A cell cycle model for the tachyzoite of Toxoplasma gondii using the Herpes simplex virus thymidine kinase

Radke, Jay R.; White, Michael

doi:10.1016/s0166-6851(98)00074-7

Cited by 81 publications

(93 citation statements)

References 32 publications

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“…All the strains were maintained by serial passage in confluent monolayers of HFF cells according to standard protocols [20]. Toxoplasma gondii strain, RH TK+ , is a transgenic isolate expressing herpes simplex virus thymidine kinase [12].…”

Section: Cell Culture and Parasite Strainsmentioning

confidence: 99%

“…Following overnight growth (8-12 h), cultures were treated with PDTC (6 h was the standard PDTC treatment) and then washed 3 times with pre-warmed growth medium (37°C) to release parasites from the growth inhibition. The growth of RH TK+ tachyzoites was synchronized with thymidine (10 μm for 4 h) as previously described [12]. Parasite growth was determined for 100 vacuoles in randomly chosen microscopic fields [21].…”

Section: Establishment Of Synchronous Cultures and Determination Of Pmentioning

confidence: 99%

“…Parasite nuclear DNA content was determined by flow cytometry using propidium iodide (PI) (Sigma, St. Louis, MO) as described previously [12] or using SYTOX Green dye (Molecular Probes #S7020, Invitrogen Corp., Carlsbad, CA) [22], which gives equivalent results to PI staining [7]. Briefly, filter-purified tachyzoites were fixed in 70% (v/v) ethanol with gentle shaking and stored at −20°C for at least 24 h prior to analysis.…”

Section: Flow Cytometry and Cell Cycle Analysismentioning

confidence: 99%

“…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 [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]).…”

Section: Introductionmentioning

confidence: 99%

“…Growth synchrony has been achieved through the use of exogenous thymidine to reversibly block tachyzoites engineered to express the herpes simplex virus thymidine kinase (RH TK+ ), an enzyme these parasites normally lack. A short treatment of RH TK+ 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].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Felipe

Lehmann

Jerome

et al. 2008

Molecular and Biochemical Parasitology

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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 T. 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, pyrrolidine dithiocarbamate. This method is an improvement over other strategies developed for this parasite 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 hours postdrug 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.

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Section: Cell Culture and Parasite Strainsmentioning

confidence: 99%

Section: Establishment Of Synchronous Cultures and Determination Of Pmentioning

confidence: 99%

Section: Flow Cytometry and Cell Cycle Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Felipe

Lehmann

Jerome

et al. 2008

Molecular and Biochemical Parasitology

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Transport and Trafficking: Toxoplasma as a Model for Plasmodium

Roos

Crawford

Donald

et al. 2007

Novartis Foundation Symposia

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Toxoplasma gondii: Laboratory Maintenance and Growth

Khan

Grigg

2017

CP Microbiology

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Toxoplasma gondii is a highly successful Apicomplexan protozoan capable of infecting any warm-blooded animal worldwide. In humans, Toxoplasma infections are life-long with approximately one-third of the world’s population chronically infected. Although normally controlled by the host immune system, T. gondii infection can lead to a variety of clinical outcomes in individuals with immature or suppressed immune systems. After penetrating the intestine, parasites rapidly disseminate throughout the body and stimulate production of the cytokines interleukin (IL)-12, IL-18, and interferon (IFN)-γ by immune cells. These cytokines play a key role in host resistance to T. gondii by promoting a strong Th1 response. Recent reports show that gut commensal bacteria can act as molecular adjuvants during T. gondii infection. Thus, T. gondii is an excellent model system to study host-pathogen interactions. This unit outlines the protocols for in vitro and in vivo maintenance and growth of T. gondii.

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A cell cycle model for the tachyzoite of Toxoplasma gondii using the Herpes simplex virus thymidine kinase

Cited by 81 publications

References 32 publications

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

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

Transport and Trafficking: Toxoplasma as a Model for Plasmodium

Toxoplasma gondii: Laboratory Maintenance and Growth

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