Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. Toxoplasma infection is a serious threat to immunocompromised individuals such as AIDS patients and organ transplant recipients. Side effects associated with current drug treatment calls for identification of new drug targets. DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen species produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host immune response. Most of the oxidative DNA damage is repaired through the base excision repair (BER) pathway, of which, the apurinic /apyrimidinic (AP) endonucleases are the rate limiting enzymes. Toxoplasma possesses two different AP endonucleases. The first, TgAPE, is a magnesium-dependent homologue of the human APE1 (hAPE1), but considerably divergent from hAPE1. The second, TgAPN, is a magnesium-independent homologue of yeast (Saccharomyces cerevisiae) APN1 and is not present in mammals. We have expressed and purified recombinant versions of TgAPE and TgAPN in E. coli and shown AP endonuclease activity. Our data shows that TgAPN is the more abundant AP endonuclease and confers protection against a DNA damaging agent when over-expressed in Toxoplasma tachyzoites. We also generated TgAPN knockdown Toxoplasma tachyzoites to establish that TgAPN is important for parasite protection against DNA damage. We have also identified pharmacological inhibitors of TgAPN in a high-throughput screen. The lead compound inhibits Toxoplasma replication at concentrations that do not have overt toxicity to the host cells. The importance of TgAPN in parasite physiology and the fact that humans lack APN1 makes TgAPN a promising candidate for drug development to treat toxoplasmosis.
|Advisor:||JR., WILLIAM J. SULLIVAN|
|Commitee:||CHOU, KAI-MING, GEORGIADIS, MILLIE M., QUEENER, SHERRY F., VASKO, MICHAEL R.|
|Department:||Pharmacology & Toxicology|
|School Location:||United States -- Indiana|
|Source:||DAI-B 75/05(E), Dissertation Abstracts International|
|Subjects:||Toxicology, Surgery, Pharmacology, Parasitology|
|Keywords:||Apicomplexan parasite, Apurinic/apyrimidinic endonucleases, Base excision repair, Toxoplasma gondii|
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