transkingdom connectivity.

The myriad microorganisms that live in close association with humans have diverse effects on physiology, yet the molecular bases for these impacts remain mostly unknown1-3. Classical pathogens often invade host tissues and modulate immune responses through interactions with human extracellular and secreted proteins (the 'exoproteome'). Commensal microorganisms may also facilitate niche colonization and shape host biology by engaging host exoproteins; however, direct exoproteome-microbiota interactions remain largely unexplored. Here we developed and validated a novel technology, BASEHIT, that enables proteome-scale assessment of human exoproteome-microbiome interactions. Using BASEHIT, we interrogated more than 1.7 million potential interactions between 519 human-associated bacterial strains from diverse phylogenies and tissues of origin and 3,324 human exoproteins. The resulting interactome revealed an extensive network of transkingdom connectivity consisting of thousands of previously undescribed host-microorganism interactions involving 383 strains and 651 host proteins. Specific binding patterns within this network implied underlying biological logic; for example, conspecific strains exhibited shared exoprotein-binding patterns, and individual tissue isolates uniquely bound tissue-specific exoproteins. Furthermore, we observed dozens of unique and often strain-specific interactions with potential roles in niche colonization, tissue remodelling and immunomodulation, and found that strains with differing host interaction profiles had divergent interactions with host cells in vitro and effects on the host immune system in vivo. Overall, these studies expose a previously unexplored landscape of molecular-level host-microbiota interactions that may underlie causal effects of indigenous microorganisms on human health and disease.

Highly multiplexed bioactivity screening reveals human and microbiota metabolome-GPCRome interactions.

The human body contains thousands of metabolites derived from mammalian cells, the microbiota, food, and medical drugs. Many bioactive metabolites act through the engagement of G-protein-coupled receptors (GPCRs); however, technological limitations constrain current explorations of metabolite-GPCR interactions. Here, we developed a highly multiplexed screening technology called PRESTO-Salsa that enables simultaneous assessment of nearly all conventional GPCRs (>300 receptors) in a single well of a 96-well plate. Using PRESTO-Salsa, we screened 1,041 human-associated metabolites against the GPCRome and uncovered previously unreported endogenous, exogenous, and microbial GPCR agonists. Next, we leveraged PRESTO-Salsa to generate an atlas of microbiome-GPCR interactions across 435 human microbiome strains from multiple body sites, revealing conserved patterns of cross-tissue GPCR engagement and activation of CD97/ADGRE5 by the Porphyromonas gingivalis protease gingipain K. These studies thus establish a highly multiplexed bioactivity screening technology and expose a diverse landscape of human, diet, drug, and microbiota metabolome-GPCRome interactions.

Commensal microbiota from patients with inflammatory bowel disease produce genotoxic metabolites.

Microbiota-derived metabolites that elicit DNA damage can contribute to colorectal cancer (CRC). However, the full spectrum of genotoxic chemicals produced by indigenous gut microbes remains to be defined. We established a pipeline to systematically evaluate the genotoxicity of an extensive collection of gut commensals from inflammatory bowel disease patients. We identified isolates from divergent phylogenies whose metabolites caused DNA damage and discovered a distinctive family of genotoxins-termed the indolimines-produced by the CRC-associated species Morganella morganii. A non-indolimine-producing M. morganii mutant lacked genotoxicity and failed to exacerbate colon tumorigenesis in mice. These studies reveal the existence of a previously unexplored universe of genotoxic small molecules from the microbiome that may affect host biology in homeostasis and disease.

High-throughput identification of autoantibodies that target the human exoproteome.

Autoantibodies that recognize extracellular proteins (the exoproteome) exert potent biological effects but are challenging to detect. Here, we developed rapid extracellular antigen profiling (REAP), a high-throughput technique for the comprehensive discovery of exoproteome-targeting autoantibodies. Patient samples are applied to a genetically barcoded yeast surface display library containing 2,688 human extracellular proteins. Antibody-coated yeast are isolated, and sequencing of barcodes is used to identify displayed antigens. To benchmark REAP's performance, we screened 77 patients with autoimmune polyglandular syndrome type 1 (APS-1). REAP sensitively and specifically detected both known and previously unidentified autoantibodies in APS-1. We further screened 106 patients with systemic lupus erythematosus (SLE) and identified numerous autoantibodies, several of which were associated with disease severity or specific clinical manifestations and exerted functional effects on cell signaling ex vivo. These findings demonstrate the utility of REAP to atlas the expansive landscape of exoproteome-targeting autoantibodies and their impacts on patient health outcomes.