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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. Woodmansee
Department of Biology, Wilmington College, Wilmington, OH 45177
Abstract
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 PDTCinduced 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 50μM PDTC 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 transcriptasepolymerase 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.
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
checkpoint 2. 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.
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.
Methods
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 143-3σ (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 transcriptasepolymerase 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 RTPCR 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.
Results
Results
PDTC treatment arrested the T. gondii cell cycle.
A
Quantitation of Tg14-3-3σ mRNA by Multiplex RT-PCR.
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.
PDTC treatment did not totally eliminate the mRNAs of any of
the three potential cell cycle regulators.
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.
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 Tg143-3σ might yet reveal evidence of transcriptional regulation of
these genes.
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.
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.
References and Notes
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.
1. Camps, M. and Boothroyd, J.C. (2001). Toxoplasma gondii: selective
killing of extracellular parasites by oxidation using pyrrolidine
dithiocarbamate. Experimental Parasitology. 98:206-214.
2. 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.
3. FastPure RNA Kit, TaKaRa Bio USA, Madison WI.
4. http://www.toxodb.org
5. C. Kvaal. St. Cloud State University, St. Cloud MN. Personal
communication.
6. http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
7. FlashGel System, Lonza Rockland Inc., Rockland ME.
8. Morgan, D.O. (2007). The Cell Cycle: Principles of Control. Sinauer
Associates Inc., Sunderland MA. 297pp.
Contact information
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.
Douglas B. Woodmansee, Ph.D.
Professor of Biology
Wilmington College
1870 Quaker Way
Wilmington, OH 45177
[email protected]
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