Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

41-POS Board 14 DEFINING THE INTIMATE INTERACTIONS BETWEEN TOXOPLASMA GONDII AND HOST ESCRT MACHINERY Hargobinder Kaur 1 ; Rebekah B Guevara 2 ; Yolanda Rivera-Cuevas 1 ; Joshua Mayoral 2 ; Louis M Weiss 2 ; Phyllis I Hanson 3 ; Vern B Carruthers 1 ; 1 University of Michigan, Microbiology and Immunology, Ann Arbor, MI, USA The endosomal sorting complexes required for transport (ESCRT) machinery remodels membranes to generate vesicles that bud away from the cytosol. A variety of intracellular pathogens exploit host ESCRTs to gain resources for replication, to protect their intracellular niche, or to exit from infected cells. Whereas mechanisms underlying virus-host ESCRT interactions are well described, precisely how other pathogens including bacteria and parasites exploit host ESCRT machinery is poorly understood. We recent reported that the protozoan parasite Toxoplasma gondii engages components of host ESCRT to convert its intracellular replicative niche (the parasitophorous vacuole, PV) into a multivesicular body-like organelle for resource acquisition from infected cells (PMIDs 34898650, 35730903, 36718630). In other work, Cygan et al. (34749525) showed that the ESCRT accessory protein apoptosis-linked gene 2 (ALG-2) is recruited to the PV. However, the basis of this recruitment was unknown. We identified the parasite PV resident dense granule protein (TgGRA8) as an ALG-2 binding protein by immunoprecipitating (IPing) ALG-2 from infected cells. This interaction was confirmed by reciprocally IPing TgGRA8, which identified ALG-2 along with ALG-2 interacting protein X (ALIX) and a constellation of other ESCRT proteins. Biochemical experiments confirmed a direct and exceptionally tight (K d <10 -12 M) interaction between ALG-2 and a synthetic peptide representing one of three ALG-2 binding elements (ABEs) in TgGRA8. Unexpectedly, TgGRA8 ABE peptide binding to ALG-2 was Ca 2+ -independent, unlike that of all other reported partners of ALG-2. Parasites lacking TgGRA8 showed a marked decrease in ALG-2 and ALIX at the PV. Re-expressing TgGRA8 restored PV recruitment of ALG-2 and ALIX, establishing a key role for TgGRA8 in recruiting these proteins to the PV. Ongoing work aims to determine if TgGRA8, ALG-2, and other ESCRT proteins including ALIX function in parasite nutrient scavenging from the host or PV membrane damage repair, a known ESCRT-dependent function for ALG-2. 2 Albert Einstein College of Medicine, Pathology, Bronx, NY, USA 3 University of Michigan, Biological Chemistry, Ann Arbor, MI, USA

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