ROLE OF MITOCHONDRIAL DNA REPAIR ENZYME IN DRUG RESISTANCE & DVL'T IN T GONDII

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The parasite Toxoplasma gondii is one of the most widespread and successful protozoan parasites of warm-blooded animals and can be pathogenic in humans. We have recently isolated a T. gondii mutant that exhibits strong resistance to several drugs including the anti-coccidians monensin and salinomycin, and the alkylating agent N-methyl-N-nitrosourea. We have determined that this drug resistance mutant is disrupted in a novel gene, TgMSH-1, which encodes a protein homologous to the mismatch repair enzyme, MutS. A directed knock-out of this gene in a wild-type T. gondii strain recapitulates the monensin-resistant phenotype, and complementation of the original mutant with a functional copy of TgMSH-1 restores drug sensitivity, indicating that the disruption of TgMSH-1 is directly responsible for conferring drug resistance in T. gondii. We have also shown that TgMSH-1 localizes to the mitochondrion of the parasite. Interestingly, in other organisms, MutS Homologs (MSHs) are believed to be involved in directing the cell to apoptosis and cell cycle arrest in response to certain streses, and cancer cells lacking MSHs are resistant to DNA damaging drugs. Responding to different stresses is key to the survival of an intracellular parasite such as T. gondii. This is most evident in the fact that certain stressors, such as pH changes, immunogenic response and mitochondrial inhibition, induce T. gondii to convert to an encysted form as to escape the immune system and other stress inducing conditions. Thus, it is our hypothesis that TgMSH-1 is central in mitochondrial stress response signaling in Toxoplasma and that it plays a role in parasite development and pathogenesis. Aim 1: Identify signaling partners of TgMHS1 in drug response. We have shown that T. gondii is sensitive to monensin in a MSH dependent manner. It is our goal to understand the role of TgMHS1 in this drug response process by identifying its functional partners and by studying the specific cellular effects of activation of this pathway. Specifically we will: + Identify and characterize proteins that directly interact with TgMSH-1. + Analyze expression and activation of signaling molecules involved in the mitochondrial stress pathway in wild type and TgMSH1 knock out parasite in response to drug treatment. + Study the role of apoptosis and cell cycle in the TgMSH-1 dependent response to drugs. Aim 2: Determine role of TgMSH1 in parasite development and pathogenesis. In an infected animal, T. gondii differentiates from the rapidly dividing tachyzoite to the encysted latent bradyzoite as to evade the immune response. While differentiation is key to the transmission and pathogenesis of this parasite, very little is known of the signaling mechanisms involved in triggering developmental changes. In tissue culture, T. gondii will differentiate to the bradyzoite form in response to low pH and mitochondrial inhibitors. Given the localization of TgMSH-1 and its potential role in stress signaling we will investigate the role of TgMSH-1 in bradyzoite development in tissue culture and in vivo. +Knock out TgMSH-1 in a T. gondii strain suitable for developmental and virulence studies. +Determine ability of mutant strain to convert to the bradyzoite stage in tissue culture. +Determine in vivo cyst formation, virulence and parasite distribution in mice infected with the knock-out strain.