Proximity-Labeling Reveals Novel Host and Parasite Proteins at the Toxoplasma Parasitophorous Vacuole Membrane

Alicja M. Cygan; Pierre M. Jean Beltran; Alma G. Mendoza; Tess C. Branon; Alice Y. Ting; Steven A. Carr; John C. Boothroyd

doi:10.1128/mBio.00260-21

mBio (Dec 2021)

Proximity-Labeling Reveals Novel Host and Parasite Proteins at the Toxoplasma Parasitophorous Vacuole Membrane

Alicja M. Cygan,
Pierre M. Jean Beltran,
Alma G. Mendoza,
Tess C. Branon,
Alice Y. Ting,
Steven A. Carr,
John C. Boothroyd

Affiliations

Alicja M. Cygan: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford California, USA
Pierre M. Jean Beltran: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA
Alma G. Mendoza: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford California, USA
Tess C. Branon: Department of Chemistry, Stanford University, Stanford, California, USA
Alice Y. Ting: Department of Chemistry, Stanford University, Stanford, California, USA
Steven A. Carr: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA
John C. Boothroyd: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford California, USA

DOI: https://doi.org/10.1128/mBio.00260-21
Journal volume & issue: Vol. 12, no. 6

Abstract

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ABSTRACT Toxoplasma gondii is a ubiquitous, intracellular parasite that envelops its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell, such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the Toxoplasma PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three parasite proteins (TGGT1_269950 [GRA61], TGGT1_215360 [GRA62], and TGGT1_217530 [GRA63]) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the Toxoplasma PVM and, given that three of the validated host proteins are components of the ESCRT (endosomal sorting complexes required for transport) machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface. IMPORTANCE Toxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation. Only a small number of parasite and host proteins present at the vacuolar membrane and exposed to the host cytosol have thus far been identified. Here, we report the identification of several novel parasite and host proteins present at the vacuolar membrane using enzyme-catalyzed proximity-labeling, significantly increasing our knowledge of the molecular players present and novel biology occurring at this crucial interface.

Published in mBio

ISSN: 2161-2129 (Print); 2150-7511 (Online)
Publisher: American Society for Microbiology
Country of publisher: United States
LCC subjects: Science: Microbiology
Website: https://journals.asm.org/journal/mbio

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