Functional Characterization of Two Thioredoxin Proteins of Toxoplasma gondii Using the CRISPR-Cas9 System

Abstract

Toxoplasmosis caused by infection with Toxoplasma gondii is an important parasitic zoonosis with a worldwide distribution. In this study, we examined the functions of two thioredoxins (namely CTrp26 and CTrx1) of T. gondii tachyzoites by generation of HA tag strains or gene deficient parasites in Type I RH strain (ToxoDB#10). Immunofluorescence analysis (IFA) was used to investigate the subcellular localization of the thioredoxins (Trxs). Results of IFA showed that both CTrp26 and CTrx1 were located in the cytoplasm of T. gondii. Functional characterizations of CTrp26 and CTrx1-deficient parasites were performed by plaque assay, intracellular replication, egress, H2O2 resistance, detection of reactive oxygen species (ROS) level and total antioxidant capacity (T-AOC) assays in vitro, as well as mouse infection in vivo. Our results showed that deletion of CTrp26 or CTrx1 did not influence the ability of T. gondii RH strain to replicate, egress, form plaque, resist H2O2 exposure, maintain the ROS level, and T-AOC, and also did not serve as virulence factors in Kunming mice. Taken together, these results provide new properties of the two Trxs. Although they are not essential for RH strain, they may have roles in other strains of this parasite due to their different expression patterns, which warrants future research.

Functional Characterization of 17 Protein Serine/Threonine Phosphatases in Toxoplasma gondii Using CRISPR-Cas9 System

“…To determine the distribution of PSPs in type Ⅰ T. gondii RH strain, C-terminal endogenous tagging was performed as previously described ( Zhang et al, 2021 ). The specific primers of each TgPSP were designed, and a fragment containing approximately 42 bp of the 3′ region of the psp gene (without the STOP codon), 6 × HA tag products were amplified by using p6 × HA-LIC-DHFR as a template.…”

Functional Characterization of 15 Novel Dense Granule Proteins in Toxoplasma gondii Using the CRISPR-Cas9 System

“…In acute infection, 100 freshly egressed tachyzoites of the RHΔ gra mutant strains and the wild-type RH strain suspended in 200 μL PBS were injected intraperitoneally (i.p.) into mice (6 mice/strain) ( 64 , 65 ). The viability of the parasites used to infect mice was determined by a plaque assay.…”

Temporal transcriptomic changes in long non-coding RNAs and messenger RNAs involved in the host immune and metabolic response during Toxoplasma gondii lytic cycle

“…The cell culture was maintained at 37 °C and 5% CO 2 . The RH (Type I) strain used in the present study was maintained in HFF cells as described previously [ 24 ].…”