Cracking the Cryptosporidium Oocyst Wall Proteins

The diarrhoeal parasite Cryptosporidium is a major cause of childhood mortality, responsible for 48,000 child deaths in 2016 alone. Cryptosporidium is transmitted by the highly durable oocysts, a structure capable of allowing parasites to survive months within the environment, providing protection against commonly used water-soluble detergents and chlorination techniques. A family of nine cysteine rich proteins known as the Cryptosporidium Oocyst Wall Proteins (COWPs, termed COWP1 to 9) are hypothesised fundamental inner oocyst wall proteins, thought to contribute to oocyst durability. However due to a lack of molecular tools to study Cryptosporidium, currently only one (COWP1) has been validated an oocyst wall protein. I hypothesised that COWPs are fundamental oocyst wall proteins, essential for oocyst formation and parasite transmission. Using CRISPR/Cas9, I fluorescently tagged five uncharacterised COWPs of Cryptosporidium parvum, confirming their localisation to the oocyst wall, either even expression throughout the structure or to a unique localisation to a band-like structure, hypothesised to be the oocyst suture. Expression of COWPs was identified exclusively in female parasites known as macrogamonts, the precursors of the oocyst. To test the essentiality of the COWPs, CRISPR/Cas9 knockouts were attempted, where I successfully generated the first documented oocyst wall protein knockout. The morphology, durability and environmental resistance of the generated COWP KO strain was investigated. This work confirmed that COWPs are indeed oocyst wall proteins, and produced fluorescent reporter strains which can be utilised in the future to help better understand the triggers of oocyst formation post-fertilisation. Furthermore, preliminary data suggests that one COWP may be the first ever identified oocyst suture marker, which if confirmed, will open possibilities to study how this structure is formed, as well as the changes which occur at the structure during parasite excystation.