Molecular signaling mechanisms controlling Cryptosporidium proliferation and development

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Project Abstract Cryptosporidium is a leading cause of diarrheal disease (cryptosporidiosis) and death among young children living in resource-poor settings. In the US, Cryptosporidium is the major cause of waterborne outbreaks linked to recreational water use. Currently, there is no fully effective drug and no vaccine to treat or prevent cryptosporidiosis. The only available US FDA approved drug, nitazoxanide has no proven efficacy in young children with weak immune status and immunocompromised individuals. Therefore, there is an urgent need to develop new drugs and vaccine to reduce the burden of cryptosporidiosis. Progress in anti-cryptosporidial drug and vaccine development has been hampered due to our limited understanding of parasite biology. The underlying reasons for this slow progress have been the unavailability of a robust method to continuously propagate Cryptosporidium, and the absence of molecular genetics to manipulate the parasite genome. We have overcome these hurdles by developing a powerful technology to manipulate the Cryptosporidium genome and propagate these genetically modified parasites in an immunocompromised mouse model system. The key advantage of this genetic system is that the entire life cycle of Cryptosporidium (both asexual and sexual stages) is completed in the mouse intestine, allowing us to unravel parasite biology (Vinayak et al 2015, Nature 523:477). We lack an understanding of the molecular signaling mechanisms that control development of parasite stages for successful completion of the complex life cycle. Signaling pathway components such as the plant-like calcium-dependent protein kinases (CDPKs) have emerged as attractive drug targets in Cryptosporidium and related parasites, due to the absence of their homologues in human host. Taking advantage of our genetic system, we have demonstrated the efficacy of selective bumped kinase inhibitors against calcium-dependent protein kinase-1 (CDPK1), thus indicating a critical role of this signaling kinase in C. parvum. Utilizing the conditional protein degradation system recently developed in our laboratory, we have demonstrated the essential role of CDPK1 in asexual proliferation and parasite survival. Moreover, we have compelling preliminary evidence that sheds light on the role of two signaling kinases in sexual developmental stages. The goal of this project is to elucidate the mechanistic role of these signaling proteins in regulating development of asexual and sexual stages in C. parvum required for parasite proliferation and transmission. Elucidation of these mechanisms will provide novel insights into the fundamental biology of Cryptosporidium, and open new avenues for development of effective therapies.