This proposal will fundamentally increase our understanding of the process by which apicomplexan parasites egress from host cells. Toxoplasma gondii is a nearly ubiquitous parasite that can infect any warm-blooded animal cell and whose prevalence in humans exceeds 30%. The most serious clinical consequences of toxoplasmosis occur during pregnancy, causing cranial malformation, seizures and ocular damage in congenitally infected neonates. Reactivation of dormant tissue cysts in immune-compromised individuals is also of growing clinical significance. Understanding the fundamental processes of host cell destruction and parasite egress will provide a rational foundation for new antiparasitic therapies. The benefits of this work for human health are further enhanced by the biological proximity of T. gondii to other burdensome apicomplexan parasites, including the malaria-causing Plasmodium species and the important waterborne parasite Cryptosporidium. The work proposed here uses diverse approaches to focus on those proteins that are involved in the Ca2+-sensitive events leading up to parasite egress from host cells. First, established cell biological and genetic techniques and a novel temperature sensitive egress mutant will be used to assign a functional role to a protein, TgDOC2.1, whose essentiality for T. gondii egress has already been demonstrated. The search for additional egress proteins is also critical for continuing to expand our understanding of parasite biology and for seeding the rational design of new anti-parasitic therapies. [--The second aim seeks to identify new components of one or multiple egress pathways in an unbiased, forward genetic fashion. Forward and genetic screening has been used very successfully in T. gondii, principally through random chemical mutagenesis.
Aim two postulates that in contrast to chemically-induced mutants screened for temperature sensitive phenotypes, inducible overexpression would make feasible the identification of distinct classes of proteins for which the generation of temperature sensitive alleles are not possible. This will be accomplished through the production of a novel inducible cDNA library. Single copy integration of ORFs into the T. gondii genome will facilitate screening and enrichment for over-expression egress mutants. Sequencing- based techniques will then be used to identify the gene contained within that clone-a gene encoding a new and critical element of parasite egress.--] In total, the proposed experiments will provide new insights into the control of a fundamental parasitic process. Using a diversity of approaches, characterizing a known component and seeking to identify new ones maximizes the potential for identification of unique therapeutic targets, guiding future drug discovery and leading to more efficient control of these medically important pathogens.
Toxoplasma gondii is a parasite infecting nearly 1 in 3 people across the globe. The symptoms of acute toxoplasmosis stem largely from repeated cycles of host cell invasion and egress that destroy host tissues and lead to inflammation and scarring. By improving our understanding the molecular basis of parasite egress from host cells, as this proposal seeks to do, we can better design therapeutic approaches targeted towards disrupting this essential process.