In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis

Abstract

Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo. Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis.

An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-κB-like signature in infected host cells

“…In contrast to microneme and rhoptry genes, there were 25 genes out of the 48 annotated and/or reported to encode dense granule and dense granule-like proteins (ToxoDB, [33]) that had higher expressions in intracellular sporozoites compared to tachyzoites (Table 10). Interestingly, these include genes for the recently characterized GRA24, GRA16, GRA28, and GRA31 [33,60,61].…”

“…Interestingly, these include genes for the recently characterized GRA24, GRA16, GRA28, and GRA31 [33,60,61]. Notably, GRA28 had the highest level of differential expression between the sporozoites and tachyzoites, with 12.5-fold higher expression in sporozoites than in tachyzoites ( q -value = 0.77% at FDR 10%).…”

“…Gene identification and gene product descriptions for Toxoplasma were obtained from ToxoDB release 29 (ToxoDB.org) and from published reports [31–33]. Metabolic Pathway Enrichment tool available on ToxoDB release 29 was used to determine enrichment in Toxoplasma differentially expressed gene sets.…”

Increased Expression of Toxoplasma Gondii GRA1 Suppresses Host Cell Apoptosis

“…15 Previously, we reported that T. gondii infection suppressed apoptosis of HeLa cells for 36hours post-infection in uninfected cells and in cells treated with actinomycin D. 16 T. gondii uses a mixture of specialized parasite secreted proteins, including ROPs, GRAs and MICs to invade cells. 17,18 Many of these parasite secreted proteins are anchored on the parasitophorous vacuole membrane (PVM) and can interact with the host cells directly. The kinase activity ROPs, such as ROP2, 19 …”

CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii

“…(2015) adapted BioID for use in T. gondii , identifying several novel protein components of the inner membrane complex (IMC). BioID has since been employed in T. gondii research to identify interactors of kinases (Gaji et al ., 2015), calmodulins (Long et al ., 2017a), and to define the protein repertoire of other cellular compartments including the parasitophorous vacuole (Nadipuram et al ., 2016), sutures of the IMC (Chen et al ., 2017), and the apical complex (Long et al ., 2017b). Here we will describe the protocol for generating a BirA gene fusions using CRISPR/Cas9 tagging (Shen et al ., 2014; Shen et al ., 2017), in vivo BirA biotin labeling and purification of biotinylated proteins from parasites, and identification of captured biotinylated proteins by mass-spectrometry.…”