Susceptibility of Toxoplasma gondii to autophagy in human cells relies on multiple interacting parasite loci

Autophagy contributes to innate immunity by targeting intracellular pathogens for elimination. Previous studies identified a non-canonical autophagy pathway that controls Toxoplasma gondii infection in a strain-dependent manner in interferon gamma-activated human cells. Ubiquitination of unknown targets recruits adaptors and LC3 to the parasitophorous vacuole, leading to membrane envelopment and stunted growth. Vacuoles containing strain types II and III are susceptible, while type I strains of T. gondii are largely resistant and hence avoid autophagy-mediated growth restriction. Here we interrogated the genetic differences in LC3 recruitment between a resistant type I (GT1) strain and a susceptible type III (CTG) strain of T. gondii. We took advantage of a previous genetic cross between these two strains to determine the LC3 recruitment phenotype of 34 unique progeny clones. Genetic linkage mapping revealed that LC3 recruitment was highly multigenic, depending on two major quantitative trait loci (QTLs) on chromosome II and VIII, as well as three minor contributing loci. Ubiquitin affinity capture followed by mass spectrometry identified several potential targets exposed at the surface of the parasitophorous vacuole, including several candidates within the major QTLs. We tested several candidates and identified the dense granule proteins MAF1 on chromosome II, and MAG1 and PSD1 on chromosome VIII, as being partially responsible for susceptibility to LC3 recruitment. Differential susceptibility is likely due to strain-specific differences in recognition of parasite molecules, rather than actively blocking recognition, thus revealing a new mechanism for cell-autonomous restriction of intracellular pathogens.