Inhibition of Toxoplasma gondii growth by pyrrolidine dithiocarbamate is cell cycle specific and leads to population synchronization

https://doi.org/10.1016/j.molbiopara.2007.09.003Get rights and content

Abstract

Successful completion of the Toxoplasma cell cycle requires the coordination of a series of complex and ordered processes that results in the formation of two daughters by internal budding. Although we now understand the order and timing of intracellular events associated with the parasite cell cycle, the molecular details of the checkpoints that regulate each step in Toxoplasma gondii division is still uncertain. In other eukaryotic cells, the use of cytostatic inhibitors that are able to arrest replication at natural checkpoints have been exploited to induce synchronization of population growth. Herein, we describe a novel method to synchronize T. gondii tachyzoites based on the reversible growth inhibition by the drug and pyrrolidine dithiocarbamate. This method is an improvement over other strategies developed for this parasites as no prior genetic manipulation of the parasite was required. RH tachyzoites blocked by pyrrolidine dithiocarbamate exhibited a near uniform haploid DNA content and single centrosome indicating that this compound arrests parasites in the G1 phase of the tachyzoite cell cycle with a minor block in late cytokinesis. Thus, these studies support the existence of a natural checkpoint that regulates passage through the G1 period of the cell cycle. Populations released from pyrrolidine dithiocarbamate inhibition completed progression through G1 and entered S phase ∼2 h post-drug release. The transit of drug-synchronized populations through S phase and mitosis followed a similar timeframe to previous studies of the tachyzoite cell cycle. Tachyzoites treated with pyrrolidine dithiocarbamate were fully viable and completed two identical division cycles post-drug release demonstrating that this is a robust method for synchronizing population growth in Toxoplasma.

Introduction

Toxoplasma gondii is the third leading cause, along with Salmonella and Listeria, of all deaths due to food-borne disease in the US [1]. Acquisition of Toxoplasma may occur through exposure to contaminated food products or through environmental sources, although recent studies indicate contaminated meat is rare and may be a minor contributor to infection in the US [2]. Inherited differences in the tachyzoite cell cycle that are manifest by distinct cell cycle length [3] influence the severity of clinical disease caused by this pathogen and may underlie differences in virulence that are characteristic of the three major genotypic lineages found in Europe and North America [3], [4], [5]. Rates of proliferation play a critical role in causing disease pathogenesis in numerous illnesses caused by other members of this phylum including parasites that are responsible for malaria and coccidiosis. Thus, understanding the mechanisms that control parasite division is an important task in the search for new approaches to combat apicomplexan-caused diseases.

T. gondii has evolved cell cycle machinery to produce different modes of replication in the definitive and intermediate hosts (schizogony and endodyogeny, respectively) [6], [7], although we do not understand how each cell cycle is regulated or how checkpoints are modified in order to switch between division schemes. Endodyogenic replication of the tachyzoite stage in the intermediate host is a binary process with a single chromosome replication followed by concurrent mitosis and parasite budding to produce new daughters. Chromsome re-replication occurs rarely, but produces viable parasites [8] and might reflect a low frequency switch to multinuclear schizogonous replication, which predominates in definitive life cycle stages. Unlike yeast cell division, tachyzoite budding is fully internal and yields two nearly equal sized daughters. This type of replication has been examined in detail by electron microscopy [9], [10] and using fluorescent markers to allow the visualization of organelle, daughter and nuclear division (reviewed in Ref. [7]). Labeling of the major steps of the tachyzoite endodyogeny in terms of conventional eukaryotic organization reveals a cell cycle composed of a primary G1 phase (60%), a bi-modal S (30%), and mitotic/cytokinetic phases (10%) (G1 > S > M), while G2 phase is either short or non-existent [3], [11], [12]. Parasites that possess a late S phase genome content (∼1.8N) are more frequent than 2N parasites [3], which are a small subfraction in asynchronous populations (estimated at 5%, [8]). These results suggest that there is a pause or slowing in late S phase that might represent a novel pre-mitotic checkpoint (equivalent to the G2 checkpoint in animal cells) associated with endodyogeny, although additional proof is needed to verify this model.

