Exit from host cells is an essential step in the life cycle of intracellular pathogens including Toxoplasma gondii, the agent of human toxoplasmosis. Related malaria parasites also egress from host cells after replication, and they additionally traverse cells by escaping from a transient vacuole in a manner similar to egress. Recent work has identified critical roles for pore-forming proteins and phospholipases in cell egress and traversal. But how these proteins facilitate egress and the extent to which they collaborate to disrupt membranes is unknown. Also, although recent reports suggest that acidification of the parasite-containing vacuole augments activity of the pore-forming protein(s), the proton pumps mediating acidification have not been identified. Precisely how acidification facilitates pore-formation and egress is also unclear. The absence of such knowledge precludes rationale design of interference strategies to alter the course of infection and disease. We will address these key gaps by identifying the proton pump(s) responsible for vacuole acidification (Aim1), defining the structural basis for pH- augmented membrane binding by the pore-forming protein Perforin-Like Protein 1 (Aim 2), and measuring pH-dependent cooperativity between Perforin-Like Protein 1 and an LCAT phospholipase for membrane disruption during egress. The proposed studies will advance the field by delivering insight that is broadly relevant to egress by apicomplexan parasites and other cytolytic pathogens.

Public Health Relevance

These studies will identify how acidifying pumps and membrane disruptive proteins promote cell exit by the human intracellular parasite Toxoplasma gondii. Our work will discovery how membrane disrupting proteins released by T. gondii are regulated and define how function together in a manner that is relevant to a broad range of microbial infections and immune-related diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI046675-20
Application #
9914201
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Mcgugan, Glen C
Project Start
1999-12-01
Project End
2022-04-30
Budget Start
2020-05-01
Budget End
2021-04-30
Support Year
20
Fiscal Year
2020
Total Cost
Indirect Cost
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
Di Cristina, Manlio; Carruthers, Vern B (2018) New and emerging uses of CRISPR/Cas9 to genetically manipulate apicomplexan parasites. Parasitology 145:1119-1126
Schultz, Aric J; Carruthers, Vern B (2018) Toxoplasma gondii LCAT Primarily Contributes to Tachyzoite Egress. mSphere 3:
Lunghi, Matteo; Spano, Furio; Magini, Alessandro et al. (2016) Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites. Curr Genet 62:31-8
Sidik, Saima M; Huet, Diego; Ganesan, Suresh M et al. (2016) A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes. Cell 166:1423-1435.e12
Bullen, Hayley E; Jia, Yonggen; Yamaryo-Botté, Yoshiki et al. (2016) Phosphatidic Acid-Mediated Signaling Regulates Microneme Secretion in Toxoplasma. Cell Host Microbe 19:349-60
Huynh, My-Hang; Carruthers, Vern B (2016) A Toxoplasma gondii Ortholog of Plasmodium GAMA Contributes to Parasite Attachment and Cell Invasion. mSphere 1:
Pszenny, Viviana; Ehrenman, Karen; Romano, Julia D et al. (2016) A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress. J Biol Chem 291:3725-46
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
Carruthers, Vern B (2015) Parasites and their heterophagic appetite for disease. PLoS Pathog 11:e1004803
Huynh, My-Hang; Boulanger, Martin J; Carruthers, Vern B (2014) A conserved apicomplexan microneme protein contributes to Toxoplasma gondii invasion and virulence. Infect Immun 82:4358-68

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