The protozoan parasite Toxoplasma gondii is an important human and veterinary pathogen. In humans, acute disease is caused by rapidly growing `tachyzoites' stage. In this context, we hypothesize that during this intense tachyzoite propagation, replication-associated DNA stress could appear to be generating fork collapse and DNA double strand breaks which should be repaired by homologous recombination repair (HRR). The hypothesis is supported by the presence of basal ?H2A.X, a marker of DSB, in the tachyzoite as well as the inhibition of its replication by the KU55933, an inhibitor of ATM, a key kinase in the HRR pathway. In addition, our analysis indicates that key molecules seem to be not present in HRR pathway in T. gondii. Differences in the HRR cascade imply an opportunity for the identification of novel and specific HRR components in T. gondii. If available, such factors could represent attractive candidates for the development of new drugs against Toxoplasmosis. Our goal is to identify novel components of HRR and analyze their importance in HRR pathway.
In specific aim 1 we will develop a methodology named iPOND to pull down components of HRR in T. gondii and their identification and analysis by bioinformatics tools. In addition, we will analyze their biological significance in tachyzoite replication and DSB sensitivity, together with some HRR components yet identified by in silico analysis (Mre11, RAD51, ATM, BRCA2, BRCT domain containing proteins).
In specific aim 2 we will design a model of HR in vivo by generating tachyzoites conditionally expressing the endonuclease I-SceI to generate DSB specifically in a plasmid incorporated by transfection. This plasmid contains two versions of GFP inactive genes in which the functional gene is expressed only after HR.
Toxoplasma gondii has achieved notoriety as a cause of life-threatening opportunistic disease in immunocompromised individuals. Recently, chronic infection was associated to schizophrenia and other neurological disorders. In addition, with the growing threat of bioterrorism this parasite is now listed as a Category B pathogen in the NIH/CDC list of priority pathogens. The proposal will produce a significant impact in elucidating the role and importance of homologous recombination repair (HRR) components in the DNA replication and DNA repair in T. gondii parasites with a great potential of identifying novel drug targets against Toxoplasma infection.
