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Toxoplasma gondii nucleus coding apicoplast protein ACP synthesis and trafficking in delayed death.

Parasitology research (2015-01-08)
Liang Wu, Jin Shen, Yupei Zhou, Xiao Wang, Lamei Wu, Xugan Jiang, Shengxia Chen
ABSTRACT

This study aimed to explore Toxoplasma gondii nucleus coding apicoplast protein acyl carrier protein (ACP) synthesis and trafficking in delayed death. The recombinant T. gondii ACP was expressed by prokaryotic expression method, and anti-ACP polyclonal antibody was obtained from rabbit immune. T. gondii "delayed death" was induced by clindamycin (CLDM), and ACP transcription was determined by real-time PCR assay. The expression of ACP with transit type (t-ACP) and mature type (m-ACP) was determined by Western blotting with anti-ACP polyclonal antibody. The mutant-expressed ACP fused with green fluorescent protein (GFP) tag was constructed by pHX-ACP-GFP. The distribution of ACP in "delayed death" was observed by ACP-GFP fusion protein with a confocal microscope. T. gondii ACP transcription and t-ACP expression had no significant decrease in the early 4 h of "delayed death," but there has been a significant decrease in 6 h. The expression of m-ACP had a significant decrease in 4 h which occurred earlier than the t-ACP expression. The number of brightly dot green fluorescence in ACP-GFP mutant decreased with prolonged time. There was very little brightly dot green fluorescence in ACP-GFP mutant when treated with CLDM for 6 h. CLDM could suppress apicoplast proliferation and induce T. gondii "delayed death"; however, it could not directly suppress nucleus coding ACP transcription and expression. T. gondii lacking of apicoplast had a barrier of transit peptide cleavage and t-ACP could not be transformed into m-ACP. The reason for the decrease in ACP expression could be due to excessive t-ACP synthesis in tachyzoites resulting in a negative feedback for the ACP coding gene transcription.

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