Toxoplasma gondii is an obligate intracellular parasite that causes severe disease in immunocompromised individuals and congenitally infected neonates. Toxoplasma also serves as a model system for the study of related apicomplexan parasites including Plasmodium falciparum, the causative agent of malaria. Intracellular survival of these organisms is critically dependent on the ability of the parasite to actively invade their host cell, establish a replication-permissive vacuole, and avoid host cell defenses. Apicomplexan parasites share a unique mechanism for invasion that involves the formation of a tight junction between the invading parasite and the host cell called the moving junction (MJ). The MJ is believed to form a stable anchor for the parasite to invade the host cell and also serve as a molecular sieve that modifies the nascent vacuole to render it non-fusogenic with the host endocytic pathway. The Toxoplasma MJ consists of micronemal AMA1 on the parasite's surface connected to a macromolecular complex of rhoptry neck proteins (RONs 2/4/5/8) that are injected into the host cell. While RON2 spans the host membrane and establishes the link to AMA1, the remaining members of the complex are surprisingly on the cytoplasmic face of the host membrane and how they function in invasion is largely unknown. We have disrupted the coccidial-specific component RON8 and shown that while not essential, this protein plays an important role in parasite invasion in vitro and in virulence in vivo. As the remaining members are conserved in the Apicomplexa and believed to be essential, this indicates that apicomplexans contain a conserved core complex that is required for invasion, which is enhanced in the coccidia via RON8. This is supported by our recent development of a conditional knockout for RON5, which shows that this protein plays a critical role in assembly of the complex and that the MJ complex is indeed essential for invasion. Our objectives in this first renewal application are to conduct an in depth functional analysis of the MJ complex and determine its architecture. Specifically, we will first focus on RON8 to determine how this component enhances invasion and links the complex to the host cell. We will then exploit the conditional knockout of RON5 to study its role in the organization and function of the MJ complex. Lastly, we will explore the architecture of the complex by determining its stoichiometry and identifying key interactions of its component proteins. These studies will open completely new insight into the mechanism by which apicomplexan parasites use this novel invasion machine to infect their mammalian hosts and cause disease.

Public Health Relevance

Toxoplasma gondii is an intracellular parasite that infects up to a third of the world's human population and causes serious disease or death in immunocompromised patients and neonates. T. gondii also serves as a model system for a number of related parasites including Plasmodium falciparum, the causative agent of malaria. This project is focused on a complex of proteins that are secreted from the parasite and are critical for apicomplexan parasites to invade their host cells. As this method of host cell entry i unique to these parasites and essential for survival, it represents a novel target for therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI064616-06A1
Application #
8297397
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Mcgugan, Glen C
Project Start
2005-12-15
Project End
2017-01-31
Budget Start
2012-02-07
Budget End
2013-01-31
Support Year
6
Fiscal Year
2012
Total Cost
$377,882
Indirect Cost
$127,882
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Nadipuram, Santhosh M; Kim, Elliot W; Vashisht, Ajay A et al. (2016) In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis. MBio 7:
Wang, Kevin; Peng, Eric D; Huang, Amy S et al. (2016) Identification of Novel O-Linked Glycosylated Toxoplasma Proteins by Vicia villosa Lectin Chromatography. PLoS One 11:e0150561
Kim, Elliot W; Nadipuram, Santhosh M; Tetlow, Ashley L et al. (2016) The Rhoptry Pseudokinase ROP54 Modulates Toxoplasma gondii Virulence and Host GBP2 Loading. mSphere 1:
Rodriguez, Jose A; Xu, Rui; Chen, Chien-Chun et al. (2015) Three-dimensional coherent X-ray diffractive imaging of whole frozen-hydrated cells. IUCrJ 2:575-83
Chen, Allan L; Kim, Elliot W; Toh, Justin Y et al. (2015) Novel components of the Toxoplasma inner membrane complex revealed by BioID. MBio 6:e02357-14
Gold, Daniel A; Kaplan, Aaron D; Lis, Agnieszka et al. (2015) The Toxoplasma Dense Granule Proteins GRA17 and GRA23 Mediate the Movement of Small Molecules between the Host and the Parasitophorous Vacuole. Cell Host Microbe 17:642-52
Silmon de Monerri, Natalie C; Yakubu, Rama R; Chen, Allan L et al. (2015) The Ubiquitin Proteome of Toxoplasma gondii Reveals Roles for Protein Ubiquitination in Cell-Cycle Transitions. Cell Host Microbe 18:621-33
Beck, Josh R; Chen, Allan L; Kim, Elliot W et al. (2014) RON5 is critical for organization and function of the Toxoplasma moving junction complex. PLoS Pathog 10:e1004025
Tonkin, Michelle L; Beck, Josh R; Bradley, Peter J et al. (2014) The inner membrane complex sub-compartment proteins critical for replication of the apicomplexan parasite Toxoplasma gondii adopt a pleckstrin homology fold. J Biol Chem 289:13962-73
Child, Matthew A; Hall, Carolyn I; Beck, Josh R et al. (2013) Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion. Nat Chem Biol 9:651-6

Showing the most recent 10 out of 27 publications