Cryptosporidium parvum is a well-recognized cause of diarrhea in humans and animals throughout the world, and is associated with a substantial degree of morbidity and mortality in patients with the acquired immunodeficiency syndrome (AIDS). At the present time, there is no effective therapy for treating or preventing infection with C. parvum. This is primarily due to a lack of understanding of the basic cellular and molecular biology of this pathogen. All protozoan parasites studied to date are dependent on purine salvage to replenish the purine nucleotides pools, and thus purine salvage pathways have been considered key targets for antiparasitic chemotherapy. In a recent study the C. parvum inosine monophosphate dehydrogenase (cplMPDH) was identified as a suppressor of mycophenolic acid sensitivity in a Toxoplasma gondii strain defective for HXGPRT, a key gene in the purine salvage pathway of T. gondii. Surprisingly, cpIMPDH was more similar to bacterial than that of eukaryotic IMPDHs. The high degree of relatedness of cpIMPDH with bacterial versions of this enzyme as compared to mammalian IMPDH suggests that this would be an excellent target for the development of new anti-cryptosporidial therapies. Importantly, there are significant differences in both kinetic parameters and inhibitor sensitivity between mammalian and microbial IMPDHs, indicating that species-specific inhibitors could be designed for use as anti-microbial therapy. The central hypothesis of this proposal is that C. parvum is absolutely dependent on a purine salvage pathway for survival, and IMPDH and adenosine transporter are essential enzymes in this biochemical pathway. To address this hypothesis the following specific aims will be addressed:
specific aim 1 : Heterologous expression of C. parvum and mammalian IMPDH enzymes in yeast;
specific aim 2 : Heterologous expression of C. parvum and mammalian adenosine transporter (AT) enzymes in yeast;
specific aim 3 : Screening of chemical libraries to identify specific inhibitors of cplMPDH and cpAT. Importantly our preliminary data support the hypothesis that that C. parvum is absolutely dependent on a purine salvage pathway for survival and that IMPDH and adenosine transporter are essential enzymes in this biochemical pathway. In addition, these studies demonstrate our ability to use heterologous expression of C. parvum genes in yeast as a means to develop a high-throughput screening assay for the identification of specific inhibitors.
