Epithelial-intrinsic restriction of Cryptosporidium

ABSTRACT Cryptosporidium infections are a leading cause of diarrheal associated death in children under the age of five. There is no vaccine or universally effective treatment for Cryptosporidium, highlighting the need for intensive study of the host immune response to infection. Cryptosporidium invades the host small intestine and establishes an intracellular vacuole on the apical surface of intestinal epithelial cells. The epithelial cell serves as both a parasite niche and a host sentinel, where the parasite is sensed by the host and immune pathways are activated to control the infection. Despite decades of study, however, the pathways and effectors that promote parasite restriction in the epithelial cell and their mechanisms of action remain elusive. Intestinal epithelial cell death and autophagy are two conserved mechanisms of host defense against enteric pathogens and recent evidence suggests that both might be important for cell-intrinsic control of Cryptosporidium. Taking advantage of advances in intestinal epithelial organoid culture, I have shown that organoid monolayers mutant for autophagy and cell death experience enhanced Cryptosporidium growth, implicating both processes in epithelial-intrinsic parasite restriction. Based on these preliminary findings and our knowledge of autophagy and cell death in defense against other enteric pathogens, I hypothesize that autophagy mediates parasite restriction by degrading the parasite vacuole and that cell death limits parasite growth by eliminating the parasite’s intracellular replicative niche. I will test these hypotheses using epithelial organoid monolayers as a model in vitro system. I will characterize the pathways that trigger both autophagy and cell death during Cryptosporidium infection and I will employ fluorescent transgenic parasites and immunofluorescence microscopy to understand how these processes impinge upon parasite growth and survival. Furthermore, I will use microscopy and flow cytometry to test whether Cryptosporidium can inhibit cell death in organoids. These studies will be the first to identify and dissect the mechanisms employed by intestinal epithelial cells to counteract Cryptosporidium intracellular survival and growth, lending crucial insights into host-pathogen conflict, conserved mechanisms of host defense, and the regulation of immune pathways relevant not only to Cryptosporidium but to other enteric pathogens and inflammatory diseases of the intestine.