Influence of microbial metabolites on mitosome function in Cryptosporidium - PROJECT SUMMARY Cryptosporidiosis is a common cause of severe, chronic diarrheal disease in immunocompromised patients and infants from resource poor settings where infections are a major cause of morbidity and mortality. The only FDA approved drug for treatment of cryptosporidiosis, nitazoxanide, has limited effectiveness in those most at risk including immunocompromised patients and infants. As an enteric pathogen, Cryptosporidium grows at the apex of the gut epithelium proximal to the microbiota and metabolites produced at this interface influence infection. In previous studies, we defined three classes of microbial metabolites that inhibit C. parvum growth: secondary bile salts, indoles, and the vitamin B6 precursor pyridoxal. Indoles inhibit oxidative phosphorylation in the host mitochondria and depolarize the remnant C. parvum mitochondrion called the mitosome. The mitosome lacks most mitochondrial functions but retains an alternative electron transport chain as well as pathways for iron- sulfur cluster and ubiquinone biosynthesis. Very little is known about the physiology of this organelle, although the inhibitory actions of indole suggest it performs essential functions. In the proposed studies, we will elucidate the role of an ADP/ATP carrier in generating the membrane potential of the mitosome and characterize the action of indole in inhibiting this pathway. In addition, we will determine the function of a putative pyridoxal phosphate transporter that may bypass salvage pathways for vitamin B6 that are absent in C. parvum. Finally, we will explore the iron sulfur cluster biosynthesis pathway, which is normally dependent on vitamin B6, by testing the essentiality of key enzymes in this pathway. To facilitate future studies, we will define import and functional pathways in the mitosome using permissive biotin ligase coupled to different organellar components. Collectively, these studies will define essential functions in the mitosome and identify potential targets that may lead to alternative treatments for cryptosporidiosis.
