The obligate intracellular protozoan Toxoplasma gondii is a leading cause of posterior uveitis in the general population, encephalitis in immunodeficient individuals, and cognitive impairment in congenitally infected children. We seek to understand how Toxoplasma prepares for and achieves cell invasion. Although it has been well recognized that many Toxoplasma invasion proteins undergo proteolytic maturation en route to apical secretory organelles, little is known about the proteases involved or where and why maturation occurs. We hypothesize that T. gondii uses a cathepsin proteases to proteolytically activate and stabilize invasion proteins within a novel multivesicular endosome (MVE), based on preliminary immunolocalization, gene knockout, and inhibitor studies, along with protease specificity analysis and in vitro processing of a putative substrate. To test this hypothesis we will: (1) Characterize defects and compensatory mechanisms in CPL knockout parasites;(2) Confirm the overlapping roles of cathepsins in proMIC maturation by genetic and inhibitor studies;and (3) Test the """"""""lock and load"""""""" hypothesis that proteolytic maturation stabilizes proMIC protein complexes. This work will illuminate basic mechanisms of secretory protein trafficking and proteolytic modulation of protein function that may be shared with related apicomplexan parasites and other eukaryotic cells that utilize regulated secretion.

Public Health Relevance

Our goal is to find new ways of interfering with Toxoplasma invasion of human cells as a strategy of controlling infections caused by this parasite. Toxoplasma proteases may be good targets for new anti-parasitic drugs;this work is designed to test the function and significance of two such proteases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI063263-05
Application #
8384858
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Rogers, Martin J
Project Start
2008-12-01
Project End
2013-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
5
Fiscal Year
2013
Total Cost
$350,011
Indirect Cost
$119,687
Name
University of Michigan Ann Arbor
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Huynh, My-Hang; Liu, Bing; Henry, Maud et al. (2015) Structural basis of Toxoplasma gondii MIC2-associated protein interaction with MIC2. J Biol Chem 290:1432-41
Dou, Zhicheng; McGovern, Olivia L; Di Cristina, Manlio et al. (2014) Toxoplasma gondii ingests and digests host cytosolic proteins. MBio 5:e01188-14
Liu, Jing; Pace, Douglas; Dou, Zhicheng et al. (2014) A vacuolar-H(+) -pyrophosphatase (TgVP1) is required for microneme secretion, host cell invasion, and extracellular survival of Toxoplasma gondii. Mol Microbiol 93:698-712
Warring, Sally D; Dou, Zhicheng; Carruthers, Vern B et al. (2014) Characterization of the chloroquine resistance transporter homologue in Toxoplasma gondii. Eukaryot Cell 13:1360-70
Blackman, Michael J; Carruthers, Vern B (2013) Recent insights into apicomplexan parasite egress provide new views to a kill. Curr Opin Microbiol 16:459-64
Dou, Zhicheng; Coppens, Isabelle; Carruthers, Vern B (2013) Non-canonical maturation of two papain-family proteases in Toxoplasma gondii. J Biol Chem 288:3523-34
Gaji, Rajshekhar Y; Huynh, My-Hang; Carruthers, Vern B (2013) A novel high throughput invasion screen identifies host actin regulators required for efficient cell entry by Toxoplasma gondii. PLoS One 8:e64693
Tomavo, Stanislas; Slomianny, Christian; Meissner, Markus et al. (2013) Protein trafficking through the endosomal system prepares intracellular parasites for a home invasion. PLoS Pathog 9:e1003629
Sloves, Pierre-Julien; Delhaye, Stephane; Mouveaux, Thomas et al. (2012) Toxoplasma sortilin-like receptor regulates protein transport and is essential for apical secretory organelle biogenesis and host infection. Cell Host Microbe 11:515-27
Hortua Triana, Miryam Andrea; Huynh, My-Hang; Garavito, Manuel F et al. (2012) Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii. Mol Biochem Parasitol 184:71-81

Showing the most recent 10 out of 17 publications