A Carbamoyl Phosphate Synthetase II (CPSII) Deletion Mutant of Toxoplasma gondii Induces Partial Protective Immunity in Mice

Abstract

Toxoplasma gondii is an obligate intracellular protozoan parasite. T. gondii primarily infection in pregnant women may result in fetal abortion, and infection in immunosuppressed population may result in toxoplasmosis. Carbamoyl phosphate synthetase II (CPSII) is a key enzyme in the de novo pyrimidine-biosynthesis pathway, and has a crucial role in parasite replication. We generated a mutant with complete deletion of CPSII via clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 in type-1 RH strain of T. gondii. We tested the intracellular proliferation of this mutant and found that it showed significantly reduced replication in vitro, though CPSII deletion did not completely stop the parasite growth. The immune responses induced by the infection of RHΔCPSII tachyzoites in mice were evaluated. During infection in mice, the RHΔCPSII mutant displayed notable defects in replication and virulence, and significantly enhanced the survival of mice compared with survival of RH-infected mice. We tracked parasite propagation from ascitic fluid in mice infected with the RHΔCPSII mutant, and few tachyzoites were observed at early infection. We also observed that the RHΔCPSII mutant induced greater accumulation of neutrophils. The mutant induced a higher level of T-helper type-1 cytokines [interferon (IFN)-γ, interleukin (IL)-12]. The mRNA levels of signal transducer and activator of transcription cellular transcription factor 1 and IFN regulatory factor 8 were significantly higher in the RHΔCPSII mutant-infected group. Together, these data suggest that CPSII is crucial for parasite growth, and that strains lack the de novo pyrimidine biosynthesis pathway and salvage pathway may become a promising live attenuated vaccine to prevent infection with T. gondii.

Keywords: CRISPR/Cas9; Toxoplasma gondii; carbamoyl phosphate synthetase II; immunization; vaccine.

FIGURE 1

Generation and characterization of a CPSII deletion mutant. (A) Schematic illustration of knocking out CPSII by CRISPR/Cas9-mediated homologous gene replacement in the RH Δku80 strain. (B) Diagnostic PCRs on a ΔCPSII clone. (C) Observation of intracellular replication of ΔCPSII RH strain with or without the addition of 200 μM uracil at 24 hpi by IFA using anti-GRA7 rabbit polyclonal antibody. (D) Intracellular replication assay of ΔCPSII RH strain with or without the addition of 200 μM uracil. The number of parasites in each parasitophorous vacuole (PV) was determined at 48 hpi. Values are shown as means ± SEM from three independent experiments. **P < 0.01 by two-way ANOVA.

FIGURE 2

Virulence test of ΔCPSII RH strain in mice. BALB/c (A) or ICR (B) mice were infected through intraperitoneal injection with 10⁴ tachyzoites of RH strain or ΔCPSII RH strain, each group comprised 10 mice. ***p < 0.001, Kaplan–Meier product limit test. (C) Diagnostic PCRs of ascitic fluid samples collected from toxoplasma-infected mice.

FIGURE 3

Parasite burden in ΔCPSII RH strain-infected mice. (A) Parasites in ascitic fluid collected from RH strain or ΔCPSII RH strain-infected ICR mice at 4 or 10 dpi were calculated by hemocytometers; each point means one mouse. (B) DNA copies of T. gondii in liver tissues 4 or 10 dpi were determined by real-time PCR based on 529 bp; each point means one mouse. ***p < 0.001 by Student’s t-test.

FIGURE 4

Percentage of neutrophils (A) and lymphocytes (B) and absolute number of neutrophils (C) and lymphocytes (D) in RH strain or ΔCPSII RH strain-infected ICR mice at 1, 4, or 10 dpi; each point means one mouse; NS, no significant, *p < 0.05, **p < 0.01 by two-way ANOVA.

FIGURE 5

IFN-γ (A) and IL-12 (B) production in RH strain or ΔCPSII RH strain-infected ICR mice at 2, 4, 8, or 10 dpi; each point means one mouse; *p < 0.05, **p < 0.01 by two-way ANOVA.

FIGURE 6

The mRNA level of stat1 and irf8 in PBS or ΔCPSII RH strain-infected ICR mice at 10 dpi was evaluated by real-time PCR; n = 5, **p < 0.01, ***p < 0.001 by Student’s t-test.