Defining Oocyst Wall Biogenesis and Function in Cryptosporidium Transmission

Cryptosporidiosis is the second leading cause of severe diarrhoeal disease in young children (estimated 79,000 years of life lost). There is no vaccine and the only approved drug is not effective for young children or immunocompromised patients. Cryptosporidium parasites are transmitted as a spore-like “oocyst” and due to their resistance to chemical disinfection (chlorination) are often water borne. There are no genetic markers or reagents available to study Cryptosporidium transmission. I propose to analyse Cryptosporidium transmission by defining the molecular mechanisms of oocyst formation using a mutagenic toolkit I developed. - First, we need to identify the components of the oocyst wall. We will use Mass Spectrometry to create the first comprehensive list of oocyst wall components and will use conditional mutagenesis to analyse their function in oocyst formation and transmission. - Second, it is critical to understand how oocyst-related genes, including Cryptosporidium Oocyst Wall Proteins (COWPs) impact transmission. We will create reporter strains to confirm expression and evaluate their function in transmission. Biochemical analysis of COWPs will reveal how their inter/intramolecular interactions contribute to oocyst structure. - Finally, regulation of the process of oocyst development is completely unknown. We will develop high-content imaging assays to screen for pharmacological inhibitors of oocyst formation.