Optical Metabolic Imaging (OMI) of Toxoplasma gondii and Cryptosporidium parvum in a 3D system

Abstract

Both T. gondii and C. parvum are intracellular, apicomplexa parasites auxotrophic for specific metabolites. Cat intestine is the definitive host of T. gondii, and cats can produce infectious oocysts, which infect humans primarily through fecal‐oral transmission. This infection can cause fatal encephalitis in immunocompromised humans. C. parvum is another intestinal parasite with a wider range of hosts who can excrete infectious oocysts in feces. These oocyts can cause fatal diarrheal illness in cows and humans, and C. parvum infection has been linked to colon cancer in humans. How T. gondii and C. parvum change host cell metabolism has not been well‐characterized to date. Currently, there are limited treatment options available for C. parvum, and the organism has been difficult thus far to culture and propagate in vitro.For this reason, mouse intestinal organoid models have been developed to simulate the conditions a parasite would encounter in vivo. It is hypothesized that the model for studying T. gondii in vitro can be easily transferred to C. parvum, given their similarities. Using mCherry fluorescent protein labelled T. gondii and cat intestinal organoids, we have modeled intestinal infection with T. gondii. The relative ease of molecular manipulations of T. gondii makes it the ideal organism with which to build a model for optical metabolic imaging (OMI) of C. parvum. OMI of mCherry‐labelled T. gondii in 2D human fibroblasts has been completed with inhibitors such as Mycocaloide B and Cytochalasin D, and the results showed changes in NADH and FAD metabolites in vitro, showing that infection with intracellular parasites impacts metabolism of host cells.T. gondii will be imaged in vitro with additional metabolic inhibitors, including atovaquone and sodium fluoroacetate. Then, OMI will be done of T. gondii in cat intestinal organoids, a 3D system, because the materials are already available and serve as a way to test the model prior to using it on C. parvum. It is anticipated that the previously obtained results will be seen again in the 3D system. This project will label C. parvum with mCherry fluorescent protein, propagate this microorganism in a 3D system and ultimately perform OMI. Thus, the model that has been successfully developed with T. gondii will be improved, then used to understand how infection with Cryptosporidium parasite influences host metabolism, particularly in organoid models. Ultimately, this better understanding will assist in the development of therapeutics for C. parvum, an area which is currently lacking.