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Genetic mapping of strain specific differences in autophagy effector recruitment to the Toxoplasma gondii parasitophorous vacuole
Author(s) -
Radke Joshua B,
Sibley L. David
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.776.5
Subject(s) - autophagy , biology , effector , toxoplasma gondii , microbiology and biotechnology , intracellular parasite , innate immune system , atg5 , vacuole , atg16l1 , genetics , immune system , intracellular , cytoplasm , antibody , apoptosis
Autophagy is a highly conserved cellular process by which cytoplasmic constituents such as damaged organelles and mis‐folded proteins are recycled. Additionally, autophagy or autophagy related genes play a critical role in the innate immune response by associating with intracellular pathogens and targeting them for elimination. Recently, our laboratory identified an innate, noncanonical autophagy (ATG) pathway that regulates T. gondii infection in a strain‐dependent manner in IFN‐γ activated human cells. The core set of ATG16L1, ATG12 and ATG5 proteins are involved in recruitment of LC3 to the PV, and yet upstream activators (i.e. Beclin and Atg14) and downstream degradation function (i.e. lysosomal fusion) are not required for this pathway. This core set of ATG protein is also necessary for the IFN‐γ‐dependent control of T. gondii strains in mouse and human cells. In humans, parasitophorous vacuoles (PV) containing susceptible strains (type II and III) of T. gondii are ubiquitinated, followed by ligation of the ATG adapters p62 and NDP52, and finally engulfed in multiple layers of host membrane resulting in the stunting of parasite growth. However, type I strains of T. gondii (type I) are largely resistant to ATG mediated growth restriction. Here we interrogated the genetic differences in autophagy effector recruitment between the resistant type I (GT1‐SNF) strain and the susceptible type III (CTG‐ARA) strain of T. gondii using 34 unique progeny isolated from a genetic cross (IxIII). We evaluated all progeny for the hIFN‐γ‐dependent recruitment of GFP‐LC3 to the PV. Genome wide analysis identified two quantitative trait loci (QTL) associated with the autophagy effector recruitment phenotype, one on Chr. II (LOD 3.35) and a second on Chr. VIII (LOD 4.05). In total, 373 genes fall within these loci (95% CI) and the two QTL account for 77% of the phenotypic variance identified in the progeny. We evaluated polymorphism, the presence of signal peptides and/or transmembrane domains and gene annotation to prioritize 10 candidate genes for further evaluation. We have evaluated subcellular localization for all priority genes and found 2 genes that localize to the PV. Current genetic studies are focused on inducible gene knockdown strategies (i.e the plant auxin inducible degron system) and genetic ablation using CRISPR/Cas9 to evaluate the role for both genes in the autophagy recruitment phenotype. Identifying the cellular basis for avoidance of ATG recruitment may reveal mechanisms of enhanced pathogenicity of T. gondii strains, and allow for selective enhancement of this pathway to affect pathogen clearance.