Increased parasite burden is a key factor in the severity of clinical toxoplasmosis and thus, pathogenesis in Toxoplasma gondii infections is caused primarily by the growth of the parasite. From recent genetic analysis it has become clear that a shortened tachyzoite cell cycle is a key virulence determinant in Toxoplasma, yet we have few molecular details about how replication is regulated in this parasite. Toxoplasma tachyzoites divide by a novel cycle where the duplication of a complex set of organelles is coordinated with an unusual bimodal S phase and a phylum-specific budding process is synchronized with, and may regulate, aspects of mitosis. The binary division of Toxoplasma tachyzoites undergoing endodyogeny offers advantages for the investigation of the apicomplexan cell cycle, although these studies will apply broadly to the growth of other pathogens in this family, such as Plasmodium, Eimeria, and Cryptosporidium, where our knowledge of parasite cell cycle mechanisms is equally deficient. In this application, we propose a comprehensive study of the mechanisms controlling the replication of virulent Type I-RH tachyzoites.
In Aim 1, we will test the hypothesis that at least four checkpoints in G1, early and late S, and mitosis regulate the RH tachyzoite cell cycle through the analysis of a large collection of temperature sensitive (ts) growth mutants (165 total), which we have produced by chemical mutagenesis.
In Aim 2, we will examine the hypothesis that known as well as unique apicomplexan proteins are required for tachyzoite checkpoint control through cosmid-based genetic complementation, which will identify the essential genes involved in specific ts-mutants. Finally, in Aim 3, we will define the role of the daughter/mitotic cytoskeletons in regulating checkpoints that control chromosome replication. In preliminary studies, we have established high throughput protocols for producing and analyzing the phenotype of cell cycle mutants and we have demonstrated robust new cosmid-based methods for genetic complementation in this parasite. These studies will provide insight into the mechanisms regulating parasite division and provide new targets upon which to disrupt parasite proliferation.

Public Health Relevance

Recent genetic analysis of parasite virulence confirms that there is an important link between increased parasite burden and disease caused by Toxoplasma gondii. The factors that control the parasite division cycle are not understood, but it is clear that the rate of progression through the parasite cell cycle is critical to parasite numbers in the host. In this proposal, we will investigate the genetic basis for cell cycle control in Toxoplasma gondii. The essential growth factors identified in these studies will be shared by other pathogens in this family, such as Plasmodium, which causes malaria, and will likely represent novel proteins responsible for parasite growth. Therefore, through this investigation of the molecular basis of the parasite cell cycle, new potential drug targets will be identified upon which novel therapies may be developed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI077662-04
Application #
8230509
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Mcgugan, Glen C
Project Start
2009-03-05
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
4
Fiscal Year
2012
Total Cost
$360,603
Indirect Cost
$76,007
Name
University of South Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
069687242
City
Tampa
State
FL
Country
United States
Zip Code
33612
Francia, Maria E; Striepen, Boris (2014) Cell division in apicomplexan parasites. Nat Rev Microbiol 12:125-36
Vinayak, Sumiti; Brooks, Carrie F; Naumov, Anatoli et al. (2014) Genetic manipulation of the Toxoplasma gondii genome by fosmid recombineering. MBio 5:e02021
Croken, Matthew McKnight; Qiu, Weigang; White, Michael W et al. (2014) Gene Set Enrichment Analysis (GSEA) of Toxoplasma gondii expression datasets links cell cycle progression and the bradyzoite developmental program. BMC Genomics 15:515
Butler, Carrie L; Lucas, Olivier; Wuchty, Stefan et al. (2014) Identifying novel cell cycle proteins in Apicomplexa parasites through co-expression decision analysis. PLoS One 9:e97625
Suvorova, Elena S; White, Michael W (2014) Transcript maturation in apicomplexan parasites. Curr Opin Microbiol 20:82-7
Radke, Joshua B; Lucas, Olivier; De Silva, Erandi K et al. (2013) ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst. Proc Natl Acad Sci U S A 110:6871-6
Suvorova, Elena S; Radke, Joshua B; Ting, Li-Min et al. (2013) A nucleolar AAA-NTPase is required for parasite division. Mol Microbiol 90:338-55
Suvorova, Elena S; Croken, Matthew; Kratzer, Stella et al. (2013) Discovery of a splicing regulator required for cell cycle progression. PLoS Genet 9:e1003305
Suvorova, Elena S; Lehmann, Margaret M; Kratzer, Stella et al. (2012) Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii. Mol Biochem Parasitol 181:7-16
Szatanek, Tomasz; Anderson-White, Brooke R; Faugno-Fusci, David M et al. (2012) Cactin is essential for G1 progression in Toxoplasma gondii. Mol Microbiol 84:566-77

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