Project Summary Babesiosis is an emerging zoonotic disease in the United States, that occurs when a patient is infected with a Babesia parasite by transmission from ticks, and is a common co-infection of Lyme disease. The disease can result from infection with various Babesia spp., resulting in everything from an asymptomatic infection to severe and life-threatening disease. Current treatment options for babesiosis either have severe side effects or a high risk of treatment failure due to the parasite developing drug resistance. The biology of Babesia parasites remains poorly studied, in part due to a lack of genetic and biological tools available. Here, we propose to establish a new approach to rapidly and efficiently identify essential targets of small molecule inhibitors in multiple Babesia spp. We will interrogate two Babesia spp. that can be cultured in vitro. We will select for Babesia divergens and Babesia bovis parasite lines that are resistant to high priority small molecule inhibitors, using chemical mutagenesis to accelerate the process and increase the likelihood of developing resistance. In order to validate and study the molecular targets of these inhibitors, we will generate the required genetic and biological tools to study essential proteins in B. divergens, and demonstrate their efficacy through the genetic validation of PhoDapi, the putative target identified by our laboratory using resistance evolution against MMV019266.