Characterization of the Toxoplasma cell cycle is greatly aided by the synchronization of population growth. Toxoplasma, and other apicomplexa parasites, naturally synchronize the division cycles of progeny that share a vacuole, although vacuolar synchrony begins to break down as parasites reach host cell lysis [8] and this feature alone is insufficient to achieve population synchrony. Metabolic depletion (serum or growth factor starvation) or treatment with growth inhibitors (e.g. hydroxyurea, aphidicoline, and colchicines) are techniques commonly employed to synchronize other eukaryotic cells [13]. Unfortunately, many growth inhibitors used in animal cells and other protozoa, such as Plasmodium [14] and Leishmania [15] are not successful in Toxoplasma. Chromosome replication is blocked in Toxoplasma by the polymerase inhibitors, aphidicoline [16] or hydroxyurea [17], however, these drugs also lead to uncoupling of daughter formation and are lethal. Growth synchrony has been achieved through the use of exogenous thymidine to reversibly block tachyzoites engineered to express the herpes simplex virus thymidine kinase (RHTK+), an enzyme parasite normally lack. A short treatment of RHTK+ tachyzoites with exogenous thymidine, which is known to cause dNTP depletion [18], arrests asynchronous parasite populations in late G1/early S phase and is presumed to act via a checkpoint that governs commitment to chromosome replication in this parasite [3], [12].

In this work, we describe a novel method to synchronize T. gondii tachyzoite populations that utilizes the antioxidant and metal chelating compound pyrrolidine dithiocarbamate (PDTC). PDTC has previously been used to eliminate extracellular parasites while leaving intracellular parasites unharmed [19]. We provide evidence that PDTC is acting on intracellular parasites to arrest growth primarily in the G1 period of the tachyzoite cell cycle, and demonstrate that a short drug treatment leads to the synchronization of tachyzoites through multiple cell division cycles.

Section snippets

Cell culture and parasite strains

Human foreskin fibroblasts (HFF) were grown in Dulbecco's modified Eagle medium (DMEM) (Gibco BRL, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (Hyclone Laboratories Inc., Logan, UT). All the strains were maintained by serial passage in confluent monolayers of HFF cells according to standard protocols [20]. T. gondii strain and RHTK+ is a transgenic isolate expressing herpes simplex virus thymidine kinase [12].

Establishment of synchronous cultures and determination of parasite growth and survivability

To measure parasite replication rates and parasite viability

PDTC inhibition of tachyzoites is cell cycle specific

The cytostatic property of growth inhibitors is often associated with arrest at a natural checkpoint in yeast and animal cells [26] and this feature has been exploited to synchronize eukaryotic cell populations. Hydroxyurea and aphidicolin are two examples of this type of compound, although these and other commonly used inhibitors have proven to be cytotoxic to Toxoplasma parasites [16], [17]. It is therefore important to identify drugs which effects on parasite growth as reversible and

Discussion

In this study, we have introduced a novel protocol for establishing synchronous Toxoplasma populations that is based on the reversible growth inhibition of the drug PDTC. PDTC protocols offer advantages over an earlier synchrony model that is dependent on ectopic expression of thymidine kinase [12] as PDTC is capable of inhibiting the growth of all major genotypic strains (types I–III, data not shown) without genetic manipulation. This model will mediate efforts to further investigate the basic

Acknowledgments

This work was support in part by grants from NIH (R01 AI48390 and NCRR P20 RR-020185) to M.W.W. We thank Dr. Gary Ward for kindly providing the monoclonal antibody against IMC1 used in these studies. We would also like to thank Dr. Doug Woodmansee for his discussions about the effects of PDTC on parasite growth. Magnolia M. Conde de Felipe is a recipient of the “MEC/FULBRIGHT” fellowship.

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