Cryptosporidium Parvum Exports Proteins To The Epithelial Host Cell

Jennifer Elizabeth Dumaine, University of Pennsylvania

Abstract

The parasite Cryptosporidium is a global cause of diarrheal disease resulting in death, malnutrition, and stunted growth in young children and animals. There is no vaccine and treatment options are limited within these patient populations, underscoring the need for the development of novel therapeutics. Cryptosporidium infects epithelial cells in a specialized intracellular, but extracytoplasmic niche. Following the establishment of infection, cells exhibit profound changes in morphology, physiology, and transcriptional activity. While it is unknown how the parasite induces these changes, host-targeted parasite effectors have been hypothesized to be driving factors, though none have been identified. We utilized genetic approaches to visualize the localization of highly polymorphic proteins and found members of the C. parvum MEDLE protein family exported to the cytosol of infected cells. The MEDLE2 protein is constitutively translocated across the lifecycle of the parasite by an export machine installed following the establishment of infection. Here we use genetic approaches to map the cellular and molecular requirements for export and define a pathway that directs proteins to the host cell across all intracellular life stages of Cryptosporidium. Proper host targeting of MEDLE2 required an intrinsically disordered structure and an N terminal signal peptide and export sequence. Introduction of ordered domains or targeted mutagenesis of these export sequences prevented targeting to the host cell. Direct expression of MEDLE2 in mammalian cells was sufficient to trigger an ER stress response, which was similarly observed during infection. Finally, MEDLE2 was also used to deliver model antigens to the host cell to drive the expansion of model antigen specific T cells, thus establishing new tools to interrogate host-pathogen interactions. Taken together, our studies reveal a Cryptosporidium secretion system capable of delivering effector proteins into the infected enterocyte. The discovery of parasite effectors that influence the host cell generates novel tools that may be utilized to generate a more complete understanding of the host-parasite interactions to inform the development of future prophylactic treatments.