PROJECT SUMMARY Giardia duodenalis (syn G. lamblia, G. intestinalis) is a common protozoan parasite that causes the diarrheal disease, Giardiasis. This disease is prevalent among individuals in low and middle-income countries (~200 million cases annually). While acute symptoms include diarrhea and vomiting, Giardiasis can also be subclinical. Long-term consequences of Giardiasis may include irritable bowel syndrome and growth stunting in children. Studies have suggested that growth stunting occurs due to impaired absorption of nutrients in the small intestine, and intestinal barrier dysfunction leading to nutrient malabsorption has been linked to infection with Giardia. However, the mechanisms underlying this phenomenon have yet to be well explored. While most research has focused on mechanisms whereby infection leads to barrier damage, it is essential to also understand the repair dynamics between bouts of active infection. This proposal examines how intestinal barrier repair signaling occurs after Giardia-induced barrier damage. I will use an established mouse malnutrition model to investigate possible mechanisms of barrier repair post-infection with Giardia. The overarching hypothesis of this proposal is that Giardia alters the production of metabolites by the gut microbiome leading to defective repair of the damaged intestinal barrier. Previous research has found that metabolites of the amino acid tryptophan can mediate intestinal barrier repair by activating the Aryl hydrocarbon receptor (AHR). Analysis of urinary metabolites in Giardia-infected children indicated reduced levels of tryptophan (Trp), along with other amino acids. Also, unpublished analyses of blood plasma metabolites in children and mice infected with Giardia yielded similar results, leading to my hypothesis of defective barrier repair in Giardiasis. To address this hypothesis and carry out the objectives of this proposal, I will quantify specific microbiota produced Trp metabolites in the plasma of infected mice by mass spectrometry. Additionally, I will analyze epithelial barrier damage and repair with various assays. To further understand the importance of AHR ligands, I will supplement the diets of infected mice with either Trp or Trp metabolites and examine weight gain, epithelial permeability, and repair dynamics. Specific assays include immunostaining for Ki67 to quantify proliferating IECs, qPCR to measure levels of IL-22 mRNA, and western blotting of villin and ezrin to determine protein levels and identify post-translational modifications related to barrier repair. Broad-spectrum antibiotics will be used to determine the role of the microbiota in converting Trp to metabolites that can activate AHR. Finally, I will utilize Ahr-deficient mouse models to identify the involvement of AHR more precisely in facilitating barrier repair in Giardiasis. Completing this project will significantly improve our knowledge of barrier repair in Giardia-infected individuals and help u...