Project Summary The human intestinal tract supports a complex microbial environment consisting of bacterial (or microbiota) and fungal (or mycobiota) constituents. Although the role of each of these communities has been a subject of multiple studies, the role of transkingdom interactions between fungi and bacteria in shaping host immunity and physiology has been much less explored. The chemical basis for such interactions, critical for the rational design of mechanistic studies with regards to host immunity and disease development, remain completely uncharted territory in the literature. We have established a genetic manipulation pipeline to identify gene transfer methodology and build a genetic tool for nonmodel human gut bacteria on a large scale. Via a multifactorial optimization of their conjugation/transformation conditions and targeting bacterial conserved 16s rRNA genes, this pipeline efficiently identified the gene transfer methods for multiple nonmodel gut bacterial commensals and set up CRISPR-based or gene insertion tools in multiple of them. This library of genetically targetable microbes comes from 5 different phyla. This genetic manipulation pipeline and this library of tractable commensals will facilitate our investigation of trans-kingdom microbiota-mycobiota interactions at the molecular level. A high throughput screening of bacterial metabolite libraries from gut bacteria identified metabolites with direct effect on intestinal mycobiota. We identified bacterial species and corresponding gene clusters responsible for the production of these metabolites. Our preliminary data suggest strong ties and specific molecular interactions between fungi and bacteria in the gut that have previously unappreciated role in microbial dynamics, metabolite production and immunity. We will utilize such bacterial strains and isogenic mutants in key biosynthetic pathways to target metabolites with mycobiota modulatory properties. We will use several mouse models and synthetic microbial communities to define the role of trans kingdom interaction between bacteria and fungi in modulating host immunity and colonization resistance in the gut. We hypothesize that metabolites from the human bacterial microbiota modulate the fungal communities in the gut to affect microbial composition, the microbiome function and immunity. In addition to revealing novel mechanisms of fungal-bacterial interaction at an unprecedented small molecule level, the results of this proposed investigation will illuminate potential new strategies for targeting of fungal pathogens