Application and verification of auxin-inducible degron system in Toxoplasma gondii Chinese type 1 strain

doi:10.12140/j.issn.1000-7423.2020.02.012

Abstract

Abstract:

Objective To regulate the expression of fluorescent proteins in Toxoplasma gondii Chinese type 1 strain using the auxin-inducible degron (AID) system.Methods The nucleotide sequence of plant auxin receptor-transport inhibitor response protein 1 (TIR1) fused with a FLAG tag at the 3′ end (TIR1-FLAG) was synthesized, and ligated into the pTub-eGFP-CAT vector to obtain the pTub-TIR1-FLAG-CAT expression plasmid. The plasmid was transfected into the Toxoplasma gondii Chinese type 1 strain by electroporation. After chloramphenicol screening and limiting dilution, positive clones were selected, in which the inserted TIR1 gene and its expression was examined by PCR and Western blotting. The verified strain clone with correct insertion was designated as the TIR1 strain. The plasmid pTub-mCherry-Ty-AID-DHFR expressing the mCherry-Ty-AID reporter gene was constructed and integrated into the genome of the TIR1 strain by electroporation, from which a clone capable of stable expression of mCherry-AID protein was obtained by pyrimethamine screening and ultimate limiting dilution, and designated as TIRI:mCherry-AID strain. The TIR1:mCherry-AID parasites were divided into the regulating group and control group, and cultured with an auxin, indole-3-acetic acid (IAA) solution (500 μmol/L) or an equal volume of ethanol for 3 h accordingly. Then the expression of mCherry fluorescent protein was detected by indirect immunofluorescence assay (IFA) and Western blotting using anti-TgGAP45 and anti-Ty-1 antibodies.Results The PCR amplification showed a specific amplification band at 1 816 bp. Western blotting showed that the anti-FLAG-Tag antibody can recognize a protein band with a relative molecular mass (M_r) of 70 000 in the TIR1 strain, and anti-Ty-1 antibody can detect a specific band with M_r of about 58 000 in the TIR1:mCherry-AID strain, which equales to the theoretical size of mCherry-Ty protein fused with AID at the C terminus; however, no specific band was detected in the auxin/IAA group. IFA assay indicated that red fluorescent protein was seen in the TIR1:mCherry-AID in the control group, but no mCherry protein was detected in the auxin/IAA group.Conclusion The AID system can regulate mCherry fluorescent protein expression in T. gondii Chinses 1 strain.

Key words: Toxoplasma gondii, Indole-3-acetic acid, Auxin-inducible degron system, mCherry fluorescent protein

CLC Number:

R382.5

Ai-xia YAN, Yong-gen JIA, Yang ZOU, Jing-jing LI, Min-jun HUANG, Jun-chao GU. Application and verification of auxin-inducible degron system in Toxoplasma gondii Chinese type 1 strain[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(2): 207-212.

Figures/Tables 3

Fig. 1

Fig. 2

Fig. 3

References 24

[1]	Mendez OA, Koshy AA . Toxoplasma gondii: entry, association, and physiological influence on the central nervous system[J]. PLoS Pathog, 2017,13(7):e1006351.
[2]	Chattopadhyay S, Mahapatra RK . Identification of adaptive inhibitors of Cryptosporidium parvum fatty acyl-coenzyme A synthetase isoforms by virtual screening[J]. Parasitol Res, 2019,118(11):3159-3171.
[3]	Warncke JD, Beck HP . Host cytoskeleton remodeling throughout the blood stages of Plasmodium falciparum[J]. Microbiol Mol Biol Rev, 2019,83(4):e00013-e00019.
[4]	Ybañez AP, Arrabis OV, Alvarez DJM , et al. Evaluation on the presence of Anaplasma, Ehrlichia, and Babesia spp. in goats (Capra hircus) in Cebu, the Philippines[J]. Vet World, 2019,12(6):774-777.
[5]	Debbou-Iouknane N, Nerín C, Amrane M , et al. In vitro anticoccidial activity of olive pulp (Olea europaea L. var. Chemlal) extract against Eimeria oocysts in broiler chickens[J]. Acta Parasitol, 2019,64(4):887-897.
[6]	Dubey JP, Ferreira LR, Martins J , et al. Oral oocyst-induced mouse model of toxoplasmosis: effect of infection with Toxoplasma gondii strains of different genotypes, dose, and mouse strains (transgenic, out-bred, in-bred) on pathogenesis and mortality[J]. Parasitology, 2012,139(1):1-13.
[7]	Shen JL, Wang L . Genotypes and main effectors of Toxoplasma gondii and their pathogenic mechanisms[J]. Chin J Parasitol Parasit Dis, 2015,33(6):429-435. (in Chinese)
	( 沈继龙, 王林 . 弓形虫的基因型及其主要效应分子的致病机制[J]. 中国寄生虫学与寄生虫病杂志, 2015,33(6):429-435.)
[8]	Li S, Prasanna X, Salo VT , et al. An efficient auxin-inducible degron system with low basal degradation in human cells[J]. Nat Methods, 2019,16(9):866-869.
[9]	Shetty A, Reim NI, Winston F . Auxin-inducible degron system for depletion of proteins in Saccharomyces cerevisiae[J]. Curr Protoc Mol Biol, 2019,128(1):e104.
[10]	Sathyan KM , McKenna BD, Anderson WD, et al. An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion[J]. Genes Dev, 2019,33(19/20):1441-1455.
[11]	Yan AX, Zou Y, Huang MJ , et al. Preparation and its application of polyclonal antibody against Toxoplasma gondii TgPRF after fusion expression in prokaryotic system[J]. Chin Trop Med, 2019,19(7):634-637. (in Chinese)
	( 闫爱霞, 邹洋, 黄敏君 , 等. 刚地弓形虫TgPRF的原核表达、多克隆抗体制备与应用[J]. 中国热带医学, 2019,19(7):634-637.)
[12]	Jia YG, Yan AX, Huang MJ , et al. CRISPR/Cas9-based localization and functional analysis of Toxoplasma gondii putative protein TGGT1₃10420[J]. Chin J Parasitol Parasit Dis, 2019,37(2):150-154, 160. (in Chinese)
	( 贾永根, 闫爱霞, 黄敏君 , 等. 基于CRISPR/Cas9技术对刚地弓形虫假定蛋白TGGT1₃10420的研究[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(2):150-154, 160.)
[13]	Pinto-Ferreira F, Mitsuka-Breganó R, Monica TC , et al. Investigation and environmental analysis of samples from outbreak of toxoplasmosis at research institution in Londrina, Paraná, Brazil, 2016[J]. Braz J Vet Parasitol: Orgao Oficial Do Colegio Brasileiro De Parasitol Vet, 2019,28(3):518-521.
[14]	van Enter BJD, Lau YL, Ling CL , et al . Seroprevalence of Toxoplasma gondii infection in refugee and migrant pregnant women along the Thailand-Myanmar border[J]. Am J Trop Med Hyg, 2017,97(1):232-235.
[15]	El Kasmi KC, Qualls JE, Pesce JT , et al. Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens[J]. Nat Immunol, 2008,9(12):1399-1406.
[16]	Stumhofer JS, Laurence A, Wilson EH , et al. Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system[J]. Nat Immunol, 2006,7(9):937-945.
[17]	Jensen KD, Wang Y, Wojno ED , et al. Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation[J]. Cell Host Microbe, 2011,9(6):472-483.
[18]	Wang C, Cheng WS, Yu Q , et al. Toxoplasma Chinese 1 strain of WH3Δrop16Ⅰ/Ⅲ/gra15Ⅱ genetic background contributes to abnormal pregnant outcomes in murine model[J]. Front Immunol, 2018,9:1222.
[19]	Meissner M, Brecht S, Bujard H , et al. Modulation of myosin A expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii[J]. Nucleic Acids Res, 2001,29(22):E115.
[20]	Armstrong CM, Goldberg DE . An FKBP destabilization domain modulates protein levels in Plasmodium falciparum[J]. Nat Methods, 2007,4(12):1007-1009.
[21]	Brown KM, Long SJ, Sibley LD . Conditional knockdown of proteins using auxin-inducible degron (AID) fusions in Toxoplasma gondii[J]. Bio Protoc, 2018,8(4):e2728.
[22]	Banaszynski LA, Chen LC, Maynard-Smith LA , et al. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules[J]. Cell, 2006,126(5):995-1004.
[23]	Jia YG, Marq JB, Bisio H , et al. Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii[J]. EMBO J, 2017,36(21):3250-3267.
[24]	Nishimura K, Fukagawa T, Takisawa H , et al. An auxin-based degron system for the rapid depletion of proteins in nonplant cells[J]. Nat Methods, 2009,6(12):917-922.