Investigating the function of Cryptosporidium transport proteins

Cryptosporidiosis is a condition caused by an infection with the parasite Cryptosporidium causing more than a million deaths per year. Cryptosporidium enters a host by ingestion, infecting the lining of the small intestines. Common symptoms include watery diarrhoea along with abdominal pain, leading to dehydration and weight loss, for most people their symptoms are self-limiting, however they can be life threatening to those with a weakened immune system. There are no current treatments that target the parasite itself, only the symptoms. During the project, we looked at Cryptosporidium membrane proteins to see if any could be used as a new therapeutic drug target. However, we were unable to overexpresses these proteins. Instead, a wellknown membrane protein was characterised to help develop the method that would have been used to characterise ligand binding for Cryptosporidium proteins. VcINDY belongs to a family of DASS (Divalent Anion Sodium Symporter) transporters and is known to transport TCA cycle intermediates. Using the binding assay Differential Scanning Fluorimetry (DSF), it was shown that VcINDY binds to succinate, fumarate, malate and alpha-ketoglutarate. Different alanine mutants were also tested to better understand which residues were important for protein-ligand binding. It was found that residues I96 and F100 are not involved in ligand binding, whereas N151 and T152 were very involved in ligand binding.