Functional Characterization of Dense Granule Proteins in Toxoplasma gondii RH Strain Using CRISPR-Cas9 System

Abstract

Infection with the apicomplexan protozoan parasite Toxoplasma gondii is an ongoing public health problem. The parasite's ability to invade and replicate within the host cell is dependent on many effectors, such as dense granule proteins (GRAs) released from the specialized organelle dense granules, into host cells. GRAs have emerged as important determinants of T. gondii pathogenesis. However, the functions of some GRAs remain undefined. In this study, we used CRISPR-Cas9 technique to disrupt 17 GRA genes (GRA11, GRA12 bis, GRA13, GRA14, GRA20, GRA21, GRA28-31, GRA33-38, and GRA40) in the virulent T. gondii RH strain. The CRISPR-Cas9 constructs abolished the expression of the 17 GRA genes. Functional characterization of single ΔGRA mutants was achieved in vitro using cell-based plaque assay and egress assay, and in vivo in BALB/c mice. Targeted deletion of these 17 GRA genes had no significant effect neither on the in vitro growth and egress of the mutant strains from the host cells nor on the parasite virulence in the mouse model of infection. Comparative analysis of the transcriptomics data of the 17 GRA genes suggest that GRAs may serve different functions in different genotypes and life cycle stages of the parasite. In sum, although these 17 GRAs might not be essential for RH strain growth in vitro or virulence in mice, they may have roles in other strains or parasite stages, which warrants further investigations.

Novel roles of dense granule protein 12 (GRA12) inToxoplasma gondiiinfection

“…While the functions of most of GRA proteins are not studied elaborately, analyses of a few of these proteins showed that GRAs play crucial roles in vacuole remodeling and nutrient uptake, 46 and immune evasion; although they are not essential for parasite growth in vitro. 31 Previous immunofluorescence and immuno-electron microscopy showed that GRA12 behave similarly to both GRA2 and GRA6, in regard to secretion into the PV and association with the IVN. 32 Consistent with previously reported data, we found that GRA12 is secreted into the PV, co-localized with GRA2 detected using polyclonal rabbit anti-GRA12 or anti-HA antibody, and is associated with the IVN.…”

“…These studies have indicated that T. gondii genome encodes more than 50 GRA proteins. While the functions of most of GRA proteins are not studied elaborately, analyses of a few of these proteins showed that GRAs play crucial roles in vacuole remodeling and nutrient uptake, and immune evasion; although they are not essential for parasite growth in vitro . Previous immunofluorescence and immuno‐electron microscopy showed that GRA12 behave similarly to both GRA2 and GRA6, in regard to secretion into the PV and association with the IVN .…”

“…Two GRA12 genes (TGME49_275850 and TGME49_288650) were identified in the ToxoDB (https://toxodb.org). The TGME49_275850 gene was renamed as GRA12bis and was found non‐essential for the parasites' lytic cycle and virulence . Moreover, GRA12 (TGME49_288650) associated with the IVN in the PV, is co‐localized with GRA2 and GRA6, however its function remains unknown .…”

“…30 The TGME49_275850 gene was renamed as GRA12bis and was found non-essential for the parasites' lytic cycle and virulence. 31 Moreover, GRA12 (TGME49_288650) associated with the IVN in the PV, is co-localized with GRA2 and GRA6, however its function remains unknown. 32 In the present study, we investigated the functions of GRA12 by deleting gra12 gene in type I RH and type II Pru strains.…”

A subcellular atlas of Toxoplasma reveals the functional context of the proteome

“…In Toxoplasma, previous efforts to identify new candidate proteins for select compartments have used the correlation of transcript abundance profiles across the cell cycle making the assumption that co-located proteins are coexpressed (Bai et al, 2018;Lacombe et al, 2019;Long et al, 2017;Sheiner et al, 2011). However, an objective assessment of the coordination of gene expression for compartment proteins has not been previously possible without comprehensive knowledge of the spatial distribution of the proteome.…”