Effects of pyrrolidine dithiocarbanate on the expression of potential cell-cycle regulators of Toxoplasma gondii

1. Effects of pyrrolidine dithiocarbanate on the expression of potential cell-cycle regulators of Toxoplasma gondii Brittney K. Fey, Ashley L. Johnson, Jessica A. Marchetti and Douglas B. WoodmanseeDepartment of Biology, Wilmington College, Wilmington, OH 45177 Abstract Results Results Methods Quantitation of Tg14-3-3σ mRNA by Multiplex RT-PCR. Toxoplasma gondii is a pathogenic intracellualr parasite belonging to the protistan Phylum Apicomplexa. The details of the cell-cycle control mechanisms of this parasite are only beginning to be studied. The compound Pyrrolidine dithiocarbamate (PDTC) can induce a reversible cell-cycle arrest in T. gondii and PDTC-induced regulation of transcription from a gene suggests the gene’s involvement in cell cycle regulation. Human foreskin fibroblasts growing in 25cm2 cell culture flasks were infected with T. gondii at a 1:1 parasite:host cell ratio. Infected cells were exposed to 50μMPDTC for 12 hours. Infected cells not exposed to PDTC were used as controls. Total RNA was isolated from both types of cultures using commercially available kits. Transcription of 3 genes encoding potential cell cycle regulators were examined by reverse transcriptase-polymerase chain reactions using commercially available reagent kits and primer sets developed by us using sequence data obtained from the T. gondii genome sequencing project (www.toxodb.org). The genes examined were a 14-3-3σ homologue, a putative cyclin gene (TgCYC2) and a putative homologue of human ménage-a-tois protein (TgMAT1). mRNAs from all 3 genes were detected both in PDTC-treated cultures and controls suggesting that the drug does not completely block transcription of any of the genes. In progress are experiments to test if PDTC induces quantitative changes in transcription of the genes. PDTC treatment arrested the T. gondii cell cycle. •  Parasites and culture conditions RH strain T. gondii was obtained from the American Type Culture Collection. Parasites were maintained by routine passage on feeder layers of human foreskin fibroblasts (HFF) grown in serum- and antibiotic-supplemented Minimal Essential Medium. The concentration of fetal bovine serum in the cell culture medium was reduced from 10% to 1% at the time of infection. •  Treatment of parasites with PDTC HFF cells were grown to confluence in 100 mm cell culture plates to which sterile coverslips had been added. The number of cells on the plate was estimated and an equal number of T. gondii tachyzoites were added to the plate. Parasites were allowed 1 hr to penetrate host cells and then the plates were washed with 3 changes of medium. Parasite cultures were allowed to grow for 24 hr, after which the medium was replaced with new medium containing 50 μM PDTC. Total RNA was isolated from the infected cultures 12 hr after the medium change using a commercial kit 3. Control cultures consisted of infected cultures growing in medium without PDTC. One control culture was harvested at the time the PDTC was added to the experimental plate (24 hr PI) and a second control culture was harvested at the same time as the experimental plate (36 hr PI). The coverslips in the plates were stained with a hematoxylin stain and examined to ensure that parasite cell division had been arrested by the PDTC treatment. • Identification of genes of interest and primer construction A literature review suggested some genes that might be of interest in this project. Putative homologues to human 14-3-3σ (Tg14-3-3) and human ménage-a-tois protein (TgMAT1) were identified using BLAST searches of the T. gondii genomic database 4. A putative T. gondii cyclin was also identified 5. Predicted mRNA sequences were acquired from the database and primer sets appropriate for detection of mRNAs by reverse transcriptase-polymerase chain reactions (RT-PCR) were generated using web-based primer design software 6. • RT-PCR All RT-PCR reactions were done using a commercial kit7. Total RNA from PDTC-treated infected cultures, control infected cultures, and uninfected HFF cells were run simultaneously. PCR products were analyzed by agarose gel electrophoresis7. We also examined the possibility of a change in the concentration of Tg14-3-3 mRNA using a multiplex RT-PCR approach. This experiment utilized the T. gondii CDK-1 homologue TgTPK2 as an internal standard. PDTC-treated and control