Project Summary Microsporidia are an early diverging group of fungal pathogens that infect a wide variety of hosts ranging from insects to humans. Encephalitozoon intestinalis is one of the main human-infecting species of microsporidia which causes diarrhea and wasting syndrome in patients and can be fatal in immunocompromised individuals. As an obligate, intracellular pathogen, E. intestinalis has evolved a highly reduced genome resulting in the loss of many regulatory and metabolic pathways, driving these parasites to rely solely on their hosts for metabolites. Upon infection, E. intestinalis replicates in a membrane bound compartment called the parasitophorous vacuole (PV) which serves as a protective barrier from the host. How E. intestinalis generates the PV membrane, acquires nutrients from the host, and develops within the PV remains elusive. E. intestinalis likely establishes a replication permissive niche and modulates host cellular processes such as vesicle trafficking, metabolite biosynthesis, and programmed cell death. Using serial block face scanning electron microscopy we have been able to gain insight into the replicative niche as well as the developmental stages of the parasite within the PV. Our data reveal that the PV nestles closely against the host mitochondria and host nucleus during infection suggesting that there are protein interactions occurring between the PV and host organelles. However, it is not known which parasite proteins localize to the PV membrane, and how these interact with host proteins or whether specific host proteins are recruited and transported to the PV compartment. Using cellular, proteomic, and structural approaches I will provide the first glimpse into the molecular and cellular features of the PV compartment and the parasites within. I will identify PV associated proteins using mass spectrometry and characterize their role in perturbing host cellular pathways and promoting parasite development (aim 1). I will also investigate the ultrastructure of the PV at high resolution including the complexes formed between the PV and host organelles and the ultrastructure of the developing parasites within the PV via cryo-electron tomography (aim 2). These results will provide insight into the mechanisms of host-parasite driven compartment formation, host cell manipulation, and parasite development.