Functional Analysis of the Role of Toxoplasma gondii Nucleoside Triphosphate Hydrolases I and II in Acute Mouse Virulence and Immune Suppression

Abstract

Bioluminescent reporter assays have been widely used to study the effect of Toxoplasma gondii on host gene expression. In the present study, we extend these studies by engineering novel reporter cell lines containing a gamma-activated sequence (GAS) element driving firefly luciferase (FLUC). In RAW264.7 macrophages, T. gondii type I strain (GT1) infection blocked interferon gamma (IFN-γ)-induced FLUC activity to a significantly greater extent than infection by type II (ME49) and type III (CTG) strains. Quantitative trait locus (QTL) analysis of progeny from a prior genetic cross identified a genomic region on chromosome XII that correlated with the observed strain-dependent phenotype. This QTL region contains two isoforms of the T. gondii enzyme nucleoside triphosphate hydrolase (NTPase) that were the prime candidates for mediating the observed strain-specific effect. Using reverse genetic analysis we show that deletion of NTPase I from a type I strain (RH) background restored the higher luciferase levels seen in the type II (ME49) strain. Rather than an effect on IFN-γ-dependent transcription, our data suggest that NTPase I was responsible for the strain-dependent difference in FLUC activity due to hydrolysis of ATP. We further show that NTPases I and II were not essential for tachyzoite growth in vitro or virulence in mice. Our study reveals that although T. gondii NTPases are not essential for immune evasion, they can affect ATP-dependent reporters. Importantly, this limitation was overcome using an ATP-independent Gaussia luciferase, which provides a more appropriate reporter for use with T. gondii infection studies.

FIG 1

Toxoplasma gondii strain-dependent inhibition of luminescence in IFN-γ-inducible FLUC reporter lines. A tandem GAS element-driven FLUC reporter was stably expressed in HeLa cells (A) or RAW264.7 macrophages (B). Reporter cell lines were plated in 96-well plates and infected with different MOIs, as indicated. At 2 h after infection, the cells were stimulated with 100 U of IFN-γ/ml for 18 h. Subsequently, the cells were lysed, and the luciferase activity was measured. The data shown are means ± standard errors of the mean (SEM) of at least three independent experiments, each with three technical replicates. Kruskal-Wallis test with Dunn's correction (***, P < 0.0001) was performed for GT1 and each of the other T. gondii strains.

FIG 2

Mapping of a single target locus on chromosome XII. (A) GT1 (type I) and ME49 (type II) parental strains and 36 progeny were tested for their ability to interfere with the GAS-FLUC in RAW264.7 macrophages. The data shown are normalized averages of three technical replicates ± standard deviations (SD). (B and C) QTL scan of normalized percent inhibition identified a single locus of ∼688 kb (TGME49_chrXII: 6134501.0.6822749) on chromosome XII defined by the two markers 181 and 193 and the progeny SF50 and SF34. Fine mapping with additional PCR markers (see Table S2 in the supplemental material) within the QTL identified two additional informative progeny (2C7C3 and SF59) and narrowed the region responsible for the observed strain difference to ∼263 kb (TGME49_chrXII: 6559896.0.6822749) comprising 36 annotated genes.

FIG 3

Generation and verification of NTPase I and II single- and double-disruptant mutants. (A) Diagram of single NTPase II (Δntpase II; RHΔhxgprt/ntpaseII::DHFR) and NTPase I (Δntpase I; RHΔhxgprt/ntpaseI::DHFR) mutants and two versions of double-knockout mutants (Δntpase II Δntpase I, RHΔhxgprt/ntpaseII::DHFR/ntpaseI::HXGPRT; Δntpase II Δntpase I v.2, RHΔhxgprt/ntpaseII::HXGPRT/ntpaseI::HXGPRT) generated by CRISPR/Cas9 guided gene disruption. The pseudogene (PG) lacks a suitable start codon and therefore is not transcribed (30). However, it shares complete sequence homology in part with either NTPase II or I for which reason the guide RNA II targets both NTPase II and the pseudogene resulting in a double cut. The type I RH strain (Δhxgprt) was the background for all the mutants described. For the locations and sequences of guides, see Table S3 in the supplemental material. (B) Diagnostic PCR of disruptant mutants with primer pair locations as indicated in panel A. For primer locations and sequences, see Table S3 in the supplemental material. (C) Western blot analysis of protein loss in NTPase disruptant mutants. GRA7 served as loading control.

FIG 4

Growth, virulence, and interference with STAT1 pathway. (A) Plaque assay on HFF monolayers examining growth of wild-type and NTPase knockout parasites. (B) Virulence of NTPase knockout strains in CD-1 mice infected intraperitoneally with 200 tachyzoites per mouse. Groups of five mice per strain were infected, and the survival was monitored. The experiment was repeated once, and the results of both experiments were plotted together. (C) A fluorescence assay performed to examine the interference of wild-type T. gondii with IRF1 protein expression as a readout for STAT1-activated transcription compared to NTPase knockout strains showed no NTPase-dependent effect. HFF monolayers were infected for 2 h and subsequently activated with 100 U of IFN-γ/ml for 5 h. Quantification of the IRF1 fluorescence of infected cells versus controls. Data points represent the mean red fluorescence in a ROI overlaying the host cell nucleus. IRF1, red; DAPI, blue; T. gondii-GFP, green. Scale bar, 10 μm. ***, P < 0.0001 (Kruskal-Wallis with Dunn's correction).

FIG 5

Influence of NTPases I and II on firefly and Gaussia luciferase reporter lines. IFN-γ-inducible tandem GAS element-driven FLUC reporter lines were infected with different MOIs for 3 h and subsequently stimulated with 100 U of IFN-γ/ml for 18 h. NTPase I and double-knockout mutants were significantly less capable at suppressing FLUC activity in both RAW264.7 macrophages (A) and HeLa cells (B) than the wild-type RH strain. Asterisks (*) indicate a P value of <0.05 between the wild type and NTPase I and double-knockout mutants. (C) The addition of ATP during cell lysis or the absence of DTT in the lysis buffer partially reversed the interference with the firefly luciferase reporter. Asterisks (*) indicate a P value of <0.05 between cell lysis with or without DTT. (D) An IFN-γ-inducible tandem GAS element-driven ATP-independent GLUC RAW264.7 macrophage reporter line showed no NTPase-dependent effect. The data shown are averages ± SEM of at least three independent experiments, each performed with three technical replicates (A, C, and D), and averages ± SD of two independent experiments with three technical replicates (B).