A new family of alpha-helical peptides regulating nitrogen metabolism and virulence in pathogenic Enterobacteria

About This Grant

Project Summary/Abstract Human intestinal organoids offer a promising new approach that aligns with the NIH Initiative's goal of prioritizing human-based research. This study will utilize this approach, along with infection and fitness mouse models, to investigate a novel family of small, hypothetical alpha-helical proteins that are highly conserved among diarrheagenic bacteria and that operate at the intersection of nitrogen metabolism and virulence. In Enteroaggregative E. coli (EAEC), the protein is tightly regulated by a master virulence regulator, suggesting an essential role in pathogenesis. EAEC damages the epithelial barrier and disrupts tight junctions in intestinal cells, leading to increased permeability and mucosal dysfunction. EAEC can also persist asymptomatically in children’s intestines, causing growth retardation and decreased physical and cognitive development, suggesting that EAEC has evolved pathogenic strategies to adapt and persist within the host. We discovered a new family of alpha-helical oligopeptides that operate upstream of central nitrogen metabolism, positively regulating hundreds of genes, including global regulators of nitrogen metabolism, ammonia transporters, operons for amino acid assimilation, and the activator AggR. Most importantly, blast analysis revealed homologs in Salmonella, Shigella, and pathogenic E. coli species. Here, we aim to further elucidate the regulatory mechanisms of these alpha-helical proteins in nitrogen metabolism and their relevance to bacterial pathogenesis. Our overall hypothesis is that these proteins play a crucial role in regulating nitrogen metabolism and virulence in EAEC, supporting bacterial adaptation and persistence in the host's intestine, and providing the energy needed to enhance virulence and cause disease. To test our hypothesis, in AIM-1, we will employ biochemical, genetic, and molecular approaches to gain mechanistic insights into the regulatory role of alpha-helix polypeptides in nitrogen metabolism and virulence in EAEC. In AIM-2, we will evaluate the impact of these proteins on EAEC fitness and pathogenesis using human intestinal organoids and malnourished mouse models of EAEC infection. Our proposed studies will uncover new regulatory mechanisms that control metabolism and virulence in pathogenic bacteria, potentially leading to new therapeutic opportunities.