samples were diluted to concentrations that resulted in similar band intensities for the TgTPK2 RT-PCR product. The band intensities of Tg14-3-3 RT-PCR products from the two types of samples were then compared. A B Figure 1. HFF cells infected with T. gondii. Panel A: 24 hr of normal growth plus 12 hr of exposure to 50 μM PDTC. No vacuole has more than 8 parasites. Panel B: 36 hr of normal growth. No vacuole has fewer than 16 parasites. Figure 5. Total RNA samples from PDTC-treated and untreated cultures were normalized for production of a TgTPK2 RT-PCR product. Examination of simultaneously amplified Tg14-3-3σ bands failed to reveal evidence of transcriptional regulation of the gene by PDTC treatment. PDTC treatment did not totally eliminate the mRNAs of any of the three potential cell cycle regulators. Discussion Cyclins and MAT protein are both essential for progression of the eukaryotic cell cycle8. We could find no evidence that PDTC eliminates transcription of either TgCYC2 or TgMAT1. It is possible that a quantitative assay like the one we used for Tg14-3-3σ might yet reveal evidence of transcriptional regulation of these genes. 14-3-3σ proteins can serve as negative regulators of eukaryotic cell cycles8. We had speculated that PDTC might arrest the T. gondii cell cycle by upregulating Tg14-3-3σ transcription. That hypothesis was not supported. Figure 2: TgMAT1. A RT-PCR product of 177 base pairs was expected if mRNA for TgMAT1 was present in the sample. A band consistent with such a product was observed in PDTC-treated cultures and both untreated controls. Introduction Pyrrolidine dithiocarbamate (PDTC) reversibly blocks cell division of the parasitic protist Toxoplasma gondii 1. Studies suggest that the blockade is the result of cell cycle arrest at the G1/S checkpoint2. We reasoned that any cell cycle regulatory protein whose expression is essential for progression through cell cycle checkpoints downstream of the arrest point would be transcriptionally blocked as a result of PDTC treatment. Indeed, we would argue that if a protein is known to be a critical cell cycle regulator in other systems and is transcriptionally regulated by PDTC treatment, then that suggests that the protein is a cell cycle regulator in T. gondii. We would predict that positive regulators would be downregulated by PDTC treatment and that negative regulators might be upregulated. We identified three T. gondii genes that might serve as cell cycle regulators and began to examine the effects of PDTC treatment on their transcription. We first determined if PDTC treatment resulted in the disappearance of mRNAs coding for the three proteins. We also conducted a pilot experiment to determine if the quantity of mRNA coding for one of the proteins was changed by PDTC treatment. References and Notes • Camps, M. and Boothroyd, J.C. (2001). Toxoplasma gondii: selective killing of extracellular parasites by oxidation using pyrrolidine dithiocarbamate. Experimental Parasitology. 98:206-214. • De Felipe, M. M. C., Lehmann, M. M., Jerome, M. E., and White, M. W. (2008). Inhibition of Toxoplasma gondii growth by pyrrolidine dithiocarbamate is cell cycle specific and leads to population synchronization. Molecular and Biochemical Parasitology. 157:22-31. • FastPure RNA Kit, TaKaRa Bio USA, Madison WI. • http://www.toxodb.org • C. Kvaal. St. Cloud State University, St. Cloud MN. Personal communication. • http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi • FlashGel System, Lonza Rockland Inc., Rockland ME. • Morgan, D.O. (2007). The Cell Cycle: Principles of Control. Sinauer Associates Inc., Sunderland MA. 297pp. Figure 3: TgCYC2. A RT-PCR product of 642 base pairs was expected if mRNA for TgCYC2 was present in the sample. A band consistent with such a product was observed in PDTC-treated cultures and both untreated controls. Contact information Douglas B. Woodmansee, Ph.D. Professor of Biology Wilmington College 1870 Quaker Way Wilmington, OH 45177 doug_woodmansee@wilmington.edu Acknowledgements This work was supported by funds provided by the Wilmington College Instructional Development and Resources Committee. Students and faculty in the Biology Research and Seminar course provided assistance and consultation. Figure 4: Tg14-3-3σ. A RT-PCR product of 202 base pairs was expected if mRNA for Tg14-3-3σ was present in the sample. A band consistent with such a product was observed in PDTC-treated cultures and both untreated controls.