OVERALL PROGRAM SUMMARY: Mucus-degrading intestinal bacteria and toxicities of hematopoietic cell transplantation The microbial community (microbiota) in the human gut plays a central role in digestion of dietary fiber, providing nutrients to the gut microbiota as well as to the host through fermentation products like short-chain fatty acids. A minority of gut bacteria can also utilize nutrients derived from mucus, a complex mixture of secreted, mucin glycoproteins that form a layer over the epithelium to protect it from microbial encroachment. The most abundant mucin utilizers are members of the genera Akkermansia and Bacteroides. Allogeneic hematopoietic cell transplantation (allo HCT) is an important treatment modality performed for a variety of benign and malignant hematological conditions. Patients receive systemic cytotoxic conditioning, followed by infusion of allogeneic hematopoietic cells. In two recent manuscripts co-authored by the Program Project Grant (PPG) Project and Core leaders, mucin-utilizing intestinal bacteria were found to contribute to intestinal graft-versus-host disease (GVHD) that is aggravated by meropenem antibiotic treatment, and neutropenic fever (NF). This is a revised PPG proposal with 3 integrated projects addressing how mucin-utilizing intestinal bacteria contribute to intestinal GVHD. Recent preliminary data from experiments performed in mice identified that Akkermansia and Bacteroides combine to compromise the colonic mucus layer, produce hypothermia in models of HCT conditioning, and aggravate intestinal GVHD. The Projects of this PPG will extend these findings, improving our understanding of how diet and metabolism modulate expression of mucin-degrading enzymes in Akkermansia and Bacteroides. Project 1 builds upon expertise in clinical allo HCT microbiome profiling and preclinical modeling to develop translational approaches. Project 2 incorporates an understanding of the natural landscape of Akkermansia genetic heterogeneity, coupled with an extensive library of functional Akkermansia mutants to deeply characterize the role of Akkermansia in intestinal GVHD. Finally, Project 3 offers a comprehensive analysis of the effects of specific dietary glycans on mucin-degrading Bacteroides and genetic underpinnings regulating expression of mucin-degrading enzymes. Supporting these are 2 unique cores. Core A will provide administrative services, microbiome- specialized biostatistical support, clinical specimen and data collection from patients undergoing allo HCT at two centers, and bionutritional support for collection of dietary data supplemented by metagenomic sequencing of fecal chloroplast DNA. Core B will perform random and targeted bacterial mutagenesis to support high throughput mutant screens as well as testing specific hypotheses, generate fluorescently labelled glycans to visualize glycan uptake and degradation at the single cell level, and build a database of mucin-degrading bacterial enzymes that wil...