Project Summary The parasite Toxoplasma gondii can cause severe disease in immunocompromised patients and fetuses and is the second leading cause of foodborne deaths in the USA. Infection starts after ingestion of oocysts shed in cat feces or after ingestion of meat contaminated with tissue cysts. Oocysts are extremely stable in the environment, resistant to the most inactivation procedures, and highly infectious. Because livestock get infected by sporozoites derived from oocysts, a vaccine based on sporozoites or sporozoite proteins could be effective in protecting livestock and humans. Currently, the only vaccine available is a live attenuated vaccine only approved for use in sheep. However, this vaccine has serious shortcomings as the genetic basis for the attenuation is not known, which poses reversion risks, and it needs to be used immediately once produced because it is based on live tachyzoites, which are not viable for long outside host cells. Despite being a critical step for Toxoplasma transmission, sporozoites inside oocysts are under-studied, largely because they are not cultivatable in vitro and difficult to access in vivo. There is, therefore, a critical need to identify sporozoite gene products that are critical for its invasion of host cells, which could lead to the discovery of vaccine targets. Furthermore, if the genetic basis for the extreme environmental resistance of sporozoites was known it might be possible to exploit this to make other Toxoplasma life stages, such as tachyzoites, or other pathogens more viable extracellularly, which could enhance the shelf-life of vaccines based on live parasites. Our overall objectives are to identify Toxoplasma genes that are specifically involved in the environmental resistance and infectivity of sporozoites. Our central hypotheses are that 1) sporozoite-specific micronemal proteins (MICs) are critical for their invasion into host cells; 2) late embryogenesis abundant proteins (LEAs), which are known to provide resistance to environmental stresses such as drought, high salinity, and freezing in a variety of organisms, determine sporozoite resistance to environmental stressors. In our first aim we will determine the role of LEAs in the protection of sporozoites against environmental stressors. In our second aim we will test the hypothesis that MICs that are specifically expressed in sporozoites are involved in the invasion of host cells. The identification of sporozoite genes that play a role in extracellular survival and attachment/invasion of host cells, will pave the way for designing vaccines that can prevent Toxoplasma infection of humans and animals.