Abstract
Schistosoma is the causative agent of schistosomiasis, a common infectious disease distributed worldwide. Our previous phosphoproteomic analysis suggested that glycogen synthase kinase 3 (GSK3), a conserved protein kinase in eukaryotes, is likely involved in protein phosphorylation of Schistosoma japonicum. Here, we aimed to identify the interacting partners of S. japonicum GSK3β (SjGSK3β) and to evaluate its role in parasite survival. Toward these ends, we determined the transcription levels of SjGSK3β at different developmental stages and identified its interacting partners of SjGSK3β by screening a yeast two-hybrid S. japonicum cDNA library. We further used RNA interference (RNAi) to inhibit the expression of SjGSK3β in adult worms in vitro and examined the resultant changes in transcription of its putative interacting proteins and in worm viability compared with those of control worms. Reverse transcription-quantitative polymerase chain analysis indicated that SjGSK3β is expressed throughout the life cycle of S. japonicum, with higher expression levels detected in the eggs and relatively higher expression level found in male worms than in female worms. By screening the yeast two-hybrid library, eight proteins were identified as potentially interacting with SjGSK3β including cell division cycle 37 homolog (Cdc37), 14-3-3 protein, tegument antigen (I(H)A), V-ATPase proteolipid subunit, myosin alkali light chain 1, and three proteins without recognized functional domains. In addition, SjGSK3β RNAi reduced the SjGSK3β gene transcript level, leading to a significant decrease in kinase activity, cell viability, and worm survival. Collectively, these findings suggested that SjGSK3β may interact with its partner proteins to influence worm survival by regulating kinase activity.
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This study was, in part or in whole, supported by the National Natural Science Foundation of China (31472187 and 31672550) and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.
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All animal experiments were carried out strictly following the guidelines for animal use with approval from Shanghai Laboratory Animal Management Committee and the Animal Care and Use Committee of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (permit number: SYXK 2016-0010).
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ESM 1
Evaluation of the self-activation of pBT3SUC-SjGSK3β plasmid. NMY32 yeast cells were transformed with different combinations of plasmids, including pNubG-Fe65 + pTSU2-APP (positive control, lane 1), pPR3N + pTSU2-APP (negative control, lane 2), pPR3N + pBT3SUC-SjGSK3β (self-activation, lane 3) and pOST1-NubI + pBT3SUC-SjGSK3β (functional evaluation, lane 4) and cultured on SD-Trp-Leu (SD-TL), SD-Trp-Leu-His (SD-TLH), and SD-Trp-Leu-His-Ade (SD-TLHA) agars plates. The number of colonies was counted. (PNG 817 kb)
ESM 2
β-galactosidase assay for colonies obtained by screening the yeast two hybrid library using pBT3SUC-SjGSK3β as the bait plasmid. A total of 34 colonies that grew on SD-TL agar were inoculated in SD-TL liquid medium and cultured for the β-galactosidase assay. The β-galactosidase activity was measured according to the optical density (OD) 615 nm and 546 nm and calculated as the OD615/OD546 ratio. (PNG 384 kb)
ESM 3
β-galactosidase assay for yeast cells containing bait plasmid transformed with prey plasmids. NYM32 yeast cells containing the pBT3SUC-SjGSK3β bait plasmid were transformed with 15 prey plasmids and cultured on SD-TL agar. Fifteen colonies that grew on SD-TL agar were inoculated in SD-TL liquid medium and cultured for the β-galactosidase assay. The β-galactosidase activity was measured based on the optical density (OD) 615 nm and 546 nm and calculated as the OD615/OD546 ratio. (PNG 238 kb)
ESM 4
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Liu, J., Li, H., Xia, T. et al. Identification of Schistosoma japonicum GSK3β interacting partners by yeast two-hybrid screening and its role in parasite survival. Parasitol Res 119, 2217–2226 (2020). https://doi.org/10.1007/s00436-020-06731-2
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DOI: https://doi.org/10.1007/s00436-020-06731-2