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Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals dramatic differences between strains.

ABSTRACT: We examined the in vivo growth, dissemination, and reactivation of strains of the protozoan parasite Toxoplasma gondii using a bioluminescence-based imaging system. Two T. gondii strains, one with a highly virulent disease phenotype in mice (S23) and the other with a 1,000-fold-lower virulence phenotype (S22), were engineered to stably express the light-emitting protein luciferase. One clone of each wild-type strain was isolated, and the two clones (S23-luc7 and S22-luc2) were found to express similar levels of luciferase. Mice were infected intraperitoneally with S23-luc7 (50 or 5 parasites) or S22-luc2 (500, 50, or 5 parasites), and the progress of the infections was examined noninvasively following injection of the substrate for luciferase, d-luciferin. In mice infected with 50 S23-luc7 parasites, the parasites grew exponentially within the peritoneal cavity (as measured by light emitted from luciferase-expressing parasites) during days 1 to 10 p.i., and this proliferation continued until there was severe disease. In mice infected with 500 S22-luc2 parasites, the parasites proliferated in a fashion similar to the S23-luc7 proliferation during days 1 to 6, but this was followed by a precipitous drop in the signal to levels below the limit of detection. Using this technique, we were also able to observe the process of reactivation of T. gondii in chronically infected mice. After treatment with dexamethasone, we detected reactivation of toxoplasmosis in mice infected with S23-luc7 and S22-luc2. During reactivation, growth of S23-luc7 was initially detected primarily in the head and neck area, while in S22-luc2-infected mice the parasites were detected primarily in the abdomen. This method has great potential for identifying important differences in the dissemination and growth of different T. gondii strains, especially strains with dramatically different disease outcomes.

SUBMITTER: Saeij JP

PROVIDER: S-EPMC547072 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals dramatic differences between strains.

Saeij Jeroen P J JP Boyle Jon P JP Grigg Michael E ME Arrizabalaga Gustavo G Boothroyd John C JC

Infection and immunity 20050201 2

We examined the in vivo growth, dissemination, and reactivation of strains of the protozoan parasite Toxoplasma gondii using a bioluminescence-based imaging system. Two T. gondii strains, one with a highly virulent disease phenotype in mice (S23) and the other with a 1,000-fold-lower virulence phenotype (S22), were engineered to stably express the light-emitting protein luciferase. One clone of each wild-type strain was isolated, and the two clones (S23-luc7 and S22-luc2) were found to express s ...[more]

PMID: 15664907

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Project description:BackgroundToxoplasma gondii has a largely clonal population in North America and Europe, with types I, II and III clonal lineages accounting for the majority of strains isolated from patients. RH, a particular type I strain, is most frequently used to characterize Toxoplasma biology. However, compared to other type I strains, RH has unique characteristics such as faster growth, increased extracellular survival rate and inability to form orally infectious cysts. Thus, to identify candidate genes that could account for these parasite phenotypic differences, we determined genetic differences and differential parasite gene expression between RH and another type I strain, GT1. Moreover, as differences in host cell modulation could affect Toxoplasma replication in the host, we determined differentially modulated host processes among the type I strains through host transcriptional profiling.ResultsThrough whole genome sequencing, we identified 1,394 single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) between RH and GT1. These SNPs/indels together with parasite gene expression differences between RH and GT1 were used to identify candidate genes that could account for type I phenotypic differences. A polymorphism in dense granule protein, GRA2, determined RH and GT1 differences in the evasion of the interferon gamma response. In addition, host transcriptional profiling identified that genes regulated by NF-ĸB, such as interleukin (IL)-12p40, were differentially modulated by the different type I strains. We subsequently showed that this difference in NF-ĸB activation was due to polymorphisms in GRA15. Furthermore, we observed that RH, but not other type I strains, recruited phosphorylated IĸBα (a component of the NF-ĸB complex) to the parasitophorous vacuole membrane and this recruitment of p- IĸBα was partially dependent on GRA2.ConclusionsWe identified candidate parasite genes that could be responsible for phenotypic variation among the type I strains through comparative genomics and transcriptomics. We also identified differentially modulated host pathways among the type I strains, and these can serve as a guideline for future studies in examining the phenotypic differences among type I strains.

Project description:In T. gondii, as well as in other model organisms, gene knock-out using CRISPR-Cas9 is a suitable tool to identify the role of specific genes. The general consensus implies that only the gene of interest is affected by the knock-out. Is this really the case? In a previous study, we generated knock-out (KO) clones of TgRH88_077450 (SRS29B; SAG1) which differed in the numbers of the integrated dihydrofolate-reductase-thymidylate-synthase (MDHFR-TS) drug-selectable marker. Clones 18 and 33 had a single insertion of MDHFR-TS within SRS29B. Clone 6 was disrupted by the insertion of a short unrelated DNA-sequence, but the marker was integrated elsewhere. In clone 30, the marker was inserted into SRS29B, and several other MDHFR-TS copies were found in the genome. KO and wild-type (WT) tachyzoites had similar shapes, dimensions, and vitality. This prompted us to investigate the impact of genetic engineering on the overall proteome patterns of the four clones as compared to the respective WT. Comparative shotgun proteomics of the five strains was performed. Overall, 3236 proteins were identified. Principal component analysis of the proteomes revealed five distinct clusters corresponding to the five strains by both iTop3 and iLFQ algorithms. Detailed analysis of the differentially expressed proteins revealed that the target of the KO, srs29B, was lacking in all KO clones. In addition to this protein, 20 other proteins were differentially expressed between KO clones and WT or between different KO clones. The protein exhibiting the highest variation between the five strains was srs36D encoded by TgRH_016110. The deregulated expression of SRS36D was further validated by quantitative PCR. Moreover, the transcript levels of three other selected SRS genes, namely SRS36B, SRS46, and SRS57, exhibited significant differences between individual strains. These results indicate that knocking out a given gene may affect the expression of other genes. Therefore, care must be taken when specific phenotypes are regarded as a direct consequence of the KO of a given gene.

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Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals dramatic differences between strains.

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Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals dramatic differences between strains.

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