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.

Keywords: CRISPR-Cas9; Toxoplasma gondii; dense granule proteins (GRAs); host-pathogen interaction; virulence.

Figure 1

Overview of the CRISPR/Cas9 system and mutation analysis of Toxoplasma gondii dense granule proteins (GRA) genes. (A) Schematic representation of CRISPR-Cas9 system used for disrupting the 17 GRAs genes by insertion of DHFR^*-Ts cassette. (B,C) Diagnostic PCR shows GRA gene disruption in the mutants compared to the wild-type strain. The KO-forward and KO-reverse primers were used to amplify the small fragment with 30 s extension time. (D,E) RT-PCR of mRNA from parental RH (WT) and GRA-deficient strains, showing that the 17 GRA's coding sequences were successfully disrupted. Marker denotes the DNA ladder.

Figure 2

Phenotypic characterization of GRA Knockouts in vitro. (A) Two hundred freshly harvested T. gondii tachyzoites of WT RH strains and GRA-deficient RH strains per well were added to monolayers of HFF cells in 6-well culture plates. After 7 days, the number of plaques caused by the parasite's proliferation was counted using a microscope. No differences were detected in the number of plaques produced by wild-type (WT) RH strain vs. any of the GRA knockout strains. (B) Representative images show the parasite egress of the parental WT RH strain and one of the GRA mutant RH strains (Δgra11). Live cell imaging showed a similar egress pattern between WT and all mutant parasites after addition of 3 μM calcium ionophore A23187 at the indicated time points.

Figure 3

Survival of BALB/c mice infected with Toxoplasma gondii wild-type or GRA-deficient RH strains. The mice were injected i.p., with 200 freshly harvested tachyzoites of the indicated strains. Ten mice were used per parasite strain. The survival time was recorded daily until all the mice have died within 7–9 days post challenge.

Figure 4

The trend charts of the distinct expression profiles of Toxoplasma gondii GRAs. (A) Time-series expression profile of 17 GRA genes of T. gondii RH strain by cell cycle phases of the parasite as described by Behnke et al. (2010). (B) Transcriptomic expression profiles of 17 GRA genes in Type I (RH and GT1), Type II (Pru and ME49), and Type III (CTG and VEG) strains. (C) Transcriptomic profiles of 17 GRA genes related to the parasite life cycle stages (oocyst, tachyzoite and bradyzoite). Expression profile of 17 GRA genes of the oocysts recovered from cat feces, at 0 day (unsporulated), 4 days (4 day sporulated), and 10 days (10 day sporulated), tachyzoites grown for 2 days in HFF cells (2 day in vitro), bradyzoites grown in HFF cells for 4 days and 8 days (4 day in vitro and 8 day in vitro), and 21 days tissue cyst-containing bradyzoites harvested from infected mouse brains (21 day in vivo). Each line represents the expression value of the corresponding gene. The data were obtained from ToxoDB (36 release) and the graph was generated using GraphPad Prism version 5.0.