Toxoplasma gondii is capable of infecting virtually all types of warm-blooded vertebrates and frequently causes food or waterborne infections in humans. Although most human infections are normally uncomplicated, they persist in a chronic state and can predispose individuals to subsequent reactivation. Current therapies can suppress parasite growth but not eliminate persistent infection, and available treatments are limited by adverse effects. As such, there is a need for increased understanding of basic parasite biology to identify new targets for therapeutic intervention. Like other apicomplexan parasites, T. gondii is an obligate intracellular parasite, and cell entry is critical for its survval. Host cell invasion is governed by calcium-regulated secretion of adhesive proteins that are discharged from micronemes, together with the concerted action of an action-myosin motor that lies beneath the plasma membrane. Although previous studies demonstrate that calcium- regulated microneme secretion is essential, the molecular details of this signaling cascade are incompletely understood. Additionally, calcium is likely to control other important aspects of parasite biology. The goal of our studies is to identify key regulatory steps in calcium signaling that are essential for survival of apicomplexan parasites. We will utilize T. gondii as a model due to its versatility for genetic, cellular, and molecular studies. Prior studies implicate protein kinase G (PKG) in controlling calcium increases and activating calcium-dependent protein kinases (CDPKs) in apicomplexan parasites. The proposed studies will test the role of guanylate cyclases in producing cyclic GMP (cGMP) to activate PKG as part of this pathway. One of the unique features of apicomplexans is that they contain a family of CDPKs that are not found in their mammalian hosts. In previous studies, we demonstrated that microneme secretion requires the activity of several canonical CDPKs, which play partially overlapping roles in controlling invasion and egress. We will extend our studies to encompass the non-canonical CDPKs encoded in the T. gondii genome. These non-canonical CDPKs differ in the number and arrangement of calcium-responsive motifs and they contain additional domains likely to govern location and/or regulation. To support these studies, we have developed novel reporter strains of T. gondii that have been engineered to detect elevated cGMP, increased calcium, and microneme secretion in response to agonists. We will use molecular, cellular, and biochemical methods to examine the roles of guanylate cyclases and non-canonical CDPKs in controlling calcium-mediated signaling in T. gondii. Efficient methods for gene disruption or inducible deletion will be used to determine the roles of individual genes in intracellular survival. Collectively, the proposed studies will define the molecular basis of calcium signaling and elucidate the roles of members of a novel family of calcium-dependent protein kinases.

Public Health Relevance

Infections caused by Toxoplasma gondii remain a significant cause of human disease in immunocompromised patients, and wide spread chronic infections raise the risk of complications in healthy patients should their immune system decline. The immediate goal of our studies is to define unique components in calcium signaling in the parasite that are not found in the host and which are essential for intracellular infection. The long-term goal of these studies is to provide fundamental knowledge as a foundation for future development of new therapeutic interventions against toxoplasmosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI034036-24
Application #
8995611
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Mcgugan, Glen C
Project Start
1993-04-01
Project End
2020-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
24
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Washington University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Shen, Bang; Brown, Kevin; Long, Shaojun et al. (2017) Development of CRISPR/Cas9 for Efficient Genome Editing in Toxoplasma gondii. Methods Mol Biol 1498:79-103
Jones, Nathaniel G; Wang, Qiuling; Sibley, L David (2017) Secreted protein kinases regulate cyst burden during chronic toxoplasmosis. Cell Microbiol 19:
Long, Shaojun; Anthony, Bryan; Drewry, Lisa L et al. (2017) A conserved ankyrin repeat-containing protein regulates conoid stability, motility and cell invasion in Toxoplasma gondii. Nat Commun 8:2236
Long, Shaojun; Brown, Kevin M; Drewry, Lisa L et al. (2017) Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii. PLoS Pathog 13:e1006379
Brown, Kevin M; Long, Shaojun; Sibley, L David (2017) Plasma Membrane Association by N-Acylation Governs PKG Function in Toxoplasma gondii. MBio 8:
Long, Shaojun; Wang, Qiuling; Sibley, L David (2016) Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9. Infect Immun 84:1262-73
Brown, Kevin M; Lourido, Sebastian; Sibley, L David (2016) Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii. J Biol Chem 291:9554-65
Jimah, John R; Salinas, Nichole D; Sala-Rabanal, Monica et al. (2016) Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption. Elife 5:
Olias, Philipp; Sibley, L David (2016) Functional Analysis of the Role of Toxoplasma gondii Nucleoside Triphosphate Hydrolases I and II in Acute Mouse Virulence and Immune Suppression. Infect Immun 84:1994-2001
Lorenzi, Hernan; Khan, Asis; Behnke, Michael S et al. (2016) Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii genomes. Nat Commun 7:10147

Showing the most recent 10 out of 79 publications