Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
Microbiology, Molecular Biology and Biochemistry
Non Technical Summary
The Apicomplexa comprise a medically important phylum of obligate intracellular parasites, including Plasmodium falciparum, the causative agent of malaria, Cryptosporidium, which causes gastrointestinal disease, and the category B agent Toxoplasma gondii, which can be pathogenic in immunocompromised hosts and the developing fetus. Drug therapies that effectively treat all stages of some of these parasites are currently lacking and in many cases existing drugs are quickly becoming ineffective due to the emergence of drug-resistant parasite strains. Thus, the discovery of new targets for drug design and the understanding of resistance mechanisms are high priorities in the studies of apicomplexan parasites. Our approach to this challenge has been to isolate and characterize T. gondii mutants that are resistant to specific antiparasitic drugs. We have recently discovered that disruption of a mitochondrial MutS homologue (MSH), TgMSH-1, directly confers multi-drug resistance in T. gondii. MSHs are critical components of the eukaryotic DNA mismatch repair machinery and are also involved in signaling cell cycle arrest and apoptosis in response to DNA damaging agents. Interestingly, we have observed that certain anti-parasitic drugs cause disruption in the expression of cell cycle markers in a TgMSH1 dependent manner. Thus, we have identified a novel pathway in T. gondii, that when induced by certain drugs leads to parasite death. It is our hypothesis that that certain drugs affect the mitochondrion of the parasite directly or indirectly and that this effect results in the activation of a signaling pathway, which includes TgMSH1 and results in parasite death. It is our goal to dissect this TgMSH1 dependent death mechanism as to characterize a novel mode of killing apicomplexan parasites. Using a combination of cell biology, biochemistry and genomic approaches we will determine the effect of TgMSH1-dependent drugs on mitochondrial function, identify signaling partners of TgMHS1 and determine the effect of TgMSH1 dependent drugs on cell cycle. The completion of these aims will elucidate the details and mechanisms of an inducible death pathway. This constitutes an innovative approach to the discovery of drug targets in this important pathogen.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
We have recently discovered that disruption of TgMSH-1, a MutS homolog (MSH) in the human and animal pathogen Toxoplasma gondii, confers resistance to various anti-parasitic drugs including monensin, salinomycin and myxothiazol. MSHs are critical components of the eukaryotic DNA mismatch repair machinery and can signal cell cycle arrest and apoptosis in response to DNA damaging agents. Accordingly, mammalian cells lacking certain MSHs are resistant to chemotherapeutic drugs, since the signaling of cell cycle arrest is disrupted. Similarly, we have observed that monensin causes gene expression changes and disruption of the cell cycle in T. gondii in a TgMHS1-dependent manner. Thus, by studying the mode of action of monensin, we have identified a novel protein in T. gondii that plays a role in an inducible death mechanism. Accordingly, we will investigate how induction of the TgMSH-1 dependent pathway leads to parasite death. Objective 1: Determine the effect of TgMSH-1-dependent drugs on mitochondrial function. Our analysis of a multi-drug resistant T. gondii mutant has shown that a mitochondrial TgMSH-1 is involved in drug-induced death. Nonetheless, we do not know the effects on the mitochondrion that leads to TgMSH-1-dependent death (question A in fig. 7). To answer this, we will use physiological and genetic analysis of the mitochondrion in the parental and resistant strains with and without drug treatment. Objective 2: Identify functional partners and domains of TgMHS1. We have shown that T. gondii dies after exposure to several drugs in a TgMSH-1 dependent manner. To understand the events involved in the TgMHS1-dependent death (question B in fig. 7), we will identify and characterize proteins that directly interact with TgMSH-1. In addition we will look at the role of different domains of TgMSH-1 in drug sensitivity. Objective 3: Determine the cellular effect of the TgMSH-1 response. To understand how TgMSH-1 is involved in drug sensitivity we need to determine the mechanisms behind TgMSH-1-dependent death (question C in fig.7). Preliminary analysis of gene expression and DNA content in monensin-treated parasites suggests that the drug is affecting the cell cycle and that this effect depends on TgMSH-1. Thus, we will focus on determine the role of cell cycle arrest in TgMSH-1-dependent death.
Project Methods
Objective 1: Determine the effect of TgMSH-1 dependent drugs on mitochondrial function: We have shown that monensin action depends on the expression of a protein localized to the mitochondrion. Thus, it is our hypothesis that monensin, directly or indirectly affects mitochondrial function, which in turn results in TgMSH-1 dependent death. To test this hypothesis, we will study the effect of monensin on mitochondrial function and integrity. Specifically, we will assay mitochondrial membrane potential using the reporter dye Safronine O, basal respiration using an oxygraph, oxidative stress using the reagent MitoSOX Red Mitochondrial Superoxide Indicator and DNA damage using mutations in the Cytochrome B gene to monitor mitochondrial DNA fidelity. Objective 2: Identify signaling partners and functional domains of TgMHS1: We have successfully epitope-tagged the endogenous TgMSH-1 gene. We have confirmed that the HA-tagged TgMSH-1 can be detected by Western analysis of whole parasite extract and it can also be purified from the extract using immuno-affinity precipitation. Using HA antibodies bound to agarose beads we will immunoprecipitate TgMSH-1 along with interacting proteins, which will be detected by PAGE-electrophoresis. Preliminary assays have identified 3 polypeptides that co-immunoprecipitate with the HA-tagged TgMSH-1 and are not seen in control immunoprecipitation with beads alone or from parasites not expressing HA tagged proteins (data not shown). We are currently performing experiments to identify those proteins, which consist of analyzing bands from the SDS-PAGE gel using liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-MS/MS). The peptides identified through LC-MS/MS will be used to determine the identity of the interacting proteins. Objective 3: Determine the cellular effect of the TgMSH-1 response. It is our goal to determine the molecular mechanism underlying the TgMSH-1-dependent death. Studies in human cell line show that certain drugs induce a G2 cell cycle arrest in an MMR-dependent fashion. Interestingly, our preliminary data shows that transcript levels of histone genes are increased during monensin treatment, a phenomenon common during cell cycle arrest. Thus, we will focus our first efforts on the effects of TgMSH-1 dependent drugs on the cell cycle of T. gondii. The cell cycle of T. gondii has been well defined using several approaches including the analysis of DNA content by flow cytometry. We will perform a similar analysis of the cell cycle in monensin-treated and untreated parasites. For this purpose, parasites are stained with the DNA label SYTOX green and the DNA content is measured using FACS sorting. The cell cycle effects observed by flow cytometry will be validated by monitoring expression of genes known to be cell cycle dependent such as TgPCNA 1 and 2, β-tubulin and Histone 2A mRNAs using qtPCR. Furthermore, we will perform a time course of gene expression changes in parasites treated with monensin, to identify other genetic events involved in TgMSH-1 dependent death.