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Principal Investigator: Vernon Bruce Carruthers
Organization: UNIVERSITY OF MICHIGAN AT ANN ARBOR
Fiscal Year: 2024
Award: $195,000
Funding agency: National Institute of Allergy and Infectious Diseases
Abstract
ESCRT protein complexes play essential roles in membrane remodeling for multivesicular body formation,
intercellular communication, formation of the nuclear envelope, and cell division. We recently showed that the
obligate intracellular parasite Toxoplasma gondii repurposes host ESCRT machinery to acquire macromolecular
resources from infected cells. Host-derived resources critically fuel rapid growth of T. gondii acute stage
parasites, which are responsible for debilitating disease in immunodeficient or congenitally infected individuals.
Although we identified one parasite effector protein (GRA14) that directly binds the ESCRT-I complex protein
TSG101 and recruits it to the parasite’s replicative compartment, this protein only accounts for ~50% of TSG101
recruitment. Using a novel combination of CRISPR/Cas9 screening, high content imaging, and machine learning-
driven image analysis, in addition to GRA14, we identified 7 new parasite secretory proteins that putatively
contribute to TSG101 recruitment. Among the newly identified parasite proteins was ROP18, which is a major
virulence factor that is best known for effector function in evading innate immunity. Importantly, ROP18 deficient
parasites showed significantly less recruitment of TSG101, thus validating it as a hit and exposing a novel
function for this parasite kinase. However, additional studies are necessary to determine how ROP18 contributes
to TSG101 recruitment and which of the other 6 hits function in such recruitment. To meet this need, in Aim 1
we will determine if the kinase activity of ROP18 is required for TSG101 recruitment to the PV and identify
ROP18-linked parasite and host proteins that regulate TSG101 recruitment. In Aim 2, we will validate other hits
from the screen and determine if they are working in conjunction with GRA14 or ROP18. Together, these studies
will identify a novel protein network responsible for engaging host ESCRT machinery to ensure the successful
replication of an important human pathogen.
Terms: <AIDS Virus><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Acute><Binding><Biology><Bipolar Affective Psychosis><Bipolar Disorder><Blindness><Body Tissues><CRISPR approach><CRISPR based approach><CRISPR editing screen><CRISPR method><CRISPR methodology><CRISPR screen><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based screen><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 screen><CRISPR/Cas9 technology><Cancers><Cas nuclease technology><Cell Body><Cell Function><Cell Physiology><Cell Process><Cell division><Cell to Cell Communication and Signaling><Cell-Cell Signaling><Cells><Cellular Function><Cellular Physiology><Cellular Process><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Collaborations><Complex><Coupled><Cytoplasm><Cytosol><Developing fetus><Disease><Disorder><Endosomes><Ensure><Environment><Fetal Development><Generalized Growth><Genetic Screening><Goals><Grafting Procedure><Groups at risk><Growth><HIV><Health><Homolog of Yeast Vacuolar Protein Sorting 23><Human><Human Immunodeficiency Viruses><Hypothetical Protein><Image><Image Analyses><Image Analysis><Immune Evasion><Individual><Infection><Ingestion><Innate Immunity><Kinases><Knowledge><LAV-HTLV-III><Link><Lipids><Lymphadenopathy-Associated Virus><Machine Learning><Maintenance><Malignant Neoplasms><Malignant Tumor><Manic-Depressive Psychosis><Mediating><Membrane><Mental disorders><Mental health disorders><Metabolic Protein Degradation><Mice><Mice Mammals><Modern Man><Molecular Interaction><Multivesicular Body><Murine><Mus><Native Immunity><Natural Immunity><Nervous System Injuries><Nervous System Trauma><Nervous System damage><Neurological Damage><Neurological Injury><Neurological trauma><Non-Specific Immunity><Nonspecific Immunity><Nuclear Envelope><Nuclear Membrane><Nutrient><Organ><Organ Transplantation><Organ Transplants><Organelles><Parasites><Pathogenicity Factors><People at risk><Persons at risk><Phenotype><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Play><Populations at Risk><Protein Phosphorylation><Protein Turnover><Proteins><Psychiatric Disease><Psychiatric Disorder><Receptosomes><Regulatory Protein Degradation><Reporting><Research><Research Resources><Residual><Residual state><Resources><Risk Factors><Role><Schizophrenia><Schizophrenic Disorders><Series><Sorting><Structure><Subcellular Process><System><T gondii><T gondii infection><T. gondii><T. gondii infection><TSG101><TSG101 gene><Testing><Tissue Growth><Tissues><Toxoplasma><Toxoplasma gondii><Toxoplasma gondii Infection><Toxoplasmosis><Transphosphorylases><VPS23><Vacuole><Vertebrate Animals><Vertebrates><Vesicle><Virulence Factors><Virus-HIV><Work><bipolar affective disorder><bipolar disease><bipolar illness><bipolar mood disorder><cartilage link protein><clustered regularly interspaced short palindromic repeats screen><congenital infection><defined contribution><dementia praecox><digital><human disease><human pathogen><hypoimmunity><image evaluation><image interpretation><imaging><immune deficiency><immune evasive><immunodeficiency><ingest><insight><intercellular communication><link protein><machine based learning><malignancy><manic depressive disorder><manic depressive illness><membrane structure><mental illness><neoplasm/cancer><neurotrauma><novel><obligate intracellular parasite><ontogeny><organ allograft><organ graft><organ xenograft><prospective><protein complex><protein degradation><psychiatric illness><psychological disorder><rapid growth><recruit><schizophrenic><secretory protein><social role><tumor susceptibility gene 101><vertebrata><virtual><vision loss><visual loss>