Toxoplasma gondii exploits the host ESCRT machinery for parasite uptake of host cytosolic proteins

Abstract

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole (PV), the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV-1 virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of TgGRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PV membrane (PVM). These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission.

Toxoplasma scavenges mammalian host organelles through usurpation of host ESCRT-III and Vps4

“…In VERO cells and HeLa cells transfected with mCherry-Vps4A EQ, enlarged endosomal vacuoles were observed, with some surrounding the PV (Fig. 6 C and D), as observed previously (Rivera-Cuevas et al, 2021), but no signal was detected inside the PV. GFP-Rab11A puncta were observed inside the PV, with no significant difference in number as compared to mock-transfected or cells transfected with Vps4A or DN of Vps4B (Fig.…”

“…A proximity-labeling study on Toxoplasma -infected fibroblasts to identify host proteins at the PV, reveals that a PVM reporter biotinylates several host ESCRT components (e.g., ALIX, ALG-2, TSG101, VPS28, CHMP4B, CC2D1A) (Cygan et al, 2021). Two concomitant studies using co-immunoprecipitation and proximity-based biotinylation identified two Toxoplasma proteins on the PVM/IVN that interact with host ESCRT: TgGRA14 (e.g., ALIX, ALG-2, TSG101, CHMP4A/B) (Rivera-Cuevas et al, 2021) and TgGRA64 (e.g., TSG101, VPS37A, VPS28, CHMP4B, ALG-2) (Mayoral et al, 2022). Some of these host ESCRT components have been shown to associate with the PV; ALIX and ALG-2 localize as puncta on the PV (Cygan et al, 2021) and TgGRA14 influences PV recruitment of TSG101 (Rivera-Cuevas et al, 2021).…”

“…Two concomitant studies using co-immunoprecipitation and proximity-based biotinylation identified two Toxoplasma proteins on the PVM/IVN that interact with host ESCRT: TgGRA14 (e.g., ALIX, ALG-2, TSG101, CHMP4A/B) (Rivera-Cuevas et al, 2021) and TgGRA64 (e.g., TSG101, VPS37A, VPS28, CHMP4B, ALG-2) (Mayoral et al, 2022). Some of these host ESCRT components have been shown to associate with the PV; ALIX and ALG-2 localize as puncta on the PV (Cygan et al, 2021) and TgGRA14 influences PV recruitment of TSG101 (Rivera-Cuevas et al, 2021). The uptake of host cytosolic proteins by Toxoplasma (Dou et al, 2014) is reduced in mammalian cells ectopically expressing the Vps4A EQ dominant negative (DN) mutant or by Δ gra14 mutant parasites (Rivera-Cuevas et al, 2021).…”

“…Some of these host ESCRT components have been shown to associate with the PV; ALIX and ALG-2 localize as puncta on the PV (Cygan et al, 2021) and TgGRA14 influences PV recruitment of TSG101 (Rivera-Cuevas et al, 2021). The uptake of host cytosolic proteins by Toxoplasma (Dou et al, 2014) is reduced in mammalian cells ectopically expressing the Vps4A EQ dominant negative (DN) mutant or by Δ gra14 mutant parasites (Rivera-Cuevas et al, 2021).…”

Efficient Cholesterol Transport in Dendritic Cells Defines Optimal Exogenous Antigen Presentation and Toxoplasma gondii Proliferation

“…In accordance with our results, a very recent study shows that T. gondii drives the recruitment of the host ESCRT machinery through the parasite effector transmembrane dense granule protein TgGRA14. The interaction between TgGRA14 and host ESCRT components allows the efficient uptake of cytosolic proteins from the host cell important for parasite survival (Rivera-Cuevas et al, 2021). Another recent work from a different group that used a proximitylabeling strategy in combination with quantitative proteomics demonstrated that three components of the host ESCRT machinery are present at the host-parasite membrane interface in infected fibroblasts (Cygan et al, 2021).…”