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. 2016 Dec 27;7(52):87351-87360.
doi: 10.18632/oncotarget.13568.

Activation of chronic toxoplasmosis by transportation stress in a mouse model

Bang Shen  1 , Yuan Yuan  1 , Jianxi Cheng  1 , Ming Pan  1 , Ningbo Xia  1 , Weichao Zhang  1 , Yifan Wang  1 , Yanqin Zhou  1 , Junlong Zhao  1   2
Affiliations

Activation of chronic toxoplasmosis by transportation stress in a mouse model

Bang Shen et al. Oncotarget. .

Abstract

Toxoplasma gondii is an obligate intracellular parasite infecting 25% of the world population and enormous number of animals. It can exist in two forms in intermediate hosts: the fast replicating tachyzoites responsible for acute infection and the slowly replicating bradyzoites responsible for life-long chronic infection. The interconversion between tachyzoites and bradyzoites plays critical roles in the transmission and pathogenesis of T. gondii. However, the molecular mechanisms that govern the interconversion are largely unknown. In this study, we established a chronic infection model in mice and examined the impact of transportation stress on the status of chronic infection. Our results demonstrated that, treating chronically infected mice with conditions mimicking transportation stress reduced the levels of several key cytokines that restrict the infection at chronic stage. Increased expression of the tachyzoite specific gene SAG1 (surface antigen 1) was detected in brain cysts of stress treated mice, indicating activation and conversion of bradyzoites to tachyzoites. Using this model, we identified fifteen toxoplasmic proteins that had significant abundance changes during stress induced cysts reactivation. These proteins serve as a basis for further investigation of the mechanisms governing bradyzoite conversion.

Keywords: Toxoplasma gondii; bradyzoite; chronic infection; reactivation; transportation stress.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declared that they have no conflicts of interest.

Figures

Figure 1
Figure 1. Relative expression levels of BAG1 and SAG1 in the brains of chronically infected mice before and after transportation stress
The T. gondii PRU strain was used to infect Kunming mice, six weeks post infection, chronically infected mice were placed under conditions mimicking transportation stress for 3 days (no treatment as negative control, 3 mice each group). Subsequently, total RNA was extracted from mice brains and RT-PCR was used to determine the mRNA levels for BAG1 and SAG1. β-tubulin was used as internal control. Means ± SD from three independent experiments were graphed, *P <0.05, student's t-test.
Figure 2
Figure 2. Cytokine level changes induced by chronic T. gondii infection
Expression levels of the 20 indicated cytokines in peripheral blood sera of uninfected vs chronically infected mice were determined by mice cytokine array chips. A. Typical results from the scanning of cytokine array chips (each had four internal replicates). The data shown were from two chips, one for an uninfected mouse (no infection) and one for a chronically infected mouse (post infection). POS1 and POS2 indicated positive controls. B. Histogram quantification of results from three independent experiments of (A). As a way for data normalization, the average level of each cytokine in uninfected mice was set to 1. Cytokine levels in chronically infected mice after data normalization were graphed. Only the ones with significant difference (compared to uninfected control) were shown. IL-9 was included as an example with no significant changes. Means ± SD, *P <0.05, student's t-test.
Figure 3
Figure 3. Cytokine level changes in chronically infected mice during transportation stress
Kunming mice chronically infected with the PRU strain were placed under transportation stress conditions, peripheral blood was taken at day 0 (before stress), 1, 2 and 3 respectively and the levels of cytokines in the sera were determined by cytokine array chips as in Figure 2. Means ± SD from three independent experiments, *P <0.05, **P <0.005, ***P <0.0001, student's t-test.
Figure 4
Figure 4. Two-dimensional gel electrophoresis to identify differentially expressed proteins during transportation stress induced activation of chronic infection
Total proteins were extracted from T. gondii cysts (also contained host materials) of chronically infected mice underwent transportation stress for indicated amount of time and subsequently used in 2D gel electrophoresis. A. SDS-PAGE analysis to check the quality of protein samples extracted from brain cysts. Protein samples (20 μg each) were separated on 8% SDS-PAGE gel and stained with coomassie blue. B-E. Two-dimensional gel electrophoresis of protein samples in (A). A total of 150 μg proteins from each sample were first separated by isoelectric focusing electrophoresis and subsequently by SDS-PAGE, stained with sliver nitrate, scanned to determine the intensity of each spot. Proteins in the 24, 48 and 72 hour samples with abundance higher than 2 fold (up-regulated) or less than 50% (down-regulated) compared with the 0 hour sample (no stress) were identified and indicated (blue arrows indicate down-regulated proteins and red arrows indicate up-regulated ones). Experiments were repeated three times independently and only the ones with significant difference were marked on these representative gels.
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