The goal of this continuing collaborative project is to a assess key subcellular and molecular events that occur in the Cryptosporidium infected host cell that may provide rational targets for drug therapy. We have shown that C, parvum induces a set of profound changes in Caco-2 cell monolayers, such as cell and monolayer increase permeability, cell death, and cytoskeletal changes. Building on these results, we have 2 major specific aims and 3 minor specific aims; 1) Characterize the host-parasite interface. The feeder organelle is a unique interface between the cytoplasms of the parasite and the host. The function of the feeder organelle is unknown. It is the likely route for nutrient utilized by the para site. It is also the likely route that any therapeutic drug must take to enter the parasite. Using a variety of techniques including microinjection, selective permeabilization, and passive permeabilization to load the host cell confocal microscopy as they transit from the host cytosol into the parasite. 2) Identify the cytoskeletal components that form the electron dense bands beneath the feeder organelle, and characterize host cytoskeletal regulation induced by the parasite. Beneath the unique feeder organelle lie electron structural building blocks, the kinetic of appearance, and the regulation f actin and other key host cytoskeletal elements in relation to these changes, using IFA techniques and PCR. 3) Determine how infection alters the apical membranes of the host, as reflected in the presence of sucrase-isomaltase, an apical brush border enzyme. The parasite causes extensive remodelling of the apical cell surface. Little is known about the functional significance of this remodelling, including that of the parasitophorous membrane. We will characterize these changes using a marker for the apical membrane surface, sucrase-isomaltase. 4) Genetically alter Caco-2 cells to prevent or retard parasite induced death. No continuous in vitro culture method exists, forcing propagation of the parasite in animals. We will introduce the bcl-2 gene into Caco-2 cells in the hopes of preventing programmed cell death, and if successful characterize the differences between parental and transformed cell lines. Early cell death in vitro may be responsible for poor cell culture propagation of C. parvum. 5) Confirm observations that the oocyst has a delivery an targeting function. We have preliminary evidence that the oocyst delivers and targets the sporozoite to the epithelial cell surface, protecting it from attack in the lumen of the gut. If true this could have major ramifications ont he design of luminally active therapeutic drugs.