The obligate intracellular parasite Toxoplasma gondii causes ocular disease in 70-90% of congenital toxo- plasmosis patients. Additionally, ocular toxoplasmosis (OT) characterized by toxoplasmic retinochoroiditis can also follow acute infection and is the most common cause of infectious uveitis in immunocompetent and im- munocompromised individuals. Disease is generally self-limiting; however, recurrent lesions are frequent. Res- olution of active lesions leads to tissue scarring, with loss of anatomical and functional integrity of the retina that can lead to permanent visual loss. There is no consensus regarding treatment of OT. Therapeutic regi- mens usually include antibiotics like pyrimethamine and sulfamethazone, combined or not with systemic corti- costeroids. Our long-term goal is to help identify novel T.gondii proteins that could be targeted with novel drugs. These are urgently needed since current drugs have toxic side effects, especially during prolonged treatment which is necessary during recurrent OT, and they do not clear the chronic cyst stages. To cause OT it is likely that T.gondii needs to disseminate to the eye and breach the blood-retinal barri- er. Parasite-mediated cell lysis can cause tissue damage; however, exacerbated inflammation is also an im- portant cause of tissue destruction. The overall objectives in this application are to determine how T.gondii reaches the eye, breaches the blood-retinal barrier and subsequently promotes development of ocular inflam- mation. T.gondii secretes effector proteins into the host cytoplasm to co-opt host cells. T.gondii-infected den- dritic cells (DCs) display hypermotility and infected DCs have enhanced transmigration across retinal epithelial cells. The central hypothesis is that T.gondii co-opts immune cells, such as DCs, as Trojan Horses to dissemi- nate to the eye and to transmigrate across the blood-retinal-barrier. Using a T.gondii CRISPR/Cas9 loss-of- function screen in mice, our lab has identified multiple T.gondii candidate genes that appear to affect its ability to colonize the eye without affecting replication efficiency. In the first aim it will be confirmed if knocking out T.gondii candidate genes that appear involved in colonization of the eye based on the CRISPR screen have a defect in colonization of the eye of infected mice. In vivo parasite replication, dissemination, colonization of the eye, and ocular inflammation, will be compared between knockout and wild-type parasites using multiple in vi- vo whole body and ocular imaging techniques on live mice over time. In the second aim an in vitro CRISPR/ Cas9 loss-of-function screen will be used to identify T.gondii genes that mediate infected leukocyte migration across polarized human retinal epithelial cells. For parasite knockouts strains generated in Aim 1, it will also be tested if they have a defect in using DCs to cross polarized retinal epithelial cells. The proposed research is significant because identification of novel T.gondii genes that determine colonization of the eye and ocular dis- ease has the potential to help with the rational design of novel therapies to treat ocular toxoplasmosis.
Ocular toxoplasmosis is the most common etiology of intraocular inflammation (uveitis) and is the most frequent sequel of congenital toxoplasmosis. To colonize the eye, Toxoplasma needs to disseminate to the eye from the site of infection and breach the blood-retinal barrier, yet the Toxoplasma proteins involved in these processes are unknown. Identifying Toxoplasma genes that determine the ability of these parasites to colonize the eye might lead to the development of new drugs for prevention of human ocular toxoplasmosis or better treatment options for recurrent ocular toxoplasmosis